Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Conduction in the right and left ventricle is differentially regulated by protein kinases and phosphatases: implications for arrhythmogenesis.

The "stress" kinases cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylate the Na+ channel Nav1.5 subunit to regulate its function. However, how the channel regulation translates to ventricular conduction is poorly understood. We hypothesized that the stress kinases positively and differentially regulate conduction in the right (RV) and the left (LV) ventricles. We applied the CaMKII blocker KN93 (2.75 µM), PKA blocker H89 (10 µM), and broad-acting phosphatase blocker calyculin (30 nM) in rabbit hearts paced at a cycle length (CL) of 150-8,000 ms. We used optical mapping to determine the distribution of local conduction delays (inverse of conduction velocity). Control hearts exhibited constant and uniform conduction at all tested CLs. Calyculin (15-min perfusion) accelerated conduction, with greater effect in the RV (by 15.3%) than in the LV (by 4.1%; P < 0.05). In contrast, both KN93 and H89 slowed down conduction in a chamber-, time-, and CL-dependent manner, with the strongest effect in the RV outflow tract (RVOT). Combined KN93 and H89 synergistically promoted conduction slowing in the RV (KN93: 24.7%; H89: 29.9%; and KN93 + H89: 114.2%; P = 0.0016) but not the LV. The progressive depression of RV conduction led to conduction block and reentrant arrhythmias. Protein expression levels of both the CaMKII-δ isoform and the PKA catalytic subunit were higher in the RVOT than in the apical LV (P < 0.05). Thus normal RV conduction requires a proper balance between kinase and phosphatase activity. Dysregulation of this balance due to pharmacological interventions or disease is potentially proarrhythmic. NEW & NOTEWORTHY We show that uniform ventricular conduction requires a precise physiological balance of the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and phosphatases, which involves region-specific expression of CaMKII and PKA. Inhibiting CaMKII and/or PKA activity elicits nonuniform conduction depression, with the right ventricle becoming vulnerable to the development of conduction disturbances and ventricular fibrillation/ventricular tachycardia.

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.

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

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 (FTMRM) 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 FTMRM 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 FTMRM 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 recovery during I-R. In contrast, spatially averaged FTMRM is not a reliable indicator of m changes during I-R.

Does the combination of hyperkalemia and KATP activation determine excitation rate gradient and electrical failure in the globally ischemic fibrillating heart?

Ventricular fibrillation (VF) in the globally ischemic heart is characterized by a progressive electrical depression manifested as a decline in the VF excitation rate (VFR) and loss of excitability, which occur first in the subepicardium (Epi) and spread to the subendocardium (Endo). Early electrical failure is detrimental to successful defibrillation and resuscitation during cardiac arrest. Hyperkalemia and/or the activation of ATP-sensitive K(+) (KATP) channels have been implicated in electrical failure, but the role of these factors in ischemic VF is poorly understood. We determined the VFR-extracellular K(+) concentration ([K(+)]o) relationship in the Endo and Epi of the left ventricle during VF in globally ischemic hearts (Isch group) and normoxic hearts subjected to hyperkalemia (HighK group) or a combination of hyperkalemia and the KATP channel opener cromakalim (HighK-Crom group). In the Isch group, Endo and Epi values of [K(+)]o and VFR were compared in the early (0-6 min), middle (7-13 min), and late (14-20 min) phases of ischemic VF. A significant transmural gradient in VFR (Endo > Epi) was observed in all three phases, whereas a significant transmural gradient in [K(+)]o (Epi > Endo) occurred only in the late phase of ischemic VF. In the Isch group, the VFR decrease and inexcitability started to occur at much lower [K(+)]o than in the HighK group, especially in the Epi. Combining KATP activation with hyperkalemia only shifted the VFR-[K(+)]o curve upward (an effect opposite to real ischemia) without changing the [K(+)]o threshold for asystole. We conclude that hyperkalemia and/or KATP activation cannot adequately explain the heterogeneous electrical depression and electrical failure during ischemic VF.

Role of KATP channel in electrical depression and asystole during long-duration ventricular fibrillation in ex vivo canine heart.

