Controlled expression of cardiac-directed adenylylcyclase type VI provides increased contractile function.

Abstract

We have previously shown that cardiac-directed expression of adenylycyclase type VI (AC(VI)) increases heart function in transgenic mice, and improves heart function and survival in murine cardiomyopathy. However, a potential problem of crossbreeding paradigms that use lines with two constitutively active transgenes is that results can be obfuscated by interactions between transgenes during growth and development. To develop a model that could be used subsequently to address this generic problem, transgenic mice with tetracycline (tet)-regulated cardiac-specific expression of AC(VI) were generated. In this transgenic strain, the expression of a tet-controlled transactivator (tTA) was under control of the rat alpha-myosin heavy chain promoter. Expression of the AC(VI) gene was driven by a tet-response element (TRE) and a minimal CMV promoter. Homogenates of hearts showed no change in AC(VI) protein content during tet suppression (doxycycline), confirming successful suppression of transgene expression. Removal of tet suppression for 10 days was associated with a 10-fold increase in cardiac AC(VI) protein content. A similar increase in mRNA was observed (Northern blot analysis). The estimated half-life of newly synthesized cardiac AC(VI) protein was 2-3 days. Isolated cardiac myocytes from animals that had tet-suppression removed for 10 days showed increased cAMP production in response to forskolin stimulation (Transgene Off: 15+/-6 fmol/microg; Transgene On: 39+/-14 fmol/microg; n=5 each group; P=0.004) and also to isoproterenol stimulation (Transgene Off: 20+/-5 fmol/microg; Transgene On: 31+/-12 fmol/microg; n=5 each group; P=0.035) and hearts isolated from these animals showed marked increased left ventricular peak dP/dt in response to dobutamine stimulation (P=0.009) indicating that inducible cardiac AC(VI) is functionally coupled and recruitable. We have generated transgenic mice with controlled cardiac-specific expression of AC(VI), provided detailed information regarding the kinetics of transgene expression and suppression and estimated the half-life of cardiac AC(VI) protein to be 2-3 days. Finally, we have shown, for the first time, that controlled cardiac-directed expression of a transgene can increase cardiac myocyte cAMP generation and left ventricular contractile function.