Cardiac Myosin Heavy Chain Research Articles

Effect of a dianthin G analogue in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

Loss of cardiomyocytes due to myocardial infarction results in ventricular remodeling which includes non-contractile scar formation, which can lead to heart failure. Stem cell therapy aims to replace the scar tissue with the functional myocardium. Mesenchymal stem cells (MSCs) are undifferentiated cells capable of self-renewal as well as differentiation into multiple lineages. MSCs can be differentiated into cardiomyocytes by treating them with small molecules and peptides. Here, we report for the first time, the role of a cyclic peptide, an analogue of dianthin G, [Glu2]-dianthin G (1) in the in vitro cardiac differentiation of rat bone marrow MSCs. In this study, [Glu2]-dianthin G (1) was synthesized using solid-phase total synthesis and characterized by NMR spectroscopy. MSCs were treated with two different concentrations (0.025 and 0.05mM) of the peptide separately for 72h and then incubated for 15days to allow the cells to differentiate into cardiomyocytes. Treated cells were analyzed for the expression of cardiac-specific genes and proteins. Results showed significant upregulation of cardiac-specific genes GATA4, cardiac troponin T (cTnT), cardiac troponin I (cTnI), cardiac myosin heavy chain, and connexin 43 in the treated MSCs compared to the untreated control. For cardiac-specific proteins, GATA4, cTnT, and Nkx2.5 were analyzed in the treated cells and were shown to have significant upregulation as compared to the untreated control. In conclusion, this study has demonstrated the cardiac differentiation potential of [Glu2]-dianthin G (1)-treated rat bone marrow MSCs in vitro both at the gene and at the protein levels. Transplantation of pre-differentiated MSCs into the infarcted myocardium may result in the efficient regeneration of cardiac cells and restoration of normal cardiac function.

Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF-1/CXCR4 signalling pathway.

AimsHomozygosity for a four‐missense single‐nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long‐living individuals. Delivery of this longevity‐associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell‐derived factor‐1 (SDF‐1). Here, we investigated if delivery of the LAV‐BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy.Methods and resultsCompared with age‐matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV‐BPIFB4 gene therapy using an adeno‐associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild‐type‐BPIFB4 exerted no benefit. Interestingly, LAV‐BPIFB4‐treated mice showed increased SDF‐1 levels in peripheral blood and myocardium and up‐regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF‐1 up‐regulation was instrumental to LAV‐BPIFB4‐induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF‐1 CXCR4 receptor.ConclusionsIn mice with type‐2 diabetes, LAV‐BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes‐induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Evidence for Progressive Shifts Towards α‐Myosin Isoform with Paradoxical Increases in Cardiac Hemodynamic Loads in Post‐Natal Healthy Rats

IntroductionThe balance of fast (α) and slow (β) cardiac myosin heavy chain (MYHC) isoforms is known to change with both aging and disease. While pathological states with increased hemodynamic loads are often accompanied by shifts toward energy‐sparing β isoforms, the underlying mechanism(s) of these changes remain poorly understood. Neonatal rats are a promising model to study such cardiac MYHC remodeling, as they show a well‐documented transition (β to α) during post‐natal development. In these experiments, MYHC‐isoform switches and in vivo cardiac structure/hemodynamic changes were evaluated during post‐natal development.MethodsCardiac echocardiographic and systemic/left ventricular (LV) hemodynamics were evaluated in Sprague‐Dawley rats (n = 61) ranging from post‐natal day (PND) 3 to adulthood. Hemodynamics were obtained before/after challenges with sodium nitroprusside (SNP, 25 μg/kg IV) and/or phenylephrine (PE, 10 μg/kg IV) to study cardiovascular responsiveness. LV’s were also collected (n = 48) for myosin isoform analysis via SDS‐PAGE MYHC separating gels.ResultsAs expected, post‐natal rats showed a progressive decline in β‐MYHC expression from PND3 (1.29 ± 0.05 α:β) to adulthood when α‐MYHC was dominant (12.78 ± 2.51 α:β). Concomitantly, postnatal rats showed progressive increases (P<0.05) in systemic/LV pressures (MAP: 46 ± 1 at <PND14 to 103 ± 2 mmHg, P<0.05), LV mass (0.16 ± 0.02g at <PND14 to 0.84 ± 017g) and diastolic dimensions (79 ± 6 at <PND14 to 288 ± 14 μL), with vastly preserved heart rates. Pressor responses to PE increased during maturation, while all age groups were sensitive to SNP‐mediated vaso‐relaxation. Ejection fraction was preserved during postnatal maturation with young pups showing lower rates of LV pressure development (dPdtmax 4052 ± 125 at <PND14 vs. 9478 ± 231 mmHg/s, P<0.05) consistent with the early expression of β‐MYHC.ConclusionsTaken together, these data provide evidence of a maturing cardiovascular system in post‐natal rats showing progressive increases in systemic/ventricular pressures and cardiac dimensions, with paradoxical shifts towards α‐MYHC isoforms. In short, in rats, the cardiac β‐MYHC isoform consistently declines over maturation despite sustained increases in systemic load.

