Muscarinic receptor stimulation fails to modify beta-adrenergic activation in left ventricular myocytes from neuregulin-1 knockout mice

The suppression of neuregulin-erbB receptor signaling in injured hearts in humans is associated with an increased risk of heart failure. One of the major biologic effects of neuregulin signaling is stimulation of acetylcholine nicotinic receptor synthesis whereas effects on muscarinic receptors are unclear. If muscarinic receptors are modified, a possible contributing mechanism for development of heart failure is a hyper-adrenergic activation due to a reduction of cardiac parasympathetic signaling. We tested the hypothesis that parasympathetic modulation of β-adrenergic stimulation is depressed in adult myocytes from transgenic mice heterozygous for knockout of the neuregulin-1 gene (KO).Methods:We evaluated isolated left ventricular myocyte contraction and intracellular Ca transients ([Ca ]i) from KO (n 48 experiments from 9 animals) and wildtype mice (WT; n 38 experiments from 9 animals). Fractional cell shortening and intracellular Ca were measured simultaneously in isolated myocytes loaded with the Ca sensitive fluorescence indicator fluo-3. Under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca ]o, 25oC), characteristics of myocyte contraction/relengthening and systolic/diastolic [Ca ]i were not different between WT and KO myocytes. β-adrenergic stimulation was then imposed by perfusion with 1 μmol/L isoproterenol. Modulation of the hyper-adrenergic state by the muscarinic agonist carbachol was then assessed. Results: The steady-state increases in fractional shortening (FS) and peak systolic [Ca ]i in response to isoproterenol were similar in both groups (WT FS: 10.0 0.74% to 16.5 0.58%; KO FS: 9.85 0.64% to 17.5 0.54%; WT peak systolic [Ca ]i: 416 20 to 623 28 nmol/L; KO peak systolic [Ca ]i: 418 23 to 651 32 nmol/L). In WT myocytes stimulated with isoproterenol, 10 and 100 μmol/L carbachol caused a dose-dependent decrease in FS (from 16.4 0.82% to 14.5 0.78% and 13.5 0.81%, respectively) and peak systolic [Ca ]i (from 604 35 to 564 32 and 542 34 nmol/L, respectively, both P 0.001). In KO myocytes, 10 and 100 μmol/L carbachol failed to attenuate either FS (from 17.3 0.79% to 16.5 0.80% and 15.9 0.84%, respectively) or peak systolic [Ca ]i (from 672 52 to 662 49 and 650 56 nmol/L, respectively, both P NS). In absence of isoproterenol, carbachol did not modify contractile function. Conclusion: In myocytes from neuregulin-1KOmice, muscarinic receptor stimulation does not attenuate the hyper-adrenergic state. These results suggest that hearts deficient in neuregulin signaling are unable to adequately counterbalance β-adrenergic activation by inhibitory parasympathetic activity. This may contribute to development of heart failure in injured hearts when neuregulin-erbB signaling is depressed.