N. Chin Lai

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.

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.

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

Scar formation occurs during the late stages of the inflammatory response but, when excessive, produces fibrosis that can lead to functional and structural damage of tissues. Here, we show that the profibrogenic agonist, transforming growth factor β1, transcriptionally decreases expression of Exchange protein activated by cAMP 1 (Epac1) in fibroblasts/fibroblast-like cells from multiple tissues (i.e., cardiac, lung, and skin fibroblasts and hepatic stellate cells). Overexpression of Epac1 inhibits transforming growth factor β1-induced collagen synthesis, indicating that a decrease of Epac1 expression appears to be necessary for the fibrogenic phenotype, an idea supported by evidence that Epac1 expression in cardiac fibroblasts is inhibited after myocardial infarction. Epac and protein kinase A, a second mediator of cAMP action, have opposite effects on migration but both inhibit synthesis of collagen and DNA by fibroblasts. Epac is preferentially activated by low concentrations of cAMP and stimulates migration via the small G protein Rap1 but inhibits collagen synthesis in a Rap1-independent manner. The regulation of Epac expression and activation thus appear to be critical for the integration of pro- and anti-fibrotic signals and for the regulation of fibroblast 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.

Increased Cardiac Adenylyl Cyclase Expression Is Associated With Increased Survival After Myocardial Infarction

Background— Cardiac-directed expression of adenylyl cyclase type VI (ACVI) in mice results in structurally normal hearts with normal basal heart rate and function but increased responses to catecholamine stimulation. We tested the hypothesis that increased left ventricular (LV) ACVI content would increase mortality after acute myocardial infarction (MI). Methods and Results— Transgenic mice with cardiac-directed ACVI expression and their transgene-negative littermates (control) underwent coronary ligation, and survival, infarct size, and LV size and function were assessed 1 to 7 days after MI. Mice with increased ACVI expression had increased survival (control 41%, ACVI 74%; P=0.004). Infarct size and myocardial apoptotic rates were similar in ACVI and control mice; however, ACVI mice had less LV dilation (P<0.001) and increased ejection fractions (P<0.03). Three days after MI, studies in isolated perfused hearts showed that basal LV +dP/dt was similar, but graded dobutamine infusion was associated with a more robust LV contractile response in ACVI mice (P<0.05). Increased LV function was associated with increases in cAMP generation (P=0.0002), phospholamban phosphorylation (P<0.04), sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) affinity for calcium (P<0.015), and reduced AV block (P=0.04). Conclusions— In acute MI, increased cardiac ACVI content attenuates adverse LV remodeling, preserves LV contractile function, and reduces mortality.

Human Oxidation-Specific Antibodies Reduce Foam Cell Formation and Atherosclerosis Progression

OBJECTIVES We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis. BACKGROUND The unregulated uptake of OxLDL by macrophage SR contributes to foam cell formation, but the importance of this pathway in vivo is uncertain. METHODS Cholesterol-fed low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated with intraperitoneal infusion of human IK17-Fab (2.5 mg/kg) 3 times per week for 14 weeks. Because anti-human antibodies developed in these mice, LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice (lacking the ability to make immunoglobulins due to loss of T- and B-cell function) were treated with an adenoviral vector encoding adenovirus expressed (Adv)-IK17-scFv or control adenovirus-enhanced green fluorescent protein vector intravenously every 2 weeks for 16 weeks. RESULTS In LDLR(-/-) mice, infusion of IK17-Fab was able to sustain IK17 plasma levels for the first 8 weeks, but these diminished afterward due to increasing murine anti-IK17 antibody titers. Despite this, after 14 weeks, a 29% decrease in en face atherosclerosis was noted compared with phosphate-buffered saline-treated mice. In LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice, sustained levels of plasma IK17-scFv was achieved by Adv-IK17-scFv-mediated hepatic expression, which led to a 46% reduction (p < 0.001) in en face atherosclerosis compared with adenovirus-enhanced green fluorescent protein vector. Importantly, peritoneal macrophages isolated from Adv-IK17-scFv treated mice had decreased lipid accumulation compared with adenovirus-enhanced green fluorescent protein-treated mice. CONCLUSIONS These data support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis and demonstrate that oxidation-specific antibodies reduce the progression of atherosclerosis, suggesting their potential in treating cardiovascular disease in humans.

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 1011 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 × 1013 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. Copyright

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.