590 results on '"Ralph Knöll"'
Search Results
2. Frequency-dependent signaling in cardiac myocytes
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Payam Haftbaradaran Esfahani, Jan Westergren, Lennart Lindfors, and Ralph Knöll
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cell geometry ,contraction frequency ,cardiac myocyte ,cardiomyocyte ,sarcomere ,signaling ,Physiology ,QP1-981 - Abstract
Background: Recent experimental data support the view that signaling activity at the membrane depends on its geometric parameters such as surface area and curvature. However, a mathematical, biophysical concept linking shape to receptor signaling is missing. The membranes of cardiomyocytes are constantly reshaped due to cycles of contraction and relaxation. According to constant-volume behavior of cardiomyocyte contraction, the length shortening is compensated by Z-disc myofilament lattice expansion and dynamic deformation of membrane between two adjacent Z-discs. Both morphological changes are strongly dependent on the frequency of contraction. Here, we developed the hypothesis that dynamic geometry of cardiomyocytes could be important for their plasticity and signaling. This effect may depend on the frequency of the beating heart and may represent a novel concept to explain how changes in frequency affect cardiac signaling.Methods: This hypothesis is almost impossible to answer with experiments, as the in-vitro cardiomyocytes are almost two-dimensional and flattened rather than being in their real in-vivo shape. Therefore, we designed a COMSOL multiphysics program to mathematically model the dynamic geometry of a human cardiomyocyte and explore whether the beating frequency can modulate membrane signal transduction. Src kinase is an important component of cardiac mechanotransduction. We first presented that Src mainly localizes at costameres. Then, the frequency-dependent signaling effect was studied mathematically by numerical simulation of Src-mediated PDGFR signaling pathway. The reaction-convection-diffusion partial differential equation was formulated to simulate PDGFR pathway in a contracting sarcomeric disc for a range of frequencies from 1 to 4 Hz. Results: Simulations exhibits higher concentration of phospho-Src when a cardiomyocyte beats with higher rates. The calculated phospho-Src concentration at 4, 2, and 1 Hz beat rates, comparing to 0 Hz, was 21.5%, 9.4%, and 4.7% higher, respectively.Conclusion: Here we provide mathematical evidence for a novel concept in biology. Cell shape directly translates into signaling, an effect of importance particularly for the myocardium, where cells continuously reshape their membranes. The concept of locality of surface-to-volume ratios is demonstrated to lead to changes in membrane-mediated signaling and may help to explain the remarkable plasticity of the myocardium in response to biomechanical stress.
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- 2022
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3. Myopalladin promotes muscle growth through modulation of the serum response factor pathway
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Maria Carmela Filomena, Daniel L. Yamamoto, Marco Caremani, Vinay K. Kadarla, Giuseppina Mastrototaro, Simone Serio, Anupama Vydyanath, Margherita Mutarelli, Arcamaria Garofalo, Irene Pertici, Ralph Knöll, Vincenzo Nigro, Pradeep K. Luther, Richard L. Lieber, Moriah R. Beck, Marco Linari, and Marie‐Louise Bang
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Skeletal muscle ,Sarcomere ,Knockout mouse ,Muscle growth ,Actin dynamics ,Serum response factor pathway ,Diseases of the musculoskeletal system ,RC925-935 ,Human anatomy ,QM1-695 - Abstract
Abstract Background Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. Methods Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. Results MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Conclusions Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
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- 2020
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4. In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model
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Alba Carreras, Luna Simona Pane, Roberto Nitsch, Katja Madeyski-Bengtson, Michelle Porritt, Pinar Akcakaya, Amir Taheri-Ghahfarokhi, Elke Ericson, Mikael Bjursell, Marta Perez-Alcazar, Frank Seeliger, Magnus Althage, Ralph Knöll, Ryan Hicks, Lorenz M. Mayr, Rosie Perkins, Daniel Lindén, Jan Borén, Mohammad Bohlooly-Y, and Marcello Maresca
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Hypercholesterolemia ,PCSK9 ,Genome editing ,Base editing ,CRISPR-Cas9 ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. Results To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. Conclusions Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
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- 2019
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5. Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation
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Tim R. Eijgenraam, Nienke M. Stege, Vivian Oliveira Nunes Teixeira, Remco de Brouwer, Elisabeth M. Schouten, Niels Grote Beverborg, Liu Sun, Daniela Später, Ralph Knöll, Kenny M. Hansson, Carl Amilon, David Janzén, Steve T. Yeh, Adam E. Mullick, Peter van der Meer, Rudolf A. de Boer, and Herman H. W. Silljé
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phospholamban ,genetic mutation ,protein aggregation ,cardiomyopathy ,heart failure ,gene therapy ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.
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- 2022
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6. Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis
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Simon Braumann, Wibke Schumacher, Nam Gyu Im, Felix Sebastian Nettersheim, Dennis Mehrkens, Senai Bokredenghel, Alexander Hof, Richard Julius Nies, Christoph Adler, Holger Winkels, Ralph Knöll, Bruce A. Freeman, Volker Rudolph, Anna Klinke, Matti Adam, Stephan Baldus, Martin Mollenhauer, and Simon Geißen
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nitro-oleic acid ,dilated cardiomyopathy ,muscle LIM protein ,myocardial fibrosis ,TGFβ ,alpha smooth muscle actin ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Nitro-oleic acid (NO2-OA), a nitric oxide (NO)- and nitrite (NO2−)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp−/−) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO2-OA on cardiac function in Mlp−/− mice both in vivo and in vitro. Mlp−/− mice were treated with NO2-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp−/− mice exhibited enhanced TGFβ signalling, fibrosis and severely reduced left ventricular systolic function. NO2-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp−/− mice. In vitro studies of TGFβ-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO2-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFβ downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO2-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFβ signaling.
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- 2021
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7. The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)—Molecular Signature Differ with the Extent of RR
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Patrícia G. Rodrigues, Daniela Miranda-Silva, Xidan Li, Cláudia Sousa-Mendes, Ricardo Martins-Ferreira, Zaher Elbeck, Adelino F. Leite-Moreira, Ralph Knöll, and Inês Falcão-Pires
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reverse remodelling ,pressure-overload ,diastolic dysfunction ,myocardial metabolism ,fibrosis ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e’ ≥ 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid β-oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.
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- 2020
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8. Epigenetics and Heart Failure
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Syeda Shegufta Ameer, Mohammad Bakhtiar Hossain, and Ralph Knöll
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epigenetics ,DNA methylation ,heart failure ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Epigenetics refers to changes in phenotypes without changes in genotypes. These changes take place in a number of ways, including via genomic DNA methylation, DNA interacting proteins, and microRNAs. The epigenome is the second dimension of the genome and it contains key information that is specific to every type of cell. Epigenetics is essential for many fundamental processes in biology, but its importance in the development and progression of heart failure, which is one of the major causes of morbidity and mortality worldwide, remains unclear. Our understanding of the underlying molecular mechanisms is incomplete. While epigenetics is one of the most innovative research areas in modern biology and medicine, compounds that directly target the epigenome, such as epidrugs, have not been well translated into therapies. This paper focuses on epigenetics in terms of genomic DNA methylation, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications. These appear to be more dynamic than previously anticipated and may underlie a wide variety of conditions, including heart failure. We also outline possible new strategies for the development of novel therapies.
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- 2020
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9. Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes
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Katharina Sielemann, Zaher Elbeck, Anna Gärtner, Andreas Brodehl, Caroline Stanasiuk, Henrik Fox, Lech Paluszkiewicz, Jens Tiesmeier, Stefan Wlost, Jan Gummert, Stefan P. Albaum, Janik Sielemann, Ralph Knöll, and Hendrik Milting
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cardiomyopathy ,lamin A/C ,RNA binding motif protein 20 ,titin ,plakophilin 2 ,DCM ,Genetics ,QH426-470 - Abstract
Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely LMNA (lamin A/C), RBM20 (RNA binding motif protein 20), TTN (titin) and PKP2 (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in RBM20 and TTN as the splice factor RBM20 targets amongst other genes TTN, leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in LMNA is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in PKP2 lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.
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- 2020
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10. Recent Advances in the Molecular Genetics of Familial Hypertrophic Cardiomyopathy in South Asian Descendants
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Jessica Kraker, Shivkumar Viswanathan, Ralph Knöll, and Sakthivel Sadayappan
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Hypertrophic Cardiomyopathy ,South Asians ,β-Myosin heavy chain ,MYH7 ,Cardiac myosin binding protein-C ,MYPBC3 ,Physiology ,QP1-981 - Abstract
The South Asian population, numbered at 1.8 billion, is estimated to comprise around 20% of the global population and 1% of the American population, and has one of the highest rates of cardiovascular disease. While South Asians show increased classical risk factors for developing heart failure, the role of population-specific genetic risk factors has not yet been examined for this group. Hypertrophic cardiomyopathy (HCM) is one of the major cardiac genetic disorders among South Asians, leading to contractile dysfunction, heart failure, and sudden cardiac death. This disease displays autosomal dominant inheritance, and it is associated with a large number of variants in both sarcomeric and non-sarcomeric proteins. South Asians, a population with large ethnic diversity, potentially carries region-specific polymorphisms. There is high variability in disease penetrance and phenotypic expression of variants associated with HCM. Thus, extensive studies are required to decipher pathogenicity and the physiological mechanisms of these variants, as well as the contribution of modifier genes and environmental factors to disease phenotypes. Conducting genotype-phenotype correlation studies will lead to improved understanding of HCM and, consequently, improved treatment options for this high-risk population. The objective of this review is to report the history of cardiovascular disease and HCM in South Asians, present previously published pathogenic variants, and introduce current efforts to study HCM using induced pluripotent stem cell-derived cardiomyocytes, next-generation sequencing, and gene editing technologies. The authors ultimately hope that this review will stimulate further research, drive novel discoveries, and contribute to the development of personalized medicine with the aim of expanding therapeutic strategies for HCM.
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- 2016
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11. Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes
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Petr G. Vikhorev, Natalia Smoktunowicz, Alex B. Munster, O’ Neal Copeland, Sawa Kostin, Cecile Montgiraud, Andrew E. Messer, Mohammad R. Toliat, Amy Li, Cristobal G. dos Remedios, Sean Lal, Cheavar A. Blair, Kenneth S. Campbell, Maya Guglin, Manfred Richter, Ralph Knöll, and Steven B. Marston
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Medicine ,Science - Abstract
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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- 2018
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12. OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency.
