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Aims In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. Methods and results We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. Conclusion Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

Aims Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce. Methods and results Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with Conclusion Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.

International audience; Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N′)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N′-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N′-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endothelium-mediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodium-hydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability.

Tetracycline antibiotics act by inhibiting bacterial protein translation. Given the bacterial ancestry of mitochondria, we tested the hypothesis that doxycycline—which belongs to the tetracycline class—reduces mitochondrial function, and results in cardiac contractile dysfunction in cultured H9C2 cardiomyoblasts, adult rat cardiomyocytes, in Drosophila and in mice. Ampicillin and carbenicillin were used as control antibiotics since these do not interfere with mitochondrial translation. In line with its specific inhibitory effect on mitochondrial translation, doxycycline caused a mitonuclear protein imbalance in doxycycline-treated H9C2 cells, reduced maximal mitochondrial respiration, particularly with complex I substrates, and mitochondria appeared fragmented. Flux measurements using stable isotope tracers showed a shift away from OXPHOS towards glycolysis after doxycycline exposure. Cardiac contractility measurements in adult cardiomyocytes and Drosophila melanogaster hearts showed an increased diastolic calcium concentration, and a higher arrhythmicity index. Systolic and diastolic dysfunction were observed after exposure to doxycycline. Mice treated with doxycycline showed mitochondrial complex I dysfunction, reduced OXPHOS capacity and impaired diastolic function. Doxycycline exacerbated diastolic dysfunction and reduced ejection fraction in a diabetes mouse model vulnerable for metabolic derangements. We therefore conclude that doxycycline impairs mitochondrial function and causes cardiac dysfunction.

Background - Variants within the alpha-tropomyosin gene (TPM1) cause dominantly inherited cardiomyopathies, including dilated (DCM), hypertrophic (HCM) and restrictive (RCM) cardiomyopathy. Here we investigated whether TPM1 variants observed in DCM and HCM patients affect cardiomyocyte physiology differently.Methods - We identified a large family with DCM carrying a recently identified TPM1 gene variant (T201M) and a child with RCM with compound heterozygote TPM1 variants (E62Q and M281T) whose family members carrying single variants show diastolic dysfunction and HCM. The effects of TPM1 variants (T201M, E62Q or M281T) and of a plasmid containing both the E62Q and M281T variants on single-cell Ca2+ transients (CaT) in HL-1 cardiomyocytes were studied. To define toxic threshold levels, we performed dose-dependent transfection of TPM1 variants. In addition, cardiomyocyte structure was studied in human cardiac biopsies with TPM1 variants.Results - Overexpression of TPM1 variants led to time-dependent progressive deterioration of CaT, with the smallest effect seen for E62Q and larger and similar effects seen for the T201M and M281T variants. Overexpression of E62Q/M281T did not exacerbate the effects seen with overexpression of a single TPM1 variant. T201M (DCM) replaced endogenous tropomyosin dose-dependently, while M281T (HCM) did not. Human cardiac biopsies with TPM1 variants revealed loss of sarcomeric structures.Conclusion - All TPM1 variants result in reduced cardiomyocyte CaT amplitudes and loss of sarcomeric structures. These effects may underlie pathophysiology of different cardiomyopathy phenotypes. (C) 2020 The Authors. Published by Elsevier B.V.

Background: The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. Methods and results: We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca2+-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca2+-sensitivity, incorporation of R278C increased Ca2+-sensitivity at low and intermediate dose, while it decreased Ca2+-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. Conclusions: Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca2+-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca2+-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

In this study, we synthesized and evaluated new photoswitchable ligands for the beta-adrenergic receptors β

Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca 2+ current density (I CaL) and steady-state inactiva-tion of I CaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMWAGEs contribute to the development of cardiovascular dysfunction in vivo, next to the wellknown low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in eft ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome. Acknowledgements We thank Marc Jans for assisting with the EM. The authors also thank Petra Bex, Rosette Beenaerts and Kanigula Mubagwa for their skillful technical assistance. The graphical abstract was created using images from Servier Medical Art Commons Attribution 3.0 Unported License (http://smart.servier.com). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. Funding This work was supported by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University (15NI06-BOF). Statement of Ethics The animal protocol was approved by the Local Ethical Committee (Ethical Commission for Animal Experimentation, UHasselt, Diepenbeek, Belgium). All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Only trained researchers, certified with a Laboratory Animal Science course according to the Federation of European Laboratory Animal Science Associations, performed animal handling and procedures.

Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have emerged as a powerful tool for disease modeling, though their immature nature currently limits translation into clinical practice. Maturation strategies increasingly pay attention to cardiac metabolism because of its pivotal role in cardiomyocyte development and function. Moreover, aberrances in cardiac metabolism are central to the pathogenesis of cardiac disease. Thus, proper modeling of human cardiac disease warrants careful characterization of the metabolic properties of iPSC-CMs. Methods Here, we examined the effect of maturation protocols on healthy iPSC-CMs applied in 23 studies and compared fold changes in functional metabolic characteristics to assess the level of maturation. In addition, pathological metabolic remodeling was assessed in 13 iPSC-CM studies that focus on hypertrophic cardiomyopathy (HCM), which is characterized by abnormalities in metabolism. Results Matured iPSC-CMs were characterized by mitochondrial maturation, increased oxidative capacity and enhanced fatty acid use for energy production. HCM iPSC-CMs presented varying degrees of metabolic remodeling ranging from compensatory to energy depletion stages, likely due to the different types of mutations and clinical phenotypes modeled. HCM further displayed early onset hypertrophy, independent of the type of mutation or disease stage. Conclusions Maturation strategies improve the metabolic characteristics of iPSC-CMs, but not to the level of the adult heart. Therefore, a combination of maturation strategies might prove to be more effective. Due to early onset hypertrophy, HCM iPSC-CMs may be less suitable to detect early disease modifiers in HCM and might prove more useful to examine the effects of gene editing and new drugs in advanced disease stages. With this review, we provide an overview of the assays used for characterization of cardiac metabolism in iPSC-CMs and advise on which metabolic assays to include in future maturation and disease modeling studies.

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-CΔC10mut) 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-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut 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-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ∼50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, SMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, SMN). Gene mutations are most often present in genes encoding the sarcomere proteins myosin heavy chain, myosin-binding protein C, and troponin T. Studies in cardiac tissue samples from patients with obstructive HCM that were obtained during myectomy surgery showed increased myofilament calcium sensitivity, increased kinetics and tension cost, and a reduction of the super-relaxed state of myosin, which is associated with an energy-conserving status of the crossbridges. The increase in myofilament calcium sensitivity is observed at a low dose of mutant protein, while the magnitude of the increase in calcium sensitivity depends on the specific mutation location. These mutation-mediated myofilament changes may underlie inefficient in vivo cardiac performance in mutation carriers. Reduced cardiac efficiency has been observed before onset of cardiac hypertrophy and at advanced disease stages. In addition, impaired diastolic function is an early disease characteristic of HCM. Our recent proteomics studies revealed increased detyrosination of microtubules, which may be a cause of diastolic dysfunction. Recent treatments that target inefficient cardiac performance, such as myosin inhibitors and metabolic drug therapies, may have the potential to prevent, delay, or even reverse disease in HCM-mutation carriers. Treatment response may depend on the specific gene mutation in SMP individuals and may explain diverse response of HCM patients to therapy. While mutation-mediated cardiomyocyte defects have become clear in past years, more research is warranted to define the cellular pathomechanisms of cardiac dysfunction in SMN patients.

Background: Genetic variants associated with cardiomyopathies (CMPs) are prevalent in the general population. In young athletes, CMPs account for roughly a quarter of sudden cardiac death, with further unexplained clustering in specific sports. Consequently, most CMPs form a contraindication for competitive sports. We hypothesized that genetic variants might (paradoxically) improve physical performance early in life while impairing cardiac function later in life. Methods: Systematic PubMed search was done to investigate whether genetic variants in genes associated with CMPs could be related to beneficial performance phenotypes. Summary: In a limited number of studies (n = 6), 2,860 individuals/subjects with genetic variants were able to outperform those without said variants, as measured by running speed (∼38 m/min in heterozygous [HET] mice, n = 6, vs. ∼32 m/min in wild type [WT] mice, n = 7, p = 0.004) and distance (966 ± 169 km HET mice vs. 561 ± 144 km WT mice, p = 0.0035, n = 10), elite athlete status in endurance athletes (n = 1,672, p = 1.43 × 10−8), maximal oxygen uptake in elite athletes (absolute difference not provided, n = 32, p = 0.005), maximal oxygen uptake in unrelated individuals (n = 473, p = 0.0025), personal records in highly trained marathon runners (2:26:28 ± 0:06:23 min HET, n = 32, vs. 2:28:53 ± 0:05:50 min without polymorphism, n = 108, p = 0.020), and peripheral muscle force contraction in patients following a cardiac rehabilitation program (absolute values not provided, n = 260). Key Message: Beneficial effects in genetic variants associated with CMPs could hypothetically play a role in the selection of young athletes, consequently explaining the prevalence of such genetic variants in athletes and the general population.

