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

Objective: 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. Methods: 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 α-myosin heavy chain promoter. Expression of the AC_VI gene was driven by a tet-response element (TRE) and a minimal CMV promoter. Results: 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/μg; Transgene On: 39±14 fmol/μg; n=5 each group; P=0.004) and also to isoproterenol stimulation (Transgene Off: 20±5 fmol/μg; Transgene On: 31±12 fmol/μg; 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. Conclusion: 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.