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Steven Marston, Cecile Montgiraud, Alex B Munster, O'Neal Copeland, Onjee Choi, Cristobal Dos Remedios, Andrew E Messer, Elisabeth Ehler, and Ralph Knöll
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Medicine ,Science - Abstract
BackgroundStudies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.ResultsWe identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.ConclusionsOBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.
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- 2015
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13. Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples
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Caroline Stanasiuk, Hendrik Milting, Sören Homm, Jan Persson, Lars Holtz, Axel Wittmer, Henrik Fox, Thorsten Laser, Ralph Knöll, Greta Marie Pohl, Lech Paluszkiewicz, Thomas Jakob, Bernd Bachmann-Mennenga, Dietrich Henzler, Steffen Grautoff, Gunter Veit, Karin Klingel, Erika Hori, Udo Kellner, Bernd Karger, Stefanie Schlepper, Heidi Pfeiffer, Jan Gummert, Anna Gärtner, and Jens Tiesmeier
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Pathology and Forensic Medicine - Abstract
Background The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS). The emergency medical service (EMS) plays an important role to save biomaterial for subsequent molecular autopsy. It is not known whether the DNA integrity of samples collected by the EMS is better suited for NGS than autopsy specimens. Material and methods DNA integrity was analyzed by standardized protocols. Fourteen blood samples collected by the EMS and biomaterials from autopsy were compared. We collected 172 autopsy samples from different tissues and blood with postmortem intervals of 14–168 h. For comparison, DNA integrity derived from blood stored under experimental conditions was checked against autopsy blood after different time intervals. Results DNA integrity and extraction yield were higher in EMS blood compared to any autopsy tissue. DNA stability in autopsy specimens was highly variable and had unpredictable quality. In contrast, collecting blood samples by the EMS is feasible and delivered comparably the highest DNA integrity. Conclusions Isolation yield and DNA integrity from blood samples collected by the EMS is superior in comparison to autopsy specimens. DNA from blood samples collected by the EMS on scene is stable at room temperature or even for days at 4 °C. We conclude that the EMS personnel should always save a blood sample of young fatal OHCA cases died on scene to enable subsequent genetic analysis.
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- 2023
14. Targeted therapies in genetic dilated and hypertrophic cardiomyopathies: from molecular mechanisms to therapeutic targets. A position paper from the Heart Failure Association (HFA) and the Working Group on Myocardial Function of the European Society of Cardiology (ESC)
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Rudolf A. de Boer, Stephane Heymans, Johannes Backs, Lucie Carrier, Andrew J.S. Coats, Stefanie Dimmeler, Thomas Eschenhagen, Gerasimos Filippatos, Lior Gepstein, Jean‐Sebastien Hulot, Ralph Knöll, Christian Kupatt, Wolfgang A. Linke, Christine E. Seidman, C. Gabriele Tocchetti, Jolanda van der Velden, Roddy Walsh, Petar M. Seferovic, Thomas Thum, Publica, Cardiologie, MUMC+: MA Med Staf Spec Cardiologie (9), RS: Carim - H02 Cardiomyopathy, HULOT, Jean-Sébastien, University of Groningen [Groningen], Maastricht University Medical Centre (MUMC), Maastricht University [Maastricht], Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Heidelberg University, German Center for Cardiovascular Research (DZHK), Berlin Institute of Health (BIH), Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), University of Warwick [Coventry], Goethe University [Germany], National and Kapodistrian University of Athens (NKUA), Technion - Israel Institute of Technology [Haifa], CIC - HEGP (CIC 1418), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Karolinska Institutet [Stockholm], AstraZeneca, Technische Universität München = Technical University of Munich (TUM), University Hospital Münster - Universitaetsklinikum Muenster [Germany] (UKM), Harvard Medical School [Boston] (HMS), Brigham & Women’s Hospital [Boston] (BWH), Howard Hughes Medical Institute [Boston] (HHMI), Howard Hughes Medical Institute (HHMI)-Harvard Medical School [Boston] (HMS), University of Naples Federico II = Università degli studi di Napoli Federico II, Vrije Universiteit Amsterdam [Amsterdam] (VU), University of Amsterdam [Amsterdam] (UvA), Serbian Academy of Sciences and Arts (SASA), University of Belgrade [Belgrade], Hannover Medical School [Hannover] (MHH), Fraunhofer Institute for Toxicology and Experimental Medicine (Fraunhofer ITEM), Fraunhofer (Fraunhofer-Gesellschaft), Cardiology, Cardiovascular Centre (CVC), Physiology, and ACS - Heart failure & arrhythmias
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Cardiomyopathy, Dilated ,GENOMIC DNA ,Cardiac & Cardiovascular Systems ,Cardiomyopathy ,Molecular biology ,RESTORES DYSTROPHIN EXPRESSION ,[SDV]Life Sciences [q-bio] ,Cardiology ,Dilated cardiomyopathy ,Heart failure ,DOUBLE-BLIND ,Gene therapy ,Humans ,GENOME-WIDE ASSOCIATION ,MUTATION ,Pharmacology ,Science & Technology ,CARDIAC MYOSIN ,Myocardium ,MOUSE MODEL ,Cardiomyopathy, Hypertrophic ,Hypertrophic cardiomyopathy ,[SDV] Life Sciences [q-bio] ,ATP TURNOVER ,Cardiovascular System & Cardiology ,Disease mechanism ,Cardiomyopathies ,Cardiology and Cardiovascular Medicine ,PHOSPHOLAMBAN ,SARCOMERE FUNCTION ,Life Sciences & Biomedicine ,PLURIPOTENT STEM-CELLS - Abstract
Genetic cardiomyopathies are disorders of the cardiac muscle, most often explained by pathogenic mutations in genes encoding sarcomere, cytoskeleton, or ion channel proteins. Clinical phenotypes such as heart failure and arrhythmia are classically treated with generic drugs, but aetiology-specific and targeted treatments are lacking. As a result, cardiomyopathies still present a major burden to society, and affect many young and older patients. The Translational Committee of the Heart Failure Association (HFA) and the Working Group of Myocardial Function of the European Society of Cardiology (ESC) organized a workshop to discuss recent advances in molecular and physiological studies of various forms of cardiomyopathies. The study of cardiomyopathies has intensified after several new study setups became available, such as induced pluripotent stem cells, three-dimensional printing of cells, use of scaffolds and engineered heart tissue, with convincing human validation studies. Furthermore, our knowledge on the consequences of mutated proteins has deepened, with relevance for cellular homeostasis, protein quality control and toxicity, often specific to particular cardiomyopathies, with precise effects explaining the aberrations. This has opened up new avenues to treat cardiomyopathies, using contemporary techniques from the molecular toolbox, such as gene editing and repair using CRISPR-Cas9 techniques, antisense therapies, novel designer drugs, and RNA therapies. In this article, we discuss the connection between biology and diverse clinical presentation, as well as promising new medications and therapeutic avenues, which may be instrumental to come to precision medicine of genetic cardiomyopathies. ispartof: EUROPEAN JOURNAL OF HEART FAILURE vol:24 issue:3 pages:406-420 ispartof: location:England status: published
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- 2022
15. The long noncoding RNA TUNAR modulates Wnt signaling and regulates human β-cell proliferation
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Ali M. Tabish, Elke Ericson, Björn Tyrberg, David M. Smith, Chandrasekhar Kanduri, Hanne Scholz, Shadab Abadpour, Tanmoy Mondal, Ralph Knöll, Magnus Althage, and Alex Xianghua Zhou
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0301 basic medicine ,medicine.medical_specialty ,Physiology ,Cell growth ,Endocrinology, Diabetes and Metabolism ,Wnt signaling pathway ,RNA ,Type 2 diabetes ,Biology ,medicine.disease ,Long non-coding RNA ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Endocrinology ,030220 oncology & carcinogenesis ,Physiology (medical) ,Internal medicine ,Diabetes mellitus ,medicine - Abstract
The discovery that long noncoding RNA TUNAR regulates β-cell proliferation may be important in designing new treatments for diabetes.
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- 2021
16. Cell shape: effects on gene expression and signaling
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Payam Haftbaradaran Esfahani and Ralph Knöll
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0303 health sciences ,Mechanotransduction ,Mechanosensation ,Cell geometry ,Biophysics ,Membrane biology ,RNA ,Review ,Biology ,Heart failure ,Transcriptome ,03 medical and health sciences ,Preload ,0302 clinical medicine ,Cell shape ,Structural Biology ,Gene expression ,Molecular Biology ,Neuroscience ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The perception of biophysical forces (mechanosensation) and their conversion into chemical signals (mechanotransduction) are fundamental biological processes. They are connected to hypertrophic and atrophic cellular responses, and defects in these processes have been linked to various diseases, especially in the cardiovascular system. Although cardiomyocytes generate, and are exposed to, considerable hemodynamic forces that affect their shapes, until recently, we did not know whether cell shape affects gene expression. However, new single-cell trapping strategies, followed by single-cell RNA sequencing, to profile the transcriptomes of individual cardiomyocytes of defined geometrical morphotypes have been developed that are characteristic for either normal or pathological (afterload or preload) conditions. This paper reviews the recent literature with regard to cell shape and the transcriptome and provides an overview of this newly emerging field, which has far-reaching implications for both biology, disease, and possibly therapy.
