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Ann-Kathrin Rahm,1– 3 Dominik Gramlich,1– 3 Teresa Wieder,1,2 Mara Elena Müller,1– 3 Axel Schoeffel,1,2 Fadwa A El Tahry,1 Patrick Most,1,2 Tanja Heimberger,1,3 Steffi Sandke,1,3 Tanja Weis,1,3 Nina D Ullrich,4 Thomas Korff,4,5 Patrick Lugenbiel,1– 3 Hugo A Katus,1– 3 Dierk Thomas1– 3 1Department of Cardiology, Medical University Hospital Heidelberg, Heidelberg, 69120, Germany; 2HCR (Heidelberg Center for Heart Rhythm Disorders), University Hospital Heidelberg, Heidelberg, 69120, Germany; 3DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University of Heidelberg, Heidelberg, 69120, Germany; 4Institute of Physiology and Pathophysiology, Department of Cardiovascular Physiology, Heidelberg University, Heidelberg, 69120, Germany; 5European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, 68167, GermanyCorrespondence: Dierk ThomasDepartment of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg, 69120, GermanyTel +49 6221 568855Fax +49 6221 565514Email dierk.thomas@med.uni-heidelberg.deAim: Effective antiarrhythmic treatment of atrial fibrillation (AF) constitutes a major challenge, in particular, when concomitant heart failure (HF) is present. HF-associated atrial arrhythmogenesis is distinctly characterized by prolonged atrial refractoriness. Small-conductance, calcium-activated K+ (KCa, SK, KCNN) channels contribute to cardiac action potential repolarization and are implicated in AF susceptibility and therapy. The mechanistic impact of AF/HF-related triggers on atrial KCa channels is not known. We hypothesized that tachycardia, stretch, β-adrenergic stimulation, and hypoxia differentially determine KCa 2.1– 2.3 channel remodeling in atrial cells.Methods: KCNN1-3 transcript levels were assessed in AF/HF patients and in a pig model of atrial tachypacing-induced AF with reduced left ventricular function. HL-1 atrial myocytes were subjected to proarrhythmic triggers to investigate the effects on Kcnn mRNA and KCa channel protein.Results: Atrial KCNN1-3 expression was reduced in AF/HF patients. KCNN2 and KCNN3 suppression was recapitulated in the corresponding pig model. In contrast to human AF, KCNN1 remained unchanged in pigs. Channel- and stressor-specific remodeling was revealed in vitro. Lower expression levels of KCNN1/KCa 2.1 were linked to stretch and β-adrenergic stimulation. Furthermore, KCNN3/KCa 2.3 expression was suppressed upon tachypacing and hypoxia. Finally, KCNN2/KCa 2.2 abundance was specifically enhanced by hypoxia.Conclusion: Reduction of KCa 2.1– 2.3 channel expression might contribute to the action potential prolongation in AF complicated by HF. Subtype-specific KCa 2 channel remodeling induced by tachypacing, stretch, β-adrenergic stimulation, or hypoxia is expected to differentially determine atrial remodeling, depending on patient-specific activation of each triggering factor. Stressor-dependent KCa 2 regulation in atrial myocytes provides a starting point for mechanism-based antiarrhythmic therapy.Keywords: atrial fibrillation, calcium, KCa channel, KCNN, remodeling, SK channel