Trabecular muscle makes up most of the ventricular wall of the human embryo. It is presumed that compaction in the foetal period changes ventricular wall morphology by converting weaker trabeculae to stronger compact muscle. Using developmental series covering the embryonic and foetal periods of human, mouse and chicken, we show ventricular morphology is determined by differential rates of positive growth of trabecular and compact layers rather than compaction. In mouse, foetal cardiomyocytes are relative weak, but adult trabecular and compact cardiomyocytes are equally strong. In foetal and adult humans, trabecular and compact myocardium exhibit a similar abundance of immunohistochemically detected vascular, mitochondrial and sarcomeric proteins. Even in human noncompaction, a congenital malformation characterized by excessive trabeculation, the trabecular and compact muscle are similar. In conclusion, trabecular and compact myocardium are equally equipped for force production and their proportions are determined not by compaction, but by differential growth rates.

Visual Abstract, Highlights • CMECs exert a direct positive effect on cardiomyocyte contraction and relaxation, which is mainly mediated by endothelial-derived NO. • Pro-inflammatory stimulation of CMECs by pre-incubation with TNF-α or interleukin-1β abrogates the positive regulatory function of these cells on cardiomyocyte contractile property. • Mechanistically, pro-inflammatory activation of CMECs leads to mitochondrial and cytoplasmic ROS accumulation that results in the scavenging of NO. • Empagliflozin directly restores the beneficial effect of CMECs on cardiomyocyte contraction and relaxation by reducing TNF-α-induced mitochondrial and cytoplasmic ROS accumulation, which leads to reinstatement of CMEC-derived NO delivery., Summary The positive findings of the EMPA-REG OUTCOME trial (Randomized, Placebo-Controlled Cardiovascular Outcome Trial of Empagliflozin) on heart failure (HF) outcome in patients with type 2 diabetes mellitus suggest a direct effect of empagliflozin on the heart. These patients frequently have HF with preserved ejection fraction (HFpEF), in which a metabolic risk-related pro-inflammatory state induces cardiac microvascular endothelial cell (CMEC) dysfunction with subsequent cardiomyocyte (CM) contractility impairment. This study showed that CMECs confer a direct positive effect on contraction and relaxation of CMs, an effect that requires nitric oxide, is diminished after CMEC stimulation with tumor necrosis factor-α, and is restored by empagliflozin. Our findings on the effect of empagliflozin on CMEC-mediated preservation of CM function suggests that empagliflozin can be used to treat the cardiac mechanical implications of microvascular dysfunction in HFpEF.

Background: Sex-differences in clinical presentation contribute to the phenotypic heterogeneity of hypertrophic cardiomyopathy (HCM) patients. While disease prevalence is higher in men, women present with more severe diastolic dysfunction and worse survival. Until today, little is known about the cellular differences underlying sex-differences in clinical presentation.Methods: To define sex-differences at the protein level, we performed a proteomic analysis in cardiac tissue obtained during myectomy surgery to relieve left ventricular outflow tract obstruction of age-matched female and male HCM patients harboring a sarcomere mutation (n = 13 in both groups). Furthermore, these samples were compared to 8 non-failing controls. Women presented with more severe diastolic dysfunction.Results: Out of 2099 quantified proteins, direct comparison of male, and female HCM samples revealed only 46 significantly differentially expressed proteins. Increased levels of tubulin and heat shock proteins were observed in female compared to male HCM patients. Western blot analyses confirmed higher levels of tubulin in female HCM samples. In addition, proteins involved in carbohydrate metabolism were significantly lower in female compared to male samples. Furthermore, we found lower levels of translational proteins specifically in male HCM samples. The disease-specificity of these changes were confirmed by a second analysis in which we compared female and male samples separately to non-failing control samples. Transcription factor analysis showed that sex hormone-dependent transcription factors may contribute to differential protein expression, but do not explain the majority of protein changes observed between male and female HCM samples.Conclusion: In conclusion, based on our proteomics analyses we propose that increased levels of tubulin partly underlie more severe diastolic dysfunction in women compared to men. Since heat shock proteins have cardioprotective effects, elevated levels of heat shock proteins in females may contribute to later disease onset in woman, while reduced protein turnover in men may lead to the accumulation of damaged proteins which in turn affects proper cellular function.

Echocardiography is a reliable and reproducible method to assess non-invasively cardiac function in clinical and experimental research. Significant progress in the development of echocardiographic equipment and transducers has led to the successful translation of this methodology from humans to rodents, allowing for the scoring of disease severity and progression, testing of new drugs, and monitoring cardiac function in genetically modified or pharmacologically treated animals. However, as yet, there is no standardization in the procedure to acquire echocardiographic measurements in small animals. This position paper focuses on the appropriate acquisition and analysis of echocardiographic parameters in adult mice and rats, and provides reference values, representative images, and videos for the accurate and reproducible quantification of left ventricular function in healthy and pathological conditions. ispartof: CARDIOVASCULAR RESEARCH vol:117 issue:1 pages:43-59 ispartof: location:England status: published

