Functional Analysis of a Homozygous MYBPC3 Null Allele in Human Pediatric Patients

Variants in cardiac myosin binding protein C (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy (HCM). In the Old Order Amish, bi-allelic inheritance of the MYBPC3 splice variant c.3330+2T>G results in severe hypertrophy and dysfunction in infancy that is uniformly fatal without heart transplantation ^1,2. Genetic ablation of cMyBP-C in mice (cMyBP-C^−/−) mirrors this rapid hypertrophy and produces a state of compensated dysfunction ³. This occurs due to loss of the ability to modulate the rate of cross-bridge recruitment and cycling in a beta-adrenergic dependent manner, which normally tunes contractile function to match circulatory demands ³. However, direct evidence from human tissue linking cMyBP-C absence to cardiac dysfunction is lacking. This is the first case report to demonstrate how the complete absence of cMyBP-C mechanistically affects sarcomere function in humans.

Left ventricular (LV) samples were obtained from patients (ages 1 and 5) with bi-allelic c.3330+2T>G variants and heart failure requiring LV assist device (LVAD). Due to the rarity of this mutation, we were only able to obtain two c.3330+2T>G samples. Results should be interpreted with caution and may not be generalizable to the broader HCM population. Measurements were performed in permeabilized myocardium, before and after protein kinase A (PKA) treatment to mimic beta-adrenergic stimulation. To distinguish effects of the c.3330+2T>G variant from typical features of end-stage HF with LVAD, we compared tissue from patients (ages 1 and 4) with end-stage HF, also on LVAD for similar duration [4–7 months]. Non-HF donor control patients (ages 9 and 22) died from anoxia and a motor vehicle crash.

Experiments were performed as described previously ^3,4. No cMyBP-C protein or fragments were present in c.3330+2T>G hearts. There were no differences in expression of other sarcomere proteins between the HF groups. Basal phosphorylation was decreased in both HF groups compared to donor, similar to previous reports (Figure D) ⁴.

Figure:

A. Representative echo (Ai) from a male child diagnosed with systolic heart failure at 5 years old, initially diagnosed with severe dilated cardiomyopathy. Genetic testing identified biallelic MYBPC3 splice variants. Following decompression via LVAD placement, marked hypertrophy of the left ventricle was noted (Aii). He underwent a successful orthotopic heart transplant, representing the oldest documented diagnosis of the biallelic hypertrophic cardiomyopathy B. Representative western blot and total protein stain show the expression of cMyBP-C in myofibrils. C. Myosin heavy chain quantification. Porcine left atrium (LA) + porcine left ventricle (LV) ~50–50% α/β-myosin. The youngest donor tissue sample showed detectable amounts of α-myosin; however, the oldest donor, c.3330+2T>G and HF myocardium did not contain detectable amounts of α-myosin, thus it is likely that myosin isoform switching is irrelevant to this study. D. Representative Pro-Q Diamond and Sypro-Ruby-stained SDS gels show the phosphorylation and expression of myofilament proteins, represented as average/heart. E. Due to procurement limitations of c.3330+2T>G patient tissue, two hearts were used from each group, with 5 myocardial fibers from each group used in the measurements. N=2 hearts, K=5 myocardial fibers per group. Myocardial fibers were incubated in 0.5U PKA (P2645, Sigma) for 1hr as described previously ³. Mechanical experiments were analyzed with a two-way linear mixed-effects model using SAS software (version 9.1.3; SAS Institute, Cary, NC). Statistical analysis was performed to provide a quantitative comparison between groups, however due to the sample size, p-values should be interpreted within the limited scope of the data set. Results are reported as mean ± SD.

HF groups had lower Fmax and Fmin compared to donor. Both c.3330+2T>G and HF hearts had left-shifted pCa50 compared to donor, indicating increased calcium sensitivity in the HF groups (Figure E).

In cMyBP-C^−/− mouse models, the predominant feature is an acceleration in the apparent rate constant of force decay (k_rel) in Phase-2 following stretch, which is representative of the rate of cross-bridge detachment ³. This aspect is mirrored in the c.3330+2T>G patient tissue, where kinetic experiments performed at submaximal [Ca²⁺] revealed that absence of cMyBP-C results in an acceleration of k_rel and a decrease in time for complete cross-bridge detachment (T_d) compared with the HF group. Compared to donor, c.3330+2T>G myocardium had a similar k_rel and T_d was decreased. k_rel is typically slowed in HF due to dephosphorylation, so the similarity in k_rel between c.3330+2T>G and donor represents a faster rate than expected for the pathophysiologic condition ⁴.

Following the detachment phase, Phase-3 shows a gradual rise in force with a rate constant k_df, which represents a rate of cross-bridge recruitment. Interpretation of k_df in this study is complicated due the advanced end-stage HF decompensation and dephosphorylation of the patient myocardium, an effect not paralleled in the cMyBP-C^−/− mouse models ³. The c.3330+2T>G myocardium exhibited a similar k_df to the HF group, which is considerably slower than donor. Previously, we reported that k_df in end-stage HF tissue is dramatically slowed by dephosphorylation of sarcomeric proteins ⁴. Thus, in this study, advanced decompensation and dephosphorylation (Panel D) may mask the effects of the lack of cMyBP-C on cross-bridge recruitment.

Modulation of cross-bridge kinetics by cMyBP-C occurs via its phosphorylation, and is absent in cMyBP-C^−/− mouse myocardium ³. PKA treatment resulted in right-shifted pCa50 in all groups, consistent with increased cTnI phosphorylation (Panel E). PKA accelerated kdf in Donor myocardium, and accelerated krel and Td in both Donor and HF, but not in c.3330+2T>G. This suggests that this variant uncouples acceleration of cross-bridge kinetics from beta-adrenergic stimulation.

This is the first case report detailing the molecular effects of a rare homozygous MYBPC3 variant in humans. The dominant effect of cMyBP-C deletion in both human and mouse is the acceleration of the k_rel, consistent with the hypothesis that absence of cMyBP-C reduces drag within the sarcomere, accelerating cross-bridge detachment and promoting rapid shortening during systole. Although mouse models mimic the acceleration in k_rel, they do not completely recapitulate the effects observed in human end-stage heart failure. In the c.3330+2T>G tissue, the k_df is dramatically slower, an effect absent in mice ³. Human-specific dephosphorylation, likely a result of chronic beta-adrenergic desensitization, blunts reattachment kinetics and reflects a pathological adaptation not seen in mouse hearts. Faster rates of shortening coupled with slowed k_df due to dephosphorylation, and a lack of response to beta-adrenergic signaling, disrupt the highly tuned stretch activation response in myocardium, leading to dramatic impairment of cardiac function. This suggests that homozygous c.3330+2T>G carriers may be ideal candidates for MYBPC3 gene replacement as re-expressing MYBPC3 could help normalize myofilament function and restore beta-adrenergic response, potentially reversing disease ⁵.

Data Availability Statement:

All original data and materials used for this study are available from the corresponding author upon reasonable request.