Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins

The direct effects of expressing hypertrophic cardiomyopathy-associated (HC M-associated) mutant troponin T (TnT) proteins on the force generation of s ingle adult cardiac myocytes have not been established. Replication-defecti ve recombinant adenovirus vectors were generated for gene transfer of HCM-a ssociated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that th e mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocy te. Interestingly, under identical experimental conditions, the ectopic exp ression of the mutant TnTs was significantly less (similar to 8% of total) than that obtained with expression of wild-type TnT (similar to 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than t hat in normal controls, suggesting that mutant TnT incorporation had no del eterious effects on sarcomeric architecture. Direct measurements of isometr ic force production in single cardiac myocytes demonstrated marked desensit ization of submaximal calcium-activated tension, with unchanged maximum ten sion generation in mutant TnT-expressing myocytes compared with control myo cytes. Collectively, these results demonstrate an impaired expression of th e mutant protein and a disabling of cardiac contraction in the submaximal r ange of myoplasmic calcium concentrations. Our functional results suggest t hat development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate forc e deficits associated with expression of I79N and R92Q in adult cardiac myo cytes.