The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout (MYBPC3 null) mice. It remains unclear whether the structural and functional phenotypes expressed in the MYBPC3 null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the MYBPC3 null background. Functional Tet-Off mice expressing WT cMyBP-C (FT-WT) were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary doxycycline administration, cMyBP-C was expressed at normal levels in FT-WT myocardium, which exhibited similar levels of steady-state force and in vivo left ventricular function as WT mice. Introduction of dietary doxycycline for four weeks resulted in a partial knockdown of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching those observed in MYBPC 3 null mice. Subsequent withdrawal of doxycycline from the diet resulted in the reexpression of cMyBP-C to levels comparable to those observed in WT mice, along with near-complete recovery of in vivo ventricular function. These results show that the cardiac phenotypes associated with MYBPC3 null mice are reversible. Our work also validates the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

Background: At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in both the fast and slow phases are known to scale with the level of activation, culminating in a single high-velocity phase at saturating Ca2+ (i.e., Vmax).

Background: Cardiac myosin binding protein-C (cMyBP-C) plays a critical role in determining the force and kinetics of contraction, primarily by modulating the probability of cross-bridge binding to actin. (46 words)

Previous studies from our laboratory have demonstrated that ablation of cMyBP-C (i.e., null myocardium) or PKA treatment of wild-type (WT) myocardium similarly accelerate cross-bridge cycling kinetics, as well as the rate of force development in living myocardium. However, these interventions differ in their effects on the rate of relaxation in living myocardium, i.e., relaxation is slowed in null myocardium and accelerated in WT myocardium treated with a β1-adrenergic agonist. Studies were done on skinned myocardium to determine the direct effects on myocardial relaxation kinetics due to ablation or PKA phosphorylation of cMyBP-C. Relaxation from steady-state Ca2+-activated force was initiated following flash photolysis of the photolabile Ca2+ chelator diazo-2. Relaxation transients were resolved into two phases: an initial linear phase followed by a double-exponential phase with rate constants k1 (i.e., reflecting the kinetics of ADP release) and k2 (i.e., reflecting the slowing of relaxation kinetics due to cooperative re-binding of cross-bridges during relaxation). Under control conditions, k1 was significantly faster while k2 was significantly slower in cMyBP-C null myocardium compared to values observed in WT myocardium at all levels of submaximal activation. In WT myocardium, PKA treatment accelerated k1 and slowed k2 to values similar to that exhibited in cMyBP-C null myocardium under control conditions. PKA treatment had no effect on k1 and k2 values in cMyBP-C null myocardium. These results indicate that myocardial relaxation is accelerated due to ablation or phosphorylation of cMyBP-C. Although enhanced cooperative cross-bridge binding due to cMyBP-C phosphorylation would be predicted to slow relaxation, the phosphorylation-dependent increase in the rate of ADP release appears to dominate in accelerating the overall rate of relaxation. Furthermore, the previous observation that the rate of relaxation is slowed in living null myocardium is presumably due to prolongation of the Ca2+ transient that is also observed in these preparations.

Background: Mutations in the MYBPC3 gene, which encodes the contractile regulatory protein cardiac myosin binding protein-C (cMyBP-C), account for approximately 40% of known cases of hypertrophic cardiomyopathy (HCM). Therefore, elucidating the pathogenicity of HCM-causing cMyBP-C mutations is critical for understanding how they contribute to the development of HCM. Objective: Our study aimed to determine the functional effects of two distinct HCM-causing mutations in MYBPC3-encoded cMyBP-C, a missense mutation in the C6 domain (W792R) and a C-terminal truncation of the C9/C10 domain (T1075 fs/5). Methods and Results: Cardiac specific transgenic mouse models were developed using a Tet-Off inducible system allowing for the controlled expression of W792R and T1075 cMyBP-C on the MYBPC3 null background. Functional cMyBP-C Tet-off (FT) mice were generated by crossing tetracycline transactivator mice with responder mice carrying the W792R and T1075 transgenes, which were compared to FT-WT controls. Short-axis M-mode echocardiography identified depressed percent ejection fraction and fractional shortening and increased left ventricular chamber size in hearts from FT-W792R and FT-T1075 mice compared to FT-WT controls. SDS-PAGE using cardiac myofibrillar fractions demonstrated a reduction in the expression of total W792R full-length and T1075 truncated cMyBP-C transgenic proteins compared to WT transgenic cMyBP-C. Immunofluorescence analysis of cardiac tissue sections revealed that the WT and W792R transgenic proteins localized in the classic cMyBP-C doublet pattern within cardiac sarcomeres. Intriguingly, the T1075 transgenic protein localized at the Z-lines within cardiac sarcomeres, suggesting a contrast in the localization patterns of cMyBP-C missense-mutated and truncated proteins. Conclusions: These results demonstrate that the W792R and T1075 cMyBP-C mutations generate cardiac contractile dysfunction in mice, although the molecular mechanisms of dysfunction differ between the two mutations, in that W792R is a missense mutation with normal localization of cMyBP-C and T1075 is a truncation mutation with reduced expression and altered sites of binding of cMyBP-C.

Loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy and impaired ventricular function in both cMyBP-C constitutive and conditional knockout mice. At present, it remains unclear whether the structural and functional phenotypes associated with the MYBPC3 null mouse are reversible. To test this idea, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of wild-type (WT) cardiac myosin binding protein-C (cMyBP-C) on the MYBPC3 null background. Functional cMyBP-C Tet-off mice were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary DOX administration, cMyBP-C expression and left ventricular function in cMyBP-C Tet-off mice were not statistically different from WT mice. Introduction of dietary doxycycline (DOX) for four weeks resulted in a partial knock-down of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching that observed in MYBPC3 null mice. Subsequent withdrawal of DOX from the diet resulted in the re-expression of cMyBP-C to levels comparable to that observed in WT mice, along with the near complete recovery of in vivo cardiac function. These results indicate that the cardiac phenotypes associated with the MYBPC3 null mouse are reversible, at least in part, and furthermore, validate the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

Cardiac myosin-binding protein-C (cMyBP-C) plays a key role in determining the force and kinetics of myocardial contraction, primarily by binding to either myosin subfragment-2 or actin or both. By modulating the probability of myosin cross-bridge binding to actin, cMyBP-C normally acts to govern the kinetics of force development and relaxation. To determine the effects on cardiac morphology and function due to a novel cMyBP-C W792R missense mutation associated with clinical hypertrophic cardiomyopathy in humans. We generated cMyBP-C-W792R heterozygous and homozygous knock-in mice to investigate the structural and functional phenotypes associated with a missense mutation within the C6-domain of cMyBP-C. Compared to wild-type, homozygous W792R knock-in hearts exhibited significant ventricular and atrial dilation. While the W792R mutant protein was expressed at equivalent levels as in wild-type mice, the extent of phosphorylation of the mutant cMyBP-C protein was markedly reduced. Measurements of steady-state force and cross-bridge cycling kinetics showed that homozygous cMyBP-C W792R skinned myocardium exhibited a significant increase in the Ca2+-sensitivity of force and accelerated cross-bridge cycling kinetics (ktr) at maximal levels of Ca2+-activation, compared to WT myocardium. Cardiac function, as assessed by transthoracic echocardiography, revealed depressed indices of systolic and diastolic ventricular function in the homozygous W792R KI mice. These results demonstrate that a mutation within the mid-region of cMyBP-C can disrupt the dynamic regulation between myosin and actin that is normally governed by cMyBP-C, thereby leading to aberrant cardiac enlargement and commensurate diminution of in vivo ventricular function.

It is well established that beta-adrenergic stimulation significantly enhances myocardial contractility, as manifested by an increases in twitch force and the rate of relaxation. These effects are believed to be due, in part, to PKA-mediated phosphorylation of TnI and MyBP-C. We tested the hypothesis that cMyBP-C contributes to the enhanced contractile state observed following beta-adrenergic stimulation by examining in vivo left ventricular (LV) pressure-volume relationships in wild-type (WT) and cMyBP-C null (cMyBP-C−/−) mice in the absence and presence of dobutamine, a beta-adrenergic agonist. Under basal conditions, cMyBP-C−/− mice exhibited significant reductions in LV fractional shortening (34.6 ± 0.8% vs. 61.0 ± 1.9%) and peak elastance (3.9 ± 0.4 mmHg/μl vs. 6.5 ± 0.8 mmHg/μl) and a marked abbreviation of the time course of LV systolic elastance (40.3 ± 1.5 ms vs. 58.7 ± 2.6 ms), compared to WT mice. Moreover, while dobutamine treatment significantly improved systolic function in the WT mice, as evidenced by marked increases in both LV fractional shortening (73.3 ± 2.0%) and peak elastance (13.4 ± 1.8 mmHg/μl) and a decreased time course of peak elastance (42.0 ± 3.3 ms), these functional indices remained unchanged in the cMyBP-C−/− hearts following beta-adrenergic stimulation. These results suggest that the absence of cMyBP-C significantly diminishes in vivo ventricular function and markedly attenuates the increase in left ventricular contractility following beta-adrenergic stimulation. (This study was supported by NIH P01 HL047053).