Sildenafil reverses the right ventricular hypertrophy induced by hypoxia but does not bring down the expression of MHC‐β to normal value

IntroductionMyosin is the major player in structure and function of cardiac muscle. There are two sub‐types of myosin heavy chain in cardiac muscle; myosin heavy chain‐α (MHC‐α) and myosin heavy chain‐β (MHC‐β). The contractile properties of the cardiac muscle are determined largely by types and the expression of the myosin heavy chain (MHC) which is altered by many physiological and pathological conditions.ObjectivesIn this study we investigated the effect of hypoxia and sildenafil treatment on myosin heavy chain isoform in hy poxia ‐ induced hypertrophied right ventricle.MethodsRight ventricular hypertrophy was induced in 45 mice by exposing them to hypoxic stimulus (11% ambient oxygen) in normobaric chamber for 20 days. The animals were distributed randomly into three groups: first group served as control (CO), second group exposed to hypoxia for 20 days without sildenafil treatment (HY) and third group was given sildenafil orally at dose of 30 mg.Kg−1.day−1 plus exposure to hypoxia for 20 days (HS). MHC isoforms were calculated using two ELISA kits containing antibodies against α and β MHC and by SDS‐PAGE (Mybiosource‐USA). The HY group in comparison with CO group, showed a significant increase in right ventricle weight/left ventricle plus septum ratio (Fulton’s ratio). HS group showed significant decrease in Fulton’s ratio compared with HY but not with CO. Expression of MHC‐β isoform was significantly increased in HY group compared with CO. There was no significant difference in MHC‐β between HY group and HS group. Expression of more beta myosin heavy in hypoxic group is beneficial to the heart since hearts with more MHC‐β have more economic contraction.Conclusionsildenafil reversed the right ventricular hypertrophy induced by hypoxia but did not bring down the expression of MHC‐β to normal value.Support or Funding InformationDeanship of research at Jordan University of Science & TechnologyEffects of hypoxia and hypoxia with sildenafil on myosin heavy chain isoforms.Expression of cardiac myosin heavy chains in different groups: Hypoxia caused expression of more MHC‐β and sildenafil failed in revering the changes caused by hypoxia. Control (CO), hypoxia for 20 days without treatment (HY) and hypoxia for 20 days plus treatment with sildenafil (HS). *** P < 0.01 HY and HS vs COFigure 1

Impact of Regulatory Light Chain Mutation (K104E) on the Atpase and Motor Properties of Human Cardiac Myosin

