In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.

BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.

Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance.

BACKGROUND: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. METHODS AND RESULTS: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. CONCLUSIONS: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Early effects of right ventricular volume overload on ventricular performance and beta-adrenergic signaling.

OBJECTIVE: Right ventricular dysfunction is a poorly understood but persistent clinical problem. This study was undertaken to evaluate ventricular performance and beta-adrenergic receptor signaling in a tricuspid regurgitation model of right ventricular overload. METHODS: Seventeen dogs were chronically instrumented with epicardial dimension transducers. By means of the shell-subtraction model, right ventricular pressure-volume relationships were evaluated in normal and right ventricular overload states. Right ventricular chamber performance was quantified by the stroke work at an end-diastolic volume relationship. RESULTS: Right ventricular volume overload caused a 28% +/- 11% and 31% +/- 9% decline in chamber performance acutely and at 1 week, respectively, whereas end-diastolic volume increased from 45 +/- 21 to 60 +/- 30 mL (P =. 019). beta-Adrenergic receptor signaling in myocardial samples was assessed, examining adenylyl cyclase and G-protein-coupled receptor kinase activity. Stimulated adenylyl cyclase activity significantly decreased, and G-protein-coupled receptor kinase activity significantly increased in both left and right ventricular samples caused by increased levels of beta-adrenergic receptor kinase 1. No change in beta-adrenergic receptor density was seen at 1 week. CONCLUSIONS: Early right ventricular overload is associated with impaired right ventricular chamber contractility, dilation, and, importantly, a biventricular alteration of beta-adrenergic receptor signaling.

Ex vivo adenovirus-mediated gene transfer to the adult rat heart.

OBJECTIVE: The ability to transfer genes to adult myocardium may have therapeutic implications for cardiac transplantation. We investigated the feasibility of adenovirus-mediated transfer of marker genes LacZ and Luciferase, as well as the potentially therapeutic gene of the human beta2-adrenergic receptor in a rat heterotopic heart transplant model. METHODS: Donor hearts were flushed with 10(12) total viral particles of one of three transgenes. Hearts were harvested at various time points after transplantation. LacZ-treated hearts were assessed by histologic staining and Luciferase-treated hearts were assayed for specific luminescence activity. Hearts treated with beta2-adrenergic receptor underwent radioligand binding assays and immunohistochemistry with the use of an antibody specific for the human beta2-adrenergic receptor. RESULTS: LacZ hearts revealed diffuse myocyte staining as opposed to none within controls at 5 days. Luciferase hearts demonstrated a mean activity of 970,000 +/- 220,000 arbitrary light units versus 500 +/- 200 for the controls (p = 0.001). Total beta2-adrenergic receptor densities (fmol/mg membrane protein) for hearts that received the beta2-adrenergic receptor transgene at 3, 5, 7, 10, and 14 days after infection were as follows: right ventricle, 488.5 +/- 126.8, 519.4 +/- 81.8,* 477.1 +/- 51.8,* 183.0 +/- 6.5,* and 82.7 +/- 19.1; left ventricle, 511.0 +/- 167.6, 1206.4 +/- 321.8,* 525.3 +/- 188.7, 183.5 +/- 18.6,* and 75.9 +/- 15.2 (*p < 0.05 vs control value of 75.6 +/- 6.4). Immunohistochemical analysis revealed diffuse staining of varying intensity within myocardial sarcolemmal membranes. CONCLUSIONS: We conclude that global overexpression of different transgenes is possible during cardiac transplantation and, ultimately, adenovirus-mediated gene transfer may provide a unique opportunity for genetic manipulation of the donor organ, potentially enhancing its function.

Alterations in hemodynamics and left ventricular contractility during carbon dioxide pneumoperitoneum.

BACKGROUND: Carbon dioxide (CO2) pneumoperitoneum has been shown to adversely affect hemodynamics in patients. This study specifically examines the potential contribution of altered left ventricular contractility (LVC) to hemodynamic changes observed during CO2 pneumoperitoneum. METHODS: In a canine model, LV volumes, LV pressure, and intrathoracic and central venous pressures were recorded both at basal intra-abdominal pressure (IAP) and after CO2 insufflation to produce IAPs of 5-25 mmHg. RESULTS: At IAPs greater than 15 mmHg, cardiac output and LV end-diastolic volume decreased. Mean arterial pressure and heart rate were unchanged. LVC, quantified using the linear Frank-Starling relationship, was not affected by increases in IAP. CONCLUSIONS: This study is the first to quantify LVC during CO2 pneumoperitoneum and demonstrates no changes in contractility over IAPs from 5 to 25 mmHg. In the dog model, any hemodynamic alterations induced by CO2 pneumoperitoneum are secondary to altered LV preload and not alterations in contractility or LV afterload.

Delivery of primary autologous skeletal myoblasts into rabbit heart by coronary infusion: a potential approach to myocardial repair.