Objective: Myocardial unloading with ventricular assist devices in patients with severe heart failure (HF) can lead to reversal of certain aspects of pathological remodeling. However, these effects do not translate into recovery of myocardial function in the human heart, possibly due to detrimental atrophic processes also elicited through unloading. We have studied the effects of long-term unloading on sarcomeric morphology and function in a small animal model of ventricular unloading, heterotopic heart transplantation (HTX) in rats.Methods: Native rat hearts were unloaded via HTX for 30 days, CMs from control and unloaded hearts were isolated (n=8 hearts/>250 individual cells/group). CM overall size was determined, sarcomere length/contractility assessed and Calcium transients as well as E-C coupling gain analyzed in patch-clamped CMs. Additionally, phosphorylation of Troponin I, indicative of sarcomere activation, was measured with western blotting.Results: CM cross-sectional area was diminished in unloaded cells by about one third (2787±345 vs 1993±230 μm2) as was cell capacitance in patched cells. Accordingly, baseline sarcomere length was significantly reduced by ~0.2μm (Figure). However, this reduction did not diminish contractile function: fractional shortening was significantly higher in unloaded CMs (8.0 ± 3 % vs 6.6 ± 2.5 % in CTR, p = 0.01). Departure velocity of the transients was similar (-135.2 ± 48 vs -119.4 ± 40 dL/dt), and return velocity was slightly increased in unloaded cells (120.7 ± 54 vs 94.0 ± 46 dL/dt, p < 0.05), indicating preserved relaxation. Calcium transient amplitudes and current-voltage relationship under basal condition and isoproterenol stimulation was not changed. Troponin I phosphorylation was elevated and may contribute to the maintenance of sarcomeric function in long-term unloaded CMs.Conclusion: Although there are limitations regarding assessment of contractility in isolated cells, we may conclude that the considerable size reduction in CMs induced by unloading does not translate into diminished contractile function or E-C coupling. [ABSTRACT FROM PUBLISHER]

Background: Promising as a treatment option for life-threatening ventricular arrhythmias, cardiac stereotactic body radiotherapy (cSBRT) has demonstrated early antiarrhythmic effects within days of treatment. The mechanisms underlying the immediate and short-term antiarrhythmic effects are poorly understood., Objective: We hypothesize that cSBRT has a direct antiarrhythmic effect on cellular electrophysiology through reprogramming of ion channel and gap junction protein expression., Methods: After exposure to 20 Gy of x-rays in a single fraction, neonatal rat ventricular cardiomyocytes were analyzed 24 and 96 hours postradiation to determine changes in conduction velocity, beating frequency, calcium transients, and action potential duration in both monolayers and single cells. In addition, the expression of gap junction proteins, ion channels, and calcium handling proteins was evaluated at protein and messenger RNA levels., Results: After irradiation with 20 Gy, neonatal rat ventricular cardiomyocytes exhibited increased beat rate and conduction velocity 24 and 96 hours after treatment. Messenger RNA and protein levels of ion channels were altered, with the most significant changes observed at the 96-hour mark. Upregulation of Cacna1c (Ca v 1.2), Kcnd3 (K v 4.3), Kcnh2 (K v 11.1), Kcnq1 (K v 7.1), Kcnk2 (K 2P 2.1), Kcnj2 (K ir 2.1), and Gja1 (Cx43) was noted, along with improved gap junctional coupling. Calcium handling was affected, with increased Ryr2 ryanodin-rezeptor 2 and Slc8a1 Na + /Ca 2+  exchanger expression and altered properties 96 hours posttreatment. Fibroblast and myofibroblast levels remained unchanged., Conclusion: cSBRT modulates the expression of various ion channels, calcium handling proteins, and gap junction proteins. The described alterations in cellular electrophysiology may be the underlying cause of the immediate antiarrhythmic effects observed after cSBRT., Competing Interests: Disclosures Dr Mages acknowledges educational support from Biotronik and Johnson & Johnson, while Dr Rahm acknowledges support from Boston Scientific, Johnson & Johnson, Abbott, and Medtronic. Dr Thomas has received lecture fees/honoraria from various pharmaceutical and medical device companies, including Abbott, AstraZeneca, Bayer Vital, Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo, Johnson & Johnson, Medtronic, Novartis, Pfizer Pharma, Sanofi-Aventis, and ZOLL CMS. Dr Lugenbiel has received lecture fees from Bayer Vital, Pfizer Pharma, and Bristol Myers Squibb as well as educational support from Boston Scientific and fees/honoraria from Johnson & Johnson and Boston Scientific. The remaining authors have declared no relevant relationships. We declare that no artificial intelligence was used in preparation of the present manuscript., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca 2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission., Methods: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca 2+ release was recorded using Fluo-4., Results: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca 2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca 2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms., Conclusions: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes., Competing Interests: Disclosures J. Laporte is a cofounder of Dynacure. The other authors report no conflicts.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca 2+ -dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca 2+ channels critical for EC coupling in close proximity, the L-type Ca 2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca 2+ -dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca 2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches., (© 2023. The Author(s).)

