Inotropic response to β‐adrenergic receptor stimulation and anti‐adrenergic effect of ACh in endothelial NO synthase‐deficient mouse hearts

Abstract

1 The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti‐adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS–/–) mice. 2 eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT‐PCR analysis. 3 In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dt_max) of eNOS–/– hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS–/–; dP/dt_max: 3700 ± 712 mmHg s⁻¹ WT vs. 4493 ± 320 mmHg s⁻¹ eNOS–/–). 4 The dobutamine (10‐300 nm)‐induced increase in LVP was enhanced in eNOS–/– hearts. In contrast, L‐type Ca²⁺ currents (I_Ca,L) in isolated cardiomyocytes of WT and eNOS–/– hearts showed no differences after β‐adrenergic stimulation. Dibutyryl‐cGMP (50 μm) reduced basal I_Ca,L in WT cells to 72 ± 12 % while eNOS–/–I_Ca,L was insensitive to the drug. The pre‐stimulated I_Ca,L (30 nm isoproterenol) was attenuated by dibutyryl‐cGMP in WT and eNOS–/– cells to the same extent. 5 The Ca²⁺ (1.5‐4.5 mm)‐induced increase in inotropy was not different between the two experimental groups and β‐adrenergic receptor density was increased by 50 % in eNOS–/– hearts. 6 The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti‐adrenergic effect of both compounds was identical in WT and eNOS–/– hearts. Measurement of I_Ca,L in isolated cardiac myocytes yielded similar results. 7 These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to β‐adrenergic stimulation and with elevated β‐adrenergic receptor density, (2) the unaltered response of I_Ca,L in eNOS–/– cardiac myocytes to β‐adrenergic stimulation suggests that endothelium‐derived NO is important in mediating the whole‐organ effects and (3) eNOS is unimportant for the anti‐adrenergic effect of ACh and adenosine.