Katie J. Sciuto

The voltage-sensitive dye di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts

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.

Detection of mitochondrial depolarization/recovery during ischaemia–reperfusion using spectral properties of confocally recorded TMRM fluorescence

•  Mitochondrial inner membrane potential (ΔΨm) 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 ΔΨm collapse during ischaemia remain controversial, in part due to difficulties in interpreting the fluorescence of potentiometric cationic probes commonly used for assessment of ΔΨm in cellular and multicellular experimental models. •  This manuscript presents a new method for monitoring ΔΨm in whole hearts based on the regular arrangement of mitochondria in cardiac myocytes, thus permitting detection of ΔΨm 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.

Blockade of CaMKII depresses conduction preferentially in the right ventricular outflow tract and promotes ischemic ventricular fibrillation in the rabbit heart

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 (dV/dtmax). At baseline, local conduction delays were similar between RV and LV, whereas the OAP dV/dtmax was lower in RV than in LV. At 2.75 μM, KN93 heterogeneously slowed conduction and reduced dV/dtmax, 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, P < 0.0001). Neither KN92 nor 1 μM KN93 depressed OAP dV/dtmax or conduction. Rabbit cardiomyocytes isolated from RVOT exhibited a significantly lower dV/dtmax than those isolated from the LV. KN93 (2.75 μM) significantly reduced dV/dtmax 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.NEW & NOTEWORTHY 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.

Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (asystole) during global ischemia, which is relevant for the success of defibrillation and resuscitation after cardiac arrest. However, the relationship between ΔΨm depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms), myosin II ATPase inhibitor blebbistatin (heart immobilizer), and the combination of blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic solution and after the contraction failed between 7 and 15 min of ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and asystole or a similar dependence of the two events on energy depletion during ischemia. Our results urge caution against the use of blebbistatin in studies addressing pathophysiology of myocardial ischemia.

β-Adrenergic stimulation and rapid pacing mutually promote heterogeneous electrical failure and ventricular fibrillation in the globally ischemic heart

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 β-adrenergic stimulation (βAS) 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 β-agonist isoproterenol (30 nM). 2,3-Butanedione monoxime (20 mM) was used to reduce motion artifact. We found that RHR and βAS 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 βAS 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) = 0 .72, P < 0.0001). We conclude that both βAS 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.

Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step

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.

di-4-ANEPPS Slows Cardiac Conduction Velocity

One of the most widely used voltage-sensitive dyes for optically mapping cardiac conduction is di-4-ANEPPS. Previous studies suggest that di-4-ANEPPS broadens the QRS; however, little is known about its effects on myocardial conduction. We hypothesized that di-4-ANEPPS suppresses cardiac conduction velocity (CV).CV was quantified in Langendorff-perfused guinea pig hearts using unipolar electrode and optical recordings. Electrode recordings from the anterior epicardium revealed that di-4-ANEPPS (7.5 µM) slowed cardiac transverse CV significantly from 23±4 cm/s to 18±3 cm/s (p<0.05). To investigate a possible concentration dependent effect of di-4-ANEPPS, CV and anisotropy was quantified using optical signals recorded from the anterior epicardium of both the right and left ventricle (RV, LV) at different concentrations of di-4-ANEPPS. Increasing the concentration of di-4-ANEPPS from 1.9 to 15 µM reduced transverse CV by 7±2 cm/s in the RV (p<0.05) (n=4) and 4±2 cm/s in the LV (p<0.05) (n=4). The decrease in longitudinal CV trended towards significance in both the RV (14±7 cm/s, p=0.08) and the LV (15±9 cm/s, p=0.07). The anisotropic ratio of CV was not affected by di-4-ANEPPS concentration. Connexin43 conductance was not significantly changed by di-4-ANEPPS at 15 µM evident from dual patch clamp experiments on HeLa cell-pairs overexpressing rat connexin43 (n=5), suggesting that decreased gap junction conductance is not the underlying mechanism.These data suggest that the perfusion of di-4-ANEPPS into whole heart tissue slows CV in both the right and left ventricles and this effect does not appear to be connexin related. Investigators should take the effect of di-4-ANEPPS on conduction into account when interpreting data obtained with this dye.