Gap junctions and their expression pattern are essential to robust function of intercellular communication and electrical propagation in cardiomyocytes. In healthy myocytes, the main cardiac gap junction protein connexin-43 (Cx43) is located at the intercalated disc providing a clear direction of signal spreading across the cardiac tissue. Dislocation of Cx43 to lateral membranes has been detected in numerous cardiac diseases leading to slowed conduction and high propensity for the development of arrhythmias. At the cellular level, arrhythmogenic diseases are associated with elevated levels of oxidative distress and gap junction remodeling affecting especially the amount and sarcolemmal distribution of Cx43 expression. So far, a mechanistic link between sustained oxidative distress and altered Cx43 expression has not yet been identified. Here, we propose a novel cell model based on murine induced-pluripotent stem cell-derived cardiomyocytes to investigate subcellular signaling pathways linking cardiomyocyte distress with gap junction remodeling. We tested the new hypothesis that chronic distress, induced by rapid pacing, leads to increased reactive oxygen species, which promotes expression of a micro-RNA, miR-1, specific for the control of Cx43. Our data demonstrate that Cx43 expression is highly sensitive to oxidative distress, leading to reduced expression. This effect can be efficiently prevented by the glutathione peroxidase mimetic ebselen. Moreover, Cx43 expression is tightly regulated by miR-1, which is activated by tachypacing-induced oxidative distress. In light of the high arrhythmogenic potential of altered Cx43 expression, we propose miR-1 as a novel target for pharmacological interventions to prevent the maladaptive remodeling processes during chronic distress in the heart.

Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

Recent advances in the technology of producing novel cardiomyocytes from induced pluripotent stem cells (iPSC-cardiomyocytes) fuel new hope for future clinical applications. The use of iPSC-cardiomyocytes is particularly promising for the therapy of cardiac diseases such as myocardial infarction, where these cells could replace scar tissue and restore the functionality of the heart. Despite successful cardiogenic differentiation, medical applications of iPSC-cardiomyocytes are currently limited by their pronounced immature structural and functional phenotype. This review focuses on gap junction function in iPSC-cardiomyocytes and portrays our current understanding around the structural and the functional limitations of intercellular coupling and viable cardiac graft formation involving these novel cardiac muscle cells. We further highlight the role of the gap junction protein connexin 43 as a potential target for improving cell-cell communication and electrical signal propagation across cardiac tissue engineered from iPSC-cardiomyocytes. Better insight into the mechanisms that promote functional intercellular coupling is the foundation that will allow the development of novel strategies to combat the immaturity of iPSC-cardiomyocytes and pave the way toward cardiac tissue regeneration.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca 2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca 2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca 2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Körner, Mosqueira, Hecker and Ullrich.)