Long-duration ventricular fibrillation (LDVF) in the globally ischemic heart is characterized by transmurally heterogeneous decline in ventricular fibrillation rate (VFR), emergence of inexcitable regions, and eventual global asystole. Rapid loss of both local and global excitability is detrimental to successful defibrillation and resuscitation during cardiac arrest. We sought to assess the role of the ATP-sensitive potassium current (I(KATP)) in the timing and spatial pattern of electrical depression during LDVF in a structurally normal canine heart. We analyzed endo-, mid-, and epicardial unipolar electrograms and epicardial optical recordings in the left ventricle of isolated canine hearts during 10 min of LDVF in the absence (control) and presence of an I(KATP) blocker glybenclamide (60 µM). In all myocardial layers, average VFR was the same or higher in glybenclamide-treated than in control hearts. The difference increased with time of LDVF and was overall significant in all layers (P < 0.05). However, glybenclamide did not significantly affect the transmural VFR gradient. In epicardial optical recordings, glybenclamide shortened diastolic intervals, prolonged action potential duration, and decreased the percentage of inexcitable area (all differences P < 0.001). During 10 min of LDVF, asystole occurred in 55.6% of control and none of glybenclamide-treated hearts (P < 0.05). In three hearts paced after the onset of asystole, there was no response to LV epicardial or atrial pacing. In structurally normal canine hearts, I(KATP) opening during LDVF is a major factor in the onset of local and global inexcitability, whereas it has a limited role in overall deceleration of VFR and the transmural VFR gradient.

Complex structure of electrophysiological gradients emerging during long-duration ventricular fibrillation in the canine heart.

Long-duration ventricular fibrillation (LDVF) in the globally ischemic heart is a common setting of cardiac arrest. Electrical heterogeneities during LDVF may affect outcomes of defibrillation and resuscitation. Previous studies in large mammalian hearts have investigated the role of Purkinje fibers and electrophysiological gradients between the endocardium (Endo) and epicardium (Epi). Much less is known about gradients between the right ventricle (RV) and left ventricle (LV) and within each chamber during LDVF. We studied the transmural distribution of the VF activation rate (VFR) in the RV and LV and at the junction of RV, LV, and septum (Sep) during LDVF using plunge needle electrodes in opened-chest dogs. We also used optical mapping to analyze the Epi distribution of VFR, action potential duration (APD), and diastolic interval (DI) during LDVF in the RV and LV of isolated hearts. Transmural VFR gradients developed in both the RV and LV, with a faster VFR in Endo. Concurrently, large VFR gradients developed in Epi, with the fastest VFR in the RV-Sep junction, intermediate in the RV, and slowest in the LV. Optical mapping revealed a progressively increasing VFR dispersion within both the LV and RV, with a mosaic presence of fully inexcitable areas after 4-8 min of LDVF. The transmural, interchamber, and intrachamber VFR heterogeneities were of similar magnitude. In both chambers, the inverse of VFR was highly correlated with DI, but not APD, at all time points of LDVF. We conclude that the complex VFR gradients during LDVF in the canine heart cannot be explained solely by the distribution of Purkinje fibers and are related to regional differences in the electrical depression secondary to LDVF.

High-precision recording of the action potential in isolated cardiomyocytes using the near-infrared fluorescent dye di-4-ANBDQBS.

The use of voltage-sensitive fluorescent dyes (VSD) for noninvasive measurement of the action potential (AP) in isolated cells has been hindered by low-photon yield of the preparation, dye toxicity, and photodynamic damage. Here we used a new red-shifted VSD, di-4-ANBDQBS, and a fast electron-multiplied charge-coupled device camera for optical AP (OAP) recording in guinea pig cardiac myocytes. Loading di-4-ANBDQBS did not alter APs recorded with micropipette. With short laser exposures (just enough to record one OAP every 1-5 min), di-4-ANBDQBS yielded fluorescent signals with very high signal-to-background ratios (change in fluorescence on depolarization/fluorescence at resting potential: 19.2 ± 4.1%) and signal-to-noise ratios (40 ± 13.2). Quantum chemical calculations comparing the ANBDQ chromophore to the conventional ANEP chromophore showed that the higher wavelength and the greater voltage sensitivity of the former have the same electro-optical origin: a longer path for electron redistribution in the excited state. OAP closely tracked simultaneously recorded electrical APs, permitting measurement of AP duration within 1% error. Prolonged laser exposure caused progressive AP duration prolongation and instability. However, these effects were alleviated or abolished by reducing the dye concentration and by perfusion with antioxidants. Thus the presented technique provides a unique opportunity for noninvasive AP recording in single cardiomyocytes.

