Adenovirus-mediated myofilament gene transfer into cardiac myocytes: Effects on myocyte structure and function.

The specific aims of this dissertation were twofold: (1) to establish an experimental model system in which cardiac contractile protein expression could be rapidly and efficiently altered to study structural and functional relationships, and (2) to assess the structural and functional consequences of ectopic expression of two mutant troponin T proteins which are linked to the disease hypertrophic cardiomyopathy using this experimental model system. In order to accomplish the first goal, two cardiac myocyte culture systems were studied. The first was cardiac myocytes derived from mouse embryonic stem cells differentiating in vitro. Infection of these cultures with recombinant adenovirus vectors led to expression of the transferred gene in ~27% of the cardiac myocytes for up to 21 days without altering cardiac myocyte differentiation or contractile function. The second system studied was adult cardiac myocytes in primary culture. Infection of these cultures with adenovirus vectors led to stable expression of the transferred gene in >90% of the myocytes for the 7 day culture period, without affecting myocyte structure or the ability of the myocytes to respond to calcium activation. Due to the greater efficiency of gene transfer and stable adult cardiac myocyte phenotype structurally and functionally, this second system was used to study the effects of expressing two mutant troponin T proteins on cardiac myocyte structure and function. Expression of each of the mutant troponin T proteins in adult cardiac myocytes led to a significant decrease in the calcium sensitivity of contraction, but did not appear to alter myocyte structure. In conclusion, an experimental model system, adult cardiac myocytes in primary culture, has been defined which can be rapidly and efficiently modified using adenovirus mediated gene transfer. Gene transfer of each of two mutant troponin T proteins resulted in a significantly decreased cardiac contractile response to calcium activation, supporting the role of these two mutant proteins in causing hypertrophic cardiomyopathy.