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Aim: Effective antiarrhythmic treatment of atrial fibrillation (AF) constitutes a major challenge, in particular, when concomitant heart failure (HF) is present. HF-associated atrial arrhythmogenesis is distinctly characterized by prolonged atrial refractoriness. Small-conductance, calcium-activated K + (K Ca , SK, KCNN ) channels contribute to cardiac action potential repolarization and are implicated in AF susceptibility and therapy. The mechanistic impact of AF/HF-related triggers on atrial K Ca channels is not known. We hypothesized that tachycardia, stretch, β-adrenergic stimulation, and hypoxia differentially determine K Ca 2.1-2.3 channel remodeling in atrial cells., Methods: KCNN1-3 transcript levels were assessed in AF/HF patients and in a pig model of atrial tachypacing-induced AF with reduced left ventricular function. HL-1 atrial myocytes were subjected to proarrhythmic triggers to investigate the effects on Kcnn mRNA and K Ca channel protein., Results: Atrial KCNN1-3 expression was reduced in AF/HF patients. KCNN2 and KCNN3 suppression was recapitulated in the corresponding pig model. In contrast to human AF, KCNN1 remained unchanged in pigs. Channel- and stressor-specific remodeling was revealed in vitro. Lower expression levels of KCNN1 /K Ca 2.1 were linked to stretch and β-adrenergic stimulation. Furthermore, KCNN3 /K Ca 2.3 expression was suppressed upon tachypacing and hypoxia. Finally, KCNN2 /K Ca 2.2 abundance was specifically enhanced by hypoxia., Conclusion: Reduction of K Ca 2.1-2.3 channel expression might contribute to the action potential prolongation in AF complicated by HF. Subtype-specific K Ca 2 channel remodeling induced by tachypacing, stretch, β-adrenergic stimulation, or hypoxia is expected to differentially determine atrial remodeling, depending on patient-specific activation of each triggering factor. Stressor-dependent K Ca 2 regulation in atrial myocytes provides a starting point for mechanism-based antiarrhythmic therapy., Competing Interests: A.K.R. report grants from Heidelberg University, fellowships from German Cardiac Society, clinician-scientist program from German Internal Medicine Society, and fellowship from German Heart Foundation/German Foundation of Heart Research, during the conduct of the study; and educational support from Boston Scientific, Johnson & Johnson, Abbott and Medtronic. Mara Elena Müller reports grants from Heidelberg University Medical Hospital, during the conduct of the study; personal fees from Heidelberg University Medical Hospital, outside the submitted work. P.L. reports receiving lecture fees from Bayer Vital and Pfizer Pharma and educational support from Boston Scientific and Johnson & Johnson. Hugo A Katus reports personal fees from Astra Zeneca, personal fees from Daiichi Sankyo, personal fees from Bayer Vital, personal fees from Roche Diagnostics, personal fees from Novo Nordisk, and personal fees from Boehringer Ingelheim, outside the submitted work. D.T. reports lecture fees/honoraria from Bayer Vital, lecture fees/honoraria from Boehringer Ingelheim Pharma, lecture fees/honoraria from Bristol-Myers Squibb, lecture fees/honoraria and study support from Daiichi Sankyo, lecture fees/honoraria from Medtronic, lecture fees/honoraria from Pfizer Pharma, personal fees from Sanofi-Aventis, lecture fees/honoraria from St. Jude Medical, lecture fees/honoraria from ZOLL CMS, grants from German Heart Foundation/German Foundation of Heart Research, research award from Joachim Siebeneicher Foundation, grants from German Research Foundation, grant from Ministry of Science, Research and the Arts Baden-Wuerttemberg, during the conduct of the study. The authors reported no other potential conflicts of interest for this work. The remaining authors have reported that they have no relationships relevant to the content of this paper to disclose., (© 2021 Rahm et al.)