Mutations in the cardiac myosin heavy chain (MYH7) as well as the essential (MYL3) and regulatory (MYL2) light chains are known to lead to cardiomyopathies with variable phenotypes, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy. We investigated the impact of the HCM-associated regulatory light chain (RLC) mutation (K104E) on myosin ATPase kinetics and in vitro motility. We expressed and purified human cardiac myosin subfragment 1 (M2B-S1) and exchanged on the human ventricular RLC, with the mutant or WT RLC. A mouse model of K104E was found to cause cardiac hypertrophy, fibrosis, and diastolic dysfunction in older animals suggesting a slow development of HCM, while variable penetrance of the mutation in human populations suggest the impact of K104E may be subtle. We found that maximum actin-activated ATPase activity and the actin-dependence of ATPase were similar in K104E and WT. Actin gliding velocities in the in vitro motility assay were slightly increased in K104E. We found that the ADP release rate constant in the presence of actin also was slightly increased in K104E compared to WT. Our results suggest that HCM-associated RLC mutations may only have a minor impact on unloaded ATPase kinetics and in vitro actin gliding, which may suggest other factors could be altered by the mutation such as mechanical load and myosin regulation. Future experiments will address the impact of the mutation in mouse cardiac vs. human cardiac myosin as well as the impact of the mutation on load-dependent motility and the off state (super-relaxed state) of cardiac myosin.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Abstract 212: Hypertrophic Cardiomyopathy Mutations With Opposite Effects on ß-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms)

Introduction: Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiovascular disease, commonly caused by mutations in beta cardiac myosin heavy chain (βMYH). We have measured the kinetics of isolated myosin proteins with different HCM mutations in βMYH and found an intriguing heterogeneity in kinetics and force production: some mutations increase whereas others decrease force and velocity; others result in no change. How these divergent molecular alterations converge to the final HCM phenotype: hypercontractility and cellular hypertrophy is still unknown. hiPSC-CMs provide a powerful tool for studying human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization, but are limited by immature phenotypes and population heterogeneity in traditional culture environments. Methods and Results: We developed a micropatterned hydrogel platform that enhances force generation and promotes both structural and molecular hiPSC-CM maturation. Using CRISPR/Cas-9 gene editing in an isogenic hiPSC line, we created hiPSCs with two different βMYH mutations (P710R and D239N) that result in opposite effects on force at the molecular level, using in vitro motility and laser trap methods. Cell morphology was quantified by both fluorescence and transmission electron microscopy (EM) and force generation measured by traction force microscopy. Despite having opposite effects at the single molecule level, both HCM lines showed increased contractile force and cellular hypertrophy compared to isogenic controls. Immunostaining for βMYH and EM revealed organizational and microstructural changes including sarcomere and myofibrillar disarray, and thickened z-discs in cells containing these mutations. Both mutations had alterations in AMPK, MAPK, and calcineurin signaling shown to regulate hypertrophy. Discussion: Divergent biomechanical alterations due to HCM mutations at the single molecule level lead to common cellular-level structural and biomechanical phenotypes through activation of common signaling pathways.

Sex-Based Mhrt Methylation Chromatinizes MeCP2 in the Heart.

SummaryIn the heart, primary microRNA-208b (pri-miR-208b) and Myheart (Mhrt) are long non-coding RNAs (lncRNAs) encoded by the cardiac myosin heavy chain genes. Although preclinical studies have shown that lncRNAs regulate gene expression and are protective for pathological hypertrophy, the mechanism underlying sex-based differences remains poorly understood. In this study, we examined DNA- and RNA-methylation-dependent regulation of pri-miR-208b and Mhrt. Expression of pri-miR-208b is elevated in the left ventricle of the female heart. Despite indistinguishable DNA methylation between sexes, the interaction of MeCP2 on chromatin is subject to RNase digestion, highlighting that affinity of the methyl-CG reader is broader than previously thought. A specialized procedure to isolate RNA from soluble cardiac chromatin emphasizes sex-based affinity of an MeCP2 co-repressor complex with Rest and Hdac2. Sex-specific Mhrt methylation chromatinizes MeCP2 at the pri-miR-208b promoter and extends the functional relevance of default transcriptional suppression in the heart.

