The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy

Abstract

AimThe aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle.MethodsMyosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate–polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain.ResultsThe fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 ± 0.034 for myectomy myosin vs. 0.305 ± 0.019 µm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 ± 5% compared with 11 ± 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30–45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 ± 0.25 vs. 3.58 ± 0.40%) and reduced relaxation rates compared with donor myocytes (TT50% = 0.32 ± 0.09 vs. 0.17 ± 0.02 s).ConclusionContractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

Reduced force production during low blood flow to the heart correlates with altered troponin I phosphorylation

“…As TnT and TnI are present in equimolar amounts in cardiac muscle, the TnT phosphoprotein signal in each homogenate was compared to the TnI phosphoprotein signal in the same sample to deduce the TnI phosphorylation stoichiometry. Generally, the phosphorylation level of TnI is higher than for TnT, in agreement with published data (Jacques et al 2008). This analysis yielded an average of 2.72 mol phosphate per mol TnI for the five samples examined, and this value was used as a guide to identify the individual TnI isoelectric variants.…”

Functions of myosin light chain-2 (MYL2) in cardiac muscle and disease

“…MLC2v dephosphorylation has also been reported in human patients carrying a rare form of familial hypertrophic cardiomyopathy (FHC) that impacts cardiac papillary muscle and adjacent ventricular muscle, based on specific MLC2v and MLCK mutations (Szczesna et al, 2001; Davis et al, 2001; Jacques et al, 2008). In addition, other MLC-2v mutations associated with this form of FHC are reported likely to be in close proximity to the Ser15 phosphorylation site (Poetter, et al, 1996).…”

Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy

“…Nevertheless, our finding (Figs. 3 and 5) that a similar contractile phenotype is seen in myocytes from six unrelated HCM patients (with different mutations in MYBPC3 or MYH7 , or neither of these), measured under identical conditions, suggests that the contractile deficiency is a common consequence of HCM, secondary to the initial abnormalities produced by the known and unknown genetic variant(s) underlying the cardiomyopathy [17]. By analogy, we suggest that similar secondary changes may account for the reduced maximum force production observed in HCM myocytes with another MYBPC3 mutation [15] or in HCM myofibrils with the MYH7 R403Q mutation [24].…”