Transient outward K + current, I to , contributes to cardiac action potential generation and is primarily carried by K v 4.3 (KCND3) channels. Two K v 4.3 isoforms are expressed in human ventricle and show differential remodeling in heart failure (HF). Lidocaine and mexiletine may be applied in selected patients to suppress ventricular arrhythmias, without effects on sudden cardiac death or mortality. Isoform-dependent effects of antiarrhythmic drugs on K v 4.3 channels and potential implications for remodeling-based antiarrhythmic management have not been assessed to date. We sought to test the hypotheses that K v 4.3 channels are targeted by lidocaine and mexiletine, and that drug sensitivity is determined in isoform-specific manner. Expression of KCND3 isoforms was quantified using qRT-PCR in left ventricular samples of patients with HF due to either ischemic or dilated cardiomyopathies (ICM or DCM). Long (K v 4.3-L) and short (K v 4.3-S) isoforms were heterologously expressed in Xenopus laevis oocytes to study drug sensitivity and effects on biophysical characteristics activation, deactivation, inactivation, and recovery from inactivation. In the present HF patient cohort KCND3 isoform expression did not differ between ICM and DCM. In vitro, lidocaine (IC 50 -K v 4.3-L: 0.8 mM; IC 50 -K v 4.3-S: 1.2 mM) and mexiletine (IC 50 -K v 4.3-L: 146 μM; IC 50 -K v 4.3-S: 160 μM) inhibited K v 4.3 with different sensitivity. Biophysical analyses identified accelerated and enhanced inactivation combined with delayed recovery from inactivation as primary biophysical mechanisms underlying K v 4.3 current reduction. In conclusion, differential effects on K v 4.3 isoforms extend the electropharmacological profile of lidocaine and mexiletine. Patient-specific remodeling of K v 4.3 isoforms may determine individual drug responses and requires consideration during clinical application of compounds targeting K v 4.3., Competing Interests: Declaration of competing interest A.K.R. reports educational support from Boston Scientific and Medtronic. D.T. reports receiving lecture fees/honoraria from Bayer Vital, Boehringer Ingelheim Pharma, Bristol-Myers Squibb, Daiichi Sankyo, Medtronic, Pfizer Pharma, Sanofi-Aventis, St. Jude Medical and ZOLL CMS. P.L. reports receiving lecture fees from Bayer Vital and Pfizer Pharma and educational support from Boston Scientific and Johnson & Johnson. B.S. reports personal fees for surgical proctoring services from Abbott. The remaining authors have reported that they have no relationships relevant to the content of this paper to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) represent the best cell source for cardiac regenerative purposes but retain an immature phenotype after differentiation with significant limitations compared to adult cardiomyocytes. Apart from an incomplete cardiomyocyte-specific structure and microarchitecture, cells show at the level of Ca 2+ signaling only slow Ca 2+ release and reuptake properties. Here, we investigated the effect of restructuring single iPSC-CMs in specially designed 3D-micro-scaffolds on cell morphology and Ca 2+ handling. Using direct laser writing, rectangular-shaped scaffolds were produced and single iPSC-CMs were seeded into these forms. Structural analyses revealed strong sarcolemmal remodeling processes and myofilament reorientation in 3D-shaped cells leading to enhanced clustered expression of L-type Ca 2+ channels and ryanodine receptors and consequently, to faster Ca 2+ transient kinetics. Spontaneous beating activity was enhanced and Ca 2+ handling was more robust compared to non-patterned cells. Overall, our data demonstrate for the first time significant improvement of Ca 2+ signaling properties in reshaped iPSC-CMs indicative of functional maturation by structural remodeling., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions., (© 2019 UICC.)

The anatomic arrangement of microvascular endothelial cells and cardiomyocytes in vivo enables close interactions among these cells. In our in vitro co-culture system, ANP and BNP expression in the mouse atrial cardiomyocyte cell line HL-1 and subsequent ANP release were significantly upregulated when co-cultured with mouse cardiac microvascular endothelial cells or exposed to endothelial cell-conditioned medium. Endothelin-1 (ET-1) activation of endothelial cells remarkably enhanced their paracrine effect on cardiomyocyte gene expression, suggesting that ET-1 stimulation of endothelial cells affects expression of fetal genes such as ANP and BNP in adult cardiomyocytes through paracrine signalling. Exposure of HL-1 cells and murine induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to authentic angiopoietin-2 (Ang2) caused a concentration-dependent decrease in ANP expression while ET-1-induced ANP expression was augmented by low but inhibited by high concentrations of Ang2. FK506-mediated inhibition of the calcineurin-NFAT pathway in the HL-1 cells selectively inhibited the stimulatory effect of the conditioned medium derived from ET-1-pre-stimulated endothelial cells on cardiomyocyte fetal gene expression. Combined with previous results indicating a crucial role for ANP and BNP in cardiac homeostasis, our findings provide further evidence that paracrine signalling by cardiac microvascular endothelial cells modulates cardiomyocyte function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB -/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB -/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.

