Disruption of adenylyl cyclase type V does not rescue the phenotype of cardiac-specific overexpression of Galphaq protein-induced cardiomyopathy

Abstract

Adenylyl cyclase (AC) is the principal effector molecule in the β-adrenergic receptor pathway. AC(V) and AC(VI) are the two predominant isoforms in mammalian cardiac myocytes. The disparate roles among AC isoforms in cardiac hypertrophy and progression to heart failure have been under intense investigation. Specifically, the salutary effects resulting from the disruption of AC(V) have been established in multiple models of cardiomyopathy. It has been proposed that a continual activation of AC(V) through elevated levels of protein kinase C could play an integral role in mediating a hypertrophic response leading to progressive heart failure. Elevated protein kinase C is a common finding in heart failure and was demonstrated in murine cardiomyopathy from cardiac-specific overexpression of G(αq) protein. Here we assessed whether the disruption of AC(V) expression can improve cardiac function, limit electrophysiological remodeling, or improve survival in the G(αq) mouse model of heart failure. We directly tested the effects of gene-targeted disruption of AC(V) in transgenic mice with cardiac-specific overexpression of G(αq) protein using multiple techniques to assess the survival, cardiac function, as well as structural and electrical remodeling. Surprisingly, in contrast to other models of cardiomyopathy, AC(V) disruption did not improve survival or cardiac function, limit cardiac chamber dilation, halt hypertrophy, or prevent electrical remodeling in G(αq) transgenic mice. In conclusion, unlike other established models of cardiomyopathy, disrupting AC(V) expression in the G(αq) mouse model is insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure.

Fig. 1.

Adenylyl cyclase type V (AC_V) disruption does not prevent cardiomyopathy in G_αq transgenic mice. A: photomicrographs showing examples of hearts from 4 groups of animals. B: G_αq and G_αq/AC_V^−/− transgenic mice show a significant increase in the cell capacity in single myocytes isolated from left ventricular free wall (*P < 0.05 compared with control; n = 9–17). C: heart, liver, and lung mass-to-body mass ratios in the 4 groups of animals. Disruption of AC_V did not prevent the increase in heart mass-to-body mass ratio in the G_αq/AC_V^−/− transgenic mice (*P < 0.05 compared with control; n = 6–9 animals). D: histologic sections with hematoxylin-eosin staining from the same hearts as in A, providing direct evidence for dilated chambers in G_αq and G_αq/AC_V^−/− mice. E: examples of M-mode ECG obtained from the 4 groups showing chamber dilatation and a decrease in cardiac contractility in G_αq and G_αq/AC_V^−/− mice. F: Western blot analysis from 4 groups of animals showing expression of β-myosin heavy chain (β-MHC) in G_αq and G_αq/AC_V^−/− mice. GAPDH was used as a loading control. Same data were obtained from 3 different sets of animals. WT, wild-type. G: both G_αq and G_αq/AC_V^−/− mice showed evidence of sinus bradycardia. G_αq and G_αq/AC_V^−/− mice showed evidence of sinus bradycardia with significant prolongation of the R-R intervals compared with control mice. Summary data for R-R, P-R, and Q-T intervals (n = 6–8 animals for each group). G_αq/AC_V^−/− mice show a significant prolongation in the R-R interval (*P < 0.05 comparing G_αq/AC_V^−/− and control mice. R-R interval was not statistical different comparing G_αq/AC_V^−/− mice to G_αq mice).

Fig. 2.

A and B: AC_V and AC_VI transcript levels normalized to GAPDH from control, G_αq, G_αq/AC_V^−/−, AC_V^−/−, and cardiac-directed expression of AC_VI transgenic animals. *P < 0.05 compared with control animals. C: cAMP concentrations (in nM) in cardiac tissues from G_αq, G_αq/AC_V^−/−, and AC_V^−/− compared with control. *P ≤ 0.05 compared with control; n = 4 for each group.

Fig. 3.

AC_V disruption does not prevent action potential (AP) prolongation in G_αq transgenic mice. A–D: examples of AP recordings from transgenic mice compared with control littermates. AP recordings from G_αq and G_αq/AC_V^−/− transgenic mice were significantly prolonged compared with control littermates. There were no statistical differences in the length of APs between G_αq/AC_V^−/− and G_αq transgenic mice. E: summary data showing AP duration at 50 and 90% repolarization (APD₅₀ and APD₉₀, in ms). *P < 0.05 compared with control animals; n = 30 to 50 cells for each group.

Fig. 4.

AC_V disruption does not prevent K⁺ current downregulation in G_αq transgenic mice. Ca²⁺-independent outward K⁺ current density from 4 different groups of animals (A, B, D, and E). Examples of current traces elicited from a holding potential of −80 mV using test potentials in duration of 2.5 s from −70 to +60 mV along 10-mV increments. C: summary data for the density of the peak outward components (*P < 0.05 comparing G_αq/AC_V^−/− and control mice; and *P < 0.05 comparing G_αq and control mice). F: summary data for the density of the sustained components (measured at the end of the pulse, *P < 0.05 comparing G_αq and G_αq/AC_V^−/− with control mice; n = 12–16 cells for each group). V, voltage.

Fig. 5.

AC_V disruption does not prevent the downregulation of the inward rectifier K⁺ current (I_K1) in G_αq transgenic mice. A: examples of current traces recorded from a holding potential of −80 mV using test potentials in duration of 2.5 s from −130 to +60 mV in 10-mV increments in control. B: after applying BaCl₂. C: after applying the BaCl₂-sensitive current. D: summary data for the BaCl₂-sensitive current density (I_K1 density) in single free wall LV myocytes isolated from the 4 groups. Inset: outward I_K1 component from each group for further clarity (*P < 0.05; n = 8–15 for each group).

Fig. 6.

Kaplan-Meier mortality curve in G_αq (n = 15) and G_αq/AC_V^−/− (n = 13) transgenic mice compared with WT and AC_V^−/− animals. Log-rank statistic calculated a P value for significant differences between the survival curves.