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- 2020
17. Myopalladin promotes muscle growth through modulation of the serum response factor pathway
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Marie Louise Bang, Daniel L. Yamamoto, Giuseppina Mastrototaro, Simone Serio, Marco Caremani, Richard L. Lieber, Ralph Knöll, Anupama Vydyanath, Marco Linari, Arcamaria Garofalo, Vinay Kumar Kadarla, Vincenzo Nigro, Irene Pertici, Maria Carmela Filomena, Margherita Mutarelli, Moriah R. Beck, Pradeep K. Luther, Filomena, Maria Carmela, Yamamoto, Daniel L, Caremani, Marco, Kadarla, Vinay K, Mastrototaro, Giuseppina, Serio, Simone, Vydyanath, Anupama, Mutarelli, Margherita, Garofalo, Arcamaria, Pertici, Irene, Knöll, Ralph, Nigro, Vincenzo, Luther, Pradeep K, Lieber, Richard L, Beck, Moriah R, Linari, Marco, and Bang, Marie-Louise
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0301 basic medicine ,Serum Response Factor ,lcsh:Diseases of the musculoskeletal system ,Actin dynamics ,Knockout mouse ,Muscle growth ,Sarcomere ,Serum response factor pathway ,Skeletal muscle ,Muscle Proteins ,Filamentous actin ,lcsh:QM1-695 ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Physiology (medical) ,Serum response factor ,Myosin ,Animals ,Humans ,Medicine ,Myocyte ,Orthopedics and Sports Medicine ,Muscle, Skeletal ,Actin ,Mice, Knockout ,Actin dynamic ,business.industry ,Original Articles ,MYPN ,lcsh:Human anatomy ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Original Article ,lcsh:RC925-935 ,business - Abstract
Background Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. Methods Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. Results MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Conclusions Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
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- 2020
18. Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations
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Jan Gummert, Anna Hucke, Alexander Goedel, Astrid Kassner, Hendrik Milting, Izabela Tuleta, Wolfgang A. Linke, Andrey Fomin, Katrin Streckfuß-Bömeke, Anna Gärtner, Norbert Hubner, Holger Reinecke, Cristobal G. dos Remedios, Lina Folsche, Alessandra Moretti, Ralph Knöll, Gerd Hasenfuss, Katharina Sielemann, Sebastiaan van Heesch, Luisa Wellers, Malte Tiburcy, Anastasia J. Hobbach, Lukas Cyganek, Wolfram H. Zimmermann, Franziska Koser, Karl-Ludwig Laugwitz, Andreas Unger, and Marion von Frieling-Salewsky
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Genetics ,0303 health sciences ,animal structures ,biology ,Cardiomyopathy ,Dilated cardiomyopathy ,General Medicine ,030204 cardiovascular system & hematology ,musculoskeletal system ,medicine.disease ,Functional recovery ,03 medical and health sciences ,0302 clinical medicine ,embryonic structures ,cardiovascular system ,biology.protein ,medicine ,Titin ,Haploinsufficiency ,tissues ,Gene ,030304 developmental biology - Abstract
Stable expression of truncated titin proteins and titin haploinsufficiency characterize TTN cardiomyopathy and represent targets for therapy.
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- 2021
19. Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to
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Andrey, Fomin, Anna, Gärtner, Lukas, Cyganek, Malte, Tiburcy, Izabela, Tuleta, Luisa, Wellers, Lina, Folsche, Anastasia J, Hobbach, Marion, von Frieling-Salewsky, Andreas, Unger, Anna, Hucke, Franziska, Koser, Astrid, Kassner, Katharina, Sielemann, Katrin, Streckfuß-Bömeke, Gerd, Hasenfuss, Alexander, Goedel, Karl-Ludwig, Laugwitz, Alessandra, Moretti, Jan F, Gummert, Cristobal G, Dos Remedios, Holger, Reinecke, Ralph, Knöll, Sebastiaan, van Heesch, Norbert, Hubner, Wolfram H, Zimmermann, Hendrik, Milting, and Wolfgang A, Linke
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Induced Pluripotent Stem Cells ,Mutation ,Heart Transplantation ,Humans ,Connectin ,Myocytes, Cardiac ,Haploinsufficiency ,Cardiomyopathies ,Tissue Donors - Abstract
Heterozygous truncating variants in
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- 2021
20. Titin M-line insertion sequence 7 is required for proper cardiac function in mice
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Jérémie Cosette, Henk Granzier, Zaher Elbeck, Joshua Strom, Simone Spinozzi, Ariane Biquand, William Lostal, Paola Tonino, Isabelle Richard, Ralph Knöll, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, University of Arizona, Karolinska Institutet [Stockholm], AstraZeneca, and Richard, Isabelle
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Cardiomyopathy, Dilated ,Sarcomeres ,Gene isoform ,Cardiac function curve ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Cardiomyopathy ,Titin ,Mex5 ,[SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics ,030204 cardiovascular system & hematology ,[SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Mice ,03 medical and health sciences ,Exon ,0302 clinical medicine ,medicine ,Animals ,Connectin ,Sarcomere organization ,Insertion sequence ,030304 developmental biology ,Heart Failure ,Is7 ,0303 health sciences ,biology ,Alternative splicing ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Cell Biology ,medicine.disease ,[SDV.BIO] Life Sciences [q-bio]/Biotechnology ,Cell biology ,Alternative Splicing ,[SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics ,DNA Transposable Elements ,biology.protein ,Protein Kinases ,Research Article - Abstract
Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.
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- 2021
21. Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere
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Barbara Franke, Jennifer R. Fleming, Siegfried Labeit, Julius Bogomolovas, Alexander Gasch, Martin Scheffner, Bruno Manso, Marija Markovic, Bernd Simon, Ju Chen, Christine Peter, Olga Mayans, Ralph Knöll, and Thomas Brunner
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Sarcomeres ,animal structures ,macromolecular substances ,X‐ray crystallography ,Biochemistry ,Sarcomere ,03 medical and health sciences ,Post-translational Modifications & Proteolysis ,0302 clinical medicine ,steered molecular dynamics simulations ,Ubiquitin ,ddc:570 ,Report ,Autophagy ,Genetics ,Molecular Biology of Disease ,Connectin ,Mechanotransduction ,Muscle, Skeletal ,Molecular Biology ,cellular signalling ,mechanotransduction ,030304 developmental biology ,Calcium signaling ,0303 health sciences ,biology ,Chemistry ,Kinase ,Ubiquitination ,musculoskeletal system ,Cell biology ,embryonic structures ,biology.protein ,Titin ,Cell Adhesion, Polarity & Cytoskeleton ,Myofibril ,tissues ,030217 neurology & neurosurgery ,Reports - Abstract
Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168‐M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross‐talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1‐mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down‐regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle., The pseudokinase domain of the titin myofilament is a node for the cross‐talk of mechanical signals and turn‐over pathways in the sarcomere, contributing to a coordinated response to cellular stress.
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- 2021
22. Control of p21Cip by BRCA1-associated protein is critical for cardiomyocyte cell cycle progression and survival
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Carla Schmidt, Gerd Hasenfuß, Henning Urlaub, Katrin Nickels, Tim Seidler, Jörg Männer, Michael Didié, Ralph Knöll, Bernhard Unsöld, Karl Toischer, Albrecht Schmidt, Cornelia Volland, Peter Schott, and Kaomei Guan
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0301 basic medicine ,Genetically modified mouse ,DNA synthesis ,Physiology ,Alpha (ethology) ,030204 cardiovascular system & hematology ,Cell cycle ,Biology ,Embryonic stem cell ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Cytoplasm ,Apoptosis ,Physiology (medical) ,Knockout mouse ,Cardiology and Cardiovascular Medicine - Abstract
Aims Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation. Methods and results General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis. Conclusion By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.
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- 2019
23. In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model
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Magnus Althage, Luna Simona Pane, Frank Seeliger, Mikael Bjursell, Ralph Knöll, Daniel Lindén, Katja Madeyski-Bengtson, Marta Perez-Alcazar, Roberto Nitsch, Mohammad Bohlooly-Y, Ryan Hicks, Alba Carreras, Marcello Maresca, Rosie Perkins, Jan Borén, Amir Taheri-Ghahfarokhi, Lorenz M. Mayr, Elke Ericson, Michelle J. Porritt, and Pinar Akcakaya
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Physiology ,Hypercholesterolemia ,Mice, Transgenic ,Plant Science ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,PCSK9 ,Base editing ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Genome editing ,Structural Biology ,Gene knockin ,Animals ,Humans ,Guide RNA ,lcsh:QH301-705.5 ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Cell Biology ,Proprotein convertase ,Cell biology ,Evolocumab ,Disease Models, Animal ,Cholesterol ,Liver ,lcsh:Biology (General) ,Humanized mouse ,Proprotein Convertase 9 ,CRISPR-Cas9 ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,Developmental Biology ,Biotechnology ,Research Article - Abstract
Background Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. Results To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. Conclusions Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia. Electronic supplementary material The online version of this article (10.1186/s12915-018-0624-2) contains supplementary material, which is available to authorized users.
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- 2019
24. The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)—Molecular Signature Differ with the Extent of RR
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Adelino F. Leite-Moreira, Inês Falcão-Pires, Daniela Miranda-Silva, Ralph Knöll, Cláudia Sousa-Mendes, Ricardo Martins-Ferreira, Xidan Li, Patrícia Rodrigues, and Zaher Elbeck
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Male ,0301 basic medicine ,Cardiac function curve ,medicine.medical_specialty ,reverse remodelling ,Diastole ,Hemodynamics ,030204 cardiovascular system & hematology ,Article ,Catalysis ,Muscle hypertrophy ,lcsh:Chemistry ,Inorganic Chemistry ,myocardial metabolism ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Fibrosis ,Internal medicine ,medicine ,Animals ,Myocytes, Cardiac ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,Pressure overload ,Ventricular Remodeling ,business.industry ,Organic Chemistry ,fibrosis ,General Medicine ,medicine.disease ,Computer Science Applications ,Mice, Inbred C57BL ,MicroRNAs ,030104 developmental biology ,medicine.anatomical_structure ,lcsh:Biology (General) ,lcsh:QD1-999 ,Ventricle ,Cardiology ,pressure-overload ,Hypertrophy, Left Ventricular ,Myocardial fibrosis ,diastolic dysfunction ,Transcriptome ,business - Abstract
This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e&rsquo, &ge, 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid &beta, oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.
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- 2020
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25. Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes
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Henrik Fox, Hendrik Milting, Lech Paluszkiewicz, Stefan P. Albaum, Jan Gummert, Caroline Stanasiuk, Andreas Brodehl, Ralph Knöll, Stefan Wlost, Jens Tiesmeier, Anna Gärtner, Janik Sielemann, Zaher Elbeck, and Katharina Sielemann
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0301 basic medicine ,Adult ,Male ,lcsh:QH426-470 ,Mutant ,Cardiomyopathy ,Muscle Proteins ,030204 cardiovascular system & hematology ,Article ,660.6 ,LMNA ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,Genetics ,medicine ,plakophilin 2 ,ARVC ,Humans ,Genetics(clinical) ,Genetic Predisposition to Disease ,titin ,Gene ,Genetics (clinical) ,Aged ,lamin A/C ,DCM ,biology ,Myocardium ,Middle Aged ,medicine.disease ,lcsh:Genetics ,030104 developmental biology ,Mutation ,biology.protein ,Titin ,Female ,Cardiomyopathies ,cardiomyopathy ,RNA binding motif protein 20 ,Lamin - Abstract
Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely \(\it LMNA\) (lamin A/C), \(\it RBM20\) (RNA binding motif protein 20), \(\it TTN\) (titin) and \(\it PKP2\) (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in \(\it RBM20\) and \(\it TTN\) as the splice factor RBM20 targets amongst other genes \(\it TTN\), leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in \(\it LMNA\) is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in \(\it PKP2\) lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.
