Data from the initial funding period of the current award indicate that increased cardiac AC type 6 (AC6), a dominant AC isoform expressed in mammalian cardiac myocytes, has protean beneficial effects on the failing left ventricle (LV). These effects, so consistent in a variety of species and pathophysiological models, must be reconciled with the dire consequences on the heart of 2-adrenergic receptor stimulation and elevations in intracellular cAMP. Logic would dictate that either: a) cAMP is not bad for the heart after all-that it is something else which leads to poor outcomes when cAMP levels are increased in the failing heart;or b) increased AC6 has beneficial cardiac effects independent of cAMP, which counterbalance its expected deleterious effects. Using pharmacological inhibitors and other approaches, our data suggest that many of the beneficial effects of increased cardiac AC6 expression do not require increased cAMP generation. Because of the inherent limitations of studies using pharmacological inhibition, we generated a catalytically inactive AC6 mutant (AC6mut) molecule by substitution of Ala for Asp at position 426 in the catalytic core. This AC6mut does not generate cAMP, but retains the cellular distribution pattern and favorable signaling effects associated with AC6. These data indicate that the beneficial effects of AC6, at least in part, are independent of cAMP. We propose now to conduct a series of experiments, both in vitro and in vivo, to establish unassailable mechanisms for the beneficial effects. The ideal inotrope would increase cardiac contractile function in the absence of 2AR stimulation or cAMP generation, improve Ca2+ handling, reduce adverse remodeling and apoptosis, and have favorable electrophysiological properties. With the exception of its effects on cAMP generation, increased cardiac AC6 expression accomplishes many of these features, and the FDA has approved an NHLBI-funded trial of AC6 gene transfer in patients with severe CHF (ClinicalTrials.gov NCT00787059). However, there are four reasons to conduct studies of the cAMP-incompetent AC6 mutant: 1) to obtain definitive mechanistic data on whether AC6's beneficial cardiac effects are unrelated to cAMP generation;2) to conduct structure-activity studies of the AC6mut molecule by generating a variety of AC6mut fragment molecules and to determine the effects of subcellular targeting of the AC6mut fragments on Ca2+ handling and Akt activation;3) to determine the cardiac effects of AC6mut in vivo and 4) to generate a suitable long term regulated expression vector encoding the optimal AC6mut fragment, and conduct preclinical gene transfer studies to determine its efficacy and safety in improving function of the failing heart. The studies are designed to uncover mechanisms, but also to have clinical relevance. For example, in clinical heart failure, agents that increase cardiac contractile function by increasing cAMP levels have failed to prolong survival. The AC6mut, based on preliminary data, preserves the beneficial effects seen with AC6, but does so in the absence of increased cAMP production, thus fulfilling features of a potentially ideal therapeutic inotrope.

Public Health Relevance

We have modified adenylyl cyclase type 6 (AC6), a molecule that regulates cardiac function. This modified AC6 (AC6mut) preserves the beneficial effects on the heart that are seen with AC6, but does so in the absence of increased cAMP production. In patients with heart failure, drugs that improve heart function by increasing cAMP levels have failed to reduce mortality. Our studies will determine if this modified AC6 molecule will be a suitable heart failure treatment.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL081741-07
Application #
8206611
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Schwartz, Lisa
Project Start
2005-09-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
7
Fiscal Year
2012
Total Cost
$350,460
Indirect Cost
$102,960
Name
Veterans Medical Research Fdn/San Diego
Department
Type
DUNS #
933863508
City
San Diego
State
CA
Country
United States
Zip Code
92161
Lai, N Chin; Gao, Mei Hua; Giamouridis, Dimosthenis et al. (2015) Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice. Hum Gene Ther 26:347-56
Lee, Kristen L; Hoey, David A; Spasic, Milos et al. (2014) Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo. FASEB J 28:1157-65
Gao, Mei Hua; Lai, Ngai Chin; Tang, Tong et al. (2013) Preserved cardiac function despite marked impairment of cAMP generation. PLoS One 8:e72151
Gao, Mei Hua; Lai, N Chin; Miyanohara, Atsushi et al. (2013) Intravenous adeno-associated virus serotype 8 encoding urocortin-2 provides sustained augmentation of left ventricular function in mice. Hum Gene Ther 24:777-85
Tang, Tong; Lai, N Chin; Wright, Adam T et al. (2013) Adenylyl cyclase 6 deletion increases mortality during sustained β-adrenergic receptor stimulation. J Mol Cell Cardiol 60:60-7
Tang, Tong; Hammond, H Kirk (2013) Gene transfer for congestive heart failure: update 2013. Transl Res 161:313-20
Timofeyev, Valeriy; Myers, Richard E; Kim, Hyo Jeong et al. (2013) Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes. Circ Res 112:1567-76
Fang, Hongfei; Lai, Ngai Chin; Gao, Mei Hua et al. (2012) Comparison of adeno-associated virus serotypes and delivery methods for cardiac gene transfer. Hum Gene Ther Methods 23:234-41
Lai, N Chin; Gao, Mei Hua; Tang, Eric et al. (2012) Pressure overload-induced cardiac remodeling and dysfunction in the absence of interleukin 6 in mice. Lab Invest 92:1518-26
Lai, N Chin; Tang, Tong; Gao, Mei Hua et al. (2012) Improved function of the failing rat heart by regulated expression of insulin-like growth factor I via intramuscular gene transfer. Hum Gene Ther 23:255-61

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