Altered contractile mechanics and Ca2+ handling contribute to cardiomyopathy pathogenesis
Keywords: Cardiomyopathy

Mutations in genes encoding sarcomeric proteins are the most common cause of inherited cardiomyopathies. However, cardiac disease caused by mutations in non-sarcomeric proteins exhibit remarkably similar phenotypes, suggesting that common modes of pathogenesis might exist. It is of particular interest whether non-sarcomeric mutations result in impaired contractile function akin to sarcomeric diseases. Accordingly, this thesis describes the effect of cardiomyopathy-causing mutations to an energy sensing protein (AMPK γ2) and a small heat shock protein (αB-crystallin) on cardiac myofilament biomechanics and Ca2+ handling.
Mutations in the regulatory γ2 subunit of AMP-activated kinase (AMPK), encoded by the PRKAG2 gene, cause a cardiomyopathy characterized by ventricular hypertrophy, electrophysiological abnormalities, and glycogen accumulation. Data from animal models in which the mutant transgene is overexpressed suggest that the electrophysiological aspects might be caused by excess glycogen, but that this is insufficient to account for all facets of the phenotype. A novel knock-in mouse expressing R299Q AMPK γ2 (homologous to the human R302Q mutation) was generated to model PRKAG2 cardiomyopathy more accurately. Assessment of the cardiac phenotype at two months to determine the early events in disease pathogenesis revealed systolic and diastolic dysfunction in mutant animals, with no excess glycogen detected. Analysis of acetyl-CoA carboxylase phosphorylation at S79 suggested increased basal AMPK activity in mutant animals. Increased sarcomeric Ca2&plus; sensitivity of contractile activation was observed in demembranated cardiac trabeculae from mutant animals, associated with decreased phosphorylation of cardiac troponin I at S23/S24 and increased phosphorylation at S150. Treatment with protein kinase A normalized this difference in Ca2&plus; sensitivity, suggesting that reduced PKA phosphorylation is the primary abnormality in mutant tissue. Cardiomyocytes from mutant animals exhibited slowed contractile and Ca2&plus; reuptake rates, associated with reduced phosphorylation of phospholamban and myosin binding protein C, as well as an increased response to isoproterenol. The lack of glycogen accumulation in these animals, combined with abnormal contractile and Ca2+ handling properties, reveals a more nuanced interpretation of the mechanism of PRKAG2 cardiomyopathy development. Further, the newly identified interaction between energy- (AMPK) and stress- (PKA) signalling networks may be of importance in numerous additional disease pathways.
This thesis clarifies the role of αB-crystallin, and its cardiomyopathy-causing mutant (R157H), in regulating cardiac muscle stiffness. Specifically, mass spectrometry data revealed that αB-crystallin forms large oligomers and binds to titin Ig domains. Nuclear magnetic resonance confirmed this binding and suggested that αB-crystallin's C-terminal is responsible for titin binding. A viscoelastic model was developed to match stress relaxation in cardiac muscle fibres. Measurements using this model indicated that αB-crystallin significantly increases myocardial stiffness and that the R157H mutation weakens this effect. Further, a 9-AA peptide from αB-crystallin's C-terminus was shown to bind titin and increase overall muscle stiffness. These results reveal a novel method of cardiac muscle stiffness regulation that might contribute to the pathogenesis of cardiomyopathy.