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- 2020
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26. An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level
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Ralph Knöll and Payam Haftbaradaran Esfahani
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Sarcomeres ,DNA, Complementary ,General Immunology and Microbiology ,General Chemical Engineering ,General Neuroscience ,Induced Pluripotent Stem Cells ,General Biochemistry, Genetics and Molecular Biology ,Fibronectins ,Rats ,Animals, Newborn ,Cell Adhesion ,Animals ,Myocytes, Cardiac ,Single-Cell Analysis ,Transcriptome ,Cell Shape - Abstract
Different types of cardiac hypertrophy have been associated with an increased volume of cardiac myocytes (CMs), along with changes in CM morphology. While the effects of cell volume on gene expression are well known, the effects of cell shape are not well understood. This paper describes a method that has been designed to systematically analyze the effects of CM morphology on gene expression. It details the development of a novel single-cell trapping strategy that is then followed by single-cell mRNA sequencing. A micropatterned chip has also been designed, which contains 3000 rectangular-shaped fibronectin micropatterns. This makes it possible to grow CMs in distinct length:width aspect ratios (AR), corresponding to different types of heart failure (HF). The paper also describes a protocol that has been designed to pick up single cells from their pattern, using a semi-automated micro-pipetting cell picker, and individually inject them into a separate lysis buffer. This has made it possible to profile the transcriptomes of single CMs with defined geometrical morphotypes and characterize them according to a range of normal or pathological conditions: hypertrophic cardiomyopathy (HCM) or afterload/concentric versus dilated cardiomyopathy (DCM) or preload/eccentric. In summary, this paper presents methods for growing CMs with different shapes, which represent different pathologies, and sorting these adherent CMs based on their morphology at a single-cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of HF.
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- 2020
27. Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes
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Chrishan J A Ramachandra, Li-Ming Gan, Katarina Ryden-Markinhuhta, Elisa A. Liehn, Malin K.B. Jonsson, Sakthivel Sadayappan, Philip Wong, Regina Fritsche-Danielson, Ying-Hsi Lin, Sauri Hernández-Reséndiz, Myu Mai Ja Kp, Hak Chiaw Tang, Jasper Chua, Winston Shim, Ratan Bhat, Ralph Knöll, Derek J. Hausenloy, and Erik Michaëlsson
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Male ,Physiology ,Human-induced pluripotent stem cells (hiPSCs) ,Left ventricular hypertrophy ,medicine.disease_cause ,Ventricular Function, Left ,AcademicSubjects/MED00200 ,Myocytes, Cardiac ,Enzyme Inhibitors ,Phosphorylation ,Myeloperoxidase ,biology ,Hypertrophic cardiomyopathy ,Cardiology ,Diastolic dysfunction ,Hypertrophy, Left Ventricular ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,Cardiac Remodelling and Heart Failure ,medicine.medical_specialty ,Induced Pluripotent Stem Cells ,Diastole ,Hypertrophic cardiomyopathy (HCM) ,Mutation, Missense ,macromolecular substances ,Exercise intolerance ,Cell Line ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Humans ,cardiovascular diseases ,Peroxidase ,Myosin Heavy Chains ,business.industry ,Original Articles ,Cardiomyopathy, Hypertrophic ,medicine.disease ,Myocardial Contraction ,Mice, Inbred C57BL ,Disease Models, Animal ,Oxidative stress ,biology.protein ,Tyrosine ,MYH7 ,business ,Carrier Proteins ,Reactive Oxygen Species ,Cardiac Myosins ,Cardiac myosin binding protein-C (MYBPC3) - Abstract
Aims Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. Methods and results Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. Conclusion This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing., Graphical Abstract
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- 2020
28. Cell shape determines gene expression: cardiomyocyte morphotypic transcriptomes
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Mohammad Bakhtiar Hossain, Zaher Elbeck, Rickard Sandberg, Payam Haftbaradaran Esfahani, Xidan Li, Sven Sagasser, Ralph Knöll, and Husain A. Talukdar
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Physiology ,Integrin ,Original Contribution ,Biology ,Mechanotransduction, Cellular ,Myocardial biology ,Cell biology ,Rats, Sprague-Dawley ,Single-cell RNA sequencing ,Gene expression and regulation ,Transcriptome ,Gene Expression Regulation ,Downregulation and upregulation ,Physiology (medical) ,Gene expression ,biology.protein ,Animals ,Cell shape ,Myocyte ,Myocytes, Cardiac ,Signal transduction ,Cardiology and Cardiovascular Medicine ,Protein kinase A ,Gene - Abstract
Cardiomyocytes undergo considerable changes in cell shape. These can be due to hemodynamic constraints, including changes in preload and afterload conditions, or to mutations in genes important for cardiac function. These changes instigate significant changes in cellular architecture and lead to the addition of sarcomeres, at the same time or at a later stage. However, it is currently unknown whether changes in cell shape on their own affect gene expression and the aim of this study was to fill that gap in our knowledge. We developed a single-cell morphotyping strategy, followed by single-cell RNA sequencing, to determine the effects of altered cell shape in gene expression. This enabled us to profile the transcriptomes of individual cardiomyocytes of defined geometrical morphotypes and characterize them as either normal or pathological conditions. We observed that deviations from normal cell shapes were associated with significant downregulation of gene expression and deactivation of specific pathways, like oxidative phosphorylation, protein kinase A, and cardiac beta-adrenergic signaling pathways. In addition, we observed that genes involved in apoptosis of cardiomyocytes and necrosis were upregulated in square-like pathological shapes. Mechano-sensory pathways, including integrin and Src kinase mediated signaling, appear to be involved in the regulation of shape-dependent gene expression. Our study demonstrates that cell shape per se affects the regulation of the transcriptome in cardiac myocytes, an effect with possible implications for cardiovascular disease. Electronic supplementary material The online version of this article (10.1007/s00395-019-0765-7) contains supplementary material, which is available to authorized users.
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- 2019
29. Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A
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Zaher Elbeck, Karine Charton, William Lostal, John E. Smith, Jack Yves Deschamps, Guillaume Corre, Isabelle Richard, Marine Faivre, Henk Granzier, Nathalie Bourg, Xidan Li, Ralph Knöll, Laurence Suel, Jochen Gohlke, Carinne Roudaut, Heather Best, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), and Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon
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0301 basic medicine ,Primates ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,Transgene ,RNA Splicing ,Muscle Proteins ,Article ,Dysferlin ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Species Specificity ,Enzyme Stability ,medicine ,Animals ,Connectin ,Tissue Distribution ,Transgenes ,Muscular dystrophy ,Muscle, Skeletal ,Regulation of gene expression ,Mice, Knockout ,Binding Sites ,biology ,Calpain ,Myocardium ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,General Medicine ,Genetic Therapy ,Dependovirus ,medicine.disease ,Cardiotoxicity ,3. Good health ,Cell biology ,MicroRNAs ,030104 developmental biology ,Gene Expression Regulation ,Muscular Dystrophies, Limb-Girdle ,[SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics ,Proteolysis ,biology.protein ,Titin ,Ectopic expression ,Titin binding ,030217 neurology & neurosurgery ,Biomarkers - Abstract
International audience; Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene ( CAPN3 ). Previous experiments using adeno-associated viral (AAV) vector–mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10 13 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.
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- 2019
30. Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes
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Petr G. Vikhorev, Natalia Smoktunowicz, Alex B. Munster, O’Neal Copeland, Sawa Kostin, Cecile Montgiraud, Andrew E. Messer, Mohammad R. Toliat, Amy Li, Cristobal G. dos Remedios, Sean Lal, Cheavar A. Blair, Kenneth S. Campbell, Maya Guglin, Manfred Richter, Ralph Knöll, and Steven B. Marston
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animal structures ,lcsh:R ,cardiovascular system ,lcsh:Medicine ,lcsh:Q ,cardiovascular diseases ,musculoskeletal system ,lcsh:Science - Abstract
Dilated cardiomyopathy (DCM) is an important cause of heart failure. Single gene mutations in at least 50 genes have been proposed to account for 25–50% of DCM cases and up to 25% of inherited DCM has been attributed to truncating mutations in the sarcomeric structural protein titin (TTNtv). Whilst the primary molecular mechanism of some DCM-associated mutations in the contractile apparatus has been studied in vitro and in transgenic mice, the contractile defect in human heart muscle has not been studied. In this study we isolated cardiac myofibrils from 3 TTNtv mutants, and 3 with contractile protein mutations (TNNI3 K36Q, TNNC1 G159D and MYH7 E1426K) and measured their contractility and passive stiffness in comparison with donor heart muscle as a control. We found that the three contractile protein mutations but not the TTNtv mutations had faster relaxation kinetics. Passive stiffness was reduced about 38% in all the DCM mutant samples. However, there was no change in maximum force or the titin N2BA/N2B isoform ratio and there was no titin haploinsufficiency. The decrease in myofibril passive stiffness was a common feature in all hearts with DCM-associated mutations and may be causative of DCM.
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- 2017
31. Genetic epidemiology of titin-truncating variants in the etiology of dilated cardiomyopathy
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Aris Alexiadis, Ali M. Tabish, Ralph Knöll, Byambajav Buyandelger, and Valerio Azzimato
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0301 basic medicine ,Titin ,Epidemiology ,Population ,Dilated cardiomyopathy ,Biophysics ,Heart failure ,Context (language use) ,Review ,Disease ,03 medical and health sciences ,Structural Biology ,medicine ,cardiovascular diseases ,education ,Molecular Biology ,Genetics ,education.field_of_study ,biology ,medicine.disease ,030104 developmental biology ,Truncating variants ,Genetic epidemiology ,cardiovascular system ,biology.protein ,Haploinsufficiency - Abstract
Heart failure (HF) is a complex clinical syndrome defined by the inability of the heart to pump enough blood to meet the body’s metabolic demands. Major causes of HF are cardiomyopathies (diseases of the myocardium associated with mechanical and/or electrical dysfunction), among which the most common form is dilated cardiomyopathy (DCM). DCM is defined by ventricular chamber enlargement and systolic dysfunction with normal left ventricular wall thickness, which leads to progressive HF. Over 60 genes are linked to the etiology of DCM. Titin (TTN) is the largest known protein in biology, spanning half the cardiac sarcomere and, as such, is a basic structural and functional unit of striated muscles. It is essential for heart development as well as mechanical and regulatory functions of the sarcomere. Next-generation sequencing (NGS) in clinical DCM cohorts implicated truncating variants in titin (TTNtv) as major disease alleles, accounting for more than 25% of familial DCM cases, but these variants have also been identified in 2–3% of the general population, where these TTNtv blur diagnostic and clinical utility. Taking into account the published TTNtv and their association to DCM, it becomes clear that TTNtv harm the heart with position-dependent occurrence, being more harmful when present in the A-band TTN, presumably with dominant negative/gain-of-function mechanisms. However, these insights are challenged by the depiction of position-independent toxicity of TTNtv acting via haploinsufficient alleles, which are sufficient to induce cardiac pathology upon stress. In the current review, we provide an overview of TTN and discuss studies investigating various TTN mutations. We also present an overview of different mechanisms postulated or experimentally validated in the pathogenicity of TTNtv. DCM-causing genes are also discussed with respect to non-truncating mutations in the etiology of DCM. One way of understanding pathogenic variants is probably to understand the context in which they may or may not affect protein–protein interactions, changes in cell signaling, and substrate specificity. In this regard, we also provide a brief overview of TTN interactions in situ. Quantitative models in the risk assessment of TTNtv are also discussed. In summary, we highlight the importance of gene–environment interactions in the etiology of DCM and further mechanistic studies used to delineate the pathways which could be targeted in the management of DCM.