Supplemental Digital Content is available in the text., Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCMSMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCMSMN). Genotype-specific differences have been reported in cardiac function. Moreover, HCMSMN patients have later disease onset and a better prognosis than HCMSMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. Methods: A proteomics screen was performed in cardiac tissue from 39 HCMSMP patients, 11HCMSMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC32373insG mouse model was used to confirm functional relevance of our proteomic findings. Results: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of total and detyrosinated α-tubulin were markedly higher in HCMSMP than in HCMSMN and controls. Higher tubulin detyrosination was also found in 2 unrelated MYBPC3 mouse models and its inhibition with parthenolide normalized contraction and relaxation time of isolated cardiomyocytes. Conclusions: Our findings indicate that microtubules and especially its detyrosination contribute to the pathomechanism of patients with HCMSMP. This is of clinical importance since it represents a potential treatment target to improve cardiac function in patients with HCMSMP, whereas a beneficial effect may be limited in patients with HCMSMN.

In recognition of the increasing health burden of cardiovascular disease, the Dutch CardioVascular Alliance (DCVA) was founded with the ambition to lower the cardiovascular disease burden by 25% in 2030. To achieve this, the DCVA is a platform for all stakeholders in the cardiovascular field to align policies, agendas and research. An important goal of the DCVA is to guide and encourage young researchers at an early stage of their careers in order to help them overcome challenges and reach their full potential. Young@Heart is part of the DCVA that supports the young cardiovascular research community. This article illustrates the challenges and opportunities encountered by young cardiovascular researchers in the Netherlands and highlights Young@Heart’s vision to benefit from these opportunities and optimise collaborations to contribute to lowering the cardiovascular disease burden together as soon as possible. Dutch contribution to the field Dutch cardiovascular alliance (DCVA) is a platform that combines stakeholders in the cardiovascular field with the goal to lower cardiovascular disease burden by 25% in 2030. Young@Heart is part of the talent pillar of DCVA and represents young researchers in the Netherlands. Young@Heart aims to provide career perspectives in academic, corporate and non-governmental settings for talented basic and clinical scientists. By providing workshops, national and international networking and funding opportunities, Young@Heart hopes to ensure a bright future for cardiovascular research in the Netherlands.

Background: It has been proposed that diabetes and chronic kidney disease cause heart failure with preserved ejection fraction (HFpEF) through low-grade inflammation and microvascular endothelial dysfunction. We have previously shown that endothelial enhancement of cardiomyocyte contraction and relaxation is mediated by NO, impaired by cytokines, and restored upon treatment with the SGLT2 inhibitor Empagliflozin. Chronic kidney disease (CKD) is linked to diabetes and HFpEF and causes systemic endothelial dysfunction. Objective: To test the hypothesis that uremic serum from patients with CKD impairs endothelial control of cardiomyocyte contraction and relaxation, and that Empagliflozin protects against this effect. Methods and Results: Co-culture of rat cardiomyocytes (CM) with human cardiac microvascular endothelial cells (CMECs) enhanced their contraction and relaxation. Pre-treatment with serum from patients with CKD abrogated these effects of CMEC, and treatment of CMECs with Empagliflozin restored the effects of CMECs on CM function. Exposure of CMECs to the uremic serum reduced endothelial NO bioavailability and increased intracellular reactive oxygen species ROS by increasing ROS generation by mitochondria. Uremic serum also increased 3-nitrotyrosine (3-NT) level, indicating scavenging of NO by ROS. Empagliflozin attenuated uremic serum-induced mitochondrial and intracellular ROS, leading to restoration of endothelial NO bioavailability. Conclusions: Exposure of CMECs to serum from CKD patients impairs CMEC-mediated enhancement of CM contraction and relaxation. Empagliflozin restores the beneficial effect of CMECs on CM function by reducing mitochondrial oxidative damage, leading to a decrease in intracellular ROS accumulation and increased endothelial NO bioavailability. Disclosure R. Juni: None. R. Al-Shama: None. D. Kuster: None. J. van der Velden: None. M. Vervloet: None. W. Paulus: None. E.C. Eringa: None. P. Koolwijk: None. V.W. van Hinsbergh: None. Funding Netherlands Heart Foundation

