Bradley M. Palmer

Male but not female mice carrying a single R403Q missense allele for cardiac α-myosin heavy chain (M-αMHC^R403Q/+and F-αMHC^R403Q/+, respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-αMHC^+/+and F-αMHC^+/+, respectively) after ∼30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-αMHC^R403Q/+and F-αMHC^R403Q/+at younger ages (10–20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to Ca²⁺activation (pCa₅₀) was significantly ( P < 0.05) enhanced in male mice independent of genotype (M-αMHC^R403Q/+: 5.70 ± 0.06, M-αMHC^+/+: 5.63 ± 0.05, F-αMHC^R403Q/+: 5.57 ± 0.03, F-αMHC^+/+: 5.54 ± 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in α-MHC^R403Q/+independent of sex (M-αMHC^R403Q/+: 29.3 ± 2.3, M-αMHC^+/+: 26.0 ± 1.4, F-αMHC^R403Q/+: 30.2 ± 2.1, F-αMHC^+/+: 26.2 ± 1.2 mN/mm²). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in αMHC^R403Q/+mice than in sex-matched controls (M-αMHC^R403Q/+: 2.26 ± 0.47, M-αMHC^+/+: 1.29 ± 0.18, F-αMHC^R403Q/+: 3.21 ± 0.33, F-αMHC^+/+: 2.52 ± 0.36 Hz). Unloaded shortening velocity was significantly enhanced in αMHC^R403Q/+and in female mice (M-αMHC^R403Q/+: 2.26 ± 0.47, M-αMHC^+/+: 1.29 ± 0.18, F-αMHC^R403Q/+: 3.21 ± 0.33, F-αMHC^+/+: 2.52 ± 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in αMHC^R403Q/+independent of sex (M-αMHC^R403Q/+: 60 ± 2 10⁻³, M-αMHC^+/+: 37 ± 3 10⁻³, F-αMHC^R403Q/+: 57 ± 3 10⁻³, F-αMHC^+/+25 ± 3 10⁻³muscle lengths/s × normalized tension). We did not find a statistically significant sex × mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for pCa₅₀and unloaded shortening velocity.

We examined cardiomyocyte intracellular calcium ([Ca²⁺]_i) dynamics and sarcomere shortening dynamics in genetic rat models of left ventricular (LV) hypertrophy associated with or without hypertension (HT) and with or without hyperactive (HA) behavior. Previous selective breeding of the spontaneously hypertensive rat (SHR) strain, which is HA and HT, with the Wistar-Kyoto (WKY) rat strain, which is not hyperactive (NA) and not hypertensive (NT), has led to two unique strains: the WKHA strain, selected for HA and NT, and the WKHT strain, selected for NA and HT. Cardiomyocytes were isolated from young adult males and females of each strain, paced at 2, 3, and 4 Hz in 1.2 mM external Ca²⁺ concentration at 37°C, and cardiomyocyte [Ca²⁺]_i and sarcomere dynamics were recorded simultaneously. Under these conditions, LV cardiomyocyte systolic and diastolic [Ca²⁺]_i dynamics and diastolic sarcomere dynamics in the WKHT were significantly enhanced compared with WKY controls, suggesting an underlying LV hypertrophic response that successfully compensated for HT in the absence of HA. LV cardiomyocyte [Ca²⁺]_i dynamics in the WKHA and SHR were strikingly similar to each other and only slightly reduced compared with WKY. LV cardiomyocyte systolic and diastolic sarcomere dynamics, on the other hand, were significantly reduced in the SHR compare with WKHA and more so in male than in female SHR. We conclude from these data that HT alone is an insufficient descriptor of the cause of LV hypertrophy and diminished LV cardiomyocyte function in the SHR rat. These data further suggest that HA (augmented by male sex) in the SHR may interact with the HT state to initiate impaired cardiomyocyte function and thereby inhibit or undermine an otherwise compensatory response that may occur with HT in the absence of HA.