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- 2017
32. Abstract 772: The Highly Prevalent 25bp Intronic Deletion in MYBPC3 is Benign Under Baseline Conditions
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Parth N Patel, Mohammad Bohlooly, Katja Madeyski-Bengtson, Christine E. Seidman, Sakthivel Sadayappan, Shiv Kumar Viswanathan, Jonathan G. Seidman, Jennifer A. Schwanekamp, Ralph Knöll, and James W. McNamara
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Cardiac function curve ,Myofilament ,medicine.medical_specialty ,Physiology ,business.industry ,Cardiomyopathy ,Hypertrophic cardiomyopathy ,medicine.disease ,Internal medicine ,Cardiology ,Medicine ,Thickening ,Cardiology and Cardiovascular Medicine ,business - Abstract
Background: Hypertrophic cardiomyopathy (HCM) affects at least 1 in 500 people worldwide, and results in the thickening of the ventricular walls and reduced cardiac function. Mutations in MYBPC3 , encoding cardiac myosin binding protein-C, are the most common cause of HCM. Previously, a highly prevalent 25bp deletion within intron 32 of MYBPC3 was described in the South Asian population. The MYBPC3 d25bp variant is present in approximately 100 million people, and encompasses a splicing branch point predicted to result in abnormal splicing of exon 33. Thus, there is a critical need to understand the mechanism by which MYBPC3 d25bp may cause cardiomyopathy. Methods: To determine the role of the 25bp deletion in vivo , knock-in humanized mice were created in which intron 32 was replaced with the human intron 32, with or without the MYBPC3 d25bp mutation. Mice were characterized at 3- and 6-months of age by echocardiography, histological, and protein analysis. The presence of aberrant exon splicing was also determined in mice carrying the MYBPC3 d25bp variant through RT-PCR and mini-gene assays. Finally, exon trapping experiments were performed to understand the mechanism behind exon skipping. Results: Under baseline conditions, MYBPC3 d25bp displayed no changes in cardiac function or morphology as measured by echocardiography (FS (%): NTG 35.3%, WT 32.8%, Het 33.7%), heart weight to body weight ratio, or histology. While exon 33 skipping was not detected by RT-PCR, the presence of an alternative splice site within exon 33 was identified in MYBPC3 d25bp mice. However, this did not affect the protein levels of cMyBP-C. Furthermore, mini-gene experiments demonstrated that the MYBPC3 d25bp mutation significantly reduced the percentage of correctly spliced transcripts (86.2% vs. 77.5%). Conclusions: These data demonstrate that the presence of the highly prevalent 25bp deletion is not sufficient to cause disease under baseline conditions. However, it is possible that the increased levels of aberrant splicing may increase the risk for developing HCM.
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- 2019
33. Myosin binding protein-C and hypertrophic cardiomyopathy: role of altered C10 domain
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Zaher Elbeck, Ralph Knöll, Mohammad Bakhtiar Hossain, and H Siga
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Physiology ,Hypertrophic cardiomyopathy ,Original Articles ,Biology ,Cardiomyopathy, Hypertrophic ,Myosins ,medicine.disease ,Domain (software engineering) ,Cell biology ,Cytoskeletal Proteins ,Myosin-binding protein C ,Physiology (medical) ,medicine ,Humans ,Cardiology and Cardiovascular Medicine ,Cardiac Myosins - Abstract
AIMS: A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-C(ΔC10mut)) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-C(ΔC10mut)in vivo are unknown. We hypothesized that expression of cMyBP-C(ΔC10mut) exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. METHODS AND RESULTS: To determine whether expression of cMyBP-C(ΔC10mut) is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-C(ΔC10mut) at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-C(ΔC10mut) (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-C(ΔC10mut), P
- Published
- 2019
34. Association of intronic DNA methylation and hydroxymethylation alterations in the epigenetic etiology of dilated cardiomyopathy
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Mohammed Arif, Zaher Elbeck, Richard C. Becker, Taejeong Song, Ali M. Tabish, Sakthivel Sadayappan, and Ralph Knöll
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0301 basic medicine ,DNA Hydroxymethylation ,Cardiomyopathy, Dilated ,Male ,Physiology ,030204 cardiovascular system & hematology ,Biology ,Hydroxylation ,Ventricular Function, Left ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,Myosin-binding protein C ,Physiology (medical) ,medicine ,Animals ,Gene Regulatory Networks ,Genetic Predisposition to Disease ,Epigenetics ,Promoter Regions, Genetic ,Gene ,Genetics ,Dilated cardiomyopathy ,DNA Methylation ,medicine.disease ,Introns ,Mice, Mutant Strains ,030104 developmental biology ,Phenotype ,DNA methylation ,Mutation ,Etiology ,cardiovascular system ,5-Methylcytosine ,CpG Islands ,Cardiology and Cardiovascular Medicine ,Carrier Proteins ,Transcriptome ,Research Article - Abstract
In this study, we investigated the role of DNA methylation [5-methylcytosine (5mC)] and 5-hydroxymethylcytosine (5hmC), epigenetic modifications that regulate gene activity, in dilated cardiomyopathy (DCM). A MYBPC3 mutant mouse model of DCM was compared with wild type and used to profile genomic 5mC and 5hmC changes by Chip-seq, and gene expression levels were analyzed by RNA-seq. Both 5mC-altered genes (957) and 5hmC-altered genes (2,022) were identified in DCM hearts. Diverse gene ontology and KEGG pathways were enriched for DCM phenotypes, such as inflammation, tissue fibrosis, cell death, cardiac remodeling, cardiomyocyte growth, and differentiation, as well as sarcomere structure. Hierarchical clustering of mapped genes affected by 5mC and 5hmC clearly differentiated DCM from wild-type phenotype. Based on these data, we propose that genomewide 5mC and 5hmC contents may play a major role in DCM pathogenesis. NEW & NOTEWORTHY Our data demonstrate that development of dilated cardiomyopathy in mice is associated with significant epigenetic changes, specifically in intronic regions, which, when combined with gene expression profiling data, highlight key signaling pathways involved in pathological cardiac remodeling and heart contractile dysfunction.
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- 2019
35. Mouse Models to Study Inherited Cardiomyopathy
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Mohammad Bohlooly-Y, Mohammad Bakhtiar Hossain, and Ralph Knöll
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business.industry ,Heart failure ,Cardiovascular research ,Cardiomyopathy ,medicine ,Inherited cardiomyopathy ,Disease ,Induced pluripotent stem cell ,medicine.disease ,Bioinformatics ,business ,Pharmacological treatment ,Cause of death - Abstract
Cardiovascular diseases, including cardiomyopathy and associated heart failure, are the number one cause of death worldwide, but our ability to interfere with these devastating diseases is limited. Cardiomyopathies, which are mainly due to genetic causes, make a significant portion of heart failure. Current pharmacological treatment of cardiovascular diseases focuses on symptoms rather than the underlying cellular mechanisms, and the gaps in our understanding of cellular mechanisms of the disease are profound. Elucidation of these mechanisms is a central issue in cardiovascular biology and important for designing new treatment for cardiovascular diseases. While significant progress has been made in using in vitro systems in deciphering the mechanisms and finding innovative solutions for cardiovascular disease treatment, including the use of induced pluripotent stem cell (iPSC) derivatives, suitable in vivo models are more difficult to develop. Among several different species, mouse models are rather inexpensive, easily manipulatable, reproducible, physiologically representative of human disease, and ethically acceptable. This chapter will provide a brief overview of genetics of heart failure, largely focusing on genetically altered mouse models and experimental approaches applicable to cardiovascular research.
- Published
- 2019
36. Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes
- Author
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O' Neal Copeland, Sawa Kostin, Cécile Montgiraud, Andrew E. Messer, Natalia Smoktunowicz, Manfred Richter, Maya Guglin, Alex B Munster, Kenneth S. Campbell, Steven B. Marston, Ralph Knöll, Amy Li, Petr G. Vikhorev, Cristobal G. dos Remedios, Cheavar A. Blair, Sean Lal, Mohammad R. Toliat, and British Heart Foundation
- Subjects
medicine.medical_specialty ,Science & Technology ,Multidisciplinary ,business.industry ,Science ,Human heart ,Dilated cardiomyopathy ,Contractile protein ,medicine.disease ,Multidisciplinary Sciences ,Contractility ,Text mining ,Internal medicine ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,Cardiology ,Medicine ,Science & Technology - Other Topics ,Myofibril ,business ,Gene - Abstract
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
- Published
- 2018
37. Abstract 373: The Molecular Consequence of a Polymorphic 25bp Deletion in Intron 32 of MYBPC3, Specific to South Asians
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Sangeetha Kandoi, Winston Shim, Jonathan G. Seidman, Ratan Bhat, Shiv Kumar Viswanathan, Ralph Knöll, Jennifer A. Schwanekamp, Rama Shanker Verma, Sakthivel Sadayappan, Parth N Patel, and Christine E. Seidman
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Genetics ,Contractility ,South asia ,Physiology ,Intron ,Cardiac myosin ,Biology ,Cardiology and Cardiovascular Medicine - Abstract
Background: MYBPC3 encodes cardiac myosin binding protein-C, which regulates sarcomeric stability and contractility. Previous work described a prevalent 25bp deletion in intron 32, MYBPC3 Δ 25bp , in the South Asian population. Occurring in an estimated 100 million carriers, this variant may be associated with cardiomyopathy and heart failure. MYBPC3 Δ25bp encompasses a splicing branch point that could affect splicing of exon 32 to exon 33, potentially resulting in an aberrantly spliced MYBPC3 transcript where exon 33 is skipped entirely. However, the mechanism by which MYBPC3 Δ 25bp associates with disease remains unclear. Methods: To determine the consequence of the 25bp deletion in vivo , mice were engineered that replaced the endogenous Mybpc3 intron 32 with either the normal human intron 32 or human intron 32 containing the 25bp deletion ( Mybpc3 Δ 25bp ) . We characterized Mybpc3 splicing in mice carrying human intron-32 and human iPSC derived cardiomyocytes (hiPSC-CM) from patients heterozygous and homozygous for MYBPC3 Δ 25bp by RT-PCR and using mini-gene assays (across 5 different mouse and human gene segments encompassing exon 32 through exon 34). Results: Echocardiography of mice 3 months of age revealed no changes in cardiac function by fractional shortening (WT: 32.8%, Het: 34.2%, Homo: 36.7%). However, homozygous Mybpc3 Δ 25bp mice had significantly smaller hearts compared to heterozygous and wildtype (3.4 vs 3.71 and 4.25 mg/g p=0.001). Mybpc3 Δ 25bp heterozygous and homozygous mice displayed minimal alternative splicing in exon 33 at baseline. Similarly, transcripts from hiPSC-CM from heterozygous and homozygous MYBPC3 Δ 25bp carriers also show minimal alternative splicing. Mini gene assays suggest that the 25bp deletion does reduce the proportion (77.5% v. 86.2%) of normally spliced transcripts. Conclusions: Taken together, these data suggest that under baseline conditions, MYBPC3 Δ 25bp results in minimal alternative splicing in both human iPSC-CMs and humanized mice harboring the 25bp deletion. While increase aberrant splicing may increase risk of hypertrophic cardiomyopathy in variant-carrying individuals, ongoing studies will determine whether other factors or gene mutations modulate the pathogenicity of the MYBPC3 Δ25bp variant.
