Altering the Receptor−Effector Ratio by Transgenic Overexpression of Type V Adenylyl Cyclase: Enhanced Basal Catalytic Activity and Function without Increased Cardiomyocyte β-Adrenergic Signalling

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Abstract
The limiting element in β-adrenergic receptor (βAR)−Gs−adenylyl cyclase (AC) signal transduction in the cardiomyocyte is not known, but it has been proposed that the level of adenylyl cyclase expression constrains βAR signaling. To alter the above equilibrium, type V AC was overexpressed in a myocyte-specific manner in the hearts of transgenic mice using the α-myosin heavy chain promoter. Expression of type V AC was ∼75% over endogenous levels as quantitated by [3H]forskolin binding. Functional activity of the transgene product was evident in cardiac membrane AC studies, where basal (45 ± 11 vs 19 ± 5 pmol min-1 mg-1) and forskolin+Mn2+ (695 ± 104 vs 386 ± 34 pmol min-1 mg-1) stimulated activities were increased compared to activities in nontransgenic (NTG) littermates. However, while isoproterenol stimulated activities were higher (74 ± 12 vs 46 ± 9.8 pmol min-1 mg-1), the fold stimulation over basal was not increased in ACV overexpressors compared to NTG (line 14.3 = 2.29 ± 0.44-fold, line 15.1 = 1.70 ± 0.1-fold, NTG = 2.62 ± 0.18-fold). Similarly, in whole cell patch-clamp studies, βAR-mediated opening of L-type Ca2+ channels was not found to be enhanced in transgenic ACV myocytes (225 ± 15 vs 216 ± 10% of basal currents). Basal and isoproterenol stimulated PKA activities were elevated in the ACV mice compared to NTG, but again the extent of stimulation over basal was not enhanced. Phosphorylated phospholamban was ∼2-fold greater in myocytes from ACV hearts compared to NTG, indicating that distal elements of the contractile cascade are activated by AC overexpression. ACV mice displayed increased heart rates and fractional shortening as assessed by echocardiography. However, in vivo hemodynamic studies revealed that heart rate and contractility responses to agonist infusion were not enhanced in ACV mice compared to NTG. We conclude that at native stoichiometries, the levels of adenylyl cyclase influence basal activities and cardiac function, but do not constrain βAR signaling in the cardiomyocyte.