Bispecific Antibody Therapy for Effective Cardiac Repair through Redirection of Endogenous Stem Cells

AbstractBone marrow stem cells (BMSCs) are a promising strategy for cardiac regenerative therapy for myocardial infarction (MI). However, cell transplantation has to overcome a number of hurdles, such as cell quality control, clinical practicality, low cell retention/engraftment, and immune reactions when allogeneic cells are used. Bispecific antibodies (BsAbs) have been developed as potential agents in cancer immunotherapy but their application is sparse in cardiovascular diseases. In the present study, BsAbs are designed by chemical cycloaddition of F(ab′)2 fragments from monoclonal anti‐CD34 and anti‐ cardiac myosin heavy chain (CMHC) antibodies, which specifically targets circulating CD34‐positive cells and injured cardiomyocytes simultaneously. It is hypothesized that intravenous administration of stem cell re‐directing (SCRD) BsAbs (anti‐CD34‐F(ab′)2–anti‐CMHC‐F(ab′)2) can home endogenous BMSCs to the injured heart for cardiac repair. The in vivo studies in a mouse model with heart ischemia/reperfusion (I/R) injury demonstrate the safety and therapeutic potency of SCRD BsAb, which supports cardiac recovery by reducing scarring, promoting angiomyogenesis, and boosting cardiac function.

Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca2+) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca2+-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca2+ activation levels and not maximal Ca2+ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca2+-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca2+ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca2+-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.

Improved Efficiency of Cardiomyocyte-Like Cell Differentiation from Rat Adipose Tissue-Derived Mesenchymal Stem Cells with a Directed Differentiation Protocol.

Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and “rosette-like structures” from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

Inhibitory effects of class I antiarrhythmic agents on Na+ and Ca2+ currents of human iPS cell-derived cardiomyocytes.

IntroductionHuman induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called “iCell cardiomyocytes” under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents.MethodsiCell cardiomyocytes were cultured for 8–14 days (38–44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp.ResultsiCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were −62.7 ± 13.7, −8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 μM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 μM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 μM, respectively.ConclusionsIn iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.

Luminex-Coupled EliFACS: A Multiparametric Method to Enumerate and Functionally Characterize Antigen-Specific T cells in Human Peripheral Blood.

We describe a Luminex-coupled EliFACS assay that integrates multiplexing technology, enzyme-linked immunospot (ELISPOT), and intracellular cytokine FACS staining for the detection of multiple parameters of antigen-specific T-cell activation in human peripheral blood. Although our protocol is for measuring T-cell responses against cardiac myosin heavy chain and myelin basic protein, the major autoantigens in myocarditis and multiple sclerosis, respectively, these methods could be used for the detection of T-cell responses to other antigens, including foreign antigens.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Atorvastatin Attenuates Myocardial Hypertrophy Induced by Chronic Intermittent Hypoxia In Vitro Partly through miR-31/PKCε Pathway.

Atorvastatin is proven to ameliorate cardiac hypertrophy induced by chronic intermittent hypoxia (CIH). However, little is known about the mechanism by which atorvastatin modulates CIH-induced cardiac hypertrophy, and whether specific hypertrophyrelated microRNAs are involved in the modulation. MiR-31 plays key roles in the development of cardiac hypertrophy induced by ischemia/hypoxia. This study examined whether miR-31 was involved in the protective role of atorvastatin against CIH-induced myocardial hypertrophy. H9c2 cells were subjected to 8-h intermittent hypoxia per day in the presence or absence of atorvastatin for 5 days. The size of cardiomyocytes, and the expression of caspase 3 and miR-31 were determined by Western blotting and RT-PCR, respectively. MiR-31 mimic or Ro 31-8220, a specific inhibitor of protein kinase C epsilon (PKCε), was used to determine the role of miR-31 in the anti-hypertrophic effect of atorvastatin on cardiomyocytes. PKCε in the cardiomyocytes with miR-31 upregulation or downregulation was detected using RT-PCR and Western blotting. The results showed that CIH induced obvious enlargement of cardiomyocytes, which was paralleled with increased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and slow/beta cardiac myosin heavy-chain (MYH7) mRNA levels. All these changes were reversed by the treatment with atorvastatin. Meanwhile, miR-31 was increased by CIH in vitro. Of note, the atorvastatin pretreatment significantly increased the mRNA and protein expression of PKCe and decreased that of miR-31. Moreover, overexpression of miR-31 abolished the anti-hypertrophic effect of atorvastatin on cardiomyocytes. Upregulation and downregulation of miR-31 respectively decreased and increased the mRNA and protein expression of PKCε. These results suggest that atorvastatin provides the cardioprotective effects against CIH probably via up-regulating PKCε and down-regulating miR-31.