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- 2018
38. Association of Cardiomyopathy With MYBPC3 D389V and MYBPC3Δ25bpIntronic Deletion in South Asian Descendants
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Sakthivel Sadayappan, Elizabeth M. McNally, Philip Wong, Michael J. Zilliox, Thriveni Sanagala, Ashish Mehta, Regina Fritsche-Danielson, Megan J. Puckelwartz, Winston Shim, U. Nalla B. Durai, Hak Chiaw Tang, Rama Shanker Verma, Gina Kuffel, Domodhar P. Suresh, Philip R. Khoury, Shiv Kumar Viswanathan, Ratan Bhat, Sangeetha Kandoi, Ralph Knöll, Richard C. Becker, Aravindakshan Jagadeesan, Chrishan J A Ramachandra, Robert E. Molokie, Jennifer A. Schwanekamp, Annie Thomas, and Lorenzo L. Pesce
- Subjects
0301 basic medicine ,Adult ,Male ,medicine.medical_specialty ,Adolescent ,Genotype ,Population ,Cardiomyopathy ,Gene mutation ,03 medical and health sciences ,Young Adult ,Internal medicine ,medicine ,Humans ,Allele ,education ,Aged ,Original Investigation ,Aged, 80 and over ,education.field_of_study ,Ejection fraction ,Asian ,business.industry ,Hypertrophic cardiomyopathy ,Stroke Volume ,Cardiomyopathy, Hypertrophic ,Middle Aged ,medicine.disease ,030104 developmental biology ,Phenotype ,Heart failure ,Mutation ,Female ,Cardiology and Cardiovascular Medicine ,business ,Carrier Proteins - Abstract
IMPORTANCE: The genetic variant MYBPC3(Δ25bp) occurs in 4% of South Asian descendants, with an estimated 100 million carriers worldwide. MYBPC3 (Δ25bp) has been linked to cardiomyopathy and heart failure. However, the high prevalence of MYBPC3(Δ25bp) suggests that other stressors act in concert with MYBPC3(Δ25bp). OBJECTIVE: To determine whether there are additional genetic factors that contribute to the cardiomyopathic expression of MYBPC3(Δ25bp). DESIGN, SETTING, ANDPARTICIPANTS: South Asian individuals living in the United States were screened for MYBPC3(Δ25bp), and a subgroup was clinically evaluated using electrocardiograms and echocardiograms at Loyola University, Chicago, Illinois, between January 2015 and July 2016. MAIN OUTCOMES AND MEASURES: Next-generation sequencing of 174 cardiovascular disease genes was applied to identify additional modifying gene mutations and correlate genotype-phenotype parameters. Cardiomyocytes derived from human-induced pluripotent stem cells were established and examined to assess the role of MYBPC3(Δ25bp). RESULTS: In this genotype-phenotype study, individuals of South Asian descent living in the United States from both sexes (36.23% female) with a mean population age of 48.92 years (range, 18-84 years) were recruited. Genetic screening of 2401 US South Asian individuals found an MYBPC3(Δ25bp)carrier frequency of 6%. A higher frequency of missense TTN variation was found in MYBPC3(Δ25bp) carriers compared with noncarriers, identifying distinct genetic backgrounds within the MYBPC3(Δ25bp) carrier group. Strikingly, 9.6% of MYBPC3(Δ25bp) carriers also had a novel MYBPC3 variant, D389V. Family studies documented D389V was in tandem on the same allele as MYBPC3(Δ25bp), and D389V was only seen in the presence of MYBPC3(Δ25bp). In contrast to MYBPC3(Δ25bp), MYBPC3(Δ25bp/D389V) was associated with hyperdynamic left ventricular performance (mean [SEM] left ventricular ejection fraction, 66.7 [0.7%]; left ventricular fractional shortening, 36.6 [0.6%]; P
- Published
- 2018
39. Desmin, desminopathy and the complexity of genetics
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Byambajav Buyandelger, Valerio Azzimato, Ralph Knöll, Ali M. Tabish, and Nina Gennebäck
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0301 basic medicine ,Pathology ,medicine.medical_specialty ,Intermediate Filaments ,Cardiomyopathy ,030204 cardiovascular system & hematology ,Biology ,medicine.disease ,Muscular Dystrophies ,Desmin ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Mutation ,medicine ,Humans ,Cardiomyopathies ,Cardiology and Cardiovascular Medicine ,Intermediate filament ,Molecular Biology - Published
- 2016
40. An inverse problem approach to identify the internal force of a mechanosensation process in a cardiac myocyte
- Author
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Ralph Knöll, Choi-Hong Lai, Byambajav Buyandelger, Kokulan Natkunam, and Serife Arif
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0301 basic medicine ,Mechanosensation ,biology ,business.industry ,Cardiac myocyte ,Health Informatics ,Structural engineering ,030204 cardiovascular system & hematology ,Inverse problem ,lcsh:Computer applications to medicine. Medical informatics ,Sarcomere ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,biology.protein ,lcsh:R858-859.7 ,Titin ,Mechanotransduction ,business ,Internal forces ,QA ,Neuroscience ,Maladaptation - Abstract
Mechanosensation and mechanotransduction are fundamental processes in understanding the link between physical stimuli and biological responses which currently still remain not well understood. The precise molecular mechanism involved in stress and strain detection in cells is unclear. Sarcomeres are the contractile machines of a cardiac myocyte and two main sarcomeric components that are directly involved in the sensation and transmission of mechanical stimuli are titin and filaments (thin and thick). Titin is known as the largest protein in biology with a mass of up to 4.2 MDa. Its flexible region (I-band region) may function as a length sensor (ε=l/l0) while its Z-disc domain may be involved in the sensation of tension and stress (Ï=F/A). Filaments act as contractile machineries by converting biochemical signals into mechanical work which in response cells either shorten or relax. Based on these considerations and a qualitative understanding of the maladaptation contribution to the development of heart failure, an inverse problem approach is taken to evaluate the contractile force in a mathematical model that describes mechanosensation in normal heart cells. Different functional forms to describe the contractile force are presented and for each of them we study the computational efficiency and accuracy of two numerical techniques. Keywords: Mechanosensation, Excitation, Contraction, Vibration model, Inverse problem approach
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- 2017
41. A role for membrane shape and information processing in cardiac physiology
- Author
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Ralph Knöll
- Subjects
Pressure overload ,medicine.medical_specialty ,Mechanotransduction ,Mechanoelectric feedback ,Membrane Fluidity ,Physiology ,Force–frequency relationship ,Clinical Biochemistry ,Action Potentials ,Heart failure ,030204 cardiovascular system & hematology ,Biology ,Mechanotransduction, Cellular ,03 medical and health sciences ,0302 clinical medicine ,Heart Conduction System ,Physiology (medical) ,Internal medicine ,Caveolae ,medicine ,Animals ,Humans ,Myocytes, Cardiac ,Electromechanic feedback ,Cytoskeleton ,Excitation Contraction Coupling ,Ion channel ,Frank Starling law of the heart ,030304 developmental biology ,0303 health sciences ,Frank–Starling law of the heart ,Invited Review ,Costameres ,Mechanosensation ,Cell Membrane ,Cardiac myocyte ,Models, Cardiovascular ,Myocardial Contraction ,Endocrinology ,Volume overload ,Pacing-induced heart failure ,Neuroscience - Abstract
While the heart is a dynamic organ and one of its major functions is to provide the organism with sufficient blood supply, the regulatory feedback systems, which allow adaptation to hemodynamic changes, remain not well understood. Our current description of mechanosensation focuses on stretch-sensitive ion channels, cytoskeletal components, structures such as the sarcomeric Z-disc, costameres, caveolae, or the concept of tensegrity, but these models appear incomplete as the remarkable plasticity of the myocardium in response to biomechanical stress and heart rate variations remains unexplained. Signaling activity at membranes depends on their geometric parameters such as surface area and curvature, which links shape to information processing. In the heart, continuous cycles of contraction and relaxation reshape membrane morphology and hence affect cardio-mechanic signaling. This article provides a brief review on current models of mechanosensation and focuses on how signaling, cardiac myocyte dynamics, and membrane shape interact and potentially give rise to a self-organized system that uses shape to sense the extra- and intracellular environment. This novel concept may help to explain how changes in frequency, and thus membrane shape, affect cardiac plasticity. One of the conclusions is that hypertrophy and associated fibrosis, which have been considered as necessary to cope with increased wall stress, can also be seen as part of complex feedback systems which use local membrane inhomogeneity in different cardiac cell types to influence whole organphysiology and which are predicted to fine-tune and thus regulate membrane-mediated signaling.
