Disruption of adenylyl cyclase type V does not rescue the phenotype of cardiac-specific overexpression of G_αq 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.

the disparate roles among adenylyl cyclase (AC) isoforms in cardiac hypertrophy and progression to heart failure have been in focus for more than two decades. AC is the principal effector molecule along the β-adrenergic receptor (β-AR) pathway, and six (AC_II, AC_III, AC_IV, AC_V, AC_VI, and AC_VII) of nine known isoforms are endogenous to the mammalian heart (33). Among these six isoforms, AC_V and AC_VI are the two predominant proteins in cardiac myocytes. A number of shared characteristics exist between AC_V and AC_VI. They exhibit 65% amino acid homology (32) and are the only two AC isoforms that are inhibited by physiological Ca²⁺ concentrations (4, 16). Similar to other isoforms, AC_V and AC_VI are sensitive to negative feedback by protein kinase A (PKA) (2, 20). AC_V and AC_VI (as well as AC_III) have been found localized primarily in caveolin-rich domains in cardiac fibroblasts (33). Despite similarities, the evidence indicates distinct physiological roles for AC_V and AC_VI, as demonstrated in null-mutant mouse models of AC_V vs. AC_VI (25, 43, 44).

In a well-described transgenic mouse with cardiac-specific overexpression of the heterotrimeric G protein α_q-subunit (G_αq), an overexpression of AC_VI attenuates hypertrophy, prevents heart failure, and reduces mortality (40), whereas an overexpression of AC_V does not (45). Moreover, the disruption of AC_V activity in murine models of age-related (48), isoproterenol-induced (29, 48), or pressure-overload cardiomyopathy (30, 31, 48) has been associated with salubrious effects to the heart. Indeed, the proposed development of novel pharmaceuticals specifically inhibiting AC_V as a potential therapy for chronic heart failure underscores the relevance of this isoform (14).

Heart failure is one of the leading causes of morbidity and mortality. Once cardiac failure develops, the condition is generally irreversible and is associated with a high mortality rate. Therefore, the molecular mechanisms that initiate or precipitate the transition to heart failure have undergone intense investigation, and there is evidence to suggest that an elevation in protein kinase C (PKC) activity (15, 17, 36, 42) may play an important role in the transition to heart failure. Specifically, increased PKC activity has been reported to be involved in the pathogenesis of cardiac hypertrophy and progression to heart failure (6, 35).

Motivated by recent reports suggesting AC_V inhibition as a possible therapeutic target in the treatment of heart failure, we directly tested whether the deletion of AC_V would have beneficial effects in G_αq-associated cardiomyopathy using comprehensive in vivo and in vitro studies. Transgenic mice with cardiac-specific overexpression of G_αq provide a clinically relevant model of heart failure with chamber dilation, decreased cardiac contractility, electrical remodeling, depressed β-AR function, and a high mortality rate. Moreover, G_αq transgenic mice exhibit an elevation of PKC level, approximately threefold compared with transgene-negative siblings (8). Furthermore, PKC is directly activated along the G_αq signaling pathway. Hence, we reasoned that transgenic mice with a cardiac-specific overexpression of G_αq would provide an ideal means to specifically test the roles of AC_V in the progression toward cardiac failure.

MATERIALS AND METHODS

All animal care and procedures were approved by the University of California, Davis Institutional Animal Care and Use Committee. Animal use was in accordance with National Institutes of Health and institutional guidelines.

Generation of AC_V gene-targeted mice.

AC_V knockout mice (AC_V^−/−) were generated as previously described (25, 44). The knockout mice were backcrossed with C57Bl/6J mice for greater than 10 generations. Two PCRs with primers specific for the mutated and wild-type alleles were used for genotype analysis. A previous study documented the absence of AC_V mRNA expression in AC_V^−/− mouse hearts, which was associated with a significant decrease in cAMP production in left ventricular (LV) homogenates after isoproterenol stimulation (44).

Transgenic mice.

AC_V^−/− mice (C57Bl/6J) were crossbred with mice with cardiac-directed expression of G_αq protein (G_αq-40 mice; FVB/N, provided by G. W. Dorn II, University of Cincinnati) (5, 47). Of note, the G_αq transgenic mice were backcrossed onto C57Bl/6J mice for greater than 10 generations before they were used for the crossbreeding to allow for the direct comparison of these lines without the confounding effects from differences in the background. Four lines emanating from this cross were studied: G_αq/AC_V^−/− (double positive), G_αq alone, AC_V^−/− alone, and control (double negative). Transgene incorporation into mouse DNA was confirmed using the PCR of tail tissue.

