172 Two mutations, one patient: which phenotype?

Methods and results Woman, 55 years old, she has as comorbidity high blood pressure and mild obesity. She came at our attention to perform screening cardiological exams after her brother, who was affected by amyloidotic TTR-related cardiomyopathy with Val30Met mutation, died for sudden cardiac death. At the first evaluation the patient is completely asymptomatic, she has not angor, dyspnoea and heartbeat. The ECG and echocardiography were negative for amyloidotic signs of heart involvement. The Tc-99-DPD scintigraphy showed no cardiac uptake (visual score = 0). To complete the diagnostic path the patient had been evaluated by a neurologist with electromyography, which was negative, and genetic test, which confirmed the presence of Val30Met mutation of TTR-gene. For this last outcome we decided to follow the patient at our clinics. In the following years the patient developed a progressive reduction of exercise tolerance and symmetric negative T waves in anterolateral and inferior lead at ECG. The echocardiogram showed a progressive medio-apical septal hypertrophy. To exclude an ischaemic cause the patient made a stress myocardial scintigraphy, which was negative for ischaemic signs, and she underwent to cardiac MRI which showed a septal thickness of 16 mm without amyloidotic radiological signs in T1-weighted and LGE sequences. For this reason, we suspected that the patient had a hypertrophic cardiomyopathy and she had been undergone another time to genetic test which confirmed the Val30Met TTR-mutation and MYBPC3 mutation. Usually, this last gene mutation for myosin binding protein C is associated with late-onset hypertrophic cardiomyopathy. Into account the new diagnosis and her sudden cardiac death family history we calculated the patient’s HCM-risk score which was under 4%, so that we did not undergo the patient to ICD implantation. Conclusions The case report is a rare example of coexistence of the transthyretin gene mutation and myosin binding protein C in the same patient. In this case to perform a correct diagnosis, it is crucial use an integrated multimodal approach including ECG, echocardiography and cardiac MRI.

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.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin–Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy–causing Double Mutation MYBPC3Δ25bp/D389V

Cardiac myosin-binding protein C (MyBPC) is a thick-filament associated regulatory protein in the sarcomere. It regulates the sensitive contractile system of the myocardium by acting as a mechanical tether, sensitizing the thin filament or modulating myosin motor activity. Mutations in the MYBPC3 gene are a frequent cause for the development of hypertrophic cardiomyopathy, the most frequent cardiac disorder. Recently, the monoallelic double mutation MYBPC3Δ25bp/D389V has been discovered as a subset of the common MYBPC3Δ25bp variant in South Asia. MYBPC3Δ25bp/D389V carriers exhibit hyperdynamic features, which are considered an early finding for the development of hypertrophic cardiomyopathy. Using correlation-guided molecular dynamics simulations sampling, we show that the D389V mutation shifts the conformational distribution of the C2 domain of MyBPC. We further applied biochemical approaches to probe the effects of the D389V mutation on structure, thermostability and protein-protein interactions of MyBPC C2. The melting temperature (Tm) of MyBPC C2 D389V is decreased by 4 to 7 °C compared to wild type while the interaction of the C0-C2 domains with myosin and actin remains unchanged. Additionally, we utilized steered molecular dynamics (SMD) simulations to investigate the altered unfolding pathway of MyBPC C2 D389V. Based on our data, we propose a pathomechanism for the development of HCM in MYBPC3Δ25bp and MYBPC3Δ25bp/D389V carriers.

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

Physiological circadian rhythm of cardiac myosin-binding protein C (cMyC) and cardiac troponin

Introduction Cardiac myosin-binding protein C (cMyC) is a novel protein biomarker of myocardial injury, with a promising role in the triage and risk stratification of patients with cardiac disease. Understanding the physiological diurnal oscillation of cMyC and cardiac troponin is important for the interpretation of single and serial measurements within the biomarker-assisted triage and risk stratification algorithms. Purpose In this study, we aim to assess and compare the physiological diurnal oscillation of cMyC and cardiac troponin cTnT and cTnI. Method Twenty-six consecutive hourly blood samples were drawn between 08.30 am and 09.30 am (+1 day) from normotensive 24 individuals without a recent history of acute myocardial infarction, for the measurement of cMyC, cardiac troponin T (Roche hs-cTnT) and I (Abbott hs-cTnI). Fitted cosinor sine regression model (with R, version 3.6.1) was used to assess the presence and significance of circadian oscillation of the biomarker, and to estimate the respective amplitude and acrophase (the time of peak activity). Amplitude and acrophase were compared across the biomarkers that exhibited significant circadian rhythm. Results Mean age was 72±7. 79% of participants (n=19) were men. All participants were free from renal disease. On population-mean cosinor analysis, hs-cTnI exhibited random diurnal oscillation, whereas significant circadian rhythm was detected for cMyC and hs-cTnT (p=0.015 and <0.001, respectively) (Figure 1). The circadian rhythm of cMyC is characterised by gradually increasing concentrations from early afternoon until early morning (acrophase 03:03 am, 95% CI 01:54–04:26 am) compared to hs-cTnT concentrations which exhibits delayed increase and a later peak (acrophase, 08:01, 95% CI 07:10–08:51 am), p=0.028 for acrophase difference (Figure 1). Diurnal rhythm remained significant after correction for possible posture-induced changes in plasma volume. To allow direct comparison between amplitudes, the measurements of cMyC and hs-TnT were normalised to the respective 08:30 am value, re-fitted cosinor model did not show significant difference between the amplitudes (amplitude ng/L, 0.12, 95% CI 0.07–0.15 vs 0.11, 95% CI 0.08–0.12, for normalised cMyC vs hs-cTnT, respectively; p=0.67). Conclusion Significant circadian rhythm exists for cMyC and hs-cTnT, with 5-hours phase difference between the two biomarkers (cMyC ahead of hs-cTnT). The cause of this rhythmic variation is unknown, but the phase difference is consistent with the previously described disparity in the release of cMyC and cTnT after iatrogenic myocardial injury, raising the possibility of an underlying diurnal variation in myocardial vulnerability. Studies are required to assess the impact of this physiological phenomenon on the performance of the biomarkers within unadjused diagnostic algorithms FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart FoundationStichting de Weijerhorst

Abstract P338: Myopeptide Improves Contractile Mechanics In A Mouse Model Of Heart Failure Expressing Phosphoablated Cardiac Myosin Binding Protein-C

Rationale: Normal heart function depends on cardiac myosin binding protein-C (cMyBP-C) phosphorylation. Its decrease is associated with heart failure (HF) by inhibiting actomyosin interactions. In absence of cMyBP-C phosphorylation, the protein is bound to myosin S2, but released when phosphorylated, allowing myosin to form cross-bridges with actin. Challenging cMyBP-C/myosin S2 interaction by myopeptide (the first 126 amino acids of myosin S2) could promote actomyosin interaction in vitro , but its ability to improve contractility in HF remains untested. Objective: To test contractile function in skinned papillary fibers of a cMyBP-C dephosphorylated mouse model using myopeptide. Methods and Results: To mimic constitutive phosphoablation, a knock-in mouse model was established to express cMyBP-C in which serines 273, 282 and 302 were mutated to alanine (cMyBP-C AAA ). Western blotting revealed 50% and 100% of cMyBP-C AAA in het and homo mouse hearts, respectively. Echocardiography showed a decreased percentage of ejection fraction (28%, p<0.01) and fractional shortening (30%, p< 0.05) in both het and homo cMyBP-C AAA mice at 3 months of age, compared to knock-in negative controls. These mice also developed diastolic dysfunction with elevated ratio of E/A and E/e’ waves. Next, pCa-force measurements using skinned papillary fibers determined that maximal force (F max ) and rate of cross-bridge formation ( k tr ) were decreased in the cMyBP-C AAA groups, compared to the control. However, administration of dose-dependent myopeptide increased F max and k tr in wild-type and cMyBP-C AAA permeabilized skinned papillary fibers without affecting myofilament Ca 2+ sensitivity. Conclusions: Myopeptide can increase contractile force and rate of cross-bridge formation by releasing cMyBP-C/myosin S2 and promoting actomyosin formation of cross-bridges, thus validating its therapeutic potential.

Abstract 123: Study Of The Role Of Myosin-binding Protein C In The Structure-function Of Intact Cardiac Muscles Through The Use Of A Sarcomere Activation Biosensor

Cardiomyopathies affect as many as 1 in 500 adults, with hypertrophic cardiomyopathy (HCM) being the most commonly inherited heart disease. Although there are various genetic mutations which cause HCM, 40-50% of mutations identified in patients with this disease are found in the cardiac myosin-binding protein C (MyBP-C) gene. Thus, understanding this protein's role in sarcomere activation is critical for the development of effective therapeutic strategies. Previous work has identified MyBP-C as a key modulator of the sarcomere through inter-myofilament signaling. Although these studies have provided valuable information on the function of MyBP-C protein, the bulk of this work has been done with either in vitro protein recombination or permeabilized muscle. Unfortunately, these systems lack significant physiological features of muscle, such as load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in HCM disease models, specifically those targeting MyBP-C, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. This new sarcomere activation biosensor reports real-time myofilament dynamics during physiological twitch contractions in live cardiac muscle. This allows for the in vivo biophysical tracking of FRET-detected conformation changes in TnC, which reflect the ensemble of regulatory events which lead to sarcomere activation. Currently, the biosensor is being utilized to investigate changes in sarcomere activation in MyBP-C knockout mice. We find that intact cardiac muscle under load shows slowed isometric twitch relaxation kinetics in MyBP-C knockout mice versus controls. In concert with thin and thick filament modifying small molecules, this biosensor system will be used to investigate sarcomere-based mechanisms of intact cardiac muscle performance in health and disease.