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- 2014
42. ESC Working Group on Myocardial Function Position Paper: how to study the right ventricle in experimental models
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Guido Tarone, Adelino F. Leite-Moreira, Denise Hilfiker-Kleiner, Johann Bauersachs, Thomas Thum, Manuel Mayr, Carlo G. Tocchetti, Leon J. De Windt, Ralph Knöll, Guido Iaccarino, Jean-Luc Balligand, Karol Kamiński, Angela Clerk, Stephane Heymans, Emilio Hirsch, and André P. Lourenço
- Subjects
Functional evaluation ,medicine.medical_specialty ,business.industry ,Cardiovascular research ,Exercise capacity ,Myocardial function ,medicine.anatomical_structure ,Right heart failure ,Ventricle ,Internal medicine ,Cardiology ,Medicine ,Position paper ,Cardiology and Cardiovascular Medicine ,business - Abstract
The right ventricle has become an increasing focus in cardiovascular research. In this position paper, we give a brief overview of the specific pathophysiological features of the right ventricle, with particular emphasis on functional and molecular modifications as well as therapeutic strategies in chronic overload, highlighting the differences from the left ventricle. Importantly, we put together recommendations on promising topics of research in the field, experimental study design, and functional evaluation of the right ventricle in experimental models, from non-invasive methodologies to haemodynamic evaluation and ex vivo set-ups.
- Published
- 2014
43. Molecular disturbance underlies to arrhythmogenic cardiomyopathy induced by transgene content, age and exercise in a truncated PKP2 mouse model
- Author
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Marta Casado, Ralph Knöll, Eduard Guasch, Maria Sanz-de la Garza, Ramon Brugada, Oscar Campuzano, Byambajav Buyandelger, Antonio Diez-Juan, Nahuel A. García, Marta Sitges, Javier Moncayo-Arlandi, Lluís Mont, La Caixa, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Fundació La Marató de TV3, Casado, Marta, and Casado, Marta [0000-0001-6457-4650]
- Subjects
0301 basic medicine ,Genetically modified mouse ,Aging ,medicine.medical_specialty ,Transgene ,Cardiomyopathy ,Plakoglobin ,Connexin ,030204 cardiovascular system & hematology ,Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Fibrosis ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Transgenes ,Molecular Biology ,Arrhythmogenic Right Ventricular Dysplasia ,Genetics (clinical) ,General Medicine ,medicine.disease ,Phenotype ,Disease Models, Animal ,030104 developmental biology ,Endocrinology ,Mutation ,Disease Progression ,Physical Endurance ,Desmin ,Plakophilins - Abstract
13 páginas, 9 tablas, 2 figuras. Contiene material suplementario., Arrhythmogenic cardiomyopathy (ACM) is a disorder characterized by a progressive ventricular myocardial replacement by fat and fibrosis, which lead to ventricular arrhythmias and sudden cardiac death. Mutations in the desmosomal gene Plakophilin-2 (PKP2) accounts for >40% of all known mutations, generally causing a truncated protein. In a PKP2-truncated mouse model, we hypothesize that content of transgene, endurance training and aging will be determinant in disease progression. In addition, we investigated the molecular defects associated with the phenotype in this model. We developed a transgenic mouse model containing a truncated PKP2 (PKP2-Ser329) and generated three transgenic lines expressing increasing transgene content. The pathophysiological features of ACM in this model were assessed. While we did not observe fibro-fatty replacement, ultrastructural defects were exhibited. Moreover, we observed transgene content-dependent development of structural (ventricle dilatation and dysfunction) and electrophysiological anomalies in mice (PR interval and QRS prolongation and arrhythmia induction). In concordance with pathological defects, we detected a content reduction and remodeling of the structural proteins Desmocollin-2, Plakoglobin, native Plakophilin-2, Desmin and β-Catenin as well as the electrical coupling proteins Connexin 43 and cardiac sodium channel (Nav1.5). Surprisingly, we observed structural but not electrophysiological abnormalities only in trained and old mice. We demonstrated that truncated PKP2 provokes ACM in the absence of fibro-fatty replacement in the mouse. Transgene dose is essential to reveal the pathology, whereas aging and endurance training trigger limited phenotype. Molecular abnormalities underlay the structural and electrophysiological defects., This work was supported by grants from “Fundació la Caixa”, Red de Investigación Cardiovascular from Instituto Salud Carlos III (RD12/0042/0044 and RD12/0042/0019), MINECO (SAF2012-39732), and Fundació Marató TV3 2014, Spain.
- Published
- 2016
44. Ventricular Assist Device Implantation Corrects Myocardial Lipotoxicity, Reverses Insulin Resistance, and Normalizes Cardiac Metabolism in Patients With Advanced Heart Failure
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Konstantinos Drosatos, Ira J. Goldberg, Ralph Knöll, Raffay S. Khan, Faisal H. Cheema, Hendrik Milting, Aalap Chokshi, Hiroo Takayama, Shuiqing Yu, P. Christian Schulze, Christine Chung, Donna Mancini, Tomoko Kato, Ulrich P. Jorde, Tuba Khawaja, Ruiping Ji, and Yoshifumi Naka
- Subjects
Adult ,Male ,medicine.medical_specialty ,medicine.medical_treatment ,Ceramides ,Severity of Illness Index ,Article ,Cell Line ,Diglycerides ,chemistry.chemical_compound ,Insulin resistance ,Physiology (medical) ,Internal medicine ,medicine ,Insulin ,Humans ,Myocytes, Cardiac ,Protein kinase B ,Protein Kinase C ,Triglycerides ,Aged ,Retrospective Studies ,Ultrasonography ,Diacylglycerol kinase ,Heart Failure ,biology ,Triglyceride ,business.industry ,Myocardium ,Fatty Acids ,Middle Aged ,Lipid Metabolism ,medicine.disease ,Insulin receptor ,Endocrinology ,chemistry ,Lipotoxicity ,Heart failure ,Ventricular assist device ,biology.protein ,Female ,Heart-Assist Devices ,Insulin Resistance ,Cardiology and Cardiovascular Medicine ,business ,Signal Transduction - Abstract
Background— Heart failure is associated with impaired myocardial metabolism with a shift from fatty acids to glucose use for ATP generation. We hypothesized that cardiac accumulation of toxic lipid intermediates inhibits insulin signaling in advanced heart failure and that mechanical unloading of the failing myocardium corrects impaired cardiac metabolism. Methods and Results— We analyzed the myocardium and serum of 61 patients with heart failure (body mass index, 26.5±5.1 kg/m 2 ; age, 51±12 years) obtained during left ventricular assist device implantation and at explantation (mean duration, 185±156 days) and from 9 control subjects. Systemic insulin resistance in heart failure was accompanied by decreased myocardial triglyceride and overall fatty acid content but increased toxic lipid intermediates, diacylglycerol, and ceramide. Increased membrane localization of protein kinase C isoforms, inhibitors of insulin signaling, and decreased activity of insulin signaling molecules Akt and Foxo were detectable in heart failure compared with control subjects. Left ventricular assist device implantation improved whole-body insulin resistance (homeostatic model of analysis–insulin resistance, 4.5±0.6–3.2±0.5; P Conclusions— Mechanical unloading after left ventricular assist device implantation corrects systemic and local metabolic derangements in advanced heart failure, leading to reduced myocardial levels of toxic lipid intermediates and improved cardiac insulin signaling.
- Published
- 2012
45. Age‐dependent changes in contractile function and passive elastic properties of myocardium from mice lacking muscle LIM protein (MLP)
- Author
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Ralph Knöll, Stefanie Klede, Hanna Schotola, Claudius Jacobshagen, Ali El-Armouche, Wolfgang A. Linke, Kaomei Guan, Tim Seidler, Bernhard Unsöld, Gerd Hasenfuss, Harald Kögler, Julius Emons, and Samuel Sossalla
- Subjects
medicine.medical_specialty ,Myofilament ,Heart Ventricles ,Gene Expression ,Muscle Proteins ,030204 cardiovascular system & hematology ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Contractility ,Mice ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Fibrosis ,Internal medicine ,medicine ,Animals ,Ventricular Function ,Myocytes, Cardiac ,RNA, Messenger ,030304 developmental biology ,Analysis of Variance ,0303 health sciences ,business.industry ,Myocardium ,Null (mathematics) ,Age Factors ,Dilated cardiomyopathy ,LIM Domain Proteins ,Papillary Muscles ,medicine.disease ,Myocardial Contraction ,Phenotype ,Endocrinology ,Echocardiography ,Heart failure ,Catecholamine ,Elasticity Imaging Techniques ,Cardiology and Cardiovascular Medicine ,business ,medicine.drug - Abstract
Aims Muscle LIM protein (MLP) null mice are often used as a model for human dilated cardiomyopathy. So far, little is known about the time course and pathomechanisms leading to the development of the adult phenotype. Methods and results We systematically analysed the contractile phenotype, myofilament calcium (Ca2+) responsiveness, passive myocardial mechanics, histology, and mRNA expression in mice aged 4 and 12 weeks. In 4-week-old animals, there was no significant difference in the force–frequency relationship (FFR) and catecholamine response of intact isolated papillary muscles between wild-type (WT) and MLP null myocardium. In 12-week-old animals, WT myocardium exhibited a significantly positive FFR, while that of MLP null mice was significantly negative, and the inotropic response to catecholamines was significantly reduced in MLP null mice. This time course of decline in contractile function was confirmed in vivo by echocardiography. Whereas at 4 weeks of age MLP null mice and WT littermates showed similar levels of SERCA2a (sarcoplasmic reticulum Ca2+ ATPase) expression, the expression was significantly lower in 12-week-old MLP null mice compared with littermate controls. Myofilament Ca2+ responsiveness was not affected by the lack of MLP, irrespective of age. Whereas in 4-week-old animals MLP null myocardium showed a trend to an increased compliance compared with the WT, myocardium of 12-week-old MLP null mice was significantly less compliant than WT myocardium. Parallel to the decrease in compliance there was an increase in fibrosis in the MLP null animals. Conclusion Our data suggest that MLP deficiency does not primarily influence myocardial contractility. A lack of MLP leads to an age-dependent impairment of excitation–contraction coupling with resulting contractile dysfunction and secondary fibrosis.
