β-Adrenergic Inhibition Prevents Action Potential and Calcium Handling Changes during Regional Myocardial Ischemia

ORIGINAL RESEARCH published: 28 August 2017 doi: 10.3389/fphys.2017.00630 β-Adrenergic Inhibition Prevents Action Potential and Calcium Handling Changes during Regional Myocardial Ischemia Shannon R. Murphy 1 , Lianguo Wang 1 , Zhen Wang 1 , Philip Domondon 2 , Di Lang 1 , Beth A. Habecker 3 , Rachel C. Myles 4 and Crystal M. Ripplinger 1* 1 Department of Pharmacology, University of California, Davis, Davis, CA, United States, 2 Department of Biomedical Engineering, University of California, Davis, Davis, CA, United States, 3 Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR, United States, 4 Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom Edited by: Tobias Opthof, Academic Medical Center, Netherlands Reviewed by: Edward Lakatta, National Institutes of Health, United States Marcella Rocchetti, University of Milano-Bicocca, Italy *Correspondence: Crystal M. Ripplinger Specialty section: This article was submitted to Cardiac Electrophysiology, a section of the journal Frontiers in Physiology Received: 23 May 2017 Accepted: 14 August 2017 Published: 28 August 2017 Citation: Murphy SR, Wang L, Wang Z, Domondon P, Lang D, Habecker BA, Myles RC and Ripplinger CM (2017) β-Adrenergic Inhibition Prevents Action Potential and Calcium Handling Changes during Regional Myocardial Ischemia. Front. Physiol. 8:630. doi: 10.3389/fphys.2017.00630 Frontiers in Physiology | www.frontiersin.org β-adrenergic receptor (β-AR) blockers may be administered during acute myocardial infarction (MI), as they reduce energy demand through negative chronotropic and inotropic effects and prevent ischemia-induced arrhythmogenesis. However, the direct effects of β-AR blockers on ventricular electrophysiology and intracellular Ca2+ handling during ischemia remain unknown. Using optical mapping of transmembrane potential (with RH237) and sarcoplasmic reticulum (SR) Ca2+ (with the low-affinity indicator Fluo-5N AM), the effects of 15 min of regional ischemia were assessed in isolated rabbit hearts (n = 19). The impact of β-AR inhibition on isolated hearts was assessed by pre-treatment with 100 nM propranolol (Prop) prior to ischemia (n = 7). To control for chronotropy and inotropy, hearts were continuously paced at 3.3 Hz and contraction was inhibited with 20 µM blebbistatin. Untreated ischemic hearts displayed prototypical shortening of action potential duration (APD80 ) in the ischemic zone (IZ) compared to the non-ischemic zone (NI) at 10 and 15 min ischemia, whereas APD shortening was prevented with Prop. Untreated ischemic hearts also displayed significant changes in SR Ca2+ handling in the IZ, including prolongation of SR Ca2+ reuptake and SR Ca2+ alternans, which were prevented with Prop pre-treatment. At 5 min ischemia, Prop pretreated hearts also showed larger SR Ca2+ release amplitude in the IZ compared to untreated hearts. These results suggest that even when controlling for chronotropic and inotropic effects, β-AR inhibition has a favorable effect during acute regional ischemia via direct effects on APD and Ca2+ handling. Keywords: ischemia, arrhythmia, beta blocker, sarcoplasmic reticulum, calcium INTRODUCTION Ventricular arrhythmias during the acute phase of myocardial infarction (MI) remain a leading cause of death (Henriques et al., 2005; Benjamin et al., 2017). Experimental studies indicate that ventricular arrhythmias peak between ∼10 and 30 min of myocardial ischemia (Curtis, 1998; de Groot and Coronel, 2004; Di Diego and Antzelevitch, 2011). Ischemia-induced arrhythmias are 1 August 2017 | Volume 8 | Article 630 Murphy et al. Murphy–Beta Block during Ischemia isolated rabbit hearts. Using a low-affinity Ca2+ indicator (Fluo5N AM, Kd ≈ 400 µM), free intra-SR Ca2+ can be directly monitored everywhere on the surface of the heart (Wang et al., 2014, 2015). Thus, SR Ca2+ release represents a rapid decrease in the signal (the inverse of the intracellular Ca2+ transient) and SR Ca2+ ATPase (SERCA) function can be directly assessed via the time constant of SR Ca2+ reuptake. Acute regional ischemia was induced by ligation of the left circumflex artery (LCA). To isolate electrophysiological effects from metabolic effects that may be secondary to negative chronotropy and inotropy induced by β-AR inhibition, hearts were continuously paced and contraction was abolished with the excitation-contraction uncoupler blebbistatin. multi-factorial and can be attributed to several changes in myocyte electrophysiology, including intracellular Ca2+ overload, extracellular K+ accumulation, slow conduction, shortening of the action potential duration (APD), increased dispersion of repolarization, and post-repolarization refractoriness (Akar and Akar, 2007; Di Diego and Antzelevitch, 2011; Coronel et al., 2012). β-adrenergic receptor (β-AR) blockers have been shown to reduce ventricular arrhythmias during the acute phase of MI (Norris et al., 1984). β-AR blockers also limit infarct size, relieve pain, and reduce early mortality when administered during acute MI (López-Sendón et al., 2004). Recent evidence suggests that when administered prior to primary percutaneous coronary intervention (PCI), intravenous β-AR blocker therapy results in improved ejection fraction and fewer major adverse cardiac events compared to PCI without β-AR blocker pre-treatment (Halkin et al., 2004; Pizarro et al., 2014). The mechanisms underlying these improved outcomes are likely multi-factorial, but may stem in part from a global reduction in oxygen demand due to reductions in heart rate and contractility, as well as inhibition of β-AR signaling in the ischemic region. Indeed, β-AR activation may be locally elevated in the ischemic region due to release of catecholamines from the ischemic cardiac sympathetic nerves (Schömig et al., 1984, 1987; Lameris et al., 2000; Killingsworth et al., 2004). This local, nonexocytotic catecholamine release is in response to intracellular acidification of the neurons and is independent of central sympathetic drive (i.e., occurs in vivo as well as in in vitro isolated ischemic hearts) (Schömig et al., 1987). Some studies indicate that the local norepinephrine (NE) concentration in the extracellular space can rise to as much as 100- to 1,000fold higher than normal plasma concentrations within 10– 30 min of no-flow ischemia or anoxia (Schömig et al., 1987; Kurz et al., 1995; Lameris et al., 2000). Significant elevation of interstitial NE has also been observed following several minutes of fibrillation-induced global ischemia (Killingsworth et al., 2004). Although less pronounced, myocardial interstitial levels of dopamine (DA, precursor to NE) and epinephrine (Epi) also rise significantly during ischemia (Schömig et al., 1984; Lameris et al., 2000). Thus, locally enhanced β-AR stimulation may exacerbate the arrhythmogenic effects of ischemia by contributing to Ca2+ overload and additional APD shortenin (...truncated)