Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

Fine tuning contractility: atrial sarcomere function in health and disease.

The molecular mechanisms of sarcomere proteins underlie the contractile function of the heart. Although our understanding of the sarcomere has grown tremendously, the focus has been on ventricular sarcomere isoforms due to the critical role of the ventricle in health and disease. However, atrial-specific or -enriched myofilament protein isoforms, as well as isoforms that become expressed in disease, provide insight into ways this complex molecular machine is fine-tuned. Here, we explore how atrial-enriched sarcomere protein composition modulates contractile function to fulfill the physiological requirements of atrial function. We review how atrial dysfunction negatively affects the ventricle and the many cardiovascular diseases that have atrial dysfunction as a comorbidity. We also cover the pathophysiology of mutations in atrial-enriched contractile proteins and how they can cause primary atrial myopathies. Finally, we explore what is known about contractile function in various forms of atrial fibrillation. The differences in atrial function in health and disease underscore the importance of better studying atrial contractility, especially as therapeutics currently in development to modulate cardiac contractility may have different effects on atrial sarcomere function.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

Mechanisms of Sarcomere Protein Mutation-Induced Cardiomyopathies.

PURPOSE OF REVIEW: The pace of identifying cardiomyopathy-associated mutations and advances in our understanding of sarcomere function that underlies many cardiomyopathies has been remarkable. Here, we aim to synthesize how these advances have led to the promising new treatments that are being developed to treat cardiomyopathies. RECENT FINDINGS: The genomics era has identified and validated many genetic causes of hypertrophic and dilated cardiomyopathies. Recent advances in our mechanistic understanding of sarcomere pathophysiology include high-resolution molecular models of sarcomere components and the identification of the myosin super-relaxed state. The advances in our understanding of sarcomere function have yielded several therapeutic agents that are now in development and clinical use to correct contractile dysfunction-mediated cardiomyopathy. New genes linked to cardiomyopathy include targets with limited clinical evidence and require additional investigation. Large portions of cardiomyopathy with family history remain genetically undiagnosed and may be due to polygenic disease.

Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects.

A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy.

BACKGROUND: Cardiomyopathy and arrhythmias are under significant genetic influence. Here, we studied a family with dilated cardiomyopathy and associated conduction system disease in whom prior clinical cardiac gene panel testing was unrevealing. METHODS: Whole-genome sequencing and induced pluripotent stem cells were used to examine a family with dilated cardiomyopathy and atrial and ventricular arrhythmias. We also characterized a mouse model with heterozygous and homozygous deletion of Mybphl. RESULTS: Whole-genome sequencing identified a premature stop codon, R255X, in the MYBPHL gene encoding MyBP-HL (myosin-binding protein-H like), a novel member of the myosin-binding protein family. MYBPHL was found to have high atrial expression with low ventricular expression. We determined that MyBP-HL protein was myofilament associated in the atria, and truncated MyBP-HL protein failed to incorporate into the myofilament. Human cell modeling demonstrated reduced expression from the mutant MYBPHL allele. Echocardiography of Mybphl heterozygous and null mouse hearts exhibited a 36% reduction in fractional shortening and an increased diastolic ventricular chamber size. Atria weight normalized to total heart weight was significantly increased in Mybphl heterozygous and null mice. Using a reporter system, we detected robust expression of Mybphl in the atria, and in discrete puncta throughout the right ventricular wall and septum, as well. Telemetric electrocardiogram recordings in Mybphl mice revealed cardiac conduction system abnormalities with aberrant atrioventricular conduction and an increased rate of arrhythmia in heterozygous and null mice. CONCLUSIONS: The findings of reduced ventricular function and conduction system defects in Mybphl mice support that MYBPHL truncations may increase risk for human arrhythmias and cardiomyopathy.

The naked mole-rat exhibits an unusual cardiac myofilament protein profile providing new insights into heart function of this naturally subterranean rodent.