- Published
- 2012
46. On Mechanosensation, Acto/Myosin Interaction, and Hypertrophy
- Author
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Steve Marston and Ralph Knöll
- Subjects
Apoptosis ,Myosins ,Telethonin ,Biology ,Contractility ,03 medical and health sciences ,Adenosine Triphosphate ,0302 clinical medicine ,Myosin ,Autophagy ,Extracellular ,Humans ,Myocyte ,Actin ,030304 developmental biology ,0303 health sciences ,Hypertrophy, Right Ventricular ,Mechanosensation ,Myocardial Contraction ,Actins ,Cell biology ,Adenosine Diphosphate ,Hypertrophy, Left Ventricular ,Cardiology and Cardiovascular Medicine ,Mechanoreceptors ,030217 neurology & neurosurgery ,Intracellular - Abstract
Current concepts of mechanosensation are general and applicable to almost every cell type. However, striated muscle cells are distinguished by their ability to generate strong forces via actin/myosin interaction, and this process is fine-tuned for optimum contractility. This aspect, unique for actively contracting cells, may be defined as "sensing of the magnitude and dynamics of contractility," as opposed to the well-known concepts of the "perception of extracellular mechanical stimuli." The acto/myosin interaction, by producing changes in ATP, ADP, Pi, and force on a millisecond timescale, may be regarded as a novel and previously unappreciated mechanosensory mechanism. In addition, sarcomeric mechanosensory structures, such as the Z-disc, are directly linked to autophagy, survival, and cell death-related pathways. One emerging example is telethonin and its ability to interfere with p53 metabolism and hence apoptosis (mechanoptosis). In this article, we introduce contractility per se as an important mechanosensory mechanism, and we differentiate extracellular from intracellular mechanosensory effects.
- Published
- 2012
47. Abstract 17978: Mlp Interacting Protein 1 (MIP1) Plays a Role for Cardiomyopathy
- Author
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Byambajav Buyandelger, Catherine Mansfield, Sawa Kostin, Onjee Choi, Rivka L. Isaacson, Kar-Lai Poon, Gudrun Knöll, Sara McSweeney, Andreas Perrot, Mohammad R Toliat, Ju Chen, Ian Gould, Enrique Lara-Pezzi, Pradeep Luther, Enrico Petretto, Hendrik Milting, Thomas Brand, and Ralph Knöll
- Subjects
Physiology (medical) ,Cardiology and Cardiovascular Medicine - Abstract
Mutations in genes involved in cardiac mechanosensation (mec), such as the muscle LIM protein (MLP) have been shown to cause heart failure. Identification of novel mec genes may provide new insights into underlying mechanisms. A yeast 2 hybrid screen identified a novel MIP, located at a chromosomal region recently implicated by GWASs in the pathogenesis of dilated cardiomyopathy (DCM), an interaction confirmed by various assays. We found that MIP1 is a stress inducible transcription factor, binds to the calcineurin promoter and activates NFAT, a known mediator of hypertrophy. Also overexpression of MIP1 in vitro promotes autophagy and activates expression of anti-apoptotic genes in cardiac myocytes. Mip1 conventional knockout animals die during gastrulation whereas Mip1 conditional knockout (cKO) animals failed to adapt to transverse aortic constriction. Four weeks after intervention, fractional shortening was significantly decreased, whereas LVEDD and LVESD were significantly increased in cKO mice compared to control littermates. TUNEL assays combined with activated caspase, whole gene expression arrays and qRT-PCR provide strong evidence that Mip1 cKO hearts are unable to undergo autophagy and as a result activate apoptotic processes which are responsible for the observed heart failure phenotype. However, overexpression of MIP1 in vivo resulted in marked cardiac hypertrophy at the organ and single cell levels, pointing to a link between autophagy and hypertrophy. As MIP1 contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus, we sequenced this gene in 575 unrelated individuals affected by DCM and identified 4 MIP1 missense mutations (T106M, G179S, A188V, Q531R) of which 2 co-segregate in two families with DCM and arrhythmia. All 4 variants show abnormalities with regards to their ability to interact with MLP, localization, structure, stability and potential to drive the calcineurin promoter. WT-MIP1 is able to efficiently protect cells from apoptosis, a property missing in all mutants. At least G179S and Q531R cause heart failure in the zebrafish. MIP1 is important for the initiation of survival pathways and autophagy upon biomechanical stress and represents a novel cardiomyopathy candidate gene.
- Published
- 2015
48. MLP (muscle LIM protein) as a stress sensor in the heart
- Author
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Keat-Eng Ng, Ching-Hsin Ku, Izabela Piotrowska, Byambajav Buyandelger, Snjezana Miocic, Sylvia Gunkel, and Ralph Knöll
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Cardiomyopathy, Dilated ,medicine.medical_specialty ,Physiology ,Clinical Biochemistry ,ved/biology.organism_classification_rank.species ,Cardiomyopathy ,Muscle Proteins ,Cardiomyocyte ,Biology ,Mechanotransduction, Cellular ,Mechanosensitivity ,Mechanoreceptor ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Humans ,Cardiac muscle ,CSRP3 ,Model organism ,Muscle stretch ,Invited Review ,Myogenesis ,ved/biology ,Myocardium ,Cardiac function ,Heart ,Dilated cardiomyopathy ,Cardiomyopathy, Hypertrophic ,LIM Domain Proteins ,medicine.disease ,Phenotype ,Cardiac myocytes ,Cardiac sarcomere ,medicine.anatomical_structure ,Endocrinology ,Cardiovascular control ,Heart failure ,Stress, Mechanical ,Gene expression ,Neuroscience - Abstract
Muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3, CRP3)) is a muscle-specific-expressed LIM-only protein. It consists of 194 amino-acids and has been described initially as a factor involved in myogenesis (Arber et al. Cell 79:221–231, 1994). MLP soon became an important model for experimental cardiology when it was first demonstrated that MLP deficiency leads to myocardial hypertrophy followed by a dilated cardiomyopathy and heart failure phenotype (Arber et al. Cell 88:393–403, 1997). At this time, this was the first genetically altered animal model to develop this devastating disease. Interestingly, MLP was also found to be down-regulated in humans with heart failure (Zolk et al. Circulation 101:2674–2677, 2000) and MLP mutations are able to cause hypertrophic and dilated forms of cardiomyopathy in humans (Bos et al. Mol Genet Metab 88:78–85, 2006; Geier et al. Circulation 107:1390–1395, 2003; Hershberger et al. Clin Transl Sci 1:21–26, 2008; Knöll et al. Cell 111:943–955, 2002; Knöll et al. Circ Res 106:695–704, 2010; Mohapatra et al. Mol Genet Metab 80:207–215, 2003). Although considerable efforts have been undertaken to unravel the underlying molecular mechanisms—how MLP mutations, either in model organisms or in the human setting cause these diseases are still unclear. In contrast, only precise knowledge of the underlying molecular mechanisms will allow the development of novel and innovative therapeutic strategies to combat this otherwise lethal condition. The focus of this review will be on the function of MLP in cardiac mechanosensation and we shall point to possible future directions in MLP research.
- Published
- 2011
49. The Sarcomeric Z-Disc and Z-Discopathies
- Author
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Byambajav Buyandelger, Max J. Lab, and Ralph Knöll
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Sarcomeres ,lcsh:Biotechnology ,Health, Toxicology and Mutagenesis ,Muscle Proteins ,lcsh:Medicine ,Review Article ,Biology ,Sarcomere ,03 medical and health sciences ,0302 clinical medicine ,Muscular Diseases ,lcsh:TP248.13-248.65 ,Genetics ,Animals ,Humans ,Mechanotransduction ,Intracellular signalling ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Mechanosensation ,lcsh:R ,General Medicine ,Anatomy ,Mechanical stability ,Molecular Medicine ,Cardiomyopathies ,Neuroscience ,030217 neurology & neurosurgery ,Biotechnology - Abstract
The sarcomeric Z-disc defines the lateral borders of the sarcomere and has primarily been seen as a structure important for mechanical stability. This view has changed dramatically within the last one or two decades. A multitude of novel Z-disc proteins and their interacting partners have been identified, which has led to the identification of additional functions and which have now been assigned to this structure. This includes its importance for intracellular signalling, for mechanosensation and mechanotransduction in particular, an emerging importance for protein turnover and autophagy, as well as its molecular links to the t-tubular system and the sarcoplasmic reticulum. Moreover, the discovery of mutations in a wide variety of Z-disc proteins, which lead to perturbations of several of the above-mentioned systems, gives rise to a diverse group of diseases which can be termed Z-discopathies. This paper provides a brief overview of these novel aspects as well as points to future research directions.
- Published
- 2011
50. Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex
- Author
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Jeroen Bakkers, Ralph Knöll, Jacek Topczewski, Padmanabhan Vakeel, Ruben Postel, and Hubrecht Institute for Developmental Biology and Stem Cell Research
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Recombinant Fusion Proteins ,Integrin ,Actinin ,Protein Serine-Threonine Kinases ,MLP ,Antigens, CD ,Laminin ,Two-Hybrid System Techniques ,Cell Adhesion ,medicine ,Dystroglycan ,Animals ,Humans ,Integrin-linked kinase ,Muscle, Skeletal ,Cell adhesion ,Molecular Biology ,Cytoskeleton ,Phylogeny ,Zebrafish ,biology ,Lim Kinases ,Skeletal muscle ,Cell Biology ,Oligonucleotides, Antisense ,Zebrafish Proteins ,Muscular dystrophy ,Myotendinous junctions ,Extracellular Matrix ,Cell biology ,Phenotype ,medicine.anatomical_structure ,loc ,β-parvin ,embryonic structures ,biology.protein ,Cancer research ,Paxillin ,ILK ,Dystrophin ,Integrin alpha Chains ,Developmental Biology - Abstract
Mechanical instability of skeletal muscle cells is the major cause of congenital muscular dystrophy. Here we show that the zebrafish lost-contact mutant, that lacks a functional integrin-linked kinase (ilk) gene, suffers from mechanical instability of skeletal muscle fibres. With genetic and morpholino knock-down experiments we demonstrate that: 1) laminin, itgalpha7, Ilk and beta-parvin are all critical for mechanical stability in skeletal muscles. 2) Ilk acts redundantly with the dystrophin/dystroglycan adhesion complex in maintaining mechanical stability of skeletal muscles. 3) Ilk protein is recruited to the myotendinous junctions, which requires the ECM component laminin and the presence of itgalpha7 in the sarcolemma. 4) Ilk, unexpectedly, is dispensable for formation of the adhesion complex. Ilk, however, is required for strengthening the adhesion of the muscle fibre with the ECM and this activity requires the presence of a functional kinase domain in Ilk. 5) We identified a novel interaction between Ilk and the mechanical stretch sensor protein MLP. Thus, Ilk is an essential intracellular component downstream of laminin and itgalpha7, providing strengthening of skeletal muscle fibre adhesion with the ECM and therefore qualified as a novel candidate gene for congenital muscular dystrophy.
- Published
- 2008
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