Bringing into focus the central domains C3-C6 of myosin binding protein C.

Myosin binding protein C (MyBPC) is a multi-domain protein with each region having a distinct functional role in muscle contraction. The central domains of MyBPC have often been overlooked due to their unclear roles. However, recent research shows promise in understanding their potential structural and regulatory functions. Understanding the central region of MyBPC is important because it may have specialized function that can be used as drug targets or for disease-specific therapies. In this review, we provide a brief overview of the evolution of our understanding of the central domains of MyBPC in regard to its domain structures, arrangement and dynamics, interaction partners, hypothesized functions, disease-causing mutations, and post-translational modifications. We highlight key research studies that have helped advance our understanding of the central region. Lastly, we discuss gaps in our current understanding and potential avenues to further research and discovery.

Hypertrophic cardiomyopathy in MYBPC3 carriers in aging.

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the myocardium, leading to arrhythmias, heart failure, and elevated risk of sudden cardiac death, particularly among the young. This inherited disease is predominantly caused by mutations in sarcomeric genes, among which those in the cardiac myosin binding protein-C3 (MYBPC3) gene are major contributors. HCM associated with MYBPC3 mutations usually presents in the elderly and ranges from asymptomatic to symptomatic forms, affecting numerous cardiac functions and presenting significant health risks with a spectrum of clinical manifestations. Regulation of MYBPC3 expression involves various transcriptional and translational mechanisms, yet the destiny of mutant MYBPC3 mRNA and protein in late-onset HCM remains unclear. Pathogenesis related to MYBPC3 mutations includes nonsense-mediated decay, alternative splicing, and ubiquitin-proteasome system events, leading to allelic imbalance and haploinsufficiency. Aging further exacerbates the severity of HCM in carriers of MYBPC3 mutations. Advancements in high-throughput omics techniques have identified crucial molecular events and regulatory disruptions in cardiomyocytes expressing MYBPC3 variants. This review assesses the pathogenic mechanisms that promote late-onset HCM through the lens of transcriptional, post-transcriptional, and post-translational modulation of MYBPC3, underscoring its significance in HCM across carriers. The review also evaluates the influence of aging on these processes and MYBPC3 levels during HCM pathogenesis in the elderly. While pinpointing targets for novel medical interventions to conserve cardiac function remains challenging, the emergence of personalized omics offers promising avenues for future HCM treatments, particularly for late-onset cases.

Effect of the Novel Myotrope Danicamtiv on Cross-Bridge Behavior in Human Myocardium.

Background Omecamtiv mecarbil (OM) and danicamtiv both increase myocardial force output by selectively activating myosin within the cardiac sarcomere. Enhanced force generation is presumably due to an increase in the total number of myosin heads bound to the actin filament; however, detailed comparisons of the molecular mechanisms of OM and danicamtiv are lacking. Methods and Results The effect of OM and danicamtiv on Ca2+ sensitivity of force generation was analyzed by exposing chemically skinned myocardial samples to a series of increasing Ca2+ solutions. The results showed that OM significantly increased Ca2+ sensitivity of force generation, whereas danicamtiv showed similar Ca2+ sensitivity of force generation to untreated preparations. A direct comparison of OM and danicamtiv on dynamic cross-bridge behavior was performed at a concentration that produced a similar force increase when normalized to predrug levels at submaximal force (pCa 6.1). Both OM and danicamtiv-treated groups slowed the rates of cross-bridge detachment from the strongly bound state and cross-bridge recruitment into the force-producing state. Notably, the significant OM-induced prolongation in the time to reach force relaxation and subsequent commencement of force generation following rapid stretch was dramatically reduced in danicamtiv-treated myocardium. Conclusions This is the first study to directly compare the effects of OM and danicamtiv on cross-bridge kinetics. At a similar level of force enhancement, danicamtiv had a less pronounced effect on the slowing of cross-bridge kinetics and, therefore, may provide a similar improvement in systolic function as OM without excessively prolonging systolic ejection time and slowing cardiac relaxation facilitating diastolic filling at the whole-organ level.

Basic science methods for the characterization of variants of uncertain significance in hypertrophic cardiomyopathy.

With the advent of next-generation whole genome sequencing, many variants of uncertain significance (VUS) have been identified in individuals suffering from inheritable hypertrophic cardiomyopathy (HCM). Unfortunately, this classification of a genetic variant results in ambiguity in interpretation, risk stratification, and clinical practice. Here, we aim to review some basic science methods to gain a more accurate characterization of VUS in HCM. Currently, many genomic data-based computational methods have been developed and validated against each other to provide a robust set of resources for researchers. With the continual improvement in computing speed and accuracy, in silico molecular dynamic simulations can also be applied in mutational studies and provide valuable mechanistic insights. In addition, high throughput in vitro screening can provide more biologically meaningful insights into the structural and functional effects of VUS. Lastly, multi-level mathematical modeling can predict how the mutations could cause clinically significant organ-level dysfunction. We discuss emerging technologies that will aid in better VUS characterization and offer a possible basic science workflow for exploring the pathogenicity of VUS in HCM. Although the focus of this mini review was on HCM, these basic science methods can be applied to research in dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic cardiomyopathy (ACM), or other genetic cardiomyopathies.

Torsional and strain dysfunction precede overt heart failure in a mouse model of dilated cardiomyopathy pathogenesis.

Detailed assessments of whole heart mechanics are crucial for understanding the consequences of sarcomere perturbations that lead to cardiomyopathy in mice. Echocardiography offers an accessible and cost-effective method of obtaining metrics of cardiac function, but the most routine imaging and analysis protocols might not identify subtle mechanical deficiencies. This study aims to use advanced echocardiography imaging and analysis techniques to identify previously unappreciated mechanical deficiencies in a mouse model of dilated cardiomyopathy (DCM) before the onset of overt systolic heart failure (HF). Mice lacking muscle LIM protein expression (MLP-/-) were used to model DCM-linked HF pathogenesis. Left ventricular (LV) function of MLP-/- and wild-type (WT) controls were studied at 3, 6, and 10 wk of age using conventional and four-dimensional (4-D) echocardiography, followed by speckle-tracking analysis to assess torsional and strain mechanics. Mice were also studied with RNA-seq. Although 3-wk-old MLP-/- mice showed normal LV ejection fraction (LVEF), these mice displayed abnormal torsional and strain mechanics alongside reduced ß-adrenergic reserve. Transcriptome analysis showed that these defects preceded most molecular markers of HF. However, these markers became upregulated as MLP-/- mice aged and developed overt systolic dysfunction. These findings indicate that subtle deficiencies in LV mechanics, undetected by LVEF and conventional molecular markers, may act as pathogenic stimuli in DCM-linked HF. Using these analyses in future studies will further help connect in vitro measurements of the sarcomere function to whole heart function.NEW & NOTEWORTHY A detailed study of how perturbations to sarcomere proteins impact whole heart mechanics in mouse models is a major yet challenging step in furthering our understanding of cardiovascular pathophysiology. This study uses advanced echocardiographic imaging and analysis techniques to reveal previously unappreciated subclinical whole heart mechanical defects in a mouse model of cardiomyopathy. In doing so, it offers an accessible set of measurements for future studies to use when connecting sarcomere and whole heart function.

The contribution of N-terminal truncated cMyBPC to in vivo cardiac function.

Cardiac myosin binding protein C (cMyBPC) is an 11-domain sarcomeric protein (C0-C10) integral to cardiac muscle regulation. In vitro studies have demonstrated potential functional roles for regions beyond the N-terminus. However, the in vivo contributions of these domains are mostly unknown. Therefore, we examined the in vivo consequences of expression of N-terminal truncated cMyBPC (C3C10). Neonatal cMyBPC-/- mice were injected with AAV9-full length (FL), C3C10 cMyBPC, or saline, and echocardiography was performed 6 wk after injection. We then isolated skinned myocardium from virus-treated hearts and performed mechanical experiments. Our results show that expression of C3C10 cMyBPC in cMyBPC-/- mice resulted in a 28% increase in systolic ejection fraction compared to saline-injected cMyBPC-/- mice and a 25% decrease in left ventricle mass-to-body weight ratio. However, unlike expression of FL cMyBPC, there was no prolongation of ejection time compared to saline-injected mice. In vitro mechanical experiments demonstrated that functional improvements in cMyBPC-/- mice expressing C3C10 were primarily due to a 35% reduction in the rate of cross-bridge recruitment at submaximal Ca2+ concentrations when compared to hearts from saline-injected cMyBPC-/- mice. However, unlike the expression of FL cMyBPC, there was no change in the rate of cross-bridge detachment when compared to saline-injected mice. Our data demonstrate that regions of cMyBPC beyond the N-terminus are important for in vivo cardiac function, and have divergent effects on cross-bridge behavior. Elucidating the molecular mechanisms of cMyBPC region-specific function could allow for development of targeted approaches to manipulate specific aspects of cardiac contractile function.

Identification of Phosphorylation and Other Post-Translational Modifications in the Central C4C5 Domains of Murine Cardiac Myosin Binding Protein C.

Cardiac myosin binding protein C (cMyBPC) is a critical multidomain protein that modulates myosin cross bridge behavior and cardiac contractility. cMyBPC is principally regulated by phosphorylation of the residues within the M-domain of its N-terminus. However, not much is known about the phosphorylation or other post-translational modification (PTM) landscape of the central C4C5 domains. In this study, the presence of phosphorylation outside the M-domain was confirmed in vivo using mouse models expressing cMyBPC with nonphosphorylatable serine (S) to alanine substitutions. Purified recombinant mouse C4C5 domain constructs were incubated with 13 different kinases, and samples from the 6 strongest kinases were chosen for mass spectrometry analysis. A total of 26 unique phosphorylated peptides were found, representing 13 different phosphorylation sites including 10 novel sites. Parallel reaction monitoring and subsequent mutagenesis experiments revealed that the S690 site (UniProtKB O70468) was the predominant target of PKA and PKG1. We also report 6 acetylation and 7 ubiquitination sites not previously described in the literature. These PTMs demonstrate the possibility of additional layers of regulation and potential importance of the central domains of cMyBPC in cardiac health and disease. Data are available via ProteomeXchange with identifier PXD031262.

Molecular characterization of linker and loop-mediated structural modulation and hinge motion in the C4-C5 domains of cMyBPC.

INTRODUCTION: The central C4 and C5 domains (C4C5) of cardiac myosin binding protein C (cMyBPC) contain a flexible interdomain linker and a cardiac-isoform specific loop. However, their importance in the functional regulation of cMyBPC has not been extensively studied. METHODS AND RESULTS: We expressed recombinant C4C5 proteins with deleted linker and loop regions and performed biophysical experiments to determine each of their structural and dynamic roles. We show that the linker and C5 loop regions modulate the secondary structure and thermal stability of C4C5. Furthermore, we provide evidence through extended molecular dynamics simulations and principle component analyses that C4C5 can adopt a completely bent or latched conformation. The simulation trajectory and interaction network analyses reveal that the completely bent conformation of C4C5 exhibits a specific pattern of residue-level interactions. Therefore, we propose a "hinge-and-latch" mechanism where the linker allows a great degree of flexibility and bending, while the loop aids in achieving a completely bent and latched conformation. Although this may be one of many bent positions that C4C5 can adopt, we illustrate for the first time in molecular detail that this type of large scale conformational change can occur in the central domains of cMyBPC. CONCLUSIONS: Our hinge-and-latch mechanism demonstrates that the linker and loop regions participate in dynamic modulation of cMyBPC's motion and global conformation. These structural and dynamic features may contribute to muscle isoform-specific regulation of actomyosin activity, and have potential implications regarding its ability to propagate or retract cMyBPC's regulatory N-terminal domains.

cMyBPC phosphorylation modulates the effect of omecamtiv mecarbil on myocardial force generation.

Omecamtiv mecarbil (OM), a direct myosin motor activator, is currently being tested as a therapeutic replacement for conventional inotropes in heart failure (HF) patients. It is known that HF patients exhibit dysregulated ß-adrenergic signaling and decreased cardiac myosin-binding protein C (cMyBPC) phosphorylation, a critical modulator of myocardial force generation. However, the functional effects of OM in conditions of altered cMyBPC phosphorylation have not been established. Here, we tested the effects of OM on force generation and cross-bridge (XB) kinetics using murine myocardial preparations isolated from wild-type (WT) hearts and from hearts expressing S273A, S282A, and S302A substitutions (SA) in the M domain, between the C1 and C2 domains of cMyBPC, which cannot be phosphorylated. At submaximal Ca2+ activations, OM-mediated force enhancements were less pronounced in SA than in WT myocardial preparations. Additionally, SA myocardial preparations lacked the dose-dependent increases in force that were observed in WT myocardial preparations. Following OM incubation, the basal differences in the rate of XB detachment (krel) between WT and SA myocardial preparations were abolished, suggesting that OM differentially affects the XB behavior when cMyBPC phosphorylation is reduced. Similarly, in myocardial preparations pretreated with protein kinase A to phosphorylate cMyBPC, incubation with OM significantly slowed krel in both the WT and SA myocardial preparations. Collectively, our data suggest there is a strong interplay between the effects of OM and XB behavior, such that it effectively uncouples the sarcomere from cMyBPC phosphorylation levels. Our findings imply that OM may significantly alter the in vivo cardiac response to ß-adrenergic stimulation.

AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice.

Decreased cardiac myosin-binding protein C (cMyBPC) expression due to inheritable mutations is thought to contribute to the hypertrophic cardiomyopathy (HCM) phenotype, suggesting that increasing cMyBPC content is of therapeutic benefit. In vitro assays show that cMyBPC N-terminal domains (NTDs) contain structural elements necessary and sufficient to modulate actomyosin interactions, but it is unknown if they can regulate in vivo myocardial function. To test whether NTDs can recapitulate the effects of full-length (FL) cMyBPC in rescuing cardiac function in a cMyBPC-null mouse model of HCM, we assessed the efficacy of AAV9 gene transfer of a cMyBPC NTD that contained domains C0C2 and compared its therapeutic potential with AAV9-FL gene replacement. AAV9 vectors were administered systemically at neonatal day 1, when early-onset disease phenotypes begin to manifest. A comprehensive analysis of in vivo and in vitro function was performed following cMyBPC gene transfer. Our results show that a systemic injection of AAV9-C0C2 significantly improved cardiac function (e.g., 52.24 ± 1.69 ejection fraction in the C0C2-treated group compared with 40.07 ± 1.97 in the control cMyBPC-/- group, P < 0.05) and reduced the histopathologic signs of cardiomyopathy. Furthermore, C0C2 significantly slowed and normalized the accelerated cross-bridge kinetics found in cMyBPC-/- control myocardium, as evidenced by a 32.41% decrease in the rate of cross-bridge detachment (krel). Results indicate that C0C2 can rescue biomechanical defects of cMyBPC deficiency and that the NTD may be a target region for therapeutic myofilament kinetic manipulation.

Strategies for targeting the cardiac sarcomere: avenues for novel drug discovery.

Introduction: Heart failure remains one of the largest clinical challenges in the United States. Researchers have continually searched for more effective heart failure treatments that target the cardiac sarcomere but have found few successes despite numerous expensive cardiovascular clinical trials. Among many reasons, the high failure rate of cardiovascular clinical trials may be partly due to incomplete characterization of a drug candidate's complex interaction with cardiac physiology.Areas covered: In this review, the authors address the issue of preclinical cardiovascular studies of sarcomere-targeting heart failure therapies. The authors consider inherent tradeoffs made between mechanistic transparency and physiological fidelity for several relevant preclinical techniques at the atomic, molecular, heart muscle fiber, whole heart, and whole-organism levels. Thus, the authors suggest a comprehensive, bottom-up approach to preclinical cardiovascular studies that fosters scientific rigor and hypothesis-driven drug discovery.Expert opinion: In the authors' opinion, the implementation of hypothesis-driven drug discovery practices, such as the bottom-up approach to preclinical cardiovascular studies, will be imperative for the successful development of novel heart failure treatments. However, additional changes to clinical definitions of heart failure and current drug discovery culture must accompany the bottom-up approach to maximize the effectiveness of hypothesis-driven drug discovery.

Myocardial-restricted ablation of the GTPase RAD results in a pro-adaptive heart response in mice.

Existing therapies to improve heart function target ß-adrenergic receptor (ß-AR) signaling and Ca2+ handling and often lead to adverse outcomes. This underscores an unmet need for positive inotropes that improve heart function without any adverse effects. The GTPase Ras associated with diabetes (RAD) regulates L-type Ca2+ channel (LTCC) current (ICa,L). Global RAD-knockout mice (gRAD-/-) have elevated Ca2+ handling and increased cardiac hypertrophy, but RAD is expressed also in noncardiac tissues, suggesting the possibility that pathological remodeling is due also to noncardiac effects. Here, we engineered a myocardial-restricted inducible RAD-knockout mouse (RADΔ/Δ). Using an array of methods and techniques, including single-cell electrophysiological and calcium transient recordings, echocardiography, and radiotelemetry monitoring, we found that RAD deficiency results in a sustained increase of inotropy without structural or functional remodeling of the heart. ICa,L was significantly increased, with RAD loss conferring a ß-AR-modulated phenotype on basal ICa,L Cardiomyocytes from RADΔ/Δ hearts exhibited enhanced cytosolic Ca2+ handling, increased contractile function, elevated sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, and faster lusitropy. These results argue that myocardial RAD ablation promotes a beneficial elevation in Ca2+ dynamics, which would obviate a need for increased ß-AR signaling to improve cardiac function.

The HCM-causing Y235S cMyBPC mutation accelerates contractile function by altering C1 domain structure.

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KOY235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.

Lost in translation: Interpreting cardiac muscle mechanics data in clinical practice.

Current inotropic therapies improve systolic function in heart failure patients but also elicit undesirable side effects such as arrhythmias and increased intracellular Ca2+ transients. In order to maintain myocyte Ca2+ homeostasis, the increased cytosolic Ca2+ needs to be actively transported back to sarcoplasmic reticulum leading to depleted ATP reserves. Thus, an emerging approach is to design sarcomere-based treatments to correct impaired contractility via a direct and allosteric modulation of myosin's intrinsic force-generating behavior -a concept that potentially avoids the "off-target" effects. To achieve this goal, various biophysical approaches are utilized to investigate the mechanistic impact of sarcomeric modulators but information derived from diverse approaches is not fully integrated into therapeutic applications. This is in part due to the lack of information that provides a coherent connecting link between biophysical data to in vivo function. Hence, our ability to clearly discern the drug-mediated impact on whole-heart function is diminished. Reducing this translational barrier can significantly accelerate clinical progress related to sarcomere-based therapies by optimizing drug-dosing and treatment duration protocols based on information obtained from biophysical studies. Therefore, we attempt to link biophysical mechanical measurements obtained in isolated cardiac muscle and in vivo contractile function.

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross-Bridge Behavior in a Murine Model of Hypercontractility.

Background Recent studies suggest that mavacamten (Myk461), a small myosin-binding molecule, decreases hypercontractility in myocardium expressing hypertrophic cardiomyopathy-causing missense mutations in myosin heavy chain. However, the predominant feature of most mutations in cardiac myosin binding protein-C ( cMyBPC ) that cause hypertrophic cardiomyopathy is reduced total cMyBPC expression, and the impact of Myk461 on cMyBPC -deficient myocardium is currently unknown. Methods and Results We measured the impact of Myk461 on steady-state and dynamic cross-bridge ( XB ) behavior in detergent-skinned mouse wild-type myocardium and myocardium lacking cMyBPC (knockout (KO)). KO myocardium exhibited hypercontractile XB behavior as indicated by significant accelerations in rates of XB detachment (krel) and recruitment (kdf) at submaximal Ca2+ activations. Incubation of KO and wild-type myocardium with Myk461 resulted in a dose-dependent force depression, and this impact was more pronounced at low Ca2+ activations. Interestingly, Myk461-induced force depressions were less pronounced in KO myocardium, especially at low Ca2+ activations, which may be because of increased acto-myosin XB formation and potential disruption of super-relaxed XB s in KO myocardium. Additionally, Myk461 slowed krel in KO myocardium but not in wild-type myocardium, indicating increased XB " on" time. Furthermore, the greater degree of Myk461-induced slowing in kdf and reduction in XB recruitment magnitude in KO myocardium normalized the XB behavior back to wild-type levels. Conclusions This is the first study to demonstrate that Myk461-induced force depressions are modulated by cMyBPC expression levels in the sarcomere, and emphasizes that clinical use of Myk461 may need to be optimized based on the molecular trigger that underlies the hypertrophic cardiomyopathy phenotype.

Sarcomere-based genetic enhancement of systolic cardiac function in a murine model of dilated cardiomyopathy.

Diminished cardiac contractile function is a characteristic feature of dilated cardiomyopathy (DCM) and many other heart failure (HF) causing etiologies. We tested the hypothesis that targeting the sarcomere to increase cardiac contractility can effectively prevent the DCM phenotype in muscle-LIM protein knockout (MLP-/-) mice. The ablation of cardiac myosin binding protein C (MYBPC3-/-) protected the MLP-/- mice from developing the DCM phenotype. We examined the in vivo cardiac function and morphology of the resultant mouse model lacking both MLP and MYBPC3 (DKO) by echocardiography and pressure-volume catheterization and found a significant reduction in hypertrophy, as evidenced by normalized wall thickness and chamber dimensions, and improved systolic function, as evidenced by enhanced ejection fraction (~26% increase compared MLP-/- mice) and rate of pressure development (DKO 7851.0 ±â€¯504.8 vs. MLP-/- 4496.4 ±â€¯196.8 mmHg/s). To investigate the molecular basis for the improved DKO phenotype we performed mechanical experiments in skinned myocardium isolated from WT and the individual KO mice. Skinned myocardium isolated from DKO mice displayed increased Ca2+ sensitivity of force generation, and significantly accelerated rate of cross-bridge detachment (+63% compared to MLP-/-) and rate of XB recruitment (+58% compared to MLP-/-) at submaximal Ca2+ activations. The in vivo and in vitro functional enhancement of DKO mice demonstrates that enhancing the sarcomeric contractility can be cardioprotective in HF characterized by reduced cardiac output, such as in cases of DCM.

Dose-Dependent Effects of the Myosin Activator Omecamtiv Mecarbil on Cross-Bridge Behavior and Force Generation in Failing Human Myocardium.

BACKGROUND: Omecamtiv mecarbil (OM) enhances systolic function in vivo by directly binding the myosin cross-bridges (XBs) in the sarcomere. However, the mechanistic details governing OM-induced modulation of XB behavior in failing human myocardium are unclear. METHODS AND RESULTS: The effects of OM on steady state and dynamic XB behavior were measured in chemically skinned myocardial preparations isolated from human donor and heart failure (HF) left ventricle. HF myocardium exhibited impaired contractile function as evidenced by reduced maximal force, magnitude of XB recruitment (Pdf), and a slowed rate of XB detachment (krel) at submaximal Ca2+ activations. Ca2+ sensitivity of force generation (pCa50) was higher in HF myocardium when compared with donor myocardium, both prior to and after OM incubations. OM incubation (0.5 and 1.0 µmol/L) enhanced force generation at submaximal Ca2+ activations in a dose-dependent manner. Notably, OM induced a slowing in krel with 1.0 µmol/L OM but not with 0.5 µmol/L OM in HF myocardium. Additionally, OM exerted other differential effects on XB behavior in HF myocardium as evidenced by a greater enhancement in Pdf and slowing in the time course of cooperative XB recruitment (Trec), which collectively prolonged achievement of peak force development (Tpk), compared with donor myocardium. CONCLUSIONS: Our findings demonstrate that OM augments force generation but also prolongs the time course of XB transitions to force-bearing states in remodeled HF myocardium, which may extend the systolic ejection time in vivo. Optimal OM dosing is critical for eliciting enhanced systolic function without excessive prolongation of systolic ejection time, which may compromise diastolic filling.

Cardiac myosin binding protein-C Ser302 phosphorylation regulates cardiac ß-adrenergic reserve.

Phosphorylation of cardiac myosin binding protein-C (MyBP-C) modulates cardiac contractile function; however, the specific roles of individual serines (Ser) within the M-domain that are targets for ß-adrenergic signaling are not known. Recently, we demonstrated that significant accelerations in in vivo pressure development following ß-agonist infusion can occur in transgenic (TG) mouse hearts expressing phospho-ablated Ser282 (that is, TGS282A) but not in hearts expressing phospho-ablation of all three serines [that is, Ser273, Ser282, and Ser302 (TG3SA)], suggesting an important modulatory role for other Ser residues. In this regard, there is evidence that Ser302 phosphorylation may be a key contributor to the ß-agonist-induced positive inotropic responses in the myocardium, but its precise functional role has not been established. Thus, to determine the in vivo and in vitro functional roles of Ser302 phosphorylation, we generated TG mice expressing nonphosphorylatable Ser302 (that is, TGS302A). Left ventricular pressure-volume measurements revealed that TGS302A mice displayed no accelerations in the rate of systolic pressure rise and an inability to maintain systolic pressure following dobutamine infusion similar to TG3SA mice, implicating Ser302 phosphorylation as a critical regulator of enhanced systolic performance during ß-adrenergic stress. Dynamic strain-induced cross-bridge (XB) measurements in skinned myocardium isolated from TGS302A hearts showed that the molecular basis for impaired ß-adrenergic-mediated enhancements in systolic function is due to the absence of protein kinase A-mediated accelerations in the rate of cooperative XB recruitment. These results demonstrate that Ser302 phosphorylation regulates cardiac contractile reserve by enhancing contractile responses during ß-adrenergic stress.

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and ß-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output.NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress.