Browsing by Author "Embry, S. L."

Objective: Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced P-adrenergic response is largely unknown. Methods: To evaluate the functional consequences of Gait overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of Galpha(i2). Results: Neither Galpha(i2) nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force (F-dev) increased after addition of 1 muM isoproterenol by 28.5 +/- 9.7 mN/mm(2) in the control group, which was unchanged from the initial response at t=0 h (23.7 +/- 3.8 mN/mm(2)). In sharp contrast, in preparations transfected with AdGalpha(i2), the response to isoproterenol was significantly attenuated (5.9 +/- 2.0 vs. 27.6 +/- 4.2 mN/mm(2), t=72 vs. 0 h, respectively, P<0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1 +/- 0.6% in the lacZ transfected myocytes, but only by 1.3 +/- 0.5% in Galpha(i2) transfected myocytes (1=72 h, P<0.01). In Galpha(i2) transfected myocytes, pertussis toxin restored beta-adrenergic responsiveness, indicating specificity of attenuation by the transgene. Conclusions: Overexpression of Galpha(i2) attenuates the positive inotropic effects of beta-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner. (C) 2002 Elsevier Science B.V. All rights reserved.

Objectives: The functional consequences of Na+/Ca2+ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. Methods and results: We investigated the Na+/K+-ATPase (NKA) inhibitor ouabain (0.5-16 mumol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% (P < 0.05). This was a result of an enhanced susceptibility to contracture after exposure to the drug (median concentration (25-75%): 4 (4-8) vs. 8 (6-16) mumol/l, P < 0.05). When analyzing relaxation before contracture, the maximum relaxation velocity was reduced (0.15 +/- 0.04 vs. 0.27 +/- 0.04 mum/s, P < 0.05) and the time from peak shortening to 90% of relaxation was increased (298 +/- 39 vs. 185 +/- 15 ms, P < 0.05). No differences in systolic and diastolic parameters were observed with the Na+ channel modulator BDF9198 (1 mumol/l). Conclusions: Inhibition of NKA by ouabain induces a combined diastolic and systolic dysfunction in NCX overexpressing rabbit myocytes. This may be the consequence of cytoplasmic Ca2+ overload due to inhibition of forward mode or induction of reverse mode Na+/Ca2+ exchange. In end-stage failing human myocardium and during digitalis treatment this mechanism may be of major importance. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.