Analysis of cardiac function by echocardiography.

M-mode measurements were used to assess systolic function as previously described (26). Measurements represent the average of twelve selected cardiac cycles from at least two separate scans performed in a random-blind manner with papillary muscles used as a point of reference to establish the consistency in level of the scan. End diastole was defined as the maximal LV diastolic dimension, and end systole was defined as the peak of posterior wall motion. Fractional shortening (FS), a measurement of systolic function, was calculated from LV dimensions as follows: FS = [(EDD − ESD)/EDD] × 100%, where EDD and ESD are LV end-diastolic and end-systolic dimensions, respectively.

Western blot analysis.

Immunoblots were performed as previously described (49). Anti-β-myosin heavy chain antibody (NOQ7.5.4D, Sigma) was used at 1:10,000 dilution. This antibody was found to be specific for β-myosin heavy chain (β-MHC) through a screening of different commercially available antibodies. Anti-GAPDH antibody (Sigma) was used as an internal loading control.

Electrocardiograph recordings.

ECG recordings were obtained using Bioamplifier (BMA 831, CWE, Ardmore, PA). Mice were placed on a temperature-controlled warming blanket at 37°C. Four consecutive 2-min time frames of ECG data were obtained from each mouse. Signals were low-pass filtered at 0.2 kHz and digitized using Digidata 1200 (Axon Instruments). A total of 100 beats were analyzed from each animal in a blinded manner.

Electrophysiology recordings from transgenic animals.

Single ventricular myocytes were isolated from transgenic and double-negative (control) sibling mice from the same littermates at 8 to 9 mo of age. All experiments were performed using the conventional whole cell patch-clamp technique at room temperature.

For action potential recordings, patch pipettes were backfilled with amphotericin (200 μg/ml). The pipette solution contained (in mM) 120 K⁺ glutamate, 25 KCl, 1 MgCl₂, 1 CaCl₂, and 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES) (pH 7.4 with KOH). The external solution contained (in mM) 138 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 0.33 NaH₂PO₄, 10 glucose, and 10 HEPES (pH 7.4 with NaOH).

For K⁺ current recording, the external solution contained 130 mM N-methylglucamine, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 μM nimodipine, 10 mM glucose, and 10 mM HEPES (pH 7.4 with HCl). The pipette solution contained (in mM) 140 KCl, 4 Mg-ATP, 1 MgCl₂, 10 EGTA, and 10 HEPES (pH 7.4 with KOH).

Chemicals were purchased from Sigma Chemical (St. Louis, MO) unless stated otherwise. All experiments were performed using 3 M KCl agar bridges. Cell capacitance was calculated as the ratio of net charge (the integrated area under the current transient) to the magnitude of the pulse (20 mV). Currents were normalized to cell capacitance to obtain the current density. The series resistance was compensated electronically. In all experiments, a series resistance compensation of ≥90% was obtained. Currents were recorded using Axopatch 200B amplifier (Axon Instrument), filtered at 2 kHz using a four-pole Bessel filter, and digitized at a sampling frequency of 10 kHz. Data analysis was carried out using custom-written software and commercially available PC-based spreadsheet and graphics software (MicroCal Origin, version 7.0).

RNA isolation, reverse transcription, and quantitative PCR.

Total RNA was isolated from LV free wall of mice with five different genotypes: wild-type, G_αq, G_αq/AC_V^−/−, AC_V^−/−, and cardiac-directed AC_VI overexpression (AC_VI transgenic), using TRIzol reagent (Invitrogen). Isolated RNA was subjected to DNase I (Invitrogen) treatment and subsequently to reverse transcription using Superscript III Reverse Transcriptase (Invitrogen), dNTPs, and oligo dT primers. Parallel reactions without the reverse transcription enzyme were also performed and used as controls to rule out the possibility of genomic DNA contamination.

