Modulation of Myosin by Cardiac Myosin Binding Protein-C Peptides Improves Cardiac Contractility in Ex-Vivo Experimental Heart Failure Models

Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of sarcomeric function. Although reduced phosphorylation of cMyBP-C has been linked to compromised contractility in heart failure patients, direct modulation of cMyBP-C to myosin using small molecules or peptides has not been reported to improve cardiac performance. Here we used previously published cMyBP-C peptides 302A and 302S (surrogates to the regulatory phosphorylation site serine 302) as tool molecules to investigate the role of cMyBP-C in modulating cardiac contraction and relaxation in experimental heart failure (HF) models in vitro. cMyBP-C peptides 302A and 302S were able to increase contractility of papillary muscle fibers isolated from a cMyBP-C phospho-ablation (cMyBP-CAAA) mouse model. In addition, 302A was able to improve the force redevelopment rate (ktr) in papillary muscle fibers from cMyBP-CAAA mice. Consistent with above findings, cMyBP-C peptides 302A and 302S were able to increase the ATPase rates in myofibrils isolated from MI rats but not from sham rats. Furthermore, in cMyBP-CAAA mouse and myocardial infarction (MI) HF models, both cMyBP-C peptides 302A and 302S were able to improve ATPase hydrolysis rates. These changes were not observed in non-transgenic (NTG) mice or sham rats, indicating the specific effects of these peptides in regulating the reduced or unphosphorylated state of cMyBP-C only under pathological conditions of heart failure. Taken together, these studies demonstrate that modulation of cMyBP-C in a reduced phosphorylation or unphosphorylated state can be a therapeutic approach to improve myosin function, sarcomere contractility and relaxation. Therefore, targeting cMyBP-C can be a differentiated approach to improve overall cardiac performance on top of standard care drugs in HF patients.

Alpha and beta myosin isoforms and human atrial and ventricular contraction

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly β-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & β-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function

ABSTRACTMutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy (HCM), a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin S2 tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven stranded beta sheet within the myosin head. Beta isoform specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

Increased VO2 peak after a structured exercise-training program is associated with reduced levels of cardiac myosin binding protein C in patients with symptomatic chronic heart failure

Background Cardiac myosin-binding protein C (cMyC), a cardiac contractile protein, is a novel biomarker of myocardial injury, rising earlier and disappearing faster than cardiac troponins. It is a promising biomarker for use in triage of patients with chest pain presenting in the emergency department. It also has prognostic significance in patients with heart failure. However, the effects of systematic exercise training on plasma levels of cMyC has previously not been evaluated. Purpose The aim of this study was to assess the effect of a 12-week exercise training program on changes in plasma levels of cMyC in patients with chronic symptomatic heart failure with reduced ejection fraction (HFrEF). The changes in plasma levels of cMyC in an intervention group, performing structured exercise programs, were compared to those in a control group, instructed to perform regular recommended exercise (RRE) according to current guidelines. Methods This was a post hoc analysis of the SMARTEX-HF trial in 215 patients with symptomatic HF with Left Ventricular Ejection Fraction (LVEF) <35% and NYHA II-III. The patients were randomly assigned to High Intensity Interval Training (HIIT, n=77), Moderate Continuous Training (MCT, n=65) or RRE, (n=73) for 12 weeks. HIIT and MCT groups constituted the intervention group (IG). Measurements and clinical data were acquired before and after the 12-week intervention. Statistical analysis We divided the patients in two groups with Δ VO2Peak above and below the median of the sample. The absolute changes of cMyC were then compared between the two groups. Mann-Whitney U test was used to compare continuous variables between the groups. Chi-squared test and Fisher exact test were used to compare categorical variables, as appropriate. A two-tailed p<0.05 was considered significant. Results There were no differences in changes of cMyC plasma levels, measured at baseline and after the intervention, between patients in the IG and RRE-group (p=0.580). When dividing the entire study population according to Δ VO2Peak higher or lower than median value 0.48 ml/kg/min, we found a statistically significant greater reduction of cMyC values after 12 weeks of exercise training for those with higher than median Delta VO2Peak values compared to those with lower values (p=0.012). This finding was even stronger for the percentage change in cMyC levels (p=0.004 between groups). Conclusion In patients with symptomatic chronic HFrEF performing a structured 12-week exercise training program, a greater increase in Δ VO2Peak is significantly associated with a reduction in cMyC, suggesting cMyC may provide a dynamic measure of cardiorespiratory state. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Central Norwegian Health authority,Norwegian University of Science and Technology Baseline characteristics Boxplot cMyC vs peak VO2