The therapeutic potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is limited by immature functional features including low impulse propagation and reduced cell excitability. Key players regulating electrical activity are voltage-gated Na + channels (Na v 1.5) and gap junctions built from connexin-43 (Cx43). Here we tested the hypothesis that enhanced Cx43 expression increases intercellular coupling and influences excitability by modulating Na v 1.5. Using transgenic approaches, Cx43 and Na v 1.5 localization and cell coupling were studied by confocal imaging. Na v 1.5 currents and action potentials (APs) were measured using the patch-clamp technique. Enhanced sarcolemmal Cx43 expression significantly improved intercellular coupling and accelerated dye transfer kinetics. Furthermore, Cx43 modulated Na v 1.5 function leading to significantly higher current and enhanced AP upstroke velocities, thereby improving electrical activity as measured by microelectrode arrays. These findings suggest a mechanistic link between cell coupling and excitability controlled by Cx43 expression in iPSC-CMs. Therefore, we propose Cx43 as novel molecular target for improving electrical properties of iPSC-CMs to match the functional properties of native myocytes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties., Methods: hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes., Results: hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed., Conclusion: We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Background: This study explored Bacopa monnieri, a medicinal Ayurvedic herb, as a cardioprotectant against ischemia/reperfusion injury using cardiac function and coronary flow as end-points., Methods: In normal isolated rat hearts, coronary flow, left ventricular developed pressure, heart rate, and functional recovery were measured using the Langendorff preparation. Hearts were perfused with either (i) Krebs-Henseleit (normal) solution, (control), or with 30, 100 μg/ml B. monnieri ethanolic extract (30 min), or (ii) with normal solution or extract for 10 min preceding no-perfusion ischemia (30 min) followed by reperfusion (30 min) with normal solution. Infarct volumes were measured by triphenyltetrazolium staining. L-type Ca 2+ -currents (I Ca, L ) were measured by whole-cell patching in HL-1 cells, a mouse atrial cardiomyocyte cell line. Cytotoxicity of B. monnieri was assessed in rat isolated ventricular myocytes by trypan blue exclusion., Results: In normally perfused hearts, B. monnieri increased coronary flow by 63 ± 13% (30 μg/ml) and 216 ± 21% (100 μg/ml), compared to control (5 ± 3%) (n = 8-10, p < 0.001). B. monnieri treatment preceding ischemia/reperfusion improved left ventricular developed pressure by 84 ± 10% (30 μg/ml), 82 ± 10% (100 μg/ml) and 52 ± 6% (control) compared to pre- ischemia/reperfusion. Similarly, functional recovery showed a sustained increase. Moreover, B. monnieri (100 μg/ml) reduced the percentage of infarct size from 51 ± 2% (control) to 25 ± 2% (n = 6-8, p < 0.0001). B. monnieri (100 μg/ml) reduced I Ca, L by 63 ± 4% in HL-1 cells. Ventricular myocyte survival decreased at higher concentrations (50-1000 μg/ml) B. monnieri., Conclusions: B. monnieri improves myocardial function following ischemia/reperfusion injury through recovery of coronary blood flow, contractile force and decrease in infarct size. Thus this may lead to a novel cardioprotectant strategy.

The orbicularis oculi are the sphincter muscles of the eyelids and are involved in modulating facial expression. They differ from both limb and extraocular muscles (EOMs) in their histology and biochemistry. Weakness of the orbicularis oculi muscles is a feature of neuromuscular disorders affecting the neuromuscular junction, and weakness of facial muscles and ptosis have also been described in patients with mutations in the ryanodine receptor gene. Here, we investigate human orbicularis oculi muscles and find that they are functionally more similar to quadriceps than to EOMs in terms of excitation-contraction coupling components. In particular, they do not express the cardiac isoform of the dihydropyridine receptor, which we find to be highly expressed in EOMs where it is likely responsible for the large depolarization-induced calcium influx. We further show that human orbicularis oculi and EOMs express high levels of utrophin and low levels of dystrophin, whereas quadriceps express dystrophin and low levels of utrophin. The results of this study highlight the notion that myotubes obtained by explanting satellite cells from different muscles are not functionally identical and retain the physiological characteristics of their muscle of origin. Furthermore, our results indicate that sparing of facial and EOMs in patients with Duchenne muscular dystrophy is the result of the higher levels of utrophin expression., (© 2016 Sekulic-Jablanovic et al.)

Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca(2+) signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca(2+) handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair. Stem Cells 2015;33:2208-2218., (© 2015 AlphaMed Press.)

