Beneficial effects of adenylyl cyclase type 6 (AC6) expression persist using a catalytically inactive AC6 mutant

Abstract

Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation.

Fig. 1.

AC6 and AC6mut expression, localization, and activity. A, expression of AC6 and AC6mut proteins. Adult cardiac myocytes were incubated with Ad.AC6mut, Ad.AC6, or Ad.Null (control) for 40 h. AC6 and AC6mut proteins were detected by anti-AC5/6 antibody in immunoblotting. B, location of transgene proteins. Double immunofluorescence staining of AC6 and AC6mut by anti-AU1 antibody (red), anti-caveolin 3 (Cav-3) antibody (green), and anti-protein disulfide-isomerase (PDI) antibody (green). Hoechst dye was used to identify the nucleus (blue). There were no apparent group differences in cellular distribution: AC6 and AC6mut proteins were present in plasma membrane (associated with caveolin), nuclear envelope, and sarcoplasmic reticulum. C, AC activity. cAMP was measured in uninfected (Con) and Ad.AC6- and Ad.AC6mut-infected cardiac myocytes before (basal) and after 10-min stimulation with isoproterenol (Iso; 10 μM) or forskolin (Fsk; 10 μM). As expected, AC6 increased cAMP generation in response to isoproterenol and forskolin stimulation. AC6mut was associated with reduced cAMP generation in response to isoproterenol (a 59% reduction) and forskolin (an 80% reduction). Bars in the graphs denote mean ± S.D. (***, p < 0.001) derived from triplicates in three independent experiments.

Fig. 2.

Cardiac myocyte cell death, apoptosis, and hypertrophy. A, phenylephrine-induced cell death. Dead cardiac myocytes were counted and expressed as a percentage of total cardiac myocytes. AC6 and AC6mut reduced PE-associated cardiac myocyte death. Bars in graphs are mean values (***, p < 0.001) derived from assessment of 800 to 1000 cells for each condition; experiments were repeated three times. B, phenylephrine-induced myocyte apoptosis. Top, apoptotic cardiac myocytes were detected using TUNEL staining and expressed as -fold increase over untreated myocytes. Bottom, TUNEL-positive cells. AC6 and AC6mut reduced PE-associated cardiac myocyte apoptosis. Bars in graphs are mean values derived from assessment of 500 to 800 cells for each condition; experiments were repeated three times. The p values are from post hoc Bonferroni t testing after one-way ANOVA (**, p < 0.01). C, phenylephrine-induced hypertrophy. Cardiac myocytes infected with Ad.AC6 or Ad.AC6mut were incubated with phenylephrine (20 μM, 44 h), and cardiac myocytes were imaged and cell area (micrometers squared) measured using MetaMorph, an integrated morphometry analysis program. AC6 and AC6mut inhibited PE-induced cardiac myocyte hypertrophy. The p values indicate comparison with control condition with PE. Bars in graphs are mean values (***, p < 0.001) derived from more than 50 cells per condition; experiments were repeated three times.

Fig. 3.

Phenylephrine-induced hypertrophy: phospholamban, CARP, and calcineurin. A, ANF mRNA was detected by qRT-PCR. AC6, but not AC6mut, reduced ANF mRNA expression after PE treatment. B, BNP mRNA was detected by qRT-PCR. BNP mRNA was increased by PE treatment in all conditions without group differences. C, CARP mRNA expression. Real-time RT-PCR was used to determine the expression of CARP, and mRNA copy number was expressed as a percentage over control (uninfected and without PE). Gene transfer of AC6 and AC6mut did not affect CARP mRNA expression. However, after incubation with PE, CARP expression was reduced by AC6 and AC6mut expression compared with the Ad.Null control (p < 0.05) or with uninfected control (**, p < 0.01). The p values are from post hoc Bonferroni t testing after one-way ANOVA. D, CARP protein expression. CARP protein was detected using anti-CARP antibody in immunoblotting and showed no group differences in the absence of PE. After PE treatment, AC6 and AC6mut expression reduced expression of CARP protein compared with both uninfected and Ad.Null controls (**, p < 0.01). E, calcineurin A protein expression. Calcineurin A protein was detected using anti-calcineurin A antibody in immunoblotting. There were no differences among groups in the basal or PE-treated conditions. F, calcineurin activity. Calcineurin activity was determined using the calcineurin cellular activity assay kit from Enzo Life Sciences. There were no differences among groups in the basal or PE-treated conditions. In all graphs, bars represent mean values of three to four experiments; error bars denote 1 S.D.

Fig. 4.

Calcium signaling. Ad.AC6mut- and Ad.AC6-infected adult rat cardiac myocytes, unstimulated or stimulated with isoproterenol (1 μM, 24 h), were analyzed by real-time [Ca²⁺]cyt imaging. Representative Ca²⁺ transients in response to caffeine stimulation were recorded with Fura-2 fluorescence. Left, unstimulated cardiac myocytes expressing AC6 or AC6mut showed no change in [Ca²⁺]cyt in response to caffeine (10 mM). Number of cardiac myocytes assessed: control, 34; AC6, 24; AC6mut, 31. Right, isoproterenol-stimulated cardiac myocytes expressing AC6 or AC6mut showed increases in the peak amplitude of Ca²⁺ transient in response to caffeine stimulation. Number of cardiac myocytes assessed: control, 19; AC6, 24; AC6mut, 17 cells. (AC6 versus Null, ***, p < 0.001; AC6mut versus Null, **, p < 0.01).

Fig. 5.

Expression and phosphorylation of PLB and cTnI proteins. A, immunodetection of total PLB protein using anti-PLB antibody in cell homogenates of adult rat cardiac myocytes. AC6 and AC6mut reduced expression of PLB. B and C, immunodetection of PLB phosphorylation at Ser16 and Thr17 using their specific antibodies in cell homogenates of adult rat cardiac myocytes unstimulated or stimulated with isoproterenol (10 μM, 24 h). AC6, but not AC6mut, increased basal PLB phosphorylation at the Ser16 site but not the Thr17 site. After isoproterenol stimulation, PLB phosphorylation at Ser16 was increased similarly in all conditions, and phosphorylation at Thr17 was increased in all conditions but was greater in AC6- than in AC6mut-infected cardiac myocytes. D, immunodetection of troponin I phosphorylation at the Ser23/24 sites using anti-phospho-cTnI antibody in cell homogenates of adult rat cardiac myocytes, unstimulated or stimulated with isoproterenol (10 μM, 24 h). AC6 and AC6mut did not increase basal cTnI phosphorylation, but isoproterenol treatment was associated with increased cTnI phosphorylation of similar degrees. Bars in all graphs show mean values from three or more experiments; error bars denote 1 S.D. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).