AIM: Calcium ions play a pivotal role in matching energy supply and demand in cardiac muscle. Mitochondrial calcium concentration is lower in animal models of heart failure with reduced ejection fraction (HFrEF), but limited information is available about mitochondrial calcium handling in heart failure with preserved ejection fraction (HFpEF).METHODS: We assessed mitochondrial Ca2+ handling in intact cardiomyocytes from Zucker/fatty Spontaneously hypertensive F1 hybrid (ZSF1)-lean (control) and ZSF1-obese rats, a metabolic risk-related model of HFpEF. A mitochondrially targeted Ca2+ indicator (MitoCam) was expressed in cultured adult rat cardiomyocytes. Cytosolic and mitochondrial Ca2+ transients were measured at different stimulation frequencies. Mitochondrial respiration and swelling, and expression of key proteins were determined ex vivo.RESULTS: At rest, mitochondrial Ca2+ concentration in ZSF1-obese was larger than in ZSF1-lean. The diastolic and systolic mitochondrial Ca2+ concentrations increased with stimulation frequency, but the steady-state levels were larger in ZSF1-obese. The half-widths of the contractile responses, the resting cytosolic Ca2+ concentration and the decay half-times of the cytosolic Ca2+ transients were higher in ZSF1-obese, likely because of a lower SERCA2a/phospholamban ratio. Mitochondrial respiration was lower, particularly with nicotinamide adenine dinucleotide (NADH) (complex I) substrates, and mitochondrial swelling was larger in ZSF1-obese.CONCLUSION: The free mitochondrial calcium concentration is higher in HFpEF owing to alterations in mitochondrial and cytosolic Ca2+ handling. This coupling between cytosolic and mitochondrial Ca2+ levels may compensate for myocardial ATP supply in vivo under conditions of mild mitochondrial dysfunction. However, if mitochondrial Ca2+ concentration is sustainedly increased, it might trigger mitochondrial permeability transition pore opening.

Mutations in genes encoding sarcomeric proteins are the most important causes of inherited cardiomyopathies, which are a major cause of mortality and morbidity worldwide. Although genetic screening procedures for early disease detection have been improved significantly, treatment to prevent or delay mutation-induced cardiac disease onset is lacking. Recent findings indicate that loss of protein quality control (PQC) is a central factor in the disease pathology leading to derailment of cellular protein homeostasis. Loss of PQC includes impairment of heat shock proteins, the ubiquitin-proteasome system, and autophagy. This may result in accumulation of misfolded and aggregation-prone mutant proteins, loss of sarcomeric and cytoskeletal proteins, and, ultimately, loss of cardiac function. PQC derailment can be a direct effect of the mutation-induced activation, a compensatory mechanism due to mutation-induced cellular dysfunction or a consequence of the simultaneous occurrence of the mutation and a secondary hit. In this review, we discuss recent mechanistic findings on the role of proteostasis derailment in inherited cardiomyopathies, with special focus on sarcomeric gene mutations and possible therapeutic applications.

Background: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). Methods: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (α-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. Results: Patients with PAH with lower RV ejection fraction ( Conclusions: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.

Key points Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+-sensitivity and reduced length-dependent activation. TNNT2p.K217del caused increased passive tension. A mutation in the gene encoding Lamin A/C (LMNAp.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy. Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. Abstract Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non-sarcomeric genes. In this study we defined the pathogenic effects of three DCM-causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non-sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane-permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+-sensitivity and impaired length-dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild-type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder. It is mainly caused by mutations in genes encoding sarcomere proteins. Mutant forms of these highly abundant proteins likely stress the protein quality control (PQC) system of cardiomyocytes. The PQC system, together with a functional microtubule network, maintains proteostasis. We compared left ventricular (LV) tissue of nine donors (controls) with 38 sarcomere mutation-positive (HCMSMP) and 14 sarcomere mutation-negative (HCMSMN) patients to define HCM and mutation-specific changes in PQC. Mutations in HCMSMP result in poison polypeptides or reduced protein levels (haploinsufficiency, HI). The main findings were 1) several key PQC players were more abundant in HCM compared to controls, 2) after correction for sex and age, stabilizing heat shock protein (HSP)B1, and refolding, HSPD1 and HSPA2 were increased in HCMSMP compared to controls, 3) &alpha, tubulin and acetylated &alpha, tubulin levels were higher in HCM compared to controls, especially in HCMHI, 4) myosin-binding protein-C (cMyBP-C) levels were inversely correlated with &alpha, tubulin, and 5) &alpha, tubulin levels correlated with acetylated &alpha, tubulin and HSPs. Overall, carrying a mutation affects PQC and &alpha, tubulin acetylation. The haploinsufficiency of cMyBP-C may trigger HSPs and &alpha, tubulin acetylation. Our study indicates that proliferation of the microtubular network may represent a novel pathomechanism in cMyBP-C haploinsufficiency-mediated HCM.