Quantitative PCR (qPCR) was performed using Applied Biosystems Fast 7900HT real-time PCR system and RT² Fast SYBR Green/ROX qPCR master mix reagent (SA Biosciences). qPCR-specific primers were custom made from SA Biosciences corresponding to mouse accession numbers: NM_001012765 for AC_V, NM_007405 for AC_VI, and NM_008084 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results were analyzed using Sequence Detection software (version 2.2.1). The quantity of AC_V and AC_VI transcripts were normalized to internal control GAPDH transcripts. qPCR was performed in triplicate to ensure quantitative accuracy.

Assessment of cAMP levels.

cAMP levels in cardiac tissues from G_αq, G_αq/AC_V^−/−, AC_V^−/−, and control mice were measured using cAMP XP Assay Kit (Cell Signaling Technology, Danvers, MA) as per the manufacturer's instructions. The hearts were rapidly excised and retrogradely perfused through the cannulation of aorta using phosphate-buffered saline to remove blood from the cardiac tissue. The tissue was then snap frozen using liquid nitrogen and stored at −80°C until use.

Survival.

To compare the rates of survival between G_αq and G_αq/AC_V^−/− groups, a Kaplan-Meier cumulative curve was used. A log-rank test calculated the statistical significance of differences between the two groups. The longevity of AC_V^−/− and double-negative control mice exceeded the duration of the experiment and were excluded from survival analysis.

Statistical analysis.

Data are presented as means ± SE where appropriate. An analysis of statistical significance was calculated using SigmaStat software. For multiple comparisons, one-way analysis of variance combined with Dunnett's test was used. The null hypothesis was rejected when P < 0.05 (two tailed).

RESULTS

AC_V disruption does not prevent cardiac hypertrophy or chamber dilation in G_αq transgenic mice.

Figure 1A shows photomicrographs of examples of whole hearts from AC_V^−/−, G_αq, G_αq/AC_V^−/−, and control mice at 6 mo of age. As expected, G_αq transgenic mice exhibit evidence of cardiomyopathy with chamber dilatation. On the other hand, AC_V^−/− animals show normal chamber size compared with control mice. More importantly, in contrast to other models of heart failure, the deletion of AC_V does not rescue the cardiomyopathic phenotype in G_αq transgenic animals. Figure 1B shows a significant increase in cell capacitance of LV myocytes isolated from both G_αq and G_αq/AC_V^−/− animals compared with AC_V^−/− and control groups at 6 mo of age. Figure 1C displays summary data for the heart, liver, and lung weight, normalized to body weight, illustrating a significant increase in the heart weight-to-body weight ratio in both G_αq and G_αq/AC_V^−/− animals compared with controls. Figure 1D illustrates the histologic sections with hematoxylin and eosin stain, comparing the four groups of animals. Both G_αq and G_αq/AC_V^−/− hearts show dilatation of all four cardiac chambers.

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).

Echocardiography was used to directly assess cardiac function in the four groups of animals. Figure 1E shows examples of M-mode echocardiogram displaying chamber dilatation and reduced FS in G_αq and G_αq/AC_V^−/− transgenic mice. Summary data for the FS, ESD, and EDD are shown in Table 1. The disruption of AC_V does not improve cardiac function in G_αq/AC_V^−/− transgenic mice.

Table 1.

Summary of echocardographic data in control, AC_V^−/−, G_αq, and G_αq/AC_V^−/− transgenic mice

	Control	Gαq	ACV−/−	Gαq/ACV−/−
n	9	7	7	6
Systolic posterior wall thickness, mm	1.5 ± 00.04	1.6 ± 00.09	1.5 ± 00.04	1.6 ± 00.08
End-diastolic posterior wall thickness, mm	1.1 ± 00.07	1.3 ± 00.08	1.1 ± 00.03	1.4 ± 00.1
ESD, mm	1.3 ± 00.01	1.9 ± 00.12*	1.4 ± 00.07	1.8 ± 10.1*
EDD, mm	2.6 ± 00.02	3.0 ± 00.11	2.8 ± 00.08	2.9 ± 00.12
FS, %	52.6	36.4*	53.6	37.2*

Values are means ± SE; n, number of mice. AC_V, adenylyl cyclase type V; ESD, left ventricular end-systolic dimension; EDD, left ventricular end-diastolic dimension; FS, fractional shortening, calculated from left ventricle dimensions as follows: FS = [(EDD − ESD)/EDD] × 100%.

^* P < 0.05 compared with control.

