1. Joshua T. Maxwell, Timothy L. Domeier*, and Lothar A. Blatter β-adrenergic Stimulation Increases the Intra-SR Ca Termination Threshold for Spontaneous Ca Release in Cardiac Myocytes ABSTRACT METHODS RESULTS In the heart, beta-adrenergic stimulation is associated with pro-arrhythmic Ca waves that occur as the result of the sarcoplasmic reticulum (SR) Ca content reaching a critical threshold level. Recently, we have shown that beta-adrenergic stimulation increases the intra-SR Ca threshold for Ca wave initiation, potentially serving as a protective mechanism against pro-arrhythmic Ca release during beta-adrenergic stimulation (Domeier et al., 2012). However, data regarding the termination of such release and details on the regulation of this process have yet to be elucidated. In this study we directly and dynamically measured the intra-SR Ca level ([Ca] SR ) at which spontaneous Ca waves terminate (termination threshold) under control conditions and during beta-adrenergic stimulation. Application of the beta-adrenergic receptor agonist isoproterenol (ISO; 1 µM) resulted in an increase in basal [Ca] SR. Importantly, in the presence of ISO the [Ca] SR at which spontaneous Ca waves terminated was also increased compared to control conditions. In addition, the depletion amplitude of spontaneous Ca waves was decreased in the presence of ISO compared to control conditions. When [Ca] SR was subsequently lowered in the presence of ISO to that observed under control conditions (by reducing extracellular Ca and partially inhibiting SERCA with cyclopiazonic acid or thapsigargin), the [Ca] SR at which spontaneous release terminated was still increased compared to control conditions. Likewise, the depletion amplitude remained decreased compared to control conditions. These data indicate that during beta-adrenergic stimulation in the heart, both the intra-SR Ca threshold at which spontaneous Ca waves initiate and terminate is increased, while the amount of Ca released during Ca waves is decreased. The Ca wave termination level may represent an important mode of altering diastolic Ca wave amplitude, and thus, the arrhythmogenic potential of the cell during acute beta-adrenergic stimulation. CONCLUSIONS Myocyte Isolation: Rabbit ventricular myocytes were enzymatically isolated and cells were kept in MEM solution with 50 μM Ca at room temperature (22-24 ◦ C) until indicator dye loading and subsequent experimentation. Ca dye loading: To directly monitor [Ca] SR the SR was loaded with the low- affinity Ca indicator fluo-5N by incubation of ventricular myocytes with 10 μM of the membrane permeant fluo-5N/AM together with 0.25% Pluronic F-127 in nominally Ca-free Tyrode solution for 2.5 h, followed by a 30 min wash, all at 37 °C. To simultaneously monitor cytosolic Ca waves, myocytes preloaded with fluo- 5N were subsequently loaded with the spectrally distinct [Ca] i indicator rhod-2/AM (5 µM) for 10 min followed by a 10 min wash. Confocal microscopy: Confocal microscopy (Nikon A1R, Nikon Corporation, Melville, NY, USA) was used to image [Ca] SR (fluo-5N) and [Ca] i (rhod-2). For simultaneous recording of [Ca] SR and [Ca] i, fluo-5N was excited at 488 nm and emission collected at 515±15 nm, while rhod-2 was excited at 543 and emission collected at wavelengths >600. Calcium wave measurements were acquired from intact myocytes during rest from 0.8-1.5 Hz stimulation in frame scan mode (60 fps at 256 x 512 pixels, 400 nm/pixel). Myocytes underwent several consecutive experimental trials consisting of electrical field stimulation of cells for a period of 30-60 s, followed by 8 s of rest. The stimulation-rest protocol was repeated at incrementally increasing pacing frequencies (0.05 to 0.2 Hz increments) to increase [Ca] SR until Ca waves were observed during the rest period. The nadir of the [Ca] SR depletion signal during a Ca wave was defined as the termination threshold. Data Presentation: Data are presented as individual observations representative of multiple recordings or as the average of multiple recordings. Rhod-2 fluorescence traces are plotted as F/F 0, where F 0 is the initial fluorescence. Changes in [Ca 2+ ] SR are presented as (F-F Min )/(F Max -F Min ). F Min is the quench- corrected (15%) fluorescence value following complete emptying of the SR with 10 mM caffeine, and F Max is taken as the maximal diastolic fluo-5N fluorescence