The long-lived, hypoxic-tolerant naked mole-rat well-maintains cardiac function over its three-decade-long lifespan and exhibits many cardiac features atypical of similar-sized laboratory rodents. For example, they exhibit low heart rates and resting cardiac contractility, yet have a large cardiac reserve. These traits are considered ecophysiological adaptations to their dank subterranean atmosphere of low oxygen and high carbon dioxide levels and may also contribute to negligible declines in cardiac function during aging. We asked if naked mole-rats had a different myofilament protein signature to that of similar-sized mice that commonly show both high heart rates and high basal cardiac contractility. Adult mouse ventricles predominantly expressed α-myosin heavy chain (97.9 ± 0.4%). In contrast, and more in keeping with humans, ß myosin heavy chain was the dominant isoform (79.0 ± 2.0%) in naked mole-rat ventricles. Naked mole-rat ventricles diverged from those of both humans and mice, as they expressed both cardiac and slow skeletal isoforms of troponin I. This myofilament protein profile is more commonly observed in mice in utero and during cardiomyopathies. There were no species differences in phosphorylation of cardiac myosin binding protein-C or troponin I. Phosphorylation of both ventricular myosin light chain 2 and cardiac troponin T in naked mole-rats was approximately half that observed in mice. Myofilament function was also compared between the two species using permeabilized cardiomyocytes. Together, these data suggest a cardiac myofilament protein signature that may contribute to the naked mole-rat's suite of adaptations to its natural subterranean habitat.

Enhanced Muscular Dystrophy from Loss of Dysferlin Is Accompanied by Impaired Annexin A6 Translocation after Sarcolemmal Disruption.

Dysferlin is a membrane-associated protein implicated in membrane resealing; loss of dysferlin leads to muscular dystrophy. We examined the same loss-of-function Dysf mutation in two different mouse strains, 129T2/SvEmsJ (Dysf(129)) and C57BL/6J (Dysf(B6)). Although there are many genetic differences between these two strains, we focused on polymorphisms in Anxa6 because these variants were previously associated with modifying a pathologically distinct form of muscular dystrophy and increased the production of a truncated annexin A6 protein. Dysferlin deficiency in the C57BL/6J background was associated with increased Evan's Blue dye uptake into muscle and increased serum creatine kinase compared to the 129T2/SvEmsJ background. In the C57BL/6J background, dysferlin loss was associated with enhanced pathologic severity, characterized by decreased mean fiber cross-sectional area, increased internalized nuclei, and increased fibrosis, compared to that in Dysf(129) mice. Macrophage infiltrate was also increased in Dysf(B6) muscle. High-resolution imaging of live myofibers demonstrated that fibers from Dysf(B6) mice displayed reduced translocation of full-length annexin A6 to the site of laser-induced sarcolemmal disruption compared to Dysf(129) myofibers, and impaired translocation of annexin A6 associated with impaired resealing of the sarcolemma. These results provide one mechanism by which the C57BL/6J background intensifies dysferlinopathy, giving rise to a more severe form of muscular dystrophy in the Dysf(B6) mouse model through increased membrane leak and inflammation.

Haploinsufficiency of MYBPC3 exacerbates the development of hypertrophic cardiomyopathy in heterozygous mice.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein-C (cMyBP-C), account for ~40% of hypertrophic cardiomyopathy (HCM) cases. Most pathological MYBPC3 mutations encode truncated protein products not found in tissue. Reduced protein levels occur in symptomatic heterozygous human HCM carriers, suggesting haploinsufficiency as an underlying mechanism of disease. However, we do not know if reduced cMyBP-C content results from, or initiates the development of HCM. In previous studies, heterozygous (HET) mice with a MYBPC3 C'-terminal truncation mutation and normal cMyBP-C levels show altered contractile function prior to any overt hypertrophy. Therefore, this study aimed to test whether haploinsufficiency occurs, with decreased cMyBP-C content, following cardiac stress and whether the functional impairment in HET MYBPC3 hearts leads to worsened disease progression. To address these questions, transverse aortic constriction (TAC) was performed on three-month-old wild-type (WT) and HET MYBPC3-truncation mutant mice and then characterized at 4 and 12weeks post-surgery. HET-TAC mice showed increased hypertrophy and reduced ejection fraction compared to WT-TAC mice. At 4weeks post-surgery, HET myofilaments showed significantly reduced cMyBP-C content. Functionally, HET-TAC cardiomyocytes showed impaired force generation, higher Ca(2+) sensitivity, and blunted length-dependent increase in force generation. RNA sequencing revealed several differentially regulated genes between HET and WT groups, including regulators of remodeling and hypertrophic response. Collectively, these results demonstrate that haploinsufficiency occurs in HET MYBPC3 mutant carriers following stress, causing, in turn, reduced cMyBP-C content and exacerbating the development of dysfunction at myofilament and whole-heart levels.

Phosphorylation of cMyBP-C affects contractile mechanisms in a site-specific manner.

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser(273), Ser(282), and Ser(302) by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala(273)-Asp(282)-Ala(302)), DAD (Asp(273)-Ala(282)-Asp(302)), and SAS (Ser(273)-Ala(282)-Ser(302)) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (~50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca(2+) sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp(273) and/or Asp(302) (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.