Background To maintain the balance between the demand of the body and supply (cardiac output), cardiac performance is tightly regulated via the parasympathetic and sympathetic nervous systems. In heart failure, cardiac output (supply) is decreased due to pathologic remodelling of the heart. To meet the demands of the body, the sympathetic system is activated and catecholamines stimulate β-adrenergic receptors (β-ARs) to increase contractile performance and cardiac output. Although this is beneficial in the acute phase, chronic β-ARs stimulation initiates a cascade of alterations at the cellular level, resulting in a diminished contractile performance of the heart. Materials and methods This narrative review includes results from previously published systematic reviews and clinical and basic research publications obtained via PubMed up to May 2015. Results We discuss the alterations that occur during sustained β-AR stimulation in diseased myocardium and emphasize the consequences of β-AR overstimulation for cardiac function. In addition, current treatment options as well as future therapeutic strategies to treat patients with heart failure to normalize consequences of β-AR overstimulation are discussed. Conclusions The heart is able to protect itself from chronic stimulation of the β-ARs via desensitization and reduced membrane availability of the β-ARs. However, ultimately this leads to an impaired downstream signalling and decreased protein kinase A (PKA)-mediated protein phosphorylation. β-blockers are widely used to prevent β-AR overstimulation and restore β-ARs in the failing hearts. However, novel and more specific therapeutic treatments are needed to improve treatment of HF in the future.

Hypertrophic cardiomyopathy (HCM) is the most frequent genetic cardiac disease with a prevalence of 1:500 to 1:200. While most patients show obstructive HCM and a relatively stable clinical phenotype (stage II), a small group of patients progresses to end-stage HCM (stage IV) within a relatively brief period. Previous research has shown sex-differences in stage II HCM with more diastolic dysfunction in female than in male patients. Moreover, female patients more often show progression to heart failure. Here we investigated if differences in functional and structural properties of the heart may underlie sex-differences in disease progression from stage II to stage IV HCM. Cardiac tissue from stage II and IV patients was obtained during myectomy (n = 54) and heart transplantation (n = 10), respectively. Isometric force was measured in membrane-permeabilized cardiomyocytes to define active and passive myofilament force development. Titin isoform composition was assessed using gel electrophoresis, and the amount of fibrosis and capillary density were determined with histology. In accordance with disease stage-dependent adverse cardiac remodeling end-stage patients showed a thinner interventricular septal wall and larger left ventricular and atrial diameters compared to stage II patients. Cardiomyocyte contractile properties and fibrosis were comparable between stage II and IV, while capillary density was significantly lower in stage IV compared to stage II. Women showed more adverse cellular remodeling compared to men at stage II, evident from more compliant titin, more fibrosis and lower capillary density. However, the disease stage-dependent reduction in capillary density was largest in men. In conclusion, the more severe cellular remodeling in female compared to male stage II patients suggests a more advanced disease stage at the time of myectomy in women. Changes in cardiomyocyte contractile properties do not explain the progression of stage II to stage IV, while reduced capillary density may underlie disease progression to end-stage heart failure.

BACKGROUND: Mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). These mutations produce dysfunctional protein that is quickly degraded and not incorporated in the myofilaments. Most patients are heterozygous and allelic expression differs between cells. We hypothesized that this would lead to cell-to-cell variation in cardiac myosin binding protein-C (cMyBP-C, encoded by MYBPC3 gene) protein levels.METHODS: Twelve HCM patients were included (six had no sarcomere mutations (HCMsmn) and served as the control group and six harbored mutations in the MYBPC3 gene (MYBPC3mut). Western blot and RNA sequencing analysis of cardiac tissue lysates were performed to detect overall cMyBP-C protein and mRNA levels. Cellular expression of cMyBP-C and α-actin was obtained by immunofluorescence staining. Quantification of cell-to-cell variation of cMyBP-C expression between cardiomyocytes was measured by determining the ratio of cMyBP-C:α-actin stained area of each cell.RESULTS: Protein and mRNA analysis revealed significantly reduced cMyBP-C levels in MYBPC3mut patients compared with HCMsmn patients (0.73 ± 0.09 vs. 1.0 ± 0.15, p CONCLUSION: This is the first study to demonstrate intercellular variation of myofilament cMyBP-C protein expression within the myocardium from HCM patients with heterozygous MYBPC3 mutations.

Significance: Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction, and myocardial disarray. Disease onset occurs between 20 and 50 years of age, thus affecting patients in the prime of their life. HCM is caused by mutations in sarcomere proteins, the contractile building blocks of the heart. Despite increased knowledge of causal mutations, the exact path from genetic defect leading to cardiomyopathy is complex and involves additional disease hits. Recent Advances: Laboratory-based studies indicate that HCM development not only depends on the primary sarcomere impairment caused by the mutation but also on secondary disease-related alterations in the heart. Here we propose a vicious mutation-induced disease cycle, in which a mutation-induced energy depletion alters cellular metabolism with increased mitochondrial work, which triggers secondary disease modifiers that will worsen disease and ultimately lead to end-stage HCM. Critical Issues: Evidence shows excessive cellular reactive oxygen species (ROS) in HCM patients and HCM animal models. Oxidative stress markers are increased in the heart (oxidized proteins, DNA, and lipids) and serum of HCM patients. In addition, increased mitochondrial ROS production and changes in endogenous antioxidants are reported in HCM. Mutant sarcomeric protein may drive excessive levels of cardiac ROS via changes in cardiac efficiency and metabolism, mitochondrial activation and/or dysfunction, impaired protein quality control, and microvascular dysfunction. Future Directions: Interventions restoring metabolism, mitochondrial function, and improved ROS balance may be promising therapeutic approaches. We discuss the effects of current HCM pharmacological therapies and potential future therapies to prevent and reverse HCM. Antioxid. Redox Signal. 31, 318-358.