To further confirm the lack of the beneficial effects of AC_V deletion in G_αq mice, we tested for the induction of a fetal gene, β-MHC isoform, a well-documented hypertrophic marker (3) using Western blot analysis. GAPDH was used as a loading control. As expected, β-MHC is expressed only in G_αq transgenic mice but not in control or AC_V^−/− animals. Consistent with the functional analysis, G_αq/AC_V^−/− mice show a persistent expression of the β-MHC protein (Fig. 1F).

AC_V disruption does not prevent sinus bradycardia in G_αq transgenic mice.

We have previously documented that cardiac-directed expression of G_αq protein resulted in sinus bradycardia (47). G_αq and G_αq/AC_V^−/− mice show sinus bradycardia with a significant prolongation of the R-R intervals (*P < 0.05 compared with control animals, Fig. 1H). In contrast, AC_V^−/− mice demonstrate an increase in basal heart rates compared with control animals (*P < 0.05, Fig. 1G). There was no difference between G_αq and G_αq /AC_V^−/− animals.

Assessment of AC_V and AC_VI expression level in AC_V and G_αq/AC_V^−/− mice.

The absence of the beneficial effects from the deletion of AC_V in G_αq-mediated cardiomyopathy compared with other previous published data in murine models of age-related (48), isoproterenol-induced (29, 48), or pressure-overload cardiomyopathy (30, 31, 48) has prompted us to examine the expression level of AC_V in G_αq and G_αq/AC_V^−/− mice (Fig. 2A). Moreover, to further ensure that AC_V knockout animals are specific for AC_V isoform, we directly assessed the transcript level of AC_VI in AC_V^−/− and G_αq/AC_V^−/− mice (Fig. 2B). Previously described cardiac-directed overexpression of AC_VI transgenic animals was used as a control for the assessment of the AC_VI transcript. Figure 2A demonstrates that the AC_V transcript is completely absent in AC_V^−/− and G_αq/AC_V^−/− animals as expected. Moreover, AC_V and AC_VI transcript levels are only slightly elevated (∼1.5-fold) in G_αq transgenic animals compared with controls (Fig. 2, A and B). The AC_VI transcript level is also slightly elevated (∼1.8-fold) in AC_V^−/− animals, supporting the previous finding that the AC_V knockout mouse is specific for the AC_V isoform. The increase in the AC_VI transcript level in AC_V^−/− animals may be secondary to compensatory responses in the knockout animals. Finally, as expected, the AC_VI transcript level is significantly elevated (>110-fold) in AC_VI transgenic animals compared with controls.

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.

Assessment of cAMP level in AC_V and G_αq/AC_V^−/− mice.

We next examined the cAMP level in the cardiac tissues from four groups of animals (Fig. 2C). cAMP levels in G_αq or AC_V^−/− animals show a trend toward a decrease compared with control, but the differences are not statistically significant. On the other hand, G_αq/AC_V^−/− exhibits a significant decrease in cAMP level compared with control. The data support the notion that the disruption of AC_V in G_αq transgenic animals results in a decrease in cAMP level without rescuing the cardiomyopathic phenotypes.

AC_V disruption does not prevent electrical remodeling in G_αq transgenic mice.

Previous studies have documented that G_αq-induced cardiomyopathy is associated with electrical remodeling with a prolongation of cardiac action potential (47). Here we tested whether the deletion of AC_V may prevent electrical remodeling in G_αq transgenic animals. Figure 3, A–D, shows recordings of action potentials from cardiomyocytes isolated from LV free wall in each of the four groups. Action potentials at 50 and 90% of repolarization (APD₅₀ and APD₉₀, respectively) recorded from both the G_αq and G_αq/AC_V^−/− groups demonstrate significant prolongation compared with those of control animals. However, there was no significant difference between the G_αq and G_αq/AC_V^−/− groups.

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.

