Date Published: August 2, 2017
Publisher: Public Library of Science
Author(s): Mei Hua Gao, N. Chin Lai, Dimosthenis Giamouridis, Young Chul Kim, Tracy Guo, H. Kirk Hammond, Guo-Chang Fan.
Increased expression of adenylyl cyclase type 6 (AC6) has beneficial effects on the heart through cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways. We previously generated a catalytically inactive mutant of AC6 (AC6mut) that has an attenuated response to β-adrenergic receptor stimulation, and, consequently, exhibits reduced myocardial cAMP generation. In the current study we test the hypothesis that cardiac-directed expression of AC6mut would protect the heart from sustained β-adrenergic receptor stimulation, a condition frequently encountered in patients with heart failure.
AC6mut mice and transgene negative siblings received osmotic mini-pumps to provide continuous isoproterenol infusion for seven days. Isoproterenol infusion caused deleterious effects that were attenuated by cardiac-directed AC6mut expression. Both groups showed reduced left ventricular (LV) ejection fraction, but the reduction was less in AC6mut mice (p = 0.047). In addition, AC6mut mice showed superior left ventricular function, manifested by higher values for LV peak +dP/dt (p = 0.03), LV peak -dP/dt (p = 0.008), end-systolic pressure-volume relationship (p = 0.003) and cardiac output (p<0.03). LV samples of AC6mut mice had more sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein (p<0.01), which likely contributed to better LV function. AC6mut mice had lower rates of cardiac myocyte apoptosis (p = 0.016), reduced caspase 3/7 activity (p = 0.012) and increased B-cell lymphoma 2 (Bcl2) expression (p = 0.0001). Mice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.
Substitution of Ala for Asp at position 426 in the catalytic core of adenylate cyclase type 6 (AC6), renders the molecule catalytically inactive, which impairs cyclic adenosine monophosphate (cAMP) generation, enabling separation of cAMP-dependent and cAMP-independent effects of AC6 in studies conducted in cultured cardiac myocytes ,,,. Transgenic mice with cardiac-directed AC6mut expression possess normal LV contractile response to isoproterenol, despite impairment of LV β-adrenergic receptor-stimulated cAMP production . This was shown to be through the beneficial effects of AC6mut on Ca2+ handling, which are predominantly cAMP-independent . The next logical question, which we address in the current study, is whether cardiac-directed expression of AC6mut might protect the heart from the deleterious consequences of sustained β-adrenergic receptor stimulation, which is often seen in association with heart failure.
The most important conclusion from our studies is that mice with cardiac-directed expression of AC6mut weather the deleterious effects of continuous isoproterenol infusion considerably better than control mice. First, after seven days of isoproterenol infusion, mice with cardiac-directed AC6mut expression exhibited superiority (vs control mice) in peak +dP/dt, peak -dP/dt, the slope of the ESPVR, and cardiac output, which are measures of systolic, diastolic and contractile function. Second, AC6mut mice, unlike control mice, did not show the expected increase in cardiac myocyte apoptosis following isoproterenol infusion. These findings indicate that AC6mut expression protects the heart against β-adrenergic receptor stimulation, and does so while preserving LV systolic and diastolic function despite lower levels of cardiac cAMP. These findings provide a rationale for proceeding to additional preclinical studies—to see whether AC6mut gene transfer has beneficial effects in pre-existing HF.