Contractile dysfunction in a mouse model expressing a heterozygous MYBPC3 mutation associated with hypertrophic cardiomyopathy.

The etiology of hypertrophic cardiomyopathy (HCM) has been ascribed to mutations in genes encoding sarcomere proteins. In particular, mutations in MYBPC3, a gene which encodes cardiac myosin binding protein-C (cMyBP-C), have been implicated in over one third of HCM cases. Of these mutations, 70% are predicted to result in C'-truncated protein products, which are undetectable in tissue samples. Heterozygous carriers of these truncation mutations exhibit varying penetrance of HCM, with symptoms often occurring later in life. We hypothesize that heterozygous carriers of MYBPC3 mutations, while seemingly asymptomatic, have subtle functional impairments that precede the development of overt HCM. This study compared heterozygous (+/t) knock-in MYBPC3 truncation mutation mice with wild-type (+/+) littermates to determine whether functional alterations occur at the whole-heart or single-cell level before the onset of hypertrophy. The +/t mice show ∼40% reduction in MYBPC3 transcription, but no changes in cMyBP-C level, phosphorylation status, or cardiac morphology. Nonetheless, +/t mice show significantly decreased maximal force development at sarcomere lengths of 1.9 µm (+/t 68.5 ± 4.1 mN/mm(2) vs. +/+ 82.2 ± 3.2) and 2.3 µm (+/t 79.2 ± 3.1 mN/mm(2) vs. +/+ 95.5 ± 2.4). In addition, heterozygous mice show significant reductions in vivo in the early/after (E/A) (+/t 1.74 ± 0.12 vs. +/+ 2.58 ± 0.43) and E'/A' (+/t 1.18 ± 0.05 vs. +/+ 1.52 ± 0.15) ratios, indicating diastolic dysfunction. These results suggest that seemingly asymptomatic heterozygous MYBPC3 carriers do suffer impairments that may presage the onset of HCM.

Cardiac Localized Polycystin-2 plays a Functional Role in Natriuretic Peptide Production and its Absence Contributes to Hypertension.

Cardiovascular complications are the most common cause of mortality in patients with autosomal dominant polycystic kidney disease (ADPKD). Hypertension is seen in 70% of patients by the age of 30 prior to decline in kidney function. The natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are released by cardiomyocytes in response to membrane stretch, increasing urinary excretion of sodium and water. Mice heterozygous for Pkd2 have attenuated NP responses and we hypothesized that cardiomyocyte-localized polycystin proteins contribute to production of NPs. Cardiomyocyte-specific knock-out models of polycystin-2 (PC2), one of the causative genes of ADPKD, demonstrate diurnal hypertension. These mice have decreased ANP and BNP expression in the left ventricle. Analysis of the pathways involved in production, maturation, and activity of NPs identified decreased transcription of CgB, PCSK6, and NFAT genes in cPC2-KOs. Engineered heart tissue with human iPSCs driven into cardiomyocytes with CRISPR/Cas9 KO of PKD2 failed to produce ANP. These results suggest that PC2 in cardiomyocytes are involved in NP production and lack of cardiac PC2 predisposes to a hypertensive volume expanded phenotype, which may contribute to the development of hypertension in ADPKD.

Reduction of Filamin C Results in Altered Proteostasis, Cardiomyopathy, and Arrhythmias.

BACKGROUND: Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. METHODS AND RESULTS: We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. CONCLUSIONS: FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

Interleukin-10 treatment attenuates pressure overload-induced hypertrophic remodeling and improves heart function via signal transducers and activators of transcription 3-dependent inhibition of nuclear factor-κB.

BACKGROUND: Inflammation plays a critical role in adverse cardiac remodeling and heart failure. Therefore, approaches geared toward inhibiting inflammation may provide therapeutic benefits. We tested the hypotheses that genetic deletion of interleukin-10 (IL-10), a potent antiinflammatory cytokine, exacerbates pressure overload-induced adverse cardiac remodeling and hypertrophy and that IL-10 therapy inhibits this pathology. METHODS AND RESULTS: Cardiac hypertrophy was induced in wild-type and IL-10 knockout mice by isoproterenol (ISO) infusion. ISO-induced left ventricular dysfunction and hypertrophic remodeling, including fibrosis and fetal gene expression, were further exaggerated in knockout mice compared with wild-type mice. Systemic recombinant mouse IL-10 administration markedly improved left ventricular function and not only inhibited but also reversed ISO-induced cardiac remodeling. Intriguingly, a very similar cardioprotective response of IL-10 was found in transverse aortic constriction-induced hypertrophy and heart failure models. In neonatal rat ventricular myocytes and H9c2 myoblasts, ISO activated nuclear factor-κB and inhibited signal transducers and activators of transcription 3 (STAT3) phosphorylation. Interestingly, IL-10 suppressed ISO-induced nuclear factor-κB activation and attenuated STAT3 inhibition. Moreover, pharmacological and genetic inhibition of STAT3 reversed the protective effects of IL-10, whereas ectopic expression of constitutively active STAT3 mimicked the IL-10 responses on the ISO effects, confirming that the IL-10-mediated inhibition of nuclear factor-κB is STAT3 dependent. CONCLUSION: Taken together, our results suggest IL-10 treatment as a potential therapeutic approach to limit the progression of pressure overload-induced adverse cardiac remodeling.

A critical function for Ser-282 in cardiac Myosin binding protein-C phosphorylation and cardiac function.

RATIONALE: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. OBJECTIVE: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during ß-adrenergic stimulation. METHODS AND RESULTS: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca(2+)-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca(2+)-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C(SAS)), Ala-273-Asp-282-Ala-302 (cMyBP-C(ADA)), or Asp-273-Ala-282-Asp-302 (cMyBP-C(DAD)), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C(SAS(t/t)), cMyBP-C(ADA(t/t)), and cMyBP-C(DAD(t/t)) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C((t/t)) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered ß-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C(SAS(t/t)) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of ß-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal ß-adrenergic responsiveness. CONCLUSIONS: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to ß-agonist stimulation.

Is haploinsufficiency a sufficient mechanism for MYBPC3 truncating mutations?

Reduced expression of MYBPC3 causes early dysfunction in human cell culture models prior to reduced cMyBP-C levels.

Sex and Gene Influence Arrhythmia Susceptibility in Murine Models of Calmodulinopathy.

BACKGROUND: Pathogenic variants in genes encoding CaM (calmodulin) are associated with a life-threatening ventricular arrhythmia syndrome (calmodulinopathy). The in vivo consequences of CaM variants have not been studied extensively and there is incomplete understanding of the genotype-phenotype relationship for recurrent variants. We investigated effects of different factors on calmodulinopathy phenotypes using 2 mouse models with a recurrent pathogenic variant (N98S) in Calm1 or Calm2. METHODS: Genetically engineered mice with heterozygous N98S pathogenic variants in Calm1 or Calm2 were generated. Differences between the sexes and affected genes were assessed using multiple physiological assays at the cellular and whole animal levels. Statistical significance among groups was evaluated using 1-way ANOVA or the Kruskal-Wallis test when data were not normally distributed. RESULTS: Calm1N98S/+ (Calm1S/+) or Calm2N98S/+ (Calm2S/+) mice exhibited sinus bradycardia and were more susceptible to arrhythmias after exposure to epinephrine and caffeine. Male Calm1S/+ mice had the most severe arrhythmia phenotype with evidence of early embryonic lethality, greater susceptibility for arrhythmic events, frequent premature beats, corrected QT prolongation, and more heart rate variability after epinephrine and caffeine than females with the same genotype. Calm2 S/+ mice exhibited a less severe phenotype, with female Calm2 S/+ mice having the least severe arrhythmia susceptibility. Flecainide was not effective in preventing arrhythmias in heterozygous CaM-N98S mice. Intracellular Ca2+ transients observed in isolated ventricular cardiomyocytes from male heterozygous CaM-N98S mice had lower peak amplitudes and slower sarcoplasmic reticulum Ca2+ release following in vitro exposure to epinephrine and caffeine, which were not observed in cardiomyocytes from heterozygous female CaM-N98S mice. CONCLUSIONS: We report heterogeneity in arrhythmia susceptibility and cardiomyocyte Ca2+ dynamics among male and female mice heterozygous for a recurrent pathogenic variant in Calm1 or Calm2, illustrating a complex calmodulinopathy phenotype in vivo. Further investigation of sex and genetic differences may help identify the molecular basis for this heterogeneity.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Aims: Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs that involves decreased blood flow velocity and reduced atrial contractility. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. The aim of this project is to define the contractile remodeling that occurs in AFib. Methods and Results: To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction in skinned single cardiomyocyte calcium-force studies. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed decreased troponin I phosphorylation, consistent with the increased calcium sensitivity that was found within these cardiomyocytes. Conclusions: With these results it can be concluded that AFib causes alterations in contraction that can be explained by both molecular changes occurring in myofilament proteins and overall myofilament protein degradation. These results provide an understanding of the contractile remodeling that occurs in AFib and provides insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.