Adrenergic supersensitivity and impaired neural control of cardiac electrophysiology following regional cardiac sympathetic nerve loss

www.nature.com/scientificreports OPEN Adrenergic supersensitivity and impaired neural control of cardiac electrophysiology following regional cardiac sympathetic nerve loss Srinivas Tapa1, Lianguo Wang1, Samantha D. Francis Stuart1, Zhen Wang1, Yanyan Jiang1, Beth A. Habecker2 & Crystal M. Ripplinger1* Myocardial infarction (MI) can result in sympathetic nerve loss in the infarct region. However, the contribution of hypo-innervation to electrophysiological remodeling, independent from MI-induced ischemia and fibrosis, has not been comprehensively investigated. We present a novel mouse model of regional cardiac sympathetic hypo-innervation utilizing a targeted-toxin (dopamine beta-hydroxylase antibody conjugated to saporin, DBH-Sap), and measure resulting electrophysiological and Ca2+ handling dynamics. Five days post-surgery, sympathetic nerve density was reduced in the anterior left ventricular epicardium of DBH-Sap hearts compared to control. In Langendorff-perfused hearts, there were no differences in mean action potential duration (APD80) between groups; however, isoproterenol (ISO) significantly shortened APD80 in DBH-Sap but not control hearts, resulting in a significant increase in APD80 dispersion in the DBH-Sap group. ISO also produced spontaneous diastolic Ca2+ elevation in DBH-Sap but not control hearts. In innervated hearts, sympathetic nerve stimulation (SNS) increased heart rate to a lesser degree in DBH-Sap hearts compared to control. Additionally, SNS produced APD80 prolongation in the apex of control but not DBH-Sap hearts. These results suggest that hypo-innervated hearts have regional super-sensitivity to circulating adrenergic stimulation (ISO), while having blunted responses to SNS, providing important insight into the mechanisms of arrhythmogenesis following sympathetic nerve loss. Myocardial infarction (MI) is the leading cause of death globally for both males and females. Though improvements in medical therapies have increased survival rates after MI, approximately 40% of patients will experience post-MI complications, including ventricular arrhythmias1–4. Arrhythmias arise due to cardiomyocyte death and subsequent electrophysiological and fibrotic remodeling, increasing the risk for irregular activation and repolarization5–8. Moreover, the sympathetic nervous system (SNS) also undergoes dramatic post-MI remodeling, which leads to altered excitability, density, distribution, and neurotransmitter content of cardiac sympathetic fibers7,9–11. This interplay between post-MI electrophysiological and sympathetic remodeling is not well understood. Clinical evidence in post-MI patients suggests that the extent of hypo-innervated surviving myocardium is a better predictor of ventricular arrhythmias and sudden cardiac death (SCD) than infarct size or ejection fraction12–16. Our previous experimental work in the mouse heart demonstrated that the infarct region remains devoid of sympathetic fibers due to the presence of chondroitin sulfate proteoglycans (CSPGs), which inhibit reinnervation11,17. We further showed that the hypo-innervated infarct region has supra-physiological β-adrenergic receptor (β-AR) responses, including dramatic action potential duration (APD) shortening, diastolic Ca2+ elevation, and premature ventricular complexes (PVCs) in response to circulating β-AR agonists. Importantly, this arrhythmogenic phenotype was almost completely reversed when sympathetic re-innervation 1 Department of Pharmacology, UC Davis School of Medicine, 2419B Tupper Hall, One Shields Ave, Davis, CA 95616, USA. 2Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, OR, USA. *email: Scientific Reports | (2020) 10:18801 | https://doi.org/10.1038/s41598-020-75903-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Effects of regional toxin application on sympathetic nerve fiber density. (A,B) Whole-heart labeling of tyrosine hydroxylase (TH) demonstrates fewer visible sympathetic nerve fibers on the anterior surface (where toxin solution is applied) of the DBH-Sap heart (B) compared to control (A). (C,D) The posterior surface is similar between control and DBH-Sap groups. (E,F) Short axis TH images of the anterior (E) and posterior (F) surface of a DBH-Sap mouse heart. (G,H) Sympathetic nerve fiber density from anterior base (G) and apex (H) regions 5 days after treatment. At the apex, the percent TH + area was significantly reduced in the epicardium of the DBH-Sap group, and there was also a main effect between groups (***p < 0.001, main effect control vs. DBHSap, two-way ANOVA, H). Data are mean ± SD; analyzed with GraphPad Prism 8.3 (GraphPad Software, San Diego, CA, USA); control: n = 3; DBH-Sap: n = 3; **p < 0.01, ***p < 0.001. of the infarct was produced with either genetic deletion or pharmacologic inhibition of the neuronal receptor for CSPGs, the protein tyrosine phosphatase receptor σ (PTPRσ)11. These data suggest an important and fundamental role for sympathetic nerve loss in post-MI arrhythmogenesis, which may be due to adrenergic super-sensitivity and subsequent supra-physiological responses of the surviving myocardium. However, because these hearts had MI and accompanying nerve loss, it was not possible to precisely assess the impact of sympathetic hypo-innervation independent of ischemia-induced electrophysiological and fibrotic remodeling. Therefore, the goal of this study was to directly investigate the role of regional sympathetic hypo-innervation on ventricular electrophysiology (EP) and C a2+ handling in the absence of infarction. Several experimental approaches have been used previously to study the cardiovascular effects of sympathetic nerve loss. Most commonly, systemic administration of reserpine or 6-hydroxydopamine (6-OHDA) has been used to deplete cardiac norepinephrine (NE) or destroy catecholaminergic neurons, respectively18,19. Reserpine, however, can also deplete catecholamines in the central nervous s ystem20, which may lead to behavioral effects, and systemic administration of either of these compounds does not produce the regional ventricular nerve loss observed following MI. To achieve regional sympathetic nerve loss, direct application of phenol to the heart has been studied21, but this method is not specific to sympathetic neurons and can also damage sensory and parasympathetic neurons as well as cardiomyocytes. Therefore, we developed a novel technique using targeted sympathetic lesioning with an anti-dopamine beta-hydroxylase antibody conjugated to the toxin saporin (antiDBH-Sap), followed by whole-heart and innervated heart optical mapping to assess electrophysiological dynamics and arrhythmogenesis. Results Anti‑DBH‑Sap targets cardiac sympathetic nerve fibers and causes regional hypo‑innervation. Anti-DBH-Sap has been used previously to lesion noradrenergic nerves in the brain and periphery to alter sympathetic control of peripheral organs includin (...truncated)