Adenylyl cyclase increases survival in cardiomyopathy.

BACKGROUND: To test the hypothesis that increased cardiac adenylyl cyclase type VI (AC(VI)) content, which results in increased cAMP generation, would increase survival in cardiomyopathy, we crossbred mice with Gq-associated cardiomyopathy and those with cardiac-directed expression of AC(VI). We also assessed myocardial hypertrophy after prolonged cardiac expression of Gq versus coexpression of Gq and AC(VI). METHODS AND RESULTS: Three experimental groups, Gq/AC (double positive), Gq, and control (double negative), were studied. Survival was increased by cardiac-directed expression of AC(VI) (P<0.0001), and Gq/AC mice had survival rates indistinguishable from control mice. Myocardial hypertrophy developed in older Gq mice but was abrogated by cardiac expression of AC(VI), as documented by the ratio of ventricular weight to tibial length (Gq, 11.93+/-0.99 mg/mm, n=11; Gq/AC, 8.00+/-0.73 mg/mm, n=9; P<0.01) and by left ventricular cardiac myocyte size (Gq, 2800+/-254 microm2, n=4; Gq/AC, 1721+/-166 microm2, n=5; P<0.01). Hearts of Gq mice were dilated, and function was impaired. Concurrent expression of AC reduced end-diastolic diameter (Gq, 4.20+/-0.15 mm, n=12; Gq/AC, 3.68+/-0.12 mm, n=7; P<0.05) and increased fractional shortening (Gq, 32+/-1%, n=12; Gq/AC, 41+/-2%, n=7; P<0.001). Cardiac myocytes from Gq/AC mice showed increased forskolin-stimulated cAMP production (Gq, 3.8+/-1.3 fmol/cell, n=5; Gq/AC, 10.7+/-2.6 fmol/cell, n=6; P<0.02), documenting increased AC function. CONCLUSIONS: Cardiac-directed expression of AC(VI) restores myocyte AC function, improves heart function, increases cAMP generation, abrogates myocardial hypertrophy, and increases survival in Gq cardiomyopathy.

Controlled expression of cardiac-directed adenylylcyclase type VI provides increased contractile function.

OBJECTIVE: We have previously shown that cardiac-directed expression of adenylycyclase type VI (AC(VI)) increases heart function in transgenic mice, and improves heart function and survival in murine cardiomyopathy. However, a potential problem of crossbreeding paradigms that use lines with two constitutively active transgenes is that results can be obfuscated by interactions between transgenes during growth and development. METHODS: To develop a model that could be used subsequently to address this generic problem, transgenic mice with tetracycline (tet)-regulated cardiac-specific expression of AC(VI) were generated. In this transgenic strain, the expression of a tet-controlled transactivator (tTA) was under control of the rat alpha-myosin heavy chain promoter. Expression of the AC(VI) gene was driven by a tet-response element (TRE) and a minimal CMV promoter. RESULTS: Homogenates of hearts showed no change in AC(VI) protein content during tet suppression (doxycycline), confirming successful suppression of transgene expression. Removal of tet suppression for 10 days was associated with a 10-fold increase in cardiac AC(VI) protein content. A similar increase in mRNA was observed (Northern blot analysis). The estimated half-life of newly synthesized cardiac AC(VI) protein was 2-3 days. Isolated cardiac myocytes from animals that had tet-suppression removed for 10 days showed increased cAMP production in response to forskolin stimulation (Transgene Off: 15+/-6 fmol/microg; Transgene On: 39+/-14 fmol/microg; n=5 each group; P=0.004) and also to isoproterenol stimulation (Transgene Off: 20+/-5 fmol/microg; Transgene On: 31+/-12 fmol/microg; n=5 each group; P=0.035) and hearts isolated from these animals showed marked increased left ventricular peak dP/dt in response to dobutamine stimulation (P=0.009) indicating that inducible cardiac AC(VI) is functionally coupled and recruitable. CONCLUSION: We have generated transgenic mice with controlled cardiac-specific expression of AC(VI), provided detailed information regarding the kinetics of transgene expression and suppression and estimated the half-life of cardiac AC(VI) protein to be 2-3 days. Finally, we have shown, for the first time, that controlled cardiac-directed expression of a transgene can increase cardiac myocyte cAMP generation and left ventricular contractile function.

Natriuretic peptide gene expression after beta-adrenergic stimulation in adult mouse cardiac myocytes.

