Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice

Abstract

We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)- associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6 month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6 month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13 months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5-6 month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype.

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

“…The expression of Mylk3 (gene ID 213435) encoding the mouse cMLCK was assessed in three to four hearts per group by quantitative (qPCR) as described in ref. 27, and at the level of protein using a cMLCK-specific antibody (16).…”

“…The paraffin-embedded longitudinal sections of the hearts stained with H&E (hematoxylin and eosin) and Masson's trichrome were examined for overall morphology and fibrosis (27). For transmission EM imaging, the hearts were fixed with 2% (vol/vol) glutaraldehyde, and the left ventricles were sectioned longitudinally for imaging as described earlier (27).…”

“…The ATPase assays on cardiac myofibrils from the right and left ventricles of ∼5-mo-old mice were performed as described previously (12). The papillary muscles of the left ventricles from Tg mice were subjected to force measurements, and the force-pCa curves were determined (27). The measurement of passive force (in pCa 8 solution) in response to muscle stretch was performed as described in Kazmierczak et al (28).…”

Late Gadolinium Enhancement for Prediction of Mutation-Positive Hypertrophic Cardiomyopathy on the Basis of Panel-Wide Sequencing

“…4, 5 Although it has been postulated that myocardial scar may be subsequent to cardiomyocyte death caused by myocardial ischemia and metabolic abnormalities, 1,6 other data have indicated that myocardial scarring could be a primary expression of HCM. 7 The development of genetic testing has enabled the identification of subgroups of HCM based on clinical and genetic information. 8 Recent data revealed that 40% of reviewed all the sequences after a 3-month interval to assess the intraobserver variability.…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Other studies from Szczesna et al's laboratory have also revealed compromised RLC phosphorylation in several different HCM models (Tg-R58Q and Tg-D166V), occurring concurrently with diminished maximal tension and altered Ca 2+ sensitivity of contraction (Abraham et al 2009;Kerrick et al 2009a;Muthu et al 2012b). Studies with HCM causing mutations in the myosin RLC suggest a correlation between the severity of cardiomyopathy phenotype and the level of RLC phosphorylation in vivo (Huang et al 2014;Kerrick et al 2009b;Muthu et al 2012b). The compromised phosphorylation of RLC mutants compared to WT suggests that diminished RLC phosphorylation observed in HCM hearts may have an important physiological role and is central to the understanding of the mutation-elicited detrimental cardiomyopathy phenotype.…”