Perspectives of a myosin motor activator agent with increased selectivity.

Clinical treatment of heart failure is still not fully solved. A novel class of agents, the myosin motor activators, acts directly on cardiac myosin resulting in an increased force generation and prolongation of contraction. Omecamtiv mecarbil, the lead molecule of this group, is now in human phase 3 displaying promising clinical performance. However, omecamtiv mecarbil is not selective to myosin, because it readily binds to and activates cardiac ryanodine receptors (RyR-2), an effect that may cause complications in case of overdose. In this study, in silico analysis was performed to investigate the docking of omecamtiv mecarbil and other structural analogues to cardiac myosin heavy chain and RyR-2 to select the structure that has a higher selectivity to myosin over RyR-2. In silico docking studies revealed that omecamtiv mecarbil has comparable affinity to myosin and RyR-2: the respective Kd values are 0.60 and 0.87 μmol/L. Another compound, CK-1032100, has much lower affinity to RyR-2 than omecamtiv mecarbil, while it still has a moderate affinity to myosin. It was concluded that further research starting from the chemical structure of CK-1032100 may result a better myosin activator burdened probably less by the RyR-2 binding side effect. It also is possible, however, that the selectivity of omecamtiv mecarbil to myosin over RyR-2 cannot be substantially improved, because similar moieties seem to be responsible for the high affinity to both myosin and RyR-2.

Mechanobiology of Myosin Mutations and Myofibril Remodeling in iPSC-Cardiomyocytes

Hypertrophic cardiomyopathies are the most prevalent form of heritable cardiovascular disease and are caused by a host of mutations that result in hypercontractility, though the largest number of known mutations are found in beta cardiac myosin heavy chain (βMYH). Myosin is a key protein responsible for muscle contraction and is arranged into a dynamic, hierarchical structure of sarcomeres and myofibrils within cardiomyocytes. We have previously measured differences in the kinetics of isolated myosin proteins with different hypertrophic cardiomyopathy mutations in βMYH, but it has been difficult to determine how these alterations manifest at the cellular level. Induced pluripotent stem cell-derived cardiomyocytes provide a powerful tool for studying specific features of human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization. We have developed a micropatterned hydrogel platform that promotes myofibril alignment, cardiomyocyte force generation, and other metrics of maturation. Using CRISPR-Cas9 gene-editing, we have created cell lines with specific βMYH mutations, including D239N and P710R. We used traction force microscopy in a preliminary study and measured increased forces in the mutant cells compared to isogenic controls, and we have observed larger cell size in one of the mutant lines in both patterned and unpatterned contexts. Infection with LifeAct adenovirus also allows for the visualization of actin structures including myofibrils and sarcomeres during cell spreading and the recovery of beating in cells recently transferred to patterned substrates. Immunostaining for βMYH revealed higher expression and more regular organization of sarcomeres in patterned cells, and we plan to use these techniques to quantify differences in myofibril and sarcomere organization in cells with βMYH mutations. This work provides valuable insight into the etiology of hypertrophic cardiomyopathy and the utility of induced pluripotent stem cell-derived cardiomyocytes as a model for human cardiac biology and disease.

Action Potential Elongation and Altered Beta-Adrenergic Responses in Cardiac Myocytes from aMHC403/+ Hypertrophic Hearts

