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Hypertrophic Cardiomyopathy (HCM) is often caused by heterozygous mutations in β-myosin heavy chain (MYH7, β-MyHC). In addition to hyper- or hypocontractile effects of HCM-mutations, heterogeneity in contractile function (contractile imbalance) among individual cardiomyocytes was observed in end-stage HCM-myocardium. Contractile imbalance might be induced by burst-like transcription, leading to unequal fractions of mutant versus wildtype mRNA and protein in individual cardiomyocytes (allelic imbalance). Until now it is not known if allelic and contractile imbalance are present early in HCM-development or rather occur in response to disease-associated remodeling. To address this question, we used patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with heterozygous MYH7-mutations R723G and G741R as models of early-stage HCM without secondary adaptions upon disease progression. R723G-hiPSC-CMs showed typical HCM-markers like hypertrophy and myofibrillar disarray. Using RNA-FISH and allele-specific single-cell-PCR, we show for both cell lines that MYH7 is transcribed in bursts. Highly variable mutant vs. wildtype MYH7-mRNA fractions in individual HCM-hiPSC-CMs indicated allelic imbalance. HCM-hiPSC-CM-lines showed functional alterations like slowed twitch contraction kinetics and reduced calcium sensitivity of myofibrillar force generation. A significantly larger variability in force generation or twitch parameters of individual HCM-hiPSC-CMs compared to WT-hiPSC-CMs indicated contractile imbalance. Our results with early-stage hiPSC-CMs strongly suggest that burst-like transcription and allelic imbalance are general features of CMs, which together with mutation-induced changes of sarcomere contraction could induce contractile imbalance in heterozygous CMs, presumably aggravating development of HCM. Genetic or epigenetic approaches targeting functional heterogeneity in HCM could lead to promising future therapies, in addition to myosin modulation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rationale: Cardiac-expressed long noncoding RNAs (lncRNAs) are important for cardiomyocyte (CM) differentiation and function. Several lncRNAs have been identified and characterized for early CM lineage commitment, however those in later CM lineage specification and maturation remain less well studied. Moreover, unique atrial / ventricular lncRNA expression has never been studied in detail., Objectives: Here, we characterized a novel ventricular myocyte-restricted lncRNA, not expressed in atrial myocytes, and conserved only in primates., Methods and Results: First, we performed single cell RNA-seq on human pluripotent stem cell derived cardiomyocytes (hPSC-CM) at the late stages of 2, 6 and 12 weeks of differentiation. Weighted correlation network analysis identified core gene modules, including a set of lncRNAs highly abundant and predominantly expressed in the human heart. A lncRNA (we call VENTHEART, VHRT) co-expressed with cardiac maturation and ventricular-specific genes MYL2 and MYH7, and was expressed in fetal and adult human ventricles, but not atria. CRISPR-mediated deletion of the VHRT gene led to impaired CM sarcomere formation and significant disruption of the ventricular CM gene program. Indeed, a similar disruption was not observed in VHRT KO hPSC-derived atrial CM, suggesting that VHRT exhibits only ventricular myocyte subtype-specific effects. Optical recordings validated that loss of VHRT significantly prolonged action potential duration at 90 % repolarization (APD 90 ) for ventricular-like, but not atrial-like, CMs., Conclusion: This reports the first lncRNA that is exclusively required for proper ventricular, and not atrial, CM specification and function., Competing Interests: Declaration of competing interest P.L.S and K.H.L are co-founders and shareholders of Ternion Biosciences., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics., Methods: We directly compared the p.β-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines., Results: Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.β-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs., Conclusions: Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a 'one-size fits all' treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy., Competing Interests: Disclosures I.M. is co-founder of EHT Technologies GmbH., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear. Here we have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, specifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers of the heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differences were observed in the phosphorylation of any of the myofilament proteins analyzed. These results highlight the importance of appropriate tissue sampling-particularly for studies aimed at elucidating disease mechanisms and biomarker discovery-in order to minimize potential variations arising from basal heterogeneity in myofilament PTMs in the heart., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the β-cardiac myosin heavy chain (β-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 microg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N(G)-nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n=8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral L-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit(+) cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process., (Copyright 2010 Elsevier Ltd. All rights reserved.)