The role of A- and B-type natriuretic peptides (ANP and BNP) in cardiac pathophysiology are of increasing interest. Isolated neonatal mouse cardiac myocytes express increased levels of ANP mRNA in the absence of growth factors in culture. Expression of ANP and BNP mRNA has not been studied in isolated adult mouse cardiac myocytes (AMCM). We examined expression of ANP and BNP mRNA in isolated AMCM with and without stimulation with beta-adrenergic receptor agonists and antagonists. AMCM were isolated and maintained in culture for 24-48 h with and without stimulation with the beta-adrenergic receptor agonist isoproterenol (Iso), the beta1-antagonist CGP20712A (CGP), or the beta2-antagonist ICI-118,551 (ICI). Northern blot analysis was performed using probes for mouse ANP and BNP mRNA. TUNEL assay was performed after beta-adrenergic receptor stimulation of AMCM. BNP mRNA expression was increased fivefold (P < 0.001) after 48 h in culture without adrenergic stimulation. BNP mRNA expression was reduced (P < 0.0001) after stimulation with Iso while ANP expression remained similar to unstimulated cells. CGP prevented the Iso reduction in BNP mRNA. Iso stimulation at doses that reduced BNP mRNA expression increased TUNEL positive nuclei, an effect blocked by the beta1-antagonist CGP. In conclusion, we have demonstrated differential gene expression of ANP and BNP in AMCM in culture. Expression of BNP mRNA increases in AMCM in culture and beta1-adrenergic receptor stimulation attenuates increased BNP gene expression and results in apoptosis.

Adenylyl cyclase type V deletion increases basal left ventricular function and reduces left ventricular contractile responsiveness to beta-adrenergic stimulation.

We tested the hypothesis that deletion of adenylyl cyclase type V (AC(V)) would be associated with decreased left ventricular (LV) contractile function and responsiveness to beta-adrenergic receptor (betaAR) stimulation. Absence of cardiac AC(V) expression was confirmed by RT-PCR and immunoblotting in AC(V)-deleted mice (AC(V) (-/-)). Compared to sibling mice with normal amounts of AC(V) (CON), basal and water-soluble forskolin derivative NKH477-stimulated cAMP production was reduced in both LV homogenates and in isolated cardiac myocytes. Basal LV +dP/dt (isolated perfused hearts) was increased (CON: 3,649 +/- 247 mmHg/s; AC(V) (-/-): 4,625 +/- 350 mmHg/s; p = 0.035, n = 10), but the potency of dobutamine on LV +dP/dt was decreased by AC(V) deletion (log EC(50): CON: -6.83 +/- 0.14 M; AC(V) (-/-): -5.99 +/- 0.15 M; p = 0.0007, n = 10). The initial rates of ATP-dependent sarcoplasmic reticulum calcium uptake, assessed in LV homogenates, showed that AC(V) deletion increased SERCA2a affinity for Ca(2+) (log EC(50): CON: -5.94 +/- 0.03 M; AC(V) (-/-): -6.09 +/- 0.02 M; p = 0.001, n = 8). AC(V) deletion is also associated with increased phospholamban phosphorylation, decreased type 1 protein phosphatase catalytic subunit content and activity, and reduced cardiac Galphas protein content. In conclusion, AC(V) deletion has a favorable effect on basal LV function despite reduced cAMP levels. Increased SERCA2a affinity for Ca(2+) and increased phospholamban phosphorylation are contributing factors. However, AC(V) deletion is associated with reduced LV contractile responsiveness to betaAR stimulation, an effect that is associated with reduced Galphas protein content and reduced cAMP generating capacity in cardiac myocytes.

Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus.

Adeno-associated viral vectors (AAV) can direct long-term gene expression in post-mitotic cells. Previous studies have established that long-term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 10(11) DNase resistant particles (drp; high-performance liquid chromatography (HPLC)-purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20-32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64-5.28 x 10(13) drp; HPLC-purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long-term expression of therapeutic genes.

Cardiac-directed expression of adenylyl cyclase and heart rate regulation.

Mice with cardiac-directed overexpression of AC(VI) show increased cardiac responsiveness to beta-adrenergic receptor stimulation but regulation of heart rate is unknown. Telemetry was used to test the hypothesis that mice overexpressing cardiac adenylyl cyclase type VI (AC(VI)) would have normal heart rate regulation. Mice overexpressing cardiac AC(VI) were generated using the alphaMHC promoter and studied 10 days after implantation of telemetry devices. Cardiac transgene AC(VI) presence and expression was verified using PCR, RT-PCR and immunoblotting. Ambulatory heart rates were assessed using time and frequency domain analysis over two 24 hour light-dark cycles. Heart rates then were assessed following pharmacological blockade. Time domain analyses showed ambulatory heart rates were unchanged (AC(VI): 597 +/- 15 (SEM) bpm, Control: 595 +/- 12 bpm; p = 0.92). Circadian heart rate variability was preserved and not different from control mice (ANOVA, p = 0.52). Frequency domain analysis of heart rate variability also was unchanged. No difference in heart rate response to pharmacological autonomic blockade was found (intrinsic heart rate: AC(VI) 622 +/- 17 bpm, control 616 +/- 16 bpm, p = 0.79). In conclusion, mice overexpressing cardiac AC(VI) have normal conscious ambulatory heart rates and normal heart rate variability. Overexpression of cardiac AC(VI) does not result in altered heart rate regulation in contrast to cardiac overexpression of other elements of the beta-adrenergic signaling pathway.