The HL-1 atrial line contains cells blocked at various developmental stages. To obtain homogeneous sub-clones and correlate changes in gene expression with functional alterations, individual clones were obtained and characterised for parameters involved in conduction and excitation-contraction coupling. Northern blots for mRNAs coding for connexins 40, 43 and 45 and calcium handling proteins (sodium/calcium exchanger, L- and T-type calcium channels, ryanodine receptor 2 and sarco-endoplasmic reticulum calcium ATPase 2) were performed. Connexin expression was further characterised by western blots and immunofluorescence. Inward currents were characterised by voltage clamp and conduction velocities measured using microelectrode arrays. The HL-1 clones had similar sodium and calcium inward currents with the exception of clone 2 which had a significantly smaller calcium current density. All the clones displayed homogenous propagation of electrical activity across the monolayer correlating with the levels of connexin expression. Conduction velocities were also more sensitive to inhibition of junctional coupling by carbenoxolone (∼ 80%) compared to inhibition of the sodium current by lidocaine (∼ 20%). Electrical coupling by gap junctions was the major determinant of conduction velocities in HL-1 cell lines. In summary we have isolated homogenous and stable HL-1 clones that display characteristics distinct from the heterogeneous properties of the original cell line.

Background: Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time., Methods: Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry., Results: In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days., Conclusions: We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention., (© 2013 International Society for Heart and Lung Transplantation Published by International Society for the Heart and Lung Transplantation All rights reserved.)

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca(2+) release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca(2+)-induced Ca(2+) release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca(2+) release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm)(-1) s(-1)). These individual photolytically triggered Ca(2+) release events were identified as Ca(2+) sparks, which originated from RyR openings. This was verified by Ca(2+) spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca(2+) release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca(2+) flux but eventless SR-Ca(2+) release through InsP3R were characterized using SR-Ca(2+) leak/SR-Ca(2+) load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca(2+) spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca(2+) flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca(2+) release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca(2+)-induced Ca(2+) release mechanism and contribute a large fraction of the Ca(2+) required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca(2+) sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression., Methods and Results: Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak., Conclusion: Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Weakening of cardiac function in patients with heart failure results from a loss of cardiomyocytes in the damaged heart. Cell replacement therapies as a way to induce myocardial regeneration in humans could represent attractive alternatives to classical drug-based approaches. However, a suitable source of precursor cells, which could produce a functional myocardium after transplantation, remains to be identified. In the present study, we isolated cardiovascular precursor cells from ventricles of human fetal hearts at 12 weeks of gestation. These cells expressed Nkx2.5 but not late cardiac markers such as α-actinin and troponin I. In addition, proliferating cells expressed the mesenchymal stem cell markers CD73, CD90, and CD105. Evidence for functional cardiogenic differentiation in vitro was demonstrated by the upregulation of cardiac gene expression as well as the appearance of cells with organized sarcomeric structures. Importantly, differentiated cells presented spontaneous and triggered calcium signals. Differentiation into smooth muscle cells was also detected. In contrast, precursor cells did not produce endothelial cells. The engraftment and differentiation capacity of green fluorescent protein (GFP)-labeled cardiac precursor cells were then tested in vivo after transfer into the heart of immunodeficient severe combined immunodeficient mice. Engrafted human cells were readily detected in the mouse myocardium. These cells retained their cardiac commitment and differentiated into α-actinin-positive cardiomyocytes. Expression of connexin-43 at the interface between GFP-labeled and endogenous cardiomyocytes indicated that precursor-derived cells connected to the mouse myocardium. Together, these results suggest that human ventricular nonmyocyte cells isolated from fetal hearts represent a suitable source of precursors for cell replacement therapies.

Rippling muscle disease is caused by mutations in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca(2+) homeostasis. In the present study we examined Ca(2+) homeostasis and excitation-contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca(2+) ](i) and 4-chloro-m-cresol-induced Ca(2+) release but reduced KCl-induced Ca(2+) influx. Detailed analysis of the voltage-dependence of Ca(2+) transients revealed a significant shift of Ca(2+) release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation-contraction coupling., (© 2011 Wiley-Liss, Inc.)