After a change in muscle length, there is an immediate intrinsic response in the amount of developed force, followed by a slower response. Although it has been well documented that the slow force response is at least in part generated by modification of calcium handling, it is unclear whether regulation at the myofilament level occurs during the slow force response. We set out to investigate myofilament calcium sensitivity and phosphorylation status of myofilament proteins after a step-wise change in cardiac muscle length. Ultra-thin right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and iontophoretically loaded with the calcium indicator bis-fura-2. Twitch force-calcium relationships and steady-state force-[Ca(2+)](i) relationships were measured at various muscle lengths at 37 degrees C using potassium induced contractures. The EC(50) significantly decreased with increase in muscle length and maximal active force development significantly increased, while no significant change in the myofilament cooperativity coefficient was found. Phosphoprotein analysis Pro-Q diamond staining as well as phosphorylation-specific antibodies revealed increased phosphorylation of tropomyosin, troponin I, and myosin light chain-2 at longer muscle lengths. Specifically, TnI phosphorylation at Ser(22/23) was increased. Since the immediate response is seen virtually instantaneously and post-translational modifications are thought not to occur within such a very short timeframe, we hypothesize that these increases in phosphorylation occur during the slow response., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts 1 week after infarction would, after 8-10 weeks: 1) improve global myocardial function, 2) contract in a Ca2+ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca2+, whereas scar tissue did not. Furthermore, the Ca2+ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca2+ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.

Myosin is a molecular motor, which interacts with actin to convert the energy from ATP hydrolysis into mechanical work. In cardiac myocytes, two myosin isoforms are expressed and their relative distribution changes in different developmental and pathophysiologic conditions of the heart. It has been realized for a long time that a shift in myosin isoforms plays a major role in regulating myocardial contractile activity. With the recent evidence implicating that alteration in myosin isoform ratio may be eventually beneficial for the treatment of a stressed heart, a new interest has developed to find out ways of controlling the myosin isoform shift. This article reviews the published data describing the role of myosin isoforms in the heart and highlighting the importance of various factors shown to influence myosin isofrom shift during physiology and disease states of the heart.

Acute myocarditis is a clinically serious disease; however, no effective treatment has been elucidated. Cyclooxygenase (COX)-2 is a key factor for progression of inflammation. Although inflammation is an essential pathological feature of acute myocarditis, the role of COX-2 in this process remains unclear. Thus, the purpose of this study was to clarify the role of COX-2 in acute myocarditis. We used a rat experimental autoimmune myocarditis (EAM) model and a specific COX-2 inhibitor in this study. Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. We administered the COX-2 inhibitor (meloxicam, 0.1 mg/kg per day) daily; the rats were killed on day 21. Echocardiograms, body and heart weight, heart rate, blood pressure, and histological and molecular examinations were performed. Cytokine expression in the hearts and cell proliferation against cardiac myosin were also analyzed. The COX-2 inhibition during the immune response (late) phase attenuated EAM development; however, the inhibition during the antigen priming (early) phase did not attenuate EAM. The COX-2 inhibitor altered Th1/Th2 cytokine balance and inhibited cell proliferation in vitro. The COX-2 inhibitor suppresses the development of EAM. COX-2 regulation is promising for treating acute myocarditis.

The precise molecular basis for myocardial stunning remains unresolved, but protein damage within the myofibril is a likely mechanism. We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify protein modifications in stunned myocardium. In isolated, perfused rabbit hearts, low-flow ischemia (1 ml/min) and reperfusion resulted in impaired left-ventricular function (rate-pressure product (RPP) after 15-min ischemia: 65 +/- 5% pre-ischemia). We have characterised the sequence of ventricular myosin-regulatory light chain (MLC-2, 18 kDa) in rabbit myocardium and identified two non-phosphorylated (P(1) and P(2)) and two phosphorylated (P(3) and P(4) at Ser-14) isoelectric point variants. MS revealed that the acidic isoelectric point post-translational modification of P(1) and P(3), resulting in P(2) and P(4) respectively, was due to deamidation of asparagine to aspartate at residue 13, adjacent to Ser-14 phosphorylation site. After 15-min ischemia and reperfusion, a 15-kDa MLC-2 fragment was detected (MLC-2(14-165)), resulting from N-terminal cleavage between Asn/Asp-13 and Ser-14 of non-phosphorylated MLC-2, which accounted for 9.8% of visible non-phosphorylated MLC-2. Subsequent 2-DE of subcellular fractions showed that the fragment was lost from the myofilament. Treatment with an OH radical scavenger, N-(2-mercaptopropionyl) glycine (MPG, 3 mmol/l), preserved contractile function (RPP: 106 +/- 9% pre-ischemia) and prevented cleavage of MLC-2. Proteolytic damage to MLC-2, related to presence of OH radicals during reperfusion, correlates with myocardial stunning and may contribute to impaired contractility.