The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice

Familial hypertrophic cardiomyopathy (FHC) is a disease caused by mutations in contractile proteins of the sarcomere. Our laboratory developed a mouse model of FHC with a mutation in the thin filament protein α-tropomyosin (TM) at amino acid 180 (Glu180Gly). The hearts of these mice exhibit dramatic systolic and diastolic dysfunction, and their myofilaments demonstrate increased calcium sensitivity. The mice also develop severe cardiac hypertrophy, with death ensuing by 6 mo. In an attempt to normalize calcium sensitivity in the cardiomyofilaments of the hypertrophic mice, we generated a chimeric α-/β-TM protein that decreases calcium sensitivity in transgenic mouse cardiac myofilaments. By mating mice from these two models together, we tested the hypothesis that an attenuation of myofilament calcium sensitivity would modulate the severe physiological and pathological consequences of the FHC mutation. These double-transgenic mice “rescue” the hypertrophic phenotype by exhibiting a normal morphology with no pathological abnormalities. Physiological analyses of these rescued mice show improved cardiac function and normal myofilament calcium sensitivity. These results demonstrate that alterations in calcium response by modification of contractile proteins can prevent the pathological and physiological effects of this disease.

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Myosin regulatory light chain mutation found in hypertrophic cardiomyopathy patients increases isometric force production in transgenic mice

Biochemical Journal ◽

10.1042/bj20111145 ◽

2012 ◽

Vol 442 (1) ◽

pp. 95-103 ◽

Cited By ~ 21

Author(s):

Katarzyna Kazmierczak ◽

Priya Muthu ◽

Wenrui Huang ◽

Michelle Jones ◽

Yingcai Wang ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Transgenic Mice ◽

Light Chain ◽

Isometric Force ◽

Force Production ◽

Regulatory Light Chain ◽

Familial Hypertrophic Cardiomyopathy ◽

Functional Changes ◽

Hypertrophic Response ◽

Isometric Force Production

FHC (familial hypertrophic cardiomyopathy) is a heritable form of cardiac hypertrophy caused by mutations in genes encoding sarcomeric proteins. The present study focuses on the A13T mutation in the human ventricular myosin RLC (regulatory light chain) that is associated with a rare FHC variant defined by mid-ventricular obstruction and septal hypertrophy. We generated heart-specific Tg (transgenic) mice with ~10% of human A13T-RLC mutant replacing the endogenous mouse cardiac RLC. Histopathological examinations of longitudinal heart sections from Tg-A13T mice showed enlarged interventricular septa and profound fibrotic lesions compared with Tg-WT (wild-type), expressing the human ventricular RLC, or non-Tg mice. Functional studies revealed an abnormal A13T mutation-induced increase in isometric force production, no change in the force–pCa relationship and a decreased Vmax of the acto-myosin ATPase. In addition, a fluorescence-based assay showed a 3-fold lower binding affinity of the recombinant A13T mutant for the RLC-depleted porcine myosin compared with WT-RLC. These results suggest that the A13T mutation triggers a hypertrophic response through changes in cardiac sarcomere organization and myosin cross-bridge function leading to abnormal remodelling of the heart. The significant functional changes observed, despite a low level of A13T mutant incorporation into myofilaments, suggest a ‘poison-peptide’ mechanism of disease.

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Abstract 1238: Myofilament Calcium Sensitization Creates Arrhythmia Susceptibility in Familial Hypertrophic Cardiomyopathy

Circulation ◽

10.1161/circ.116.suppl_16.ii_252 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Franz Baudenbacher ◽

Veniamin Y Sidorov ◽

Tilmann Schober ◽

Nagesh Chopra ◽

Raghav Venkataraman ◽

...

Keyword(s):

Ventricular Tachycardia ◽

Hypertrophic Cardiomyopathy ◽

Action Potential ◽

Ventricular Arrhythmias ◽

Calcium Sensitivity ◽

Familial Hypertrophic Cardiomyopathy ◽

Arrhythmogenic Substrate ◽

Sarcomeric Mutations ◽

Underlying Mechanisms ◽

Myofilament Calcium Sensitivity

Although sudden cardiac death is a common feature in hypertrophic cardiomyopathy (HCM), the underlying mechanisms are unclear. We recently reported that some but not all mice expressing troponin mutations associated with HCM display increased myofilament calcium sensitivity. Here, we used these mice as well as acute challenge with the Ca 2+ sensitizing agent EMD57033 (EMD) in isolated mouse and rabbit hearts to test the hypothesis that myofilament sensitization leads directly to arrhythmia susceptibility. HCM mice with Ca 2+ sensitizing troponin mutations displayed significantly higher rate of ventricular ectopy (TnT-I79N 10±3, TnT-F110I 11±5, ssTnI 15±4 PVC/h) than mice expressing non-sensitizing troponins (TnT-R278C 1±1, TnT-WT 1±1, non-Tg 2±1 PVC/h, n=5–14 per group, p<0.01). Myofilament sensitization by troponin mutations or EMD shortened the effective refractory period and the ventricular action potential of isolated perfused mouse and rabbit hearts, and caused early afterdepolarizations and triggered beats after fast pacing trains. The action potential shortening was attributable to increased cytosolic Ca 2+ buffering by the Ca 2+ sensitized myofilaments, which resulted in decreased systolic and increased end-diastolic Ca 2+ during fast pacing. Optical mapping demonstrated that Ca 2+ sensitization slowed the ventricular conduction velocity and increased the size of the vulnerable window for ventricular tachycardia both in mouse and rabbit hearts, resulting in a significantly higher incidence of sustained ventricular tachycardia in Ca 2+ sensitized compared with control hearts (5/6 vs 0/6, p<0.05). Conclusion: Myofilament Ca 2+ sensitization alters Ca 2+ buffering to render hearts susceptible to ventricular arrhythmias by creating both an arrhythmogenic substrate and trigger. These results identify a novel mechanism linking sarcomeric mutations to susceptibility to ventricular arrhythmias and raise the prospect of a molecular approach to stratifying arrhythmia risk in HCM.

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