We have previously shown that cardiac action potential prolongation in G_αq transgenic animals results, at least in part, from a decrease in the outward K⁺ current and the inward rectifier K⁺ current (I_K1) and that the salubrious effects of AC_VI overexpression in G_αq proteins transgenic mice are associated with the upregulation of both the outward K⁺ current and I_K1 (47). Here we directly examined the effects of AC_V disruption on the K⁺ current in the G_αq cardiomyopathy model. Figure 4, A–D, demonstrates examples of outward K⁺ current recorded from each of the four groups. The G_αq and G_αq/AC_V^−/− groups show a significant decrease in the both the transient outward K⁺ current (I_to) and the sustained K⁺ current densities (Fig. 4, B and D). Summary data for I_to and the sustained K⁺ current densities are presented in Fig. 4, C and F, respectively. There was no difference between the recorded densities of I_to and the sustained outward K⁺ current from the G_αq and G_αq/AC_V^−/− groups. Similarly, I_K1 within the G_αq and G_αq/AC_V^−/− groups were very similar and significantly smaller than the current densities in AC_V^−/− and control animals (Fig. 5).

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).

AC_V disruption did not improve survival in G_αq transgenic mice.

Finally, Kaplan-Meier mortality analyses were performed comparing the G_αq and G_αq/AC_V^−/− animals (Fig. 6). There were no significant differences in the survival between the two groups of animals, consistent with our data showing a lack of improvement in cardiomyopathy and electrical remodeling in G_αq transgenic animals by the disruption of AC_V (P = 0.349).

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.

DISCUSSION

The present study directly tested the possible beneficial 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. Our study provides new evidence that, in contrast to other models of cardiomyopathy, AC_V disruption did not improve survival or cardiac function, limit cardiac chamber dilation, or prevent electrical remodeling in G_αq transgenic mice. Indeed, the findings are relevant and important in view of the proposed development of novel pharmaceuticals specifically inhibiting AC_V as a potential therapy for chronic heart failure.

AC expression and its implications in cardiomyopathy.

The initially attractive idea of restoring cardiac contractility in heart failure by stimulating β-AR pathways has not been shown to reduce mortality. Indeed, the administration of dobutamine (23), a β₁-AR agonist, or milrinone (34), a phosphodiesterase inhibitor, in clinical trials of patients with severe heart failure resulted in increases in mortality. Similarly, cardiac-directed expression of G_αs, β₁-AR, or β₂-AR proteins in murine models all demonstrate sustained increases in heart rate, decreased contractility, cardiomyopathy, and increased mortality (7, 9, 11, 21, 27, 39).

Remarkably, unlike the overexpression or stimulation of β-ARs, the overexpression of AC does not progress toward cardiac hypertrophy. The overexpression of AC_VI prevents heart failure in the G_αq transgenic model (40) and, additionally, does not affect the basal heart rate or heart function compared with control animals (47). This rescue is associated with a restoration of heart function, the prevention of electrical remodeling, and a marked reduction in the mortality. The mechanisms underlying the observed beneficial effects remain unclear, but several proposals exist (37).

The increased expression of AC_V, in contrast to AC_VI, does not prevent cardiac hypertrophy in the G_αq-model of cardiomyopathy (45). AC_V overexpression either slightly increases heart rate (46) in basal conditions or has no effects (10). On the other hand, the disruption of AC_V has demonstrated benefits in several animals of cardiomyopathy. In the age-related model, older control mice have a greater disposition toward cardiomyocyte apoptosis, fibrosis, and decreased longevity compared with AC_V knockout mice (48). Data taken from the pressure-overload model show that the LV ejection fraction (29, 30) improves in AC_V knockout compared with control mice. Furthermore, this improvement is associated with reduced cardiac apoptosis in the AC_V knockout compared with control mice (48). An ablation of AC_V with a subsequent isoproterenol infusion results in an increased expression of Bcl-2, a suppressor of apoptosis in the Akt signaling pathway (31). Indeed, AC_V has been viewed as a potential therapeutic target for chronic heart failure (14).

Our data indicate that the role of the AC_V isoform may be diminished in G_αq-mediated cardiomyopathy compared with other animal models of heart failure. The beneficial effects associated with AC_VI overexpression in the G_αq model cannot be explained by suppression of AC_V because an absence of AC_V did not rescue the phenotype. The reported beneficial effects from disrupting AC_V expression were not present in our experiments, and this absence distinguishes G_αq overexpression from a set of cardiomyopathy models.