Background: One of the first clinically detectable alterations in heart function in hypertrophic cardiomyopathy (HCM) is a decline in diastolic function. Diastolic dysfunction is caused by changes in intrinsic properties of cardiomyocytes or an increase in fibrosis. We investigated whether clinical and cellular parameters of diastolic function are different between male and female patients with HCM at the time of myectomy. Methods and Results: Cardiac tissue from the interventricular septum of patients with HCM (27 women and 44 men) was obtained during myectomy preceded by echocardiography. At myectomy, female patients were 7 years older than male patients and showed more advanced diastolic dysfunction than men evident from significantly higher values for E/e′ ratio, left ventricular filling pattern, tricuspid regurgitation velocity, and left atrial diameter indexed for body surface. Whereas most male patients (56%) showed mild (grade I) diastolic dysfunction, 50% of female patients showed grade III diastolic dysfunction. Passive tension in HCM cardiomyocytes was comparable with controls, and myofilament calcium sensitivity was higher in HCM compared with controls, but no sex differences were observed in myofilament function. In female patients with HCM, titin was more compliant, and more fibrosis was present compared with men. Differences between female and male patients with HCM remained significant after correction for age. Conclusions: Female patients with HCM are older at the time of myectomy and show greater impairment of diastolic function. Furthermore, left ventricular and left atrial remodeling is increased in women when corrected for body surface area. At a cellular level, HCM women showed increased compliant titin and a larger degree of interstitial fibrosis.

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)–mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.

Hypertrophic cardiomyopathy (HCM), the most common genetic cardiac disorder, is frequently caused by mutations in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Moreover, HCM is the leading cause of sudden cardiac death (SCD) in young athletes. Interestingly, SCD is more likely to occur in male than in female athletes. However, the pathophysiological mechanisms leading to sex-specific differences are poorly understood. Therefore, we studied the effect of sex and exercise on functional properties of the heart and sarcomeres in mice carrying a MYBPC3 point mutation (G A transition in exon 6) associated with human HCM. Echocardiography followed by isometric force measurements in left ventricular (LV) membrane-permeabilized cardiomyocytes was performed in wild-type (WT) and heterozygous (HET) knock-in mice of both sex (N = 5 per group) in sedentary mice and mice that underwent an 8-week voluntary wheel-running exercise protocol. Isometric force measurements in single cardiomyocytes revealed a lower maximal force generation (F max) of the sarcomeres in male sedentary HET (13.0 ± 1.1 kN/m(2)) compared to corresponding WT (18.4 ± 1.8 kN/m(2)) male mice. Exercise induced a higher F max in HET male mice, while it did not affect HET females. Interestingly, a low cardiac troponin I bisphosphorylation, increased myofilament Ca(2+)-sensitivity, and LV hypertrophy were particularly observed in exercised HET females. In conclusion, in sedentary animals, contractile differences are seen between male and female HET mice. Male and female HET hearts adapted differently to a voluntary exercise protocol, indicating that physiological stimuli elicit a sexually dimorphic cardiac response in heterozygous MYBPC3-targeted knock-in mice.

Background: Myocardial infarction (MI) leads to proteolytic cleavage of cMyBP-C (hC0C1f) and decreased contractility. Results: hC0C1f can incorporate into the human cardiac sarcomere, depressing force generation and increasing tension cost. Conclusion: Interaction between hC0C1f and both actin and α-tropomyosin causes disruption of intact cMyBP-C function. Significance: Proteolytic cleavage of cMyBP-C is sufficient to cause contractile dysfunction following MI., Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

