Haploinsufficiency of MYBPC3 exacerbates the development of hypertrophic cardiomyopathy in heterozygous mice

Abstract

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein-C (cMyBP-C), account for ~40% of hypertrophic cardiomyopathy (HCM) cases. Most pathological MYBPC3 mutations encode truncated protein products not found in tissue. Reduced protein levels occur in symptomatic heterozygous human HCM carriers, suggesting haploinsufficiency as an underlying mechanism of disease. However, we do not know if reduced cMyBP-C content results from, or initiates the development of HCM. In previous studies, heterozygous (HET) mice with a MYBPC3 C'-terminal truncation mutation and normal cMyBP-C levels show altered contractile function prior to any overt hypertrophy. Therefore, this study aimed to test whether haploinsufficiency occurs, with decreased cMyBP-C content, following cardiac stress and whether the functional impairment in HET MYBPC3 hearts leads to worsened disease progression. To address these questions, transverse aortic constriction (TAC) was performed on three-month-old wild-type (WT) and HET MYBPC3-truncation mutant mice and then characterized at 4 and 12weeks post-surgery. HET-TAC mice showed increased hypertrophy and reduced ejection fraction compared to WT-TAC mice. At 4weeks post-surgery, HET myofilaments showed significantly reduced cMyBP-C content. Functionally, HET-TAC cardiomyocytes showed impaired force generation, higher Ca(2+) sensitivity, and blunted length-dependent increase in force generation. RNA sequencing revealed several differentially regulated genes between HET and WT groups, including regulators of remodeling and hypertrophic response. Collectively, these results demonstrate that haploinsufficiency occurs in HET MYBPC3 mutant carriers following stress, causing, in turn, reduced cMyBP-C content and exacerbating the development of dysfunction at myofilament and whole-heart levels.

Keywords: Cardiac myosin binding protein-C; Haploinsufficiency; Hypertrophic cardiomyopathy; MYBPC3; Mouse models.

Fig. 1

TAC-induced pressure-overload causes more severe hypertrophy in HET hearts compared to WT. (A) Representative pulse-wave Doppler of blood-flow velocities across the transverse aorta following sham and TAC procedures. Quantification of transverse aortic blood flow velocities shows significant increases in both WT and HET mice following TAC compared to sham controls. (B) Hypertrophy as assessed by the ratio of heart weight to body weight shows significant increases in HET hearts following TAC compared to WT-TAC controls. (C) SYPRO-Ruby-stained SDS-PAGE shows separation of myosin heavy chain isoforms. Elevated β-MHC indicates hypertrophic remodeling in both WT-TAC and HET-TAC hearts. (D & E) Gene expression levels of the hypertrophic markers MYH7 and NPPA relative to GAPDH show significant increase in expression of both genes in WT-TAC and HET-TAC hearts compared to sham controls. * P < 0.05 compared to sham control; # P < 0.05 compared to genotype control.

Fig. 2

Heterozygous mice have systolic deficits compared to WT following TAC. (A) Systolic function derived from parasternal long axis M-mode imaging shows significantly reduced ejection fraction in HET-TAC mice compared to WT-TAC mice at 4 and 12 weeks following surgery. As expected, both TAC groups show significantly impaired systolic function compared to sham controls. (B & C) Diastolic parameters E/A measured by pulse-wave Doppler and E’/A’ showed significant alterations in both WT-TAC and HET-TAC hearts. * P < 0.05 compared to sham control; # P < 0.05 compared to genotype control.

Fig. 3

Levels of cMyBP-C are transiently reduced in HET hearts. (A) Western blot analysis of myofilament fractions showed significantly reduced cMyBP-C at 4 weeks post-surgery in both HET-sham and HET-TAC groups with this alteration not observed at 12 weeks. (B) Phosphorylation of cMyBP-C at Ser-302, as assessed by antibodies that recognize site-specific phosphorylated serines, shows significant increase in cMyBP-C phosphorylation at 4 weeks in both WT-TAC and HET-TAC groups, as well as HET-sham in Ser-273 and Ser-282 sites. By 12 weeks, phosphorylated cMyBP-C levels were significantly lower at Ser-273 in both WT-TAC and HET-TAC groups. (C) The phosphorylation status of cTnI serine 23/24 was assessed by Western blot and revealed significant hyperphosphorylation at 4 weeks in both TAC groups compared to sham controls. At 12 weeks, HET-sham showed significantly elevated cTnI phosphorylation. * P < 0.05 compared to sham control; # P < 0.05 compared to genotype control.

Fig. 4

Heterozygous cardiomyocytes show reduced force development and blunted length-dependent changes in force and calcium sensitivity. (A & B) Force-pCa curves generated at SL 1.9μm and SL 2.3μm. HET-sham cardiomyocytes showed a significant deficit in force development. At 12 weeks, both WT-TAC and HET-TAC groups showed impaired function. Two-way ANOVA at both SL detected significant genotype-specific effects at 12 weeks. (C) Length-dependent increase in force development was attenuated in HET-TAC compared to WT-TAC, as well as a significant genotype-specific effect at 4 weeks. This effect was not observed at 12 weeks. (D) Maximal force was not attenuated in WT(t/t) cardiomyocytes compared to non-transgenic WT controls, whereas HET cardiomyocytes showed dysfunction as reported previously. * P < 0.05 compared to sham control; # P < 0.05 compared to genotype control.

Fig. 5

Calcium sensitivity of force development was significantly increased in both HET-sham and HET-TAC groups at 4 weeks, with HET-TAC showing significant sensitization compared to WT TAC. (A & B) Calcium sensitivity of force development at short SL (A) and long SL (B) was increased in HET-sham compared to WT-sham at 4 weeks, with significant genotype-specific effects observed at both time points, including significant desensitization at 12 weeks at long SL in both TAC groups compared to sham control. This effect was not observed at 12 weeks (C). * P < 0.05 compared to sham control; # P < 0.05 compared to genotype control.

Fig. 6

RNA-Seq shows clusters of genes that are differentially regulated in HET hearts after sham or TAC surgery. Heat maps representing normalized fold-change gene expression per million reads are illustrated between groups for the genes significantly altered in HET-sham (HS) versus WT-sham (WS) (A) and in HET-TAC (HT) versus WT-TAC (WT) (B). The fold change, up or down, is represented in the keys for the respective panels.