The absence of benefits from the deletion of AC_V in G_αq-mediated cardiomyopathy prompted a closer examination of cardiomyopathy models. The models of cardiomyopathy can be arranged from pronounced to subtle salutary effects from AC_V ablation as follows: isoproterenol induced, age related, pressure overload, and G_αq mediated. The disruption of AC_V in isoproterenol-induced cardiomyopathy results in improved FS, decreased cardiac mass, and reduced cardiomyocyte apoptosis compared with those in control mice (44). In age-related decline in LV diastolic function, disrupting AC_V decreased cardiomyocyte apoptosis and transgenic mice lived 30% longer than sibling controls (50). Pressure-overloaded AC_V^−/− mice show less cardiomyocyte apoptosis than control animals (30), though the disruption of AC_V in aortic banding does not prevent the progression to hypertrophy (48).

Administering isoproterenol to control mice produced a slight elevation of PKC expression; however, this effect was diminutive compared with PKA activation (36). Disrupting the PKA regulatory subunit-RIIβ in mice increases longevity and reduces body size, mirroring the beneficial effects of disrupting AC_V in age-related cardiomyopathy (38). PKA attenuation has been suggested as part of a mechanism for the observed benefits of AC_V disruption in age-related cardiomyopathy (50). Constitutively active PKCε parallels the progression to cardiomyopathy with age (13), but the intrinsic elevation of PKC has only been hypothesized in age-related cardiomyopathy. Elevated PKC activity in pressure overload has been reported (15, 17, 42), in particular, PKCα and PKCδ but not PKCε (1). Although the rising levels of catecholamine in the blood plasma (12, 41) are suggestive of downstream AC activation, PKA activation precedes hypertrophy in aortic banding and is not clearly linked with the development of hypertrophy (24). Overexpressing G_αq leads to a prominent elevation in PKC levels (particularly PKCα) (7). PKCα can stimulate AC_V [isoforms ζ (22), α (18), and γ (18) interact with AC_V], but our results show that cardiac hypertrophy manifests despite any possible interaction between PKC and AC_V.

Outcomes of disrupting AC_V in the G_αq transgenic model of cardiomyopathy.

Electrical remodeling is well documented in several models of heart failure including human heart failure. The downregulation of K⁺ channel expression is common to cardiac hypertrophy and failure and is associated with a prolonged action potential. A prolonged action potential, in turn, may be involved in the pathogenesis of cardiac hypertrophy through increasing intracellular Ca²⁺ entering mostly through L-type Ca²⁺ channels during the plateau phase. Our previous data demonstrate that the beneficial effects of AC_VI overexpression in the G_αq transgenic model are associated with the prevention of electrical modeling of outward K⁺ current and I_K1 (47). AC_V deletion, however, does not reproduce these beneficial effects in the G_αq transgenic model; outward K⁺ current and I_K1 remain unchanged between G_αq and G_αq/AC_V^−/− mice. The two groups of animals, G_αq and G_αq/AC_V^−/−, displayed a similar prolongation of the action potentials and sinus bradycardia. The mechanisms of increase mortality in G_αq and G_αq/AC_V^−/− mice may be related to the documented prolongation in APD and possibly brady- or tachyarrhythmias. However, other mechanisms may be involved including progressive heart failure.

Interestingly, the AC_V^−/− group shows a slight, but statistically significant, decrease in R-R interval compared with the control animals. A minor deviation in heart rates in AC_V^−/− mice compared with the control animals has previously been reported (28, 29, 43). A decreasing effect of acetylcholine receptors in AC_V^−/− mice compared with control mice was proposed to explain this deviation (28). The activation of the parasympathetic system as an explanation for heart rate deviations between AC_V^−/− and control mice appears reasonable since acetylcholine receptors have been found to be colocalized with AC in macromolecular complexes in cardiomyocytes (19).

Conclusion.

Our results demonstrate that unlike in age-related, isoproterenol-induced, and pressure-overload models, a disruption of AC_V in the G_αq transgenic model provides no benefits in preventing the development of cardiomyopathy. The disruption of the constant activation of AC_V by PKC in G_αq transgenic mouse model may be insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-85727 and HL-85844 and a Veterans Affairs merit review grant (to N. Chiamvimonvat); NHLBI Grants P01-HL-66941, HL-81741, and HL-88426 and a Veterans Affairs merit review grant (to H. K. Hammond); American Heart Association Western Affiliates beginning grant-in-aid (to T. Tang); and American Heart Association Western Affiliates postdoctoral fellowship award (to H. Qiu).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).