Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca2+ sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca2+ sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca2+ transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E′/A′, and higher E/E′ ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca2+ sensitivity, faster Ca2+ transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca2+ sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca2+ transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.
► Absence of left ventricular hypertrophy in heterozygous Mybpc3-targeted knock-in mice. ► Myofilament Ca2+ sensitization in heterozygous Mybpc3-targeted knock-in mice. ► Diastolic dysfunction independent of left ventricular hypertrophy. ► Hypertrophic cardiomyopathy as a model of diastolic heart failure.
cMyBP-C, cardiac myosin-binding protein C; cTnI, cardiac troponin I; CSQ, calsequestrin; HCM, hypertrophic cardiomyopathy; Het, heterozygous Mybpc3-targeted knock-in mice; KI, homozygous Mybpc3-targeted knock-in mice; KO, homozygous Mybpc3-targeted knock-out mice; LVH, left ventricular hypertrophy; max F, maximal Ca2+-activated force; MYBPC3, human cardiac myosin-binding protein C gene; Mybpc3, mouse cardiac myosin-binding protein C gene; NCX, Na+/Ca2+ exchanger; nH, Hill coefficient; pCa50, log of [Ca2+] required for 50% of maximal activation; PKA, cAMP-dependent protein kinase A; PLB, phospholamban; SERCA2, SR-Ca2+ ATPase; SL, sarcomere length; SR, sarcoplasmic reticulum; Ca2+ sensitivity; Ca2+ transient; Diastolic dysfunction; Hypertrophy; Mouse model
Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca2+-activated force was much lower in HCM myocytes (14 ± 1 kN/m2) than in donor myocytes (23 ± 3 kN/m2; P < 0.01), though cross-bridge kinetics (ktr) during maximal Ca2+ activation were 10% faster in HCM myocytes. Myofibrillar Ca2+ sensitivity in HCM myocytes (pCa50 = 6.40 ± 0.05) was higher than for donor myocytes (pCa50 = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca2+ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca2+-activated force and high Ca2+ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.
► The passive stiffness of skinned HCM cardiac myocytes was similar to that of normal (donor) myocytes. ► Maximum Ca-activated force production was reduced by 40% in HCM vs donor myocytes. ► This loss of force could contribute to systolic dysfunction in HCM hearts. ► Myofibrillar Ca sensitivity was higher in HCM than in donor myocytes. ► The enhanced Ca sensitivity could compensate for the smaller maximum force but would tend to cause diastolic dysfunction. ► These characteristics were common to all HCM patients studied, suggesting the changes were secondary consequence of the underlying genetic variants.
Hypertrophic cardiomyopathy; Skinned cardiac myocytes; Viscoelasticity; Ca2+ sensitivity; Cross-bridge kinetics