Modulation of ventricular function through gene transfer <em>in vivo</em>

R Hajjar

U Schmidt

T Matsui

J Guerrero

Cardiovascular Research Center and Heart Failure and Cardiac Transplantation Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129; and ^{Boston University School of Medicine, Boston, MA 02118}

^{To whom reprint requests should be addressed at: Cardiovascular Research Center, Massachusetts General Hospital, East, 149 13th Street, 4th Floor, Room 4207, Charlestown, MA 02129.}

Communicated by Alexander Leaf, Massachusetts General Hospital, Charlestown, MA

Received 1997 Nov 27; Accepted 1998 Feb 26.

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^{ATPase (SERCA2a). By using this approach, rat hearts were transduced}in vivo with 5 × 10^{pfu of recombinant adenoviral vectors carrying cDNA for either PL, β-galactosidase (β-gal), or modified green fluorescent 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.βgal. 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.βgal (92.6 ± 5.9 mmHg, n = 6). Both peak rate of pressure rise and pressure fall (+3, 210 ± 298 mmHg/s, −2, 117 ± 178 mmHg/s, n = 8) were decreased in hearts overexpressing PL compared with uninfected hearts (+5, 225 ± 136 mmHg/s, −3, 805 ± 97 mmHg/s, n = 6) or hearts infected with Ad.βgal (+5, 108 ± 167 mmHg/s, −3, 765 ± 121 mmHg/s, 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.βgal (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.

Keywords: sarcoplasmic reticulum, phospholamban, calcium, Ca^{ATPase, adenovirus}

Abstract

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^{ATPase (SERCA2a) pump regulates the uptake of Ca}^{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 PL/SERCA2a 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 transfer has been previously achieved predominantly by direct injection into the myocardium or perfusion of an isolated coronary segment (7, 8). Either approach results in focal overexpression of the transgene and is therefore unlikely to effectively modulate global cardiac function. In this study we used a catheter-based technique to achieve highly effective transgene expression in rat heart in vivo. In vivo overexpression of PL resulted in profound physiological alterations in cardiac function including a decrease in left ventricular systolic pressure and an increase in diastolic pressure and a prolonged isovolumic relaxation. As in single cells overexpressing PL, these effects mimic abnormalities seen in experimental and human heart failure. Our data suggest that global adenoviral gene transfer to rodent hearts in vivo may be a useful tool for studying the molecular mechanisms regulating cardiac function. Overexpression of PL in particular creates an acquired phenotype that recapitulates many abnormalities seen in human heart failure and may provide a useful model for testing therapeutic interventions.

All data presented as mean ± SD. LVSP, left ventricular systolic pressure; LVDP, left ventricular end-diastolic pressure; HR, heart rate; +dP/dt, maximal rate of pressure rise; −dP/dt, maximal rate of pressure fall; τ, time constant of relaxation; n, number of hearts.

Acknowledgments

This work was supported in part by grants from the National Institutes of Health: HL 50361 and HL 57623 (R.J.H.); HL 54202, HL 59521, and AI 40970 (A.R.); and HL 49574 and HL 52249 (J.K.G.), and by donations to the Katherine Catani Memorial Fund (A.R.).

Acknowledgments

ABBREVIATIONS

PL	phospholamban
EGFP	modified green fluorescent protein
SR	sarcoplasmic reticulum
SERCA2a	SR Ca^ATPase
Ad	adenovirus
β-gal	β-galactosidase
LVSP	left ventricular systolic pressure

ABBREVIATIONS

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