"Heart Oddity": Intrinsically Reduced Excitability in the Right Ventricle Requires Compensation by Regionally Specific Stress Kinase Function.

The traditional view of ventricular excitation and conduction is an all-or-nothing response mediated by a regenerative activation of the inward sodium channel, which gives rise to an essentially constant conduction velocity (CV). However, whereas there is no obvious biological need to tune-up ventricular conduction, the principal molecular components determining CV, such as sodium channels, inward-rectifier potassium channels, and gap junctional channels, are known targets of the "stress" protein kinases PKA and calcium/calmodulin dependent protein kinase II (CaMKII), and are thus regulatable by signal pathways converging on these kinases. In this mini-review we will expose deficiencies and controversies in our current understanding of how ventricular conduction is regulated by stress kinases, with a special focus on the chamber-specific dimension in this regulation. In particular, we will highlight an odd property of cardiac physiology: uniform CV in ventricles requires co-existence of mutually opposing gradients in cardiac excitability and stress kinase function. While the biological advantage of this peculiar feature remains obscure, it is important to recognize the clinical implications of this phenomenon pertinent to inherited or acquired conduction diseases and therapeutic interventions modulating activity of PKA or CaMKII.

Modulation of the sinus rate during ventricular fibrillation.

BACKGROUND: During supraventricular and ventricular tachycardia, the arterial baroreflex predominates with minimal contribution from the cardiopulmonary reflex. To our knowledge, the role of the arterial baroreflex gain (BRG) during and immediately following termination of ventricular fibrillation (VF) has not been characterized. OBJECTIVE: We hypothesized that (1) arterial BRG correlated with sinus node cycle length (SNCL) changes during VF, and that (2) the greater the arterial BRG, the greater the blood pressure (BP) recovery following successful defibrillation. METHODS: Arterial BRG was assessed in 18 patients referred for the implantation of a defibrillator incorporating an atrial lead. The average SNCL was measured during the 5 seconds prior to VF induction and the last 5 seconds during VF before defibrillation. Percent SNCL change (%DeltaSNCL) was determined. Arterial BP recovery was calculated as the difference in mean BP following defibrillation compared to during VF. RESULTS: Arterial BRG ranged between -3 and 18 ms/mmHg. During VF, SNCL shortened in 11 patients (group A, mean %DeltaSNCL =-15%), and surprisingly lengthened in seven patients (group B, mean %DeltaSNCL = 5%). There was no correlation between %DeltaSNCL and arterial BRG. In fact, arterial BRG in group A was lower when compared with group B (P = 0.075). Similarly, there was no correlation between arterial BRG and BP recovery. CONCLUSIONS: We found no correlation between arterial BRG and %DeltaSNCL during VF, or BP recovery following defibrillation. Our findings of SNCL lengthening in 7 of 18 patients suggest that in some patients, arterial BRG plays a minor role during VF with a greater contribution from the cardiopulmonary BRG.

Spatiotemporal relationship between intracellular Ca2+ dynamics and wave fragmentation during ventricular fibrillation in isolated blood-perfused pig hearts.

Normal "master-slave" relationship between the action potential (AP) and intracellular Ca2+ transient (Ca(i)T) is sometimes altered during ventricular fibrillation (VF). The nature of AP/Ca(i)T dissociation during VF and its role in inducing wavebreaks (WBs) remain unclear. We simultaneously mapped AP (RH237) and Ca(i)T (Rhod-2) during VF in blood-perfused pig hearts. We computed AP and Ca(i)T dominant frequency (DF) and Ca(i)T delay in each AP cycle. We identified WBs as singularity points in AP phase movies and sites of conduction block (CB) as sites where an AP wavefront failed to propagate. We analyzed spatiotemporal relationship between abnormal AP/Ca(i)T sequences and CB sites. We used a calcium chelator (BAPTA-AM) to abolish Ca(i)T and test its involvement in WB formation. During VF, the DF difference between AP and Ca(i)T was <10% of the respective values in 95% of pixels, and 80% of all Ca(i)T upstrokes occurred during the initial 25% of the excitation cycle. Aberrant sequences of AP and Ca(i)T occurred almost exclusively near CB sites but could be traced to normal wavefront sequences away from CB sites. Thus, apparent AP/Ca(i)T dissociation was largely attributable to spatial uncertainty of the absolute position of block of each wave. BAPTA-AM reduced Ca(i)T amplitude to 30.5+/-12.9% of control and the DF of AP from 12.2+/-1.6 to 10.4+/-1.3 Hz (P<0.01), but did not significantly alter WB incidence (0.76+/-0.19 versus 0.72+/-0.19 SP/mm2). These results do not support presence of spontaneous, non-voltage-gated Ca(i)Ts during VF and suggest that AP/Ca(i)T dissociation is a consequence rather than a cause of wave fragmentation.

Spatial distribution of fibrosis governs fibrillation wave dynamics in the posterior left atrium during heart failure.

Heart failure (HF) commonly results in atrial fibrillation (AF) and fibrosis, but how the distribution of fibrosis impacts AF dynamics has not been studied. HF was induced in sheep by ventricular tachypacing (220 bpm, 6 to 7 weeks). Optical mapping (Di-4-ANEPPS, 300 frames/sec) of the posterior left atrial (PLA) endocardium was performed during sustained AF (burst pacing) in Langendorff-perfused HF (n=7, 4 micromol/L acetylcholine; n=3, no acetylcholine) and control (n=6) hearts. PLA breakthroughs were the most frequent activation pattern in both groups (72.0+/-4.6 and 90.2+/-2.7%, HF and control, respectively). However, unlike control, HF breakthroughs preferentially occurred at the PLAs periphery near the pulmonary vein ostia, and their beat-to-beat variability was greater than control (1.93+/-0.14 versus 1.47+/-0.07 changes/[beats/sec], respectively, P<0.05). On histological analysis (picrosirius red), the area of diffuse fibrosis was larger in HF (23.4+/-0.4%) than control (14.1+/-0.6%; P<0.001, n=4). Also the number and size of fibrous patches were significantly larger and their location was more peripheral in HF than control. Computer simulations using 2-dimensional human atrial models with structural and ionic remodeling as in HF demonstrated that changes in AF activation frequency and dynamics were controlled by the interaction of electrical waves with clusters of fibrotic patches of various sizes and individual pulmonary vein ostia. During AF in failing hearts, heterogeneous spatial distribution of fibrosis at the PLA governs AF dynamics and fractionation.

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia.

While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salvaging the myocardium after an ischemic attack. Here we used neonatal rat ventricular myocytes (NRVMs) subjected to 20-30 min of simulated ischemia followed by 1 hour of "reperfusion". Using different combinations of spectrally-compatible fluorescent indicators, we analyzed the relative timing of the following events: (1) abnormal increase in cytoplasmic [Ca2+] (TCaCy); (2) abnormal increase in mitochondrial [Ca2+] (TCaMi); (3) loss of mitochondrial inner membrane potential (ΔΨm) indicating mitochondrial permeability transitions (TMPT); (4) sacrolemmal permeabilization (SP) to the normally impermeable small fluorophore TO-PRO3 (TSP). In additional experiments we also analyzed the timing of abnormal uptake of Zn2+ into the cytoplasm (TZnCy) relative to TCaCy and TSP. We focused on those NRVMs which survived anoxia, as evidenced by at least 50% recovery of ΔΨm and the absence of detectable SP. In these cells, we found a consistent sequence of critical events in the order, from first to last, of TCaCy, TCaMi, TMPT, TSP. After detecting TCaCy and TCaMi, abrupt switches between 1.1 mM and nominally zero [Ca2+] in the perfusate quickly propagated to the cytoplasmic and mitochondrial [Ca2+]. Depletion of the sarcoplasmic reticulum with ryanodine (5 µM)/thapsigargin (1 µM) accelerated all events without changing their order. In the presence of ZnCl2 (10-30 µM) in the perfusate we found a consistent timing sequence TCaCy < TZn ≤ TSP. In some cells ZnCl2 interfered with Ca2+ uptake, causing "steps" or "gaps" in the [Ca2+]Cy curve, a phenomenon never observed in the absence of ZnCl2. Together, these findings suggest an evolving permeabilization of NRVM's sarcolemma during reoxygenation, in which the expansion of the pore size determines the timing of critical events, including TMPT.

Influence of the skeletal muscle activity on time and frequency domain properties of the body surface ECG during evolving ventricular fibrillation in the pig.