observed during electrical pacing in the presence of 1  M ISO. Statistical comparisons: Comparisons were performed using Student’s t-test for unpaired data, with significance set at P < 0.05. “n” refers to the number of individual experimental trials. This work was supported by National Institutes of Health grants HL62231, HL80101 and HL101235 (to L.A.B.) and F32HL090211 and 1K01AG041208 (to T.L.D.) and the Leducq Foundation (to L.A.B). ACKNOWLEDGEMENTS Isoproterenol increases the intra-SR initiation threshold of spontaneous Ca waves Figure 1: Simultaneous [Ca] i (top traces, black) and [Ca] SR (bottom traces, gray) measurements under control conditions (A & B), during beta-adrenergic stimulation with isoproterenol (ISO, 1 μM; C), in the presence of ISO and cyclopiazonic acid (CPA, 3 μM; D), and after application of 250 μM caffeine (E). Field stimulation was followed by a period of rest during which spontaneous Ca waves were observed (arrow denotes electrically-induced Ca transient, star denotes spontaneous Ca wave). The dashed lines represent: no wave control (1), wave control (2), and no wave ISO (3) SR Ca levels. (Taken from Domeier, Maxwell, and Blatter; 2012) Figure 2: [Ca] SR traces show an electrically induced SR Ca depletion transient (arrow) followed by a period of rest to observe spontaneous Ca waves (wave denoted by star). This protocol was applied under (A) control conditions, and repeated in the presence of (B) isoproterenol (ISO; 1 µM), (C) ISO plus cyclopiazonic acid (CPA; 3 µM), and (D) during addition of caffeine (Caff; 250 µM). The dashed lines indicate Ca wave termination thresholds in control (1), ISO (2), ISO+CPA (3) and ISO+CPA+Caff (4). Correlation between pre-wave [Ca] SR and Ca wave termination threshold Summary of the intra-SR termination threshold of spontaneous Ca waves Figure 3: Pre-wave [Ca] SR (squares) and [Ca] SR at the wave nadir (termination threshold; circles) in control (CTL; n=23), ISO (n=22), ISO+CPA/TG (n=8), and ISO+CPA/TG+Caff (n=8). *P<0.01 vs. CTL, # P<0.05 vs. CTL Figure 4: Ca wave depletion amplitudes at matched average pre-wave [Ca] SR in the absence (CTL) and presence of ISO (ISO+CPA/TG). *P<0.01 vs. CTL. Summary of the depletion amplitude of spontaneous Ca waves at matched [Ca] SR 2 s 0.5 [Ca] SR (F-F Min /F Max -F Min ) 1.0 1.4 [Ca] i (F/F 0 ) A B C D E 3 2 1 2 mM [Ca] o, 1 µM ISO 1 mM [Ca] o, 3 µM CPA 250 Caff Isoproterenol increases the intra-SR termination threshold of spontaneous Ca waves 0.4 0.5 0.6 0.7 0.8 0.9 1 [Ca] SR ((F-F Min )/(F Max -F Min )) CTL ISO CPA/TG ISO CPA/TG Caffeine ISO * * # * * 0 0.1 0.2 0.3 ∆[Ca] SR ((F-F Min )/(F Max -F Min )) * CTL ISO 2 mM [Ca] o, 1 µM ISO 1 mM [Ca] o, 3 µM CPA 250 Caff 0.3 [Ca] SR ((F-F Min )/(F Max -F Min )) 1.0 1 s 1 2 4 3 ABC D REFERENCES Domeier TL, Maxwell JT, Blatter LA. β-adrenergic stimulation increases the intra-sarcoplasmic reticulum Ca 2+ threshold for Ca 2+ wave generation. J. Physiol. 590(Pt 23): 6093-108, 2012 Figure 5: Scatter plot, average values (± SEM) and linear regression lines of pre-wave [Ca] SR and [Ca] SR at the nadir of Ca depletion (termination threshold) during spontaneous Ca waves under control conditions (CTL; open symbols; n=23) and in the presence of ISO (filled symbols; n=30). Rush University, Department of Molecular Biophysics & Physiology, Chicago, IL 60612 * Present address: The University of Missouri, Department of Medical Pharmacology and Physiology, Columbia, MO 65212 Wave Nadir [Ca] SR ((F-F Min )/(F Max -F Min )) Pre-wave [Ca] SR ((F-F Min )/(F Max -F Min )) 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0.70.80.91 R = 0.82 R = 0.72 In the presence of beta-adrenergic stimulation the SR Ca level at which Ca waves initiate and terminate are both increased compared to control Ca wave depletion amplitudes are increased during beta-adrenergic stimulation due to increased SR Ca load. However, when SR Ca loads are experimentally matched the Ca wave depletion amplitude is decreased during beta-adrenergic stimulation The thresholds for the initiation and termination of spontaneous Ca waves represent key points of regulation of spontaneous pro-arrhythmic Ca release in cardiac myocytes The observed increases in the initiation and termination thresholds of spontaneous Ca waves during beta-adrenergic stimulation potentially represent protective mechanisms against arrhythmogenic Ca release