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle. It is caused by a mutation of the dystrophin gene and characterized by a progressive loss of skeletal muscle function. Most patients also develop a dystrophic cardiomyopathy, resulting in dilated hypertrophy and heart failure, but the cellular mechanisms leading to the deterioration of cardiac function remain elusive. In the present study, we tested whether defective excitation-contraction (E-C) coupling contributes to impaired cardiac performance. "E-C coupling gain" was determined in cardiomyocytes from control and dystrophin-deficient mdx mice. To this end, L-type Ca2+ currents (ICaL) were measured with the whole cell patch-clamp technique, whereas Ca2+ transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle changes of E-C coupling in mdx cells despite matched Ca2+ loading of the sarcoplasmic reticulum (SR). However, lowering the extracellular Ca2+ concentration, a maneuver used to unmask latent E-C coupling problems, was surprisingly much better tolerated by mdx myocytes, suggesting a hypersensitive E-C coupling mechanism. Challenging the SR Ca2+ release by slow elevations of the intracellular Ca2+ concentration resulted in Ca2+ oscillations after a much shorter delay in mdx cells. This is consistent with an enhanced Ca2+ sensitivity of the SR Ca2+-release channels [ryanodine receptors (RyRs)]. The hypersensitivity could be normalized by the introduction of reducing agents, indicating that the elevated cellular ROS generation in dystrophy underlies the abnormal RyR sensitivity and hypersensitive E-C coupling. Our data suggest that in dystrophin-deficient cardiomyocytes, E-C coupling is altered due to potentially arrhythmogenic changes in the Ca2+ sensitivity of redox-modified RyRs.

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca(2+) spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca(2+) sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca(2+) responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca(2+) transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca(2+) spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca(2+) signals resulted in mitochondrial Ca(2+) accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca(2+) signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.

Estrogens modify contraction of vascular smooth muscle and cardiomyocytes, but suggestions that they confer protective effects on the cardiovascular system remain controversial. The negative inotropic effects of estrogens are a consequence of L-type Ca2+ channel inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that membrane-associated estrogen receptors (ER)-alpha and -beta are involved. We measured the effect of estrogens on Ca2+ current (ICaL) in isolated ventricular cardiomyocytes of wild-type (WT), ERalpha knockout (ERalphaKO), and ERbetaKO mice using the whole cell patch-clamp technique at 37 degrees C. No differences in current densities or inactivation profiles of ICaL were found under control conditions in WT, ERalphaKO, and ERbetaKO cardiomyocytes, suggesting that absence of either ER has no effect on functional properties of ICaL. In all groups, application of raloxifene (2 microM) or 17alpha- or 17beta-estradiol (50 microM) reduced ICaL (P < 0.001). Raloxifene decreased ICaL by 44 +/- 9% (mean +/- SE) in WT (n = 5), 34 +/- 5% in ERalphaKO (n = 5), and 30 +/- 5% in ERbetaKO mice (n = 8). 17alpha-Estradiol reduced ICaL by 41 +/- 10% in WT (n = 4), 34 +/- 12% in ERalphaKO (n = 7), and 38 +/- 8% in ERbetaKO mice (n = 7). 17beta-Estradiol inhibited ICaL by 31 +/- 4% in WT (n = 4), 28 +/- 6% in ERalphaKO (n = 3), and 42 +/- 3% in ERbetaKO mice (n = 5). Decreases in cell shortening occurred in parallel with these findings. Our results suggest that inhibition of ICaL and the decrease in contraction by estrogens do not depend on ERalpha or ERbeta.

Oestrogen can modify the contractile function of vascular smooth muscle and cardiomyocytes. The negative inotropic actions of oestrogen on the heart and coronary vasculature appear to be mediated by L-type Ca(2+) channel (Ca(v)1.2) inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that oestrogen directly inhibits the cardiovascular L-type Ca(2+) current, I(CaL). The effect of oestrogen on I(CaL) was measured in Ca(v)1.2-transfected HEK-293 cells using the whole-cell patch-clamp technique. The current revealed typical activation and inactivation profiles of nifedipine- and cadmium-sensitive I(CaL). Oestrogen (50 microM) rapidly reduced I(CaL) by 50% and shifted voltage-dependent activation and availability to more negative potentials. Furthermore, oestrogen blocked the Ca(2+) channel in a rate-dependent way, exhibiting higher efficiency of block at higher stimulation frequencies. Our data suggest that oestrogen inhibits I(CaL) through direct interaction of the steroid with the channel protein.

Background: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro., Methods: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry., Results: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes., Conclusion: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.