Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation

Abstract

Background and purpose: Muscarinic stimulation increases myofilament Ca2+ sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca 2+ sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC). Experimental approach: Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified. Key results: Carbachol (10 mmol·L -1 ) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 mmol·L -1 isoproterenol (20 Ϯ 1.5% and 56 Ϯ 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M2 receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment. Conclusion and implications:In failing ventricular muscle, muscarinic receptor activation, most likely via M2 receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca 2+ sensitivity. Enhancement of myofilament Ca 2+ sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.

NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors

“…Any inotropic response was expressed as an increase in maximal development of force [(dF/dt) max ]. As in failing adult rat hearts (Hussain et al, 2009), carbachol concentration-dependently increased contractility, reaching a maximum about 40% above basal (see Supplemental Fig. 1) with a half maximal effective concentration of about 1 mM.…”

“…In addition to the finding that NSC27366 is a competitive antagonist at mAChR, we show that in neonatal rat hearts the stimulation of M 2 mAChR or A 1 R induced ROCK-dependent positive inotropic responses, which are usually absent in adult myocardium (Hussain et al, 2009). Because such effects reoccur in failing rat myocardium and also have been reported in diseased human myocardium (Gergs et al, 2009;Hussain et al, 2009), it might be speculated that such coupling is part of the well-known fetal gene program being reinitiated in the failing heart (Katz, 1990).…”

“…Exposure to ACh improves survival after both acute and chronic myocardial infarction (Katare et al, 2010). Moreover, cholinergic activation of M 2 mAChR increases contractility in ventricular muscle from rats with chronic heart failure (Hussain et al, 2009). This positive inotropic effect is likely mediated by increased myosin light chain-2 (MLC-2) phosphorylation and enhanced Ca 21 sensitivity of the myofilaments.…”

“…We report herein that, as in failing rat hearts (Hussain et al, 2009), carbachol induces a positive inotropic response in neonatal rat heart ventricles via activation of M 2 mAChR in a ROCKdependent manner. Thus, to investigate the role of RhoGTPases, we used NSC23766, a small molecule that inhibits the activation of Rac1 by hindering its binding to the Rac-specific GEFs Tiam1 and Trio without altering the access of the related RhoA or Cdc42 to their specific GEFs (Gao et al, 2004).…”

“…This positive inotropic effect is likely mediated by increased myosin light chain-2 (MLC-2) phosphorylation and enhanced Ca 21 sensitivity of the myofilaments. As the carbachol (2-[(aminocarbonyl) oxy]-N,N,N-trimethylethanaminium chloride) evoked positive inotropic response as well as myosin light chain-2 (MLC-2) phosphorylation were attenuated by pertussis toxin treatment or the inhibition of the Rho-dependent kinase (ROCK), the activation of G i/o -proteins and the ROCK pathway are involved (Hussain et al, 2009).…”