Modulation of ventricular function through gene transfer in vivo

Abstract

We used a catheter-based technique to achieve generalized cardiac gene transfer in vivo and to alter cardiac function by overexpressing phospholamban (PL) which regulates the activity of the sarcoplasmic reticulum Ca 2؉ ATPase (SERCA2a). By using this approach, rat hearts were transduced in vivo with 5 ؋ 10 9 pfu of recombinant adenoviral vectors carrying cDNA for either PL, ␤-galactosidase (␤-gal), or modified green f luorescent protein (EGFP). Western blot analysis of ventricles obtained from rats transduced by Ad.PL showed a 2.8-fold increase in PL compared with hearts transduced by Ad.␤gal. Two days after infection, rat hearts transduced with Ad.PL had lower peak left ventricular pressure (58.3 ؎ 12.9 mmHg, n ‫؍‬ 8) compared with uninfected hearts (92.5 ؎ 3.5 mmHg, n ‫؍‬ 6) or hearts infected with Ad.␤gal (92.6 ؎ 5.9 mmHg, n ‫؍‬ 6). Both peak rate of pressure rise and pressure fall (؉3, 210 ؎ 298 mmHg͞s, ؊2, 117 ؎ 178 mmHg͞s, n ‫؍‬ 8) were decreased in hearts overexpressing PL compared with uninfected hearts (؉5, 225 ؎ 136 mmHg͞s, ؊3, 805 ؎ 97 mmHg͞s, n ‫؍‬ 6) or hearts infected with Ad.␤gal (؉5, 108 ؎ 167 mmHg͞s, ؊3, 765 ؎ 121 mmHg͞s, n ‫؍‬ 6). The time constant of left ventricular relaxation increased significantly in hearts overexpressing PL (33.4 ؎ 3.2 ms, n ‫؍‬ 8) compared with uninfected hearts (18.5 ؎ 1.0 ms, n ‫؍‬ 6) or hearts infected with Ad.␤gal (20.8 ؎ 2.1 ms, n ‫؍‬ 6). These differences in ventricular function were maintained 7 days after infection. These studies open the prospect of using somatic gene transfer to modulate overall cardiac function in vivo for either experimental or therapeutic applications.The regulation of intracellular calcium is intimately related to the systolic and diastolic function of cardiac cells (1, 2). The sarcoplasmic reticulum (SR), which releases calcium during systole and takes it up during diastole, plays an integral part in controlling the synchronized movement of calcium in myocardial cells. The SR Ca 2ϩ ATPase (SERCA2a) pump regulates the uptake of Ca 2ϩ into the SR during diastole. The function of the SERCA2a pump is regulated in turn by phospholamban (PL) (3). In its unphosphorylated form, PL inhibits the SERCA2a pump whereas in its phosphorylated form, this inhibition is relieved. A decrease in SERCA2a activity has been identified in a number of animal models of heart failure and in human heart failure and an increase in the relative ratio of PL to SERCA2a appears to be an important characteristic of both experimental and human heart failure (3, 4). We have previously modeled such alteration in the PL͞SERCA2a ratio by using adenoviral gene transfer to cardiocytes in vitro. Adenoviral overexpression of PL in vitro recapitulates many of the physiological abnormalities seen in heart failure, including prolonged relaxation and decreased contractile function. In contrast, overexpression of SERCA2a enhances relaxation and contractility of normal cardiomyocytes and rescues myocytes overexpressing PL from their abnormal phenotype (5, 6). Cardiac gene transf...

In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo

“…Whilst the majority of the transgene-positive cells were distributed throughout the ventricle, a third of the transgene-positive myocytes were distributed along the path of needle insertion. The results of all three studies 12,13 employing high pressure are consistent, suggesting that this is a truly reproducible method of gene delivery. The resulting grossly unphysiological pressure in the coronary artery that we have demonstrated presumably increases the fenestrations between the capillary endothelial cells, allowing transit of the adenovirus to the cardiac myocytes.…”

“…Using a similar model to Hajjar et al, 12 who performed experiments in the rat, we have demonstrated that intraventricular injection of recombinant adenovirus results in relatively efficient gene transfer. Although, in contrast to other interventions, this technique required an open chest, there were no associated alterations in baseline haemodynamics to suggest an artifactual increase in transfection efficiency.…”

“…Changing the vehicle had no significant effect on gene transfer (mean number of positive cells per section 129 ± 61, n = 4, P = NS versus high-pressure delivery). This procedure has been described in the literature as intracoronary delivery, 12 however we noted that there are numerous positive myocytes at the point of needle insertion. With both the normal buffer and the nominal calcium-free buffer, approximately a third of the transgene positive cells were in the vicinity of the needle track (35% and 31%, respectively).…”

“…High pressure adenoviral gene delivery was achieved using a modification to the approach of Hajjar et al 12 The right carotid artery was cannulated as before, and a 0.014 high fidelity pressure wire (RADI Medical System, Uppsala, Sweden) was manipulated into the left ventricle. The pressure wire was connected to a RADI Medical Systems PGA10, and left ventricular pressure was recorded using the MacLab system.…”

“…10,11 These studies show that the coronary flow rate, absolute amount of virus, time spent in the coronary circulation and composition of perfusate were major factors affecting intracoronary gene transfer 10 and increasing capillary permeability leads to improvements in gene transfer efficiency. 11 A related model of intracoronary gene delivery has confirmed feasibility provided transcapillary pressure is increased by simultaneous occlusion of the aorta and pulmonary artery in the rat, 12 and more recently, the rabbit. 13 It is upon this background that we attempted to resolve these discrepancies by intracoronary delivery of recombinant adenovirus, in combination with manipulations to increase capillary permeability and dwell time.…”