Diagnosis of myocardial infarction (MI) is based on ST-segment elevation on electrocardiographic evaluation and/or elevated plasma cardiac troponin (cTn) levels. However, troponins lack the sensitivity required to detect the onset of MI at its earliest stages. Therefore, to confirm its viability as an ultra-early biomarker of MI, this study investigates the release kinetics of cardiac myosin binding protein-C (cMyBP-C) in a porcine model of MI and in two human cohorts. Release kinetics of cMyBP-C were determined in a porcine model of MI ( n = 6, pigs, either sex) by measuring plasma cMyBP-C level serially from 30 min to 14 days after coronary occlusion, with use of a custom-made immunoassay. cMyBP-C plasma levels were increased from baseline (76 ± 68 ng/l) at 3 h (767 ± 211 ng/l) and peaked at 6 h (2,418 ± 780 ng/l) after coronary ligation. Plasma cTnI, cTnT, and myosin light chain-3 levels were all increased 6 h after ligation. In a cohort of patients ( n = 12) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy, cMyBP-C was significantly increased from baseline (49 ± 23 ng/l) in a time-dependent manner, peaking at 4 h (560 ± 273 ng/l). In a cohort of patients with non-ST segment elevation MI ( n = 176) from the SYNERGY trial, cMyBP-C serum levels were significantly higher (7,615 ± 4,514 ng/l) than those in a control cohort (416 ± 104 ng/l; n = 153). cMyBP-C is released in the blood rapidly after cardiac damage and therefore has the potential to positively mark the onset of MI.

Cardiac myosin-binding protein C (cMyBP-C) research has been characterized by two waves. Initial interest was piqued by its discovery in 1973 as a contaminant of myosin preparations from skeletal muscle. The second wave started in 1995 by the discovery that mutations in the gene encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM). In this review, we will address what is known of cMyBP-C's role as a regulator of contraction as well as its role in HCM.

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that has been suggested to regulate cardiac contraction via its amino terminal (N’) region. However, the necessity of the N’-C0-C1f region (domains C0 through C1 and the first 17 residues of the M-domain) of cMyBP-C in regulating cardiac function in vivo has not been elucidated. Hypothesis: The N’-C0-C1f region of cMyBP-C is critical for normal cardiac function in vivo . Methods and Results: Transgenic mice with 80±4% expression of a truncated cMyBP-C missing the N’-C0-C1f region (cMyBP-C 110kDa ) were generated and characterized at 3-months of age. cMyBP-C 110kDa animals exhibited cardiac hypertrophy as suggested by an increased heart weight to body weight ratio (5.0±0.1 mg/g NTG vs. 6.9±0.1 mg/g cMyBP-C 110kDa , p110kDa hearts compared to hearts from non-transgenic (NTG) littermates. Intriguingly, increased phosphorylation of cMyBP-C at Ser-282 and Ser-302, sites important for cMyBP-C’s regulation of actomyosin interactions, was observed in cMyBP-C 110kDa hearts compared to controls. Electron microscopy revealed normal sarcomere structure in cMyBP-C 110kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110kDa hearts. Short-axis M-mode echocardiography indicated a significant elevation in left ventricular internal diameter and a significant reduction in fractional shortening (31±5% NTG vs. 16±3% cMyBP-C 110kDa , p=0.0003) in cMyBP-C 110kDa hearts compared to controls. Finally, global longitudinal strain analysis revealed abnormal wall motion in cMyBP-C 110kDa hearts. Based upon these data, we propose that the N’-region of cMyBP-C is a critical regulator of actomyosin interactions and controls aberrant contraction kinetics within the cardiac sarcomere. Conclusion: The N’-C0-C1f region of cMyBP-C regulates cardiac contractility and is necessary for maintaining normal cardiac function in vivo .

Background— Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results— Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3 mut ; n=17), mutation negative HCM patients without an identified sarcomere mutation (HCM mn ; n=11), and nonfailing donors (n=12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3 mut by 33±5%, and similar in HCM mn compared with donor. cMyBP-C phosphorylation in MYBPC3 mut was similar to donor, whereas it was significantly lower in HCM mn . Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3 mut and HCM mn, compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m 2 , respectively), and higher myofilament Ca 2+ -sensitivity (EC 50 =2.5±0.2, 2.4±0.2, and 3.0±0.2 μmol/L, respectively). The sarcomere length-dependent increase in Ca 2+ -sensitivity was significantly smaller in both patient groups compared with donor (ΔEC 50 : 0.46±0.04, 0.37±0.05, and 0.75±0.07 μmol/L, respectively). Protein kinase A treatment restored myofilament Ca 2+ -sensitivity and length-dependent activation in both patient groups to donor values. Conclusions— Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression.

Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation.

Since the completion of the Human Genome Project and the advent of the large scaled unbiased '-omics' techniques, the field of systems biology has emerged. Systems biology aims to move away from the traditional reductionist molecular approach, which focused on understanding the role of single genes or proteins, towards a more holistic approach by studying networks and interactions between individual components of networks. From a conceptual standpoint, systems biology elicits a 'back to the future' experience for any integrative physiologist. However, many of the new techniques and modalities employed by systems biologists yield tremendous potential for integrative physiologists to expand their tool arsenal to (quantitatively) study complex biological processes, such as cardiac remodelling and heart failure, in a truly holistic fashion. We therefore advocate that systems biology should not become/stay a separate discipline with '-omics' as its playing field, but should be integrated into physiology to create 'Integrative Physiology 2.0'.