Myocardial repair after injury is limited because the adult heart cannot regenerate. We propose using autologous skeletal muscle cells (myoblasts) as a source of reserve cells for repair of regions of damaged myocardium. This report examines two potential methods for the transfer of cells to the myocardium: selective coronary catheterization, and myoblast infusion or myoblast injection directly into the left ventricular wall. Autologous, primary rabbit skeletal myoblasts were harvested, were transduced ex vivo with adenoviruses expressing the Escherichia coli beta-galactosidase (beta-gal) gene, and were infused selectively into the coronary circulation or injected directly into the myocardial wall. After either delivery method, beta-gal expression was detectable at the earliest times examined (3 days) and persisted for several weeks. The method of delivery influenced the spatial pattern of beta-gal expression. After direct injection, a localized concentration of myoblasts that decreased with distance from the injection site was visible primarily in the myocardial layer of the ventricle, although occasional staining could be detected in other layers. After coronary infusion, discrete punctate or linear foci of beta-gal expression were found throughout the distribution of the left coronary circulation in all cardiac layers. After infusion or injection, beta-gal-positive cells were seen in direct physical apposition to cardiocytes; interestingly, beta-gal could be detected also in some branched cells with clear cross-striations. Autologous myoblasts survived with no obvious dysrhythmic effects despite their presence in extensive or discrete loci in the myocardium. These observations provide the first evidence that myoblast transfer is possible by catheter-based methods, and they create the basis for studies to investigate the functional consequences of myoblast infusion in damaged heart.

The in vivo quantification of myocardial performance in rabbits: a model for evaluation of cardiac gene therapy.

Evaluating cardiac gene therapy in the intact animal requires an index of cardiac function capable of detecting regional differences in contractility in a load-independent fashion. Potentially load-insensitive measures of ventricular performance were therefore evaluated in 10 open- and closed-chested, anesthetized rabbits. LV transmural pressure and myocardial segment length were measured using micromanometry and sonomicrometry during steady-state and transient inferior vena caval occlusion, over a range of inotropic and loading conditions. For each intervention, segmental stroke work was calculated as the area within the left ventricular transmural pressure-length loops at a given end-diastolic segment length during inferior vena caval occlusion; regression analysis was applied to obtain the linear Frank-Starling relationship. In both open- and closed-chested states, these relationships were highly linear (r = 0.97 +/- 0.1) and reproducible. The slope of the linear relationship between segmental stroke work and end-diastolic segment length increased significantly with calcium and epinephrine infusions (P < 0.05 v control) but was not significantly altered by decreased afterload or increased afterload (P > 0.4). The x-intercept was not significantly altered by changes in intropy or afterload (P > 0.4). These data validate the linear Frank-Starling relationship and the slope, MW, as a load-insensitive index of contractility in the intact rabbit. This study presents a novel approach to the quantification of regional cardiac function in smaller animals.

Ventricular and myocardial efficiencies during acute aortic regurgitation in conscious dogs.

BACKGROUND: Alterations in cardiac efficiency may signal pathologic stresses and energetic adaptation during aortic regurgitation (AR). METHODS AND RESULTS: LV systolic function, left coronary blood flow, and AVO2 difference were measured in conscious dogs to assess LV and contractile efficiencies at baseline, 1 day (n=10), 1 week (n=10), and 3 weeks (n=8) of AR. LV systolic function was assessed by the preload recruitable stroke work relationship. Total LV Efficiency (TEFF=SWxHeart Rate/MVO2) and contractile efficiency (CEFF=1/the slope of the MVO2 -pressure-volume area relationship) and steady-state potential energy (PVA-SWxHR), mechanical coupling efficiency (MCE=SWxHR/PVA) were calculated. LV systolic function decreased by 17% at 1 day (P<.05) and by 24% at 3 weeks (P<.05). CEFF decreased from 58+/-8% to 38+/-10% (P<.05) at 1 day, normalized at 1 week, and decreased to 28+/-14% at 3 weeks (P<.05). TEFF was not altered at 1 day and 1 week but decreased by 3 weeks (P<.05). MCE trended downward from baseline of 47+/-5%, reaching significance at 3 weeks (34+/-6%, P<.05). CONCLUSIONS: CEFF decreases acutely, indicating diminished economy of myocardial contraction. CEFF normalizes at 1 week, suggesting adequate compensation. TEFF is not altered in early AR. By 3 weeks, LV systolic dysfunction is accompanied by depressed TEFF, mechanical coupling, and CEFF signaling the onset of decompensated AR. Thus, volume overload of acute AR resulted in early compensation at the expense of myocardial efficiency with subsequent global dysfunction characterized by depressed LV systolic mechanics, mechanical, coupling, and contractile efficiencies.

Mechanical determinants of myocardial oxygen consumption in conscious dogs.

A new practical descriptor of metabolic to mechanical myocardial energy transfer (MET), termed the virtual work model, was evaluated in 32 conscious dogs and in 8 isolated canine hearts. An index of total mechanical energy expenditure (TME) was calculated as the sum of external energy (stroke work) and an internal energy index of heat (left ventricular end-diastolic volume times left ventricular mean ejection pressure). Physiological comparison of TME (x-axis) and myocardial oxygen consumption (MVO2; y-axis) yielded highly linear MET relationships (mean r = 0.93 +/- 0.07), with an average slope of 0.86 +/- 0.39 (SD) and a y-intercept of 9.1 +/- 6.4 mW/ml myocardium. The linear MVO2-TME relationship did not vary under steady-state vs. dynamic vena caval occlusion, increased heart rate, increased afterload, or increased inotropic state with calcium infusion. Compared with five other indexes of myocardial energetics, the virtual work model of MET was the most linear, the most practical in not requiring determination of the end-systolic pressure-volume relationship, and the most accurate predictor of MVO2 under normal and altered hemodynamic conditions.