H. Kirk Hammond

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.

Normal and abnormal heart rate responses to exercise

N A RECENT STUDY’ we found impaired heart rate responses to maximal exercise in a subgroup of patients with coronary heart disease. Thallium scintigraphy demonstrated more myocardial scar in patients not limited by angina. The idea that the amount of viable myocardium may be an important factor in determining the chronotropic response to exercise was provocative and stimulated a review of the literature. Immediately other questions arose. Are patients with third degree atrioventricular block able to increase their cardiac output during exercise without adequate heart rate responses? The importance of the autonomic nervous system during exercise is unknown. Are cardiac transplantation patients able to exercise normally with denervated hearts? During exercise, inotropic factors may not be important in patients with chronic heart failure. Are these patients more dependent on chronotropic factors to increase their work capacity? Of the many factors ultimately important in determining the cardiac output, the heart rate is certainly the easiest to measure. By analysis of the heart rate response to exercise in a variety of disease states we felt that the interrelationships of inotropic state, stroke volume, autonomic dysfunction, and myocardial disease could be clarified. This paper reviews the normal and abnormal heart rate responses to exercise.

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.

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.

Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials

The current published clinical literature on angiogenic gene therapy for the treatment of myocardial ischemia does not include a single randomized, placebo-controlled trial. Based on current clinical literature, it is an unproven therapy. Successful animal studies combined with published reports of good outcomes in patients enrolled in uncontrolled trials has led to the expectation that angiogenic gene therapy will ultimately become a clinical reality. The next important landmark in the field will be the publication of data showing a favorable effect of angiogenic gene transfer in placebo-controlled, blinded clinical trials.