We first identified the origin of the RPc factor. Since the efficacy of RPc cardioprotection is critically dependent on vagal activity and muscarinic mechanisms, we hypothesized that the humoral RPc factor is produced by the internal organ(s) which receive rich parasympathetic innervation. 30 min of myocardial ischaemia followed by 120 min of reperfusion resulted in a mean infarct size of 53±2%. Significant cardioprotection was established by application of the RPc stimulus (15 min occlusion of femoral arteries followed by 10 min of reperfusion) as evident from a marked reduction in infarct size (32±2%, p<0.01). Subdiaphragmatic vagotomy abolished RPc cardioprotection (infarct size 50±3%; p<0.01) while selective electrical stimulation of posterior gastric brunch of subdiaphragmatic vagus nerve was sufficient to establish (i.e. mimic RPc) cardioprotection (infarct size 30±2%, p<0.01). These data suggest that the circulating factor(s) of RPc cardioprotection is produced and released into the systemic circulation by the visceral organ(s) innervated by subdiaphragmatic efferent fibers of the vagus nerve.
We next hypothesized that the gut hormone glucagon-like peptide-1 (GLP-1), which is known to have potent beneficial effects on the heart in diabetic patients and in the context of cardiac I/R injury, plays the central role in the RPc cardioprotection, since GLP-1 release is controlled by vagus nerve. RPc failed to establish cardioprotection in conditions of cervical or subdiaphragmatic vagotomy, or GLP-1R blockade with GLP-1R antagonist Exendin(9-39). Exendin(9-39) also prevented RPc-induced phosphorylation of pro-survival kinase AKT in the heart. Cardioprotection induced by GLP-1R agonist Exendin-4 was preserved following cervical vagotomy, but was abolished in conditions of M3 muscarinic receptor blockade. The GLP-1 plasma levels increased in response to RPc. These data strongly suggest that GLP-1 functions as a humoral factor of RPc cardioprotection. This requires intact vagal innervation of the visceral organs and recruitment of GLP-1R-mediated signalling. Cardioprotection induced by GLP-1R agonism is mediated by AKT-dependent mechanism and involves activation of M3 muscarinic receptors.
One of the most common complications of myocardial ischaemia is a sustained ventricular tachyarrhythmia. We found that systemic GLP-1R activation with Exendin-4 reduces the arrhythmic burden in the rat model of I/R injury, and this effect is blocked by M3 muscarinic receptor antagonist 4DAMP. Exendin-4 also effectively reversed the effects of β-adrenoceptor agonists dobutamine and isoprenaline on ventricular action potential duration both in vivo and in isolated heart. The antiarrhythmic effects of GLP-1R activation were blocked by atropine and NOS inhibitors. Thus, the beneficial effects of GLP-1 on the heart (acutely recruitable by RPc) are indirect and mediated via release of acetylcholine and nitric oxide and their actions on coronary vessels and cardiomyocytes.
Capitalizing on our previous studies that demonstrated the crucial role of autonomic parasympathetic outflow to the heart and activity of vagal preganglionic neurones of the dorsal motor nucleus of the vagus nerve (DVMN) in cardioprotection, we manipulated these neural mechanisms to slow down the remodelling process and improve left ventricular (LV) function. We targeted vagal preganglionic neurones of the DVMN to express light-sensitive optogenetic actuators (Channelrhodopsin variant ChIEF) and determined the effect of selective optical stimulation of vagal C-fibre efferents on LV function and exercise capacity in a rat model of myocardial infarction (MI)-induced heart failure. The development of post-MI LV dysfunction in rats was associated with a marked reduction in exercise capacity (calculated in Joules from the distance that animals were able to run on a treadmill). Optogenetic stimulation of vagal C-fibre efferents expressing ChIEF significantly enhanced exercise capacity in sham-operated animals (105±11 vs 56±8 J in sham-operated rats expressing control virus eGFP; p=0.001) and preserved exercise capacity in animals with LV dysfunction (56±4 vs 28±3 J in post-MI rats expressing eGFP; p=0.02). Development of LV dysfunction was characterized by marked reduction of ejection fraction (33±3 vs 50±5% in sham-operated animals; p=0.01); E/A ratio (0.99±0.09 vs 1.20±0.10; p=0.04) and LV deceleration slope (3580±350 vs 2470±290 mm/s/s; p=0.02). Optogenetic stimulation of DVMN neurones expressing ChIEF resulted in significant improvements in ejection fraction (49±3 vs 33±3%; p=0.005) E/A ratio (1.2±0.1 vs 0.9±0.1; p=0.03) and deceleration slope (-2670±260 vs -3580±350 mm/s/s; p=0.04). Complementary improvements were also recorded in the maximum and minimum differentials of LV pressure. Thus, using optogenetics for highly selective recruitment of vagal efferent projections from the DVMN, we demonstrated that vagal C-fibre efferents exert trophic effects on LV function that can be exploited to slow the progression of heart failure developing after myocardial infarction.