AIM OF THE STUDY: To evaluate influence of the skeletal muscle activity (SMA) on time and frequency domain properties of ECG during VF. MATERIALS AND METHODS: We studied the first 9min of electrically induced VF (N=7). We recorded Lead II ECG, 247 unipolar epicardial ventricular electrograms (UEGs) and 3 bipolar skeletal electromyograms (EMGs) near the positions of the ECG electrodes (sampling rate, 500Hz). We reconstructed ECG (RECG) from UEGs using forward-solution transformation matrix. Spectral properties of ECG, RECG, UEGs and MEGs were assessed in the range 2-250Hz by the median frequency (MF) and the upper limit of frequency range containing 99% of spectral energy (Flim(99)). Scaling exponent of ECG, RECG and EMGs was calculated in the ranges of 1-8 and 5-20 sampling intervals (ScE1-8 and ScE5-20, respectively). RESULTS: We observed non-monotonic increases in MF and Flim(99) of the ECG, but not UEGs and RECG, at 1-5min of VF. Maximum values of MF and Flim(99) in ECG, UEGs and RECG were (in Hz): 32+/-29 and 166+/-67; 11+/-2 and 36+/-7; 10+/-2 and 32+/-6, respectively. The transient increases in the high-frequency content of the ECG were correlated with enhanced activity in EMGs, characterized by an almost uniform spectrum in the range 2-250Hz (MF=92+/-29; Flim(99)=245+/-4Hz). Peak values of ScE(1-8) were the highest in EMGs (1.95+/-0.04), intermediate in the ECG (1.59+/-0.26), and the lowest in RECG (1.088+/-0.007). CONCLUSION: SMA significantly contributes to ECG during VF and can bias metrics used for assessment of VF organization.

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.

Mechanisms of wave fractionation at boundaries of high-frequency excitation in the posterior left atrium of the isolated sheep heart during atrial fibrillation.

BACKGROUND: High-frequency fractionated electrograms recorded during atrial fibrillation (AF) in the posterior left atrium (PLA) and elsewhere are being used as target sites for catheter ablation. We tested the hypothesis that highly periodic electric waves emerging from AF sources at or near the PLA give rise to the most fractionated activity in adjacent locations. METHODS AND RESULTS: Sustained AF was induced in 8 isolated sheep hearts (0.5 micromol/L acetylcholine). Endocardial videoimaging (DI-4-ANEPPS) and electric mapping of the PLA enabled spatial characterization of dominant frequencies (DFs) and a regularity index (ratio of DF to total power). Regularity index showed that fractionation was lowest within the area with the maximal DF (DFmax domain; 0.19+/-0.02) and highest within a band of &3 mm (0.16+/-0.02; P=0.047) at boundaries with lower-frequency domains. The numbers of spatiotemporal periodic episodes (25.9+/-2.3) and rotors per experiment (1.9+/-0.7) were also highest within the DFmax domain. Most commonly, breakthrough waves at the PLA traveled toward the rest of the atria (76.8+/-8.1% outward versus 23.2+/-8.1% inward; P<0.01). In both experiments and simulations with an atrial ionic model, fractionation at DFmax boundaries was associated with increased beat-to-beat variability of conduction velocity and directionality with wavebreak formation. CONCLUSIONS: During stable AF, the PLA harbors regular, fast, and highly organized activity; the outer limit of the DFmax domain is the area where the most propagation pattern variability and fractionated activity occur. These new concepts introduce a new perspective in the clinical use of high-frequency fractionated electrograms to localize sources of AF precisely at the PLA and elsewhere.

Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium.

BACKGROUND: Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been used successfully for optical mapping in cardiac cells and tissues. However, their utility for probing electrical activity deep inside the myocardial wall and in blood-perfused myocardium has been limited because of light scattering and high absorption by endogenous chromophores and hemoglobin at blue-green excitation wavelengths. OBJECTIVE: The purpose of this study was to characterize two new styryl dyes--di-4-ANBDQPQ (JPW-6003) and di-4-ANBDQBS (JPW-6033)--optimized for blood-perfused tissue and intramural optical mapping. METHODS: Voltage-dependent spectra were recorded in a model lipid bilayer. Optical mapping experiments were conducted in four species (mouse, rat, guinea pig, and pig). Hearts were Langendorff perfused using Tyrode's solution and blood (pig). Dyes were loaded via bolus injection into perfusate. Transillumination experiments were conducted in isolated coronary-perfused pig right ventricular wall preparations. RESULTS: The optimal excitation wavelength in cardiac tissues (650 nm) was >70 nm beyond the absorption maximum of hemoglobin. Voltage sensitