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.

Trends in Management of Anal Fissures.

BACKGROUND: It is unclear how patients with anal fissures are treated in real-world settings, particularly since patients may not see colorectal surgeons. This study describes trends in treatment with medical therapies (calcium-channel blockers [CCBs], nitroglycerin [NTG], and narcotics) and surgical treatments. METHODS: Cohorts were created within the TriNetX database platform using codes for anal fissures and surgical interventions. Demographics were compared between patients that received surgical intervention within 1 year of diagnosis, CCB or NTG within 1 year (or preoperatively), or narcotics within 30 days or postoperatively vs those who did not. RESULTS: 121,213 patients were included of which 4.0% had surgical intervention. Factors associated with surgical intervention were male sex (OR 1.40), White race (OR 1.17), and Hispanic ethnicity (OR 1.11). Male patients were more likely to undergo sphincterotomy (OR 1.49). Female (OR 1.27), non-Hispanic (OR 1.34), and White patients (OR 1.41) were more likely to have chemodenervation. Regarding nonoperatively managed patients, non-Hispanic (OR .91) and White patients (OR .89) were less likely to receive CCB/NTG. Male (OR 1.21), non-Hispanic (OR 1.08), and Black patients (OR 1.20) were more likely to receive narcotics. Male patients that required surgery were more likely to be prescribed CCB/NTG preoperatively (OR 1.27). Non-Hispanic surgical patients were more likely to receive narcotics (OR 1.84). DISCUSSION: Male fissure patients were more likely to undergo surgical intervention other than chemodenervation. Differences in the rates of surgery and medical therapy (especially narcotics) between races and ethnicities require exploration to enhance the care of patients with anal fissures.

Burn Injuries From TikTok Challenges: A Brief Report.

Social media (e.g., TikTok) challenge is a relatively new phenomenon wherein a user creates and posts videos performing an often-dangerous task. The ease of access and availability of social media in recent times make teens and young adults susceptible to these viral Internet challenges and accidental injury. The severity, morbidity, and mortality of burn injuries from social media challenges have not readily been documented in the medical literature. In this brief report, we present three cases of accidental burns after attempting social media challenges involving boiling water or flame. The injuries ranged from superficial partial thickness burns to 24% total body surface area (TBSA) full thickness burns. Online challenges show the potential for severe injury and disability and underlie the importance of awareness and education of the public, further research into the usage of TikTok and other media platforms, and early referral to the American Burn Association recognized center.

Can perioperative electroencephalogram and adverse hemodynamic events predict neurodevelopmental outcomes in infants with congenital heart disease?

OBJECTIVE: The study objective was to characterize preoperative and postoperative continuous electroencephalogram metrics and hemodynamic adverse events as predictors of neurodevelopment in congenital heart disease infants undergoing cardiac surgery. METHODS: From 2010 to 2021, 320 infants underwent congenital heart disease surgery at our institution, of whom 217 had perioperative continuous electroencephalogram monitoring and were included in our study. Neurodevelopment was assessed in 76 patients by the Bayley Scales of Infant and Toddler Development, 3rd edition, consisting of cognitive, communication, and motor scaled scores. Patient and procedural factors, including hemodynamic adverse events, were included by means of the likelihood of covariate selection in our predictive model. Median (25th, 75th percentile) follow-up was 1.03 (0.09, 3.44) years with 3 (1, 6) Bayley Scales of Infant and Toddler Development, 3rd Edition evaluations per patient. RESULTS: Median age at index surgery was 7 (4, 23) days, and 81 (37%) were female. Epileptiform discharges, encephalopathy, and abnormality (lethargy and coma) were more prevalent on postoperative continuous electroencephalograms, compared with preoperative continuous electroencephalograms (P < .005). In 76 patients with Bayley Scales of Infant and Toddler Development, 3rd edition evaluations, patients with diffuse abnormality (P = .009), waveform discontinuity (P = .007), and lack of continuity (P = .037) on preoperative continuous electroencephalogram had lower cognitive scores. Patients with synchrony (P < .005) on preoperative and waveform continuity (P = .009) on postoperative continuous electroencephalogram had higher fine motor scores. Patients with postoperative adverse events had lower cognitive (P < .005) and gross motor scores (P < .005). CONCLUSIONS: Phenotypic patterns of perioperative continuous electroencephalogram metrics are associated with late-term neurologic injury in infants with congenital heart disease requiring surgery. Continuous electroencephalogram metrics can be integrated with hemodynamic adverse events in a predictive algorithm for neurologic impairment.

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.

Early results of bare metal extension stent for thoracoabdominal aortic dissection.

Objectives: Debakey type I and IIIb aortic dissections are complicated by extension along the full length of the aorta. Over the long term, the thoracoabdominal aorta in these patients often continues to degenerate, requiring endovascular or open repair. The purpose of this investigation is to determine the early clinical outcome on aortic remodeling using a composite thoracic stent graft and thoracoabdominal bare metal extension stenting strategy. Methods: From April 2019 to April 2021, 73 patients with Debakey I/IIIb aortic dissection underwent endovascular stent graft repair of the descending thoracic aorta and repair of the thoracoabdominal aorta using bare metal extension stenting. Preoperative and follow-up surveillance computed tomography imaging scans were analyzed. Results: Fifty-three (73%) patients had a Debakey I aortic dissection, and 50 (69%) patients underwent surgery during the chronic (time to surgery >30 days) dissection phase. Mortality at 30 days was 4% (3 hyperacute patients). Stroke occurred in 3 (4%), paraparesis in 2 (2.7%), and acute renal failure requiring dialysis occurred in 2 (2.7%) patients. On postoperative and follow-up computed tomography, there was a significant increase in false lumen thrombosis (P < .001). This coincided with a significant increase in true lumen fraction suggestive of positive aortic remodeling (P < .001) at the time of latest follow-up. Conclusions: Altering the course of aortic remodeling, with placement of a dissection stent in the thoracoabdominal aorta simultaneous with descending thoracic aortic repair may promote true lumen re-expansion and false lumen thrombosis during acute and chronic dissection phases.

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.

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.