Modifications in thick filament protein content and performance are thought to underlie contraction-relaxation dysfunction in human heart failure. It has been found that myofibrillar Mg.ATPase is reduced in failing myocardium, which may be due in part to the reduction in α-myosin heavy chain (MHC) isoform content from ∼5–10% in normal myocardium to <2% in failing myocardium. The physiological importance of this seemingly small amount of α-MHC appears substantiated by the development of cardiopathologies in humans with mutated α-MHC at normal abundance. Therefore, the replacement of α-MHC by β-MHC (possessing slower actomyosin enzymatic kinetics) may underlie to a significant degree the reduced myocardial shortening velocity and reduced relaxation function in human heart failure. The atrial isoform of myosin essential light chain (ELC) may replace up to 25% of the ventricular isoform in failing ventricles and in so doing promotes myocardial shortening velocity. An elevated accumulation of the higher performing atrial-ELC, unlike the reduced content of the higher performing α-MHC, is therefore considered a compensatory response in heart failure. Phosphorylation of the myofilament proteins myosin regulatory light chain and troponin-I are both reduced in heart failure and collectively result in an elevated myofilament sensitivity to calcium activation, which inhibits relaxation function. These and other modifications in thick filament proteins, as discussed in this review, directly affect mechanical power output and relaxation function of the myocardium and thereby may be considered to cause or in some cases to compensate for the otherwise ineffective myocardial performance in heart failure.

The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from heterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament ( V_RTF) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal V_RTF was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production ( b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (pCa₅₀ 5.64 ± 0.02) and significantly enhanced in R453C/+ (5.82 ± 0.03) compared with WT (5.58 ± 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level.

We examined the effect of cardiac myosin binding protein-C (cMyBP-C) on contractile efficiency in isovolumically contracting left ventricle (LV) and on internal viscosity and oscillatory work production in skinned myocardial strips. A 6-week diet of 0.15% 6- n -propyl-2-thiouracil (PTU) was fed to wild-type (+/+ _PTU ) and homozygous-truncated cMyBP-C (t/t _PTU ) mice starting at age ≈8 weeks and leading to a myosin heavy chain (MHC) isoform profile of 10% α-MHC and 90% β-MHC in both groups. Western blot analysis confirmed that cMyBP-C was present in the +/+ _PTU and effectively absent in the t/t _PTU . Total LV mechanical energy per beat was quantified as pressure-volume area (PVA). O ₂ consumption (Vo ₂ ) per beat was plotted against PVA at varying LV volumes. The reciprocal of the slope of the linear Vo ₂ -PVA relation represents the contractile efficiency of converting O ₂ to mechanical energy. Contractile efficiency was significantly enhanced in t/t _PTU (26.1±2.6%) compared with +/+ _PTU (17.1±1.6%). In skinned myocardial strips, maximum isometric tension was similar in t/t _PTU (18.7±2.1 mN · mm ⁻² ) and +/+ _PTU (21.9±4.0 mN · mm ⁻² ), but maximum oscillatory work induced by sinusoidal length perturbations occurred at higher frequencies in t/t _PTU (7.31±1.17 Hz) compared with +/+ _PTU (4.48±0.60 Hz) and was significantly more sensitive to phosphate concentration in the t/t _PTU . Under rigor conditions, the internal viscous load was significantly lower in the t/t _PTU compared with +/+ _PTU , ie, ≈40% lower at 1 Hz. These results collectively suggest that contractile efficiency is enhanced in the t/t _PTU , probably through a reduced loss of mechanical energy by a viscous load normally provided by cMyBP-C and through a gain of phosphate-dependent oscillatory work normally inhibited by cMyBP-C.

Despite advances in the molecular biology of cardiac myosin binding protein-C (cMyBP-C), little is understood about its precise role in muscle contraction, particularly in the intact heart. We tested the hypothesis that cMyBP-C is central to the time course and magnitude of left ventricular systolic elastance (chamber stiffening), and assessed mechanisms for this influence in intact hearts, trabeculae, and skinned fibers from wild-type (+/+) and homozygous truncated cMyBP-C (t/t) male mice. cMyBP-C protein was not detected by gel electrophoresis or Western blot in t/t myocardium. cMyBP-C t/t ventricles displayed reduced peak elastance, but more strikingly a marked abbreviation of the systolic elastance time course, which peaked earlier (27.6±2.1 ms) than in +/+ controls (47.8±1.6 ms). Control hearts reached only 42±4% of maximum elastance at the onset of ejection, with substantial further stiffening during ejection. This contrasted to t/t mutants, which reached 77±3% of peak elastance before ejection of peak. These unusual findings were not observed in alternative models involving severe cardiomyopathy, but were recapitulated in a cMyBP-C null mouse. The abbreviated elastance time course and lower peak were consistent with earlier time-to-peak trabecular tension, increased unloaded shortening velocity in t/t skinned muscle strips, and dramatically reduced myofilament stiffness at diastolic calcium concentrations. These results provide novel insights into the role of cMyBP-C in myocardial systolic mechanics. Abnormal sarcomere shortening velocity and abbreviated muscle stiffening may underlie development of cardiac dysfunction associated with deficient incorporation of cMyBP-C.