Optical Mapping of Sarcoplasmic Reticulum Ca 2+ in the Intact Heart

Abstract

Sarcoplasmic Reticulum Ca 2+ and Cardiac Alternans 1411 severity, and concordance of cardiac alternans in myocardial tissue. Thus, it is difficult to extrapolate directly findings in isolated cells to the intact heart. Furthermore, arrhythmogenic behavior, such as spatially discordant alternans and conse-quent ventricular fibrillation (VF), are inherently tissue-level phenomena and thus can only be studied in the intact heart. Experimental investigations into the mechanisms of Ca 2+ alternans in the intact heart and the role of Ca 2+ in arrhyth-mogenesis have predominantly used wide-field optical map-ping that can record signals over multiple sites but, until now, have been limited to dual mapping of transmembrane potential (V m) and intracellular Ca 2+ and so have been un-able to examine SR Ca 2+ kinetics directly. In this study, we report for the first time simultaneous mapping of V m and [Ca 2+ ] SR across the surface of the intact heart and use this novel approach to investigate the role of SR Ca 2+ in cardiac alternans and VF. We further investigated the role of RyR re-fractoriness by sensitizing the RyR with low-concentration (200 μmol/L) caffeine and the combined effects of sensi-tized RyR and increased SR Ca 2+ -ATPase (SERCA) activ-ity through β-adrenergic receptor (β-AR) stimulation with isoproterenol (ISO, 100 nmol/L). Methods An expended Methods section is provided in the online-only Data Supplement. All procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis, and adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male New Zealand white rabbits (n=27) were anesthetized with an intravenous injection of pentobarbital sodium (50 mg/kg). Hearts were removed rapidly and Langendorff perfused as described previously. 16 An ECG was recorded continuously, and pacing was from the base of the left ventricular epicardium. To monitor intra-SR free [Ca 2+ ] optically, hearts were loaded with the low-affinity Ca 2+ indicator Fluo-5N AM (Invitrogen, Carlsbad, CA) for 60 minutes at room temperature. 17 Hearts were subsequently stained with the voltage-sensitive dye RH237 (Invitrogen, Carlsbad, CA). All experiments were performed at 37°C. In a separate set of hearts (n=3), dual optical mapping of V m with RH237 and intracellu-lar Ca 2+ with Rhod-2 AM (Invitrogen, Carlsbad, CA) was performed as previously described. 16 To induce alternans and ventricular arrhyth-mia, the pacing cycle length (PCL) was decremented in 10 ms steps until loss of 1:1 capture or induction of VF (Online Figure III). In a subset of animals, either low-concentration caffeine (200 μmol/L; n=6) to sensitize RyR 18 or ISO (100 nmol/L; n=3) to stimulate β-AR was added to the perfusate. Data analysis was performed using 2 commercially available anal-ysis programs (BV_Analyze, Brainvision, Tokyo, Japan and Optiq, Cairn, United Kingdom). V m and [Ca