Calcium/calmodulin-dependent protein kinase II (CaMKII) regulates the principle ion channels mediating cardiac excitability and conduction, but how this regulation translates to the normal and ischemic heart remains unknown. Diverging results on CaMKII regulation of Na channels further prevent predicting how CaMKII activity regulates excitability and conduction in the intact heart. To address this deficiency, we tested the effects of the CaMKII blocker KN93 (1 and 2.75 μM) and its inactive analog KN92 (2.75 μM) on conduction and excitability in the left (LV) and right (RV) ventricles of rabbit hearts during normal perfusion and global ischemia. We used optical mapping to determine local conduction delays and the optical action potential (OAP) upstroke velocity (d/d). At baseline, local conduction delays were similar between RV and LV, whereas the OAP d/d was lower in RV than in LV. At 2.75 μM, KN93 heterogeneously slowed conduction and reduced d/d, with the largest effect in the RV outflow tract (RVOT). This effect was further exacerbated by ischemia, leading to recurrent conduction block in the RVOT and early ventricular fibrillation (at 6.7 ± 0.9 vs. 18.2 ± 0.8 min of ischemia in control, < 0.0001). Neither KN92 nor 1 μM KN93 depressed OAP d/d or conduction. Rabbit cardiomyocytes isolated from RVOT exhibited a significantly lower d/d than those isolated from the LV. KN93 (2.75 μM) significantly reduced d/d in cells from both locations. This led to frequency-dependent intermittent activation failure occurring predominantly in RVOT cells. Thus CaMKII blockade exacerbates intrinsically lower excitability in the RVOT, which is proarrhythmic during ischemia. We show that calcium/calmodulin-dependent protein kinase II (CaMKII) blockade exacerbates intrinsically lower excitability in the right ventricular outflow tract, which causes highly nonuniform chamber-specific slowing of conduction and facilitates ventricular fibrillation during ischemia. Constitutive CaMKII activity is necessary for uniform and safe ventricular conduction, and CaMKII block is potentially proarrhythmic.

BACKGROUND Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. OBJECTIVE To test the hypothesis that the voltage-sensitive dye di-4-ANEPPS modulates cardiac impulse propagation. METHODS Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of di-4-ANEPPS on gap junctional conductance. RESULTS In electrical mapping experiments, di-4-ANEPPS (7.5 μM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of di-4-ANEPPS was dose dependent. di-4-ANEPPS had no detectable effect on connexin 43–mediated gap junctional conductance in transfected HeLa cells. CONCLUSION Our results demonstrate that the voltage-sensitive dye di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using di-4-ANEPPS in cardiac preparations.

Key points• Mitochondrial inner membrane potential ( m ) collapse during myocardial ischaemia is one of the key events determining the physiological consequences of ischaemic attack in terms of post-ischaemic arrhythmias and cell survival.• Timing and pattern of m collapse during ischaemia remain controversial, in part due to difficulties in interpreting the fluorescence of potentiometric cationic probes commonly used for assessment of m in cellular and multicellular experimental models.• This manuscript presents a new method for monitoring m in whole hearts based on the regular arrangement of mitochondria in cardiac myocytes, thus permitting detection of m collapse using spectral analysis of fluorescence.• The proposed method will help to ascertain the role of mitochondrial function in acute cardiovascular conditions, such as acute myocardial infarction or sudden cardiac arrest.Abstract Timing and pattern of mitochondrial potential ( m ) depolarization during no-flow ischaemia-reperfusion (I-R) remain controversial, at least in part due to difficulties in interpreting the changes in the fluorescence of m -sensitive dyes such as TMRM. The objective of this study was to develop a new approach for interpreting confocal TMRM signals during I-R based on spatial periodicity of mitochondrial packaging in ventricular cardiomyocytes. TMRM fluorescence (F TMRM ) was recorded from Langendorff-perfused rabbit hearts immobilized with blebbistatin using either a confocal microscope or an optical mapping system. The hearts were studied under normal conditions, during mitochondrial uncoupling using the protonophore FCCP, and during I-R. Confocal images of F TMRM were subjected to spatial Fourier transform which revealed distinct peaks at a spatial frequency of ∼2 μm −1 . The area under the peak (MPA) progressively decreased upon application of increasing concentrations of FCCP (0.3-20 μM), becoming undetectable at 5-20 μM FCCP. During ischaemia, a dramatic decrease in MPA, reaching the low/undetectable level comparable to that induced by 5-20 μM FCCP, was observed between 27 and 69 min of ischaemia. Upon reperfusion, a heterogeneous MPA recovery was observed, but not a de novo MPA decrease. Both confocal and wide-field imaging registered a consistent decrease in spatially averaged F TMRM in the presence of 5 μM FCCP, but no consistent change in this parameter during I-R. We conclude that MPA derived from confocal images provides a sensitive and specific indicator of significant mitochondrial depolarization or

bayama J, Zaitsev A. ␤-Adrenergic stimulation and rapid pacing mutually promote heterogeneous electrical failure and ventricular fibrillation in the globally ischemic heart. Am J Physiol Heart Circ Physiol 308: H1155-H1170, 2015. First published February 20, 2015 doi:10.1152/ajpheart.00768.2014.-Global ischemia, catecholamine surge, and rapid heart rhythm (RHR) due to ventricular tachycardia or ventricular fibrillation (VF) are the three major factors of sudden cardiac arrest (SCA). Loss of excitability culminating in global electrical failure (asystole) is the major adverse outcome of SCA with increasing prevalence worldwide. The roles of catecholamines and RHR in the electrical failure during SCA remain unclear. We hypothesized that both ␤-adrenergic stimulation (␤AS) and RHR accelerate electrical failure in the globally ischemic heart. We performed optical mapping of the action potential (OAP) in the right ventricular (RV) and left (LV) ventricular epicardium of isolated rabbit hearts subjected to 30-min global ischemia. Hearts were paced at a cycle length of either 300 or 200 ms, and either in the presence or in the absence of ␤-agonist isoproterenol (30 nM). 2,3-Butanedione monoxime (20 mM) was used to reduce motion artifact. We found that RHR and ␤AS synergistically accelerated the decline of the OAP upstroke velocity and the progressive expansion of inexcitable regions. Under all conditions, inexcitability developed faster in the LV than in the RV. At the same time, both RHR and ␤AS shortened the time to VF (TVF) during ischemia. Moreover, the time at which 10% of the mapped LV area became inexcitable strongly correlated with TVF (R 2 ϭ 0 .72, P Ͻ 0.0001). We conclude that both ␤AS and RHR are major factors of electrical depression and failure in the globally ischemic heart and may contribute to adverse outcomes of SCA such as asystole and recurrent/persistent VF.

A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60–80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (TSP -TMPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5–20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H2O2 (200–800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.