Mei Hua Gao

Adenylylcyclase Increases Responsiveness to Catecholamine Stimulation in Transgenic Mice

BACKGROUND The cellular content of cAMP generated by activation of adenylylcyclase (AC) through the beta-adrenergic receptor (betaAR) is a key determinant of a cells response to catecholamine stimulation. We tested the hypothesis that increased AC content, independently of betaAR number, increases responsiveness to catecholamine stimulation in vivo. METHODS AND RESULTS Transgenic mice with cardiac-directed expression of ACVI showed increased transgene AC expression but no change in myocardial betaAR number or G-protein content. When stimulated through the betaAR, cardiac function was increased, and cardiac myocytes showed increased cAMP production. In contrast, basal cAMP and cardiac function were normal, and long-term transgene expression was not associated with abnormal histological findings or deleterious changes in cardiac function. CONCLUSIONS The amount of AC sets a limit on cardiac beta-adrenergic signaling in vivo, and increased AC, independent of betaAR number and G-protein content, provides a means to regulate cardiac responsiveness to betaAR stimulation. Overexpressing an effector (AC) does not alter transmembrane signaling except when receptors are activated, in contrast to receptor/G-protein overexpression, which yields continuous activation and has detrimental consequences. Our findings establish the importance of AC content in modulating beta-adrenergic signaling in the heart, suggesting a new target for safely increasing cardiac responsiveness to betaAR stimulation.

Cardiac-Directed Adenylyl Cyclase Expression Improves Heart Function in Murine Cardiomyopathy

BACKGROUND We tested the hypothesis that increased cardiac myocyte adenylyl cyclase (AC) content increases cardiac function and response to catecholamines in cardiomyopathy. METHODS AND RESULTS Transgenic mice with cardiac-directed expression of AC type VI (ACVI) were crossbred with mice with cardiomyopathy induced by cardiac-directed Gq expression. Gq mice had dilated left ventricles, reduced heart function, decreased cardiac responsiveness to catecholamine stimulation, and impaired beta-adrenergic receptor (betaAR)-dependent and AC-dependent cAMP production. Gq/AC mice showed improved basal cardiac function in vivo (P=0.01) and ex vivo (P<0.0005). When stimulated through the betaAR, cardiac responsiveness was increased (P=0.02), and cardiac myocytes showed increased cAMP production in response to isoproterenol (P=0.03) and forskolin (P<0.0001). CONCLUSIONS Increasing myocardial ACVI content in cardiomyopathy restores cAMP-generating capacity and improves cardiac function and responsiveness to betaAR stimulation.

Intracoronary Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function in Heart Failure

Background—We tested the hypothesis that intracoronary delivery of an adenovirus encoding adenylyl cyclase type VI (Ad.ACVI) would be associated with increased left ventricular (LV) function in pigs with congestive heart failure. Methods and Results—Pigs (52±6 kg; n=16) underwent placement of pacemakers, LV pressure transducers, and left atrial and aortic catheters. Physiological and echocardiographic studies were obtained from conscious animals 13 days later, and pacing was initiated (220 bpm). Seven days later, measures of LV function were reduced, documenting severe LV dysfunction and dilation. Pigs then received intracoronary Ad.ACVI (1.4×1012 vp; n=7) or saline (PBS; n=9) (randomized, blinded), with concomitant infusion of nitroprusside (50 μg/min, 6.4 minutes) to increase gene transfer. Pacing was continued for 14 days, and final studies were obtained. The a priori key end point was change in LV dP/dt during isoproterenol infusion (pre-Ad.ACVI value minus value after 21 days of pacing). Pigs receiving Ad.ACVI showed a smaller decrease in both LV +dP/dt (P =0.0014) and LV −dP/dt (P =0.0008). Serial echocardiography showed that Ad.ACVI treatment was associated with increased LV function and reduced LV dilation and that end-systolic wall stress was reduced. AC-stimulated cAMP production was increased 1.7-fold in LV samples from Ad.ACVI-treated pigs (P =0.006), and B-type natriuretic peptide was reduced (0.035). Gene transfer was confirmed by polymerase chain reaction. Conclusions—ACVI gene transfer increases LV function and attenuates deleterious LV remodeling in congestive heart failure.

Adenylyl Cyclase Increases Survival in Cardiomyopathy

Background—To test the hypothesis that increased cardiac adenylyl cyclase type VI (ACVI) 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 ACVI. We also assessed myocardial hypertrophy after prolonged cardiac expression of Gq versus coexpression of Gq and ACVI. 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 ACVI (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 ACVI, 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 &mgr;m2, n=4; Gq/AC, 1721±166 &mgr;m2, 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 ACVI restores myocyte AC function, improves heart function, increases cAMP generation, abrogates myocardial hypertrophy, and increases survival in Gq cardiomyopathy.

Intracoronary Delivery of Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function and cAMP-Generating Capacity

BackgroundWe tested the hypothesis that the intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (ACVI) would increase cardiac function in pigs. Methods and ResultsLeft ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after the intracoronary delivery of histamine followed by the intracoronary delivery of an adenovirus encoding lacZ (control) or ACVI (1.4×1012 vp). Animals that had received ACVI gene transfer showed increases in peak LV dP/dt (average increase of 1267±807 mm Hg/s;P =0.0002) and cardiac output (average increase of 39±20 mL · kg−1 · min−1;P <0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving ACVI gene transfer showed increased ACVI protein content (P =0.0007) and stimulated cAMP production (P =0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks (P <0.0002). ConclusionsIntracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.

Adenylyl Cyclase Type 6 Deletion Decreases Left Ventricular Function via Impaired Calcium Handling

Background— Adenylyl cyclases (ACs) are a family of effector molecules for G-protein–coupled receptors. The 2 ACs most abundantly expressed in cardiac myocytes are types 5 (AC5) and 6 (AC6), which have 65% amino acid homology. It has been speculated that coexpression of 2 AC types in cardiac myocytes represents redundancy, but the specific role of AC6 in cardiac physiology and its differences from AC5 remain to be defined. Methods and Results— We generated transgenic mice with targeted deletion of AC6. Deletion of AC6 was associated with reduced left ventricular contractile function (P=0.026) and relaxation (P=0.041). The absence of AC6 was associated with a 48% decay in &bgr;-adrenergic receptor–stimulated cAMP production in cardiac myocytes (P=0.003) and reduced protein kinase A activity (P=0.015). In addition, phospholamban phosphorylation was reduced (P=0.015), sarcoplasmic reticulum Ca2+-ATPase activity was impaired (P<0.0001), and cardiac myocytes showed marked abnormalities in calcium transient formation (P=0.001). Conclusions— The combination of impaired cardiac cAMP generation and calcium handling that result from AC6 deletion underlies abnormalities in left ventricular function. The biochemical and physiological consequences of AC6 deletion reveal it to be an important effector molecule in the adult heart, serving unique biological functions not replicated by AC5.

Activation of cardiac adenylyl cyclase expression increases function of the failing ischemic heart in mice.

OBJECTIVES This study sought to evaluate whether increased left ventricular (LV) adenylyl cyclase VI (AC(VI)) expression, at a time when severe congestive heart failure (CHF) was present, would increase function of the actively failing heart. BACKGROUND Increased LV AC(VI) content markedly reduces mortality and increases LV function after acute myocardial infarction (MI) in mice. However, the effects of increased cardiac AC(VI) content in the setting of severe heart failure caused by ischemic cardiomyopathy are unknown. METHODS Mice with cardiac-directed and regulated expression of AC(VI) underwent coronary artery ligation to induce severe CHF 5 weeks later. AC(VI) expression was then activated in 1 group (AC-On) but not the other (AC-Off). Multiple measures of LV systolic and diastolic function were obtained 5 weeks later, and LV samples were assessed for alterations in calcium and beta-adrenergic receptor signaling, apoptosis, and cardiac troponin I phosphorylation. RESULTS The LV systolic and diastolic function was increased 5 weeks after activation of AC(VI) expression. Improved LV function was associated with normalization of cardiac troponin I phosphorylation and reduced apoptosis. CONCLUSIONS Activation of cardiac AC(VI) expression in mice with ischemic cardiomyopathy and severe CHF improves function of the failing heart.

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.

Reduced Collagen Deposition in Infarcted Myocardium Facilitates Induced Pluripotent Stem Cell Engraftment and Angiomyogenesis for Improvement of Left Ventricular Function

OBJECTIVES The purpose of this study was to assess the effect of scar tissue composition on engraftment of progenitor cells into infarcted myocardium. BACKGROUND Scar tissue formation after myocardial infarction creates a barrier that severely compromises tissue regeneration, limiting potential functional recovery. METHODS In vitro: A tricell patch (Tri-P) was created from peritoneum seeded and cultured with induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, and mouse embryonic fibroblasts. The expression of fibrosis-related molecules from mouse embryonic fibroblasts and infarcted heart was measured by Western blot and quantitative reverse transcriptase polymerase chain reaction. In vivo: A Tri-P was affixed over the entire infarcted area 7 days after myocardial infarction in mice overexpressing adenylyl cyclase 6 (AC6). Engraftment efficiency of progenitor cells in hearts of AC6 mice was compared with that of control wild-type (WT) mice using a combination of in vivo bioluminescence imaging, post-mortem ex vivo tissue analysis, and the number of green fluorescent protein-positive cells. Echocardiography of left ventricular (LV) function was performed weekly. Hearts were harvested for analysis 4 weeks after Tri-P application. Mouse embryonic fibroblasts were stimulated with forskolin before an anoxia/reoxygenation protocol. Fibrosis-related molecules were analyzed. RESULTS In AC6 mice, infarcted hearts treated with Tri-P showed significantly higher bioluminescence imaging intensity and numbers of green fluorescent protein-positive cells than in WT mice. LV function improved progressively in AC6 mice from weeks 2 to 4 and was associated with reduced LV fibrosis. CONCLUSIONS Application of a Tri-P in AC6 mice resulted in significantly higher induced pluripotent stem cell engraftment accompanied by angiomyogenesis in the infarcted area and improvement in LV function.

Adenylyl Cyclase Type VI Increases Akt Activity and Phospholamban Phosphorylation in Cardiac Myocytes

Increased expression of adenylyl cyclase VI has beneficial effects on the heart, but strategies that increase cAMP production in cardiac myocytes usually are harmful. Might adenylyl cyclase VI have beneficial effects unrelated to increased β-adrenergic receptor-mediated signaling? We previously reported that adenylyl cyclase VI reduces cardiac phospholamban expression. Our focus in the current studies is how adenylyl cyclase VI influences phospholamban phosphorylation. In cultured cardiac myocytes, increased expression of adenylyl cyclase VI activates Akt by phosphorylation at serine 473 and threonine 308 and is associated with increased nuclear phospho-Akt. Activated Akt phosphorylates phospholamban, a process that does not require β-adrenergic receptor stimulation or protein kinase A activation. These previously unrecognized signaling events would be predicted to promote calcium handling and increase contractile function of the intact heart independently of β-adrenergic receptor activation. We speculate that phospholamban phosphorylation, through activation of Akt, may be an important mechanism by which adenylyl cyclase VI increases the function of the failing heart.