Biophysical and Physiologic Characterization of Thin Filament Cardiomyopathies

Abstract

Regulatory thin filament proteins of the cardiac sarcomere, alpha-tropomyosin (Tm) and cardiac troponin T (cTnT), are essential to myofilament activation and cardiac function. Mutations in these proteins cause hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). This work explores the genotype-phenotype link in a novel DCM mutation in Tm and determines the role of CaMKII in a well-studied HCM mutation in cTnT. The DCM-linked Tm mutation D230N causes a bimodal clinical phenotype with severe forms of disease in young patients and mild to moderate disease in adults. We hypothesized that this age-dependent phenotype was due to differential interactions of D230N-Tm with fetal and adult cTnT isoforms. Our in vitro studies showed that D230N decreases the calcium sensitivity in the presence of adult cTnT, and even more so in the presence of fetal cTnT, suggesting that the D230N mutation may cause DCM in a cTnT-isoform dependent manner. To understand the mechanistic links between primary mutational effects and ventricular remodeling we created a transgenic mouse model and found that left ventricular dilatation occurs early and results in functional impairments and altered calcium handling. Our laboratory previously established a mouse line to study a well-documented HCM mutation cTnT-R92W that also demonstrates downstream effects in calcium handling. While the R92W-cTnT mutation myocellular dysfunction at two months, there was improvement by six months that correlated with a presumed compensatory mechanism involving increased CaMKII phosphorylation of phospholamban. We hypothesized that while initially compensatory, long-term over-activation of CaMKII contributes to R92W FHC pathophysiology. We tested this hypothesis using genetic CaMKII inhibition in R92W mice. We found that CaMKII inhibition decreased SERCA2a calcium uptake activity due to decreased phosphorylation of phospholamban. Additionally, CaMKII inhibition significantly altered atrial remodeling in R92W mice associated with long-standing diastolic dysfunction. These studies suggest that CaMKII plays a role in R92W pathophysiology. Moreover, these findings indicate that CaMKII inhibition is protective in R92W HCM pathogenesis and may be a valuable therapeutic target in this disease. These two studies elucidate important aspects of thin filament cardiomyopathy pathogenesis, providing a new mechanism and disease model for DCM as well as a potential therapeutic target for HCM.