Cardiac-directed expression of a catalytically inactive adenylyl cyclase 6 protects the heart from sustained β-adrenergic stimulation

Abstract

Objectives: 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.

Methods and results: 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).

Conclusion: Mice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.

Fig 1. LV function.

A. After seven days of continuous isoproterenol infusion, mice with cardiac-directed AC6mut expression exhibited a 25% greater LV peak +dP/dt (p = 0.03), indicating increased LV systolic function. B. LV peak -dP/dt was also superior in mice with cardiac-directed AC6mut expression (31% increase, p = 0.008), indicating increased LV diastolic function. C. LV developed pressure also was superior in mice with cardiac-directed AC6mut expression. D. The slope of the LV end-systolic pressure-volume relationship (ESPVR), a load-independent measure of LV contractility, was also 2-fold steeper in mice with cardiac-directed AC6mut expression (p = 0.003) E. Cardiac output was 41% higher in AC6mut mice (p<0.03). F. Heart rate showed no group difference. Data are from individual animals, collected without knowledge of group identity; mean±SE are also indicated; p value from Student’s t-test, unpaired, 2-tailed.

Fig 2. LV cAMP, PKA Activity and Signaling Proteins.

A. Cyclic AMP Generation. LV membranes from AC6mut (M) and control mice (C) were used to measure cAMP production before (No Iso, n = 5) and after 7d isoproterenol infusion (7d Iso, n = 5). Membranes were stimulated with isoproterenol (Iso; 10 μM, 10 min) or NKH477 (NKH, a water-soluble direct AC stimulant; 10 μM, 10 min). Cyclic AMP production was lower in LV from AC6mut than from control mice before and after 7d isoproterenol infusion. B. PKA Activity in isolated cardiac myocytes before (No Iso, n = 5) and after 7d isoproterenol infusion (7d Iso, n = 5). Cardiac myocytes were stimulated with isoproterenol (Iso; 10 μM, 10 min) or NKH477 (NKH, 10 μM, 10 min). PKA activity (μmol/mg/min) was lower in cardiac myocytes from AC6mut (M) than from control mice (C) before isoproterenol infusion, but no group differences were seen after 7d isoproterenol infusion. C. There was no group difference in total or phosphorylated PLB, total or phosphorylated troponin-I or CREB phosphorylation after 7d of continuous isoproterenol infusion. However, there were pre-Iso group differences in total PLB (reduced in AC6mut, p<0.003), and in P-TnI (reduced in AC6mut, p = 0.005). In these instances, 7d isoproterenol was associated with reduced levels in the control animals, such that there no longer were group differences. D. LV SERCA2a protein showed group differences (2-Way ANOVA: Interaction: p = 0.29; Iso: p = 0.44; Gene: p = 0.002). Seven days Iso increased LV SERCA2a in AC6mut (M) mice vs control (p = 0.01). In A and B, P values from Student’s t-test (unpaired, 2-tailed); In D, p value from 2-way followed by Sidak’s multiple comparison test. In all Figures displaying immunoblotting, summary data are normalized to GAPDH.

Fig 3. LV hypertrophy.

A. LV-to-Tibial length (LV/TL) ratios were increased by 7d isoproterenol in both groups (2-Way ANOVA: Interaction: p = 0.80; Iso: p<0.0001; Gene: p = 0.43) and differences were seen within groups (Control: p<0.02; AC6mut: p = 0.0001; Sidak’s multiple comparison test). There were no between-group differences before or 7d after continuous isoproterenol infusion. B. Calcineurin activity (PO4 release) in LV samples showed an overall group difference (2-way ANOVA: Interaction: p<0.04; Iso: p = 0.085; Gene: p = 0.04). A 7d isoproterenol infusion resulted in increased calcineurin activity in control mice (p = 0.03), not seen in AC6mut mice; AC6mut mice showed lower calcineurin activity vs control mice after 7d isoproterenol (p<0.01). Between-group comparisons were made using Sidak’s multiple comparison test. C. LV phosphorylation of NFATC3 (p = 0.001) and NFATC4 (p<0.001) were higher after isoproterenol infusion in AC6mut (Mut) mice. D. LV FHL1 protein content (L graph) was 3-fold greater in control than in AC6mut mice 7d after isoproterenol infusion (p<0.01), although FHL1 content was similar in both groups before isoproterenol infusion. LV FHL1 mRNA (R graph) was higher in control than in AC6mut mice after 7d isoproterenol. In C & D, p values are from Student’s t-test (unpaired, 2-tailed). In Figures displaying immunoblotting, summary data are normalized to GAPDH.

Fig 4. Cardiac myocyte apoptosis.

A. Upper Panel: Example of TUNEL positive nucleus in photomicrographs from confocal microscopic images of transmural LV sections. Nuclei were stained with DAPI (blue) and apoptotic nuclei with broken DNA were labeled with FITC (green). Lower Panel: Quantitative analysis of cardiac myocyte apoptosis in AC6mut vs control mice before (Pre) and after continuous isoproterenol infusion showed group differences (2-Way ANOVA: Interaction: p = 0.17; Iso: p = 0.03; Gene: p<0.03). Cardiac myocyte apoptosis was increased >2.1-fold in control mice after isoproterenol infusion (p = 0.03), but was not increased in LV samples from mice with cardiac-directed AC6mut expression. Control mice showed more apoptosis vs AC6mut mice after 7d isoproterenol (p<0.03). Between-group comparisons made using Sidak’s multiple comparison test. B. After 7d isoproterenol infusion, LV caspase 3/7 activity was greater in control mice (Con) than in AC6mut mice (p = 0.012, Student’s t-test, unpaired, 2-tailed). C. LV Bcl2 protein content showed group differences (2-Way ANOVA: Interaction: p = 0.001; Iso: p<0.0001; Gene: p = 0.19). Seven days after continuous isoproterenol infusion, LV Bcl2 was increased in AC6mut mice (M), compared to control mice (p<0.0001). Bcl2 was increased after 7d isoproterenol infusion in AC6mut (p<0.004). Between-group comparisons made using Sidak’s multiple comparison tests. In all graphs mean ±SE are shown. In C, summary data are normalized to GAPDH.

Fig 5. LV fibrosis.

A. LV collagen deposition LV sections using Masson trichrome staining. B. LV collagen deposition showed group differences (2-Way ANOVA: Interaction: p = 0.30; Iso: p<0.0001; Gene: p = 0.30). Collagen deposition was increased following 7d isoproterenol in control (p = 0.001) and in AC6mut mice (p<0.02). Between-group comparisons made using Sidak’s multiple comparison test. C. LV periostin protein expression was greater in control mice than in AC6mut mice after 7d isoproterenol infusion (p<0.005). Periostin protein was undetectable prior to isoproterenol infusion. D. LV mRNA expression of periostin, and collagen 1 (Col 1) were detected using RT-PCR. Compared to control mice, 7d isoproterenol infusion was associated with reduced LV periostin mRNA (p = 0.008) and reduced collagen-1 mRNA (p = 0.03; data normalized to GADPH mRNA). In C & D, p values from Student’s t-test (unpaired, 2-tailed).