Familial hypertrophic cardiomyopathy is an inherited heart condition associated with mutations in the genes encoding sarcomeric proteins, such as the cardiac myosin heavy chain protein (MHC). Patients experience arrhythmias and sudden death. Morphological changes include myocyte hypertrophy, myofibrillar disarray, and fibrosis. Although differences in cardiac function are well described, the exact mechanism contributing to the increased arrhythmogenicity is poorly understood. We studied the heterozygous mouse model αMHC403/+ expressing the human disease-causing mutation Arg403Gln. Electrophysiology experiments were performed on ventricular myocytes isolated from 40-week-old mutant mice and their negatively genotyped littermates. Using current clamp mode we characterised the action potentials (APs). Resting membrane potentials wer not significantly different (−66.6 ± 1.6 [n = 7] vs −70.6 ± 0.8 mV [n = 6], mutant vs non–mutant cardiac myocytes), nor the AP amplitudes (83.8 ± 7.9 vs 86.7 ± 9.3 mV), but the AP duration (APD) at 90% repolarisation was significantly longer for the αMHC403/+ cardiomyocytes (APD90 = 99.3 ± 14.1 ms [n = 7]) compared with the non–mutant mice (32.3 ± 3.5 ms [n = 6]; p<0.05). The response to isoproterenol (Iso) was also altered. In control myocytes there was ∼34% increase in APD90 (n = 4) in the presence of 100 nM Iso. In contrast in mutant cardiomyocytes Iso significantly shortened the APD. The effect was evident with 10 nM Iso but more pronounced in the presence of 100 nM Iso (APD90 = 54.9 ± 7.5 ms [n = 6]). At low-frequency stimulation (1 Hz) extra action potentials were common in mutant but absent in non–mutant cells. These findings indicate a complex mechanism behind the increased arrhythmic activity in hypertrophic hearts, which needs further investigation.

20-Hydroxyecdysone attenuates cardiac remodeling in spontaneously hypertensive rats

BackgroundEcdysteroids, a group of steroid hormones found in insects and many plants, have been shown to prevent various changes in mammalian tissues after female sex hormone deprivation. PurposeTo examine whether an ecdysteroid, 20-hydroxyecdysone (20-HE), exhibits regulatory or protective roles in the cardiovascular system. Study design/methodBlood pressure and cardiac function were evaluated in spontaneously hypertensive rats (SHR) during and after daily treatment with 20-HE for six weeks. ResultsThe progressive increase in systolic blood pressure with age in SHR rats was significantly lower in animals treated with either 5 or 10mg/kg body weight of 20-HE. However, treatment with 20-HE did not diminish the increase in diastolic pressure. Echocardiography after six weeks of treatment demonstrated that the left ventricular chamber of SHR rats treated with 20-HE was smaller than that of SHR controls, while contractility was not affected by 20-HE. Histological images also demonstrated a decrease in cardiomyocyte cross-sectional area in 20-HE treated groups. Interestingly, treatment with 20-HE caused a shift in cardiac myosin heavy chain towards more β-isoforms. SHR rats treated with 20-HE also exhibited a decrease in seminal vesicular weight and an increase in testicular weight, especially at a dose of 10mg/kg body weight. This finding suggests a possible anti-androgenic effect of 20-HE. ConclusionOur finding reveal that 20-HE has a beneficial effect on reducing blood pressure and consequently preventing dilated cardiac hypertrophy in SHR rats.

Digenic inheritance of mutations in the cardiac troponin (TNNT2) and cardiac beta myosin heavy chain (MYH7) as the cause of severe dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is characterized by ventricular dilation and depressed myocardial performance. It is a genetically heterogeneous disorder associated with mutations in over 60 genes. We carried out whole exome sequencing in combination with cardiomyopathy-related gene-filtering on two affected family members to identify the possible causative mutation in a consanguineous Iranian family with DCM.Two novel variants in cardiomyopathy-related genes were identified: c.247 A > C; p.N83H in the Troponin T Type 2 gene (TNNT2) and c.2863G > A; p.D955N in the Myosin Heavy Polypeptide 7 gene (MYH7). Sanger sequencing and co-segregation analysis in the remaining family members supported the coexistence of these digenic mutations in affected members of the family. Carriers of either variant alone were asymptomatic.In summary, we find that digenic inheritance of two novel variants in DCM related genes is associated with a severe form of DCM. Exome sequencing has been shown to be very useful in identifying pathogenic mutations in cardiomyopathy families, and this report emphasizes the importance of comprehensive screening of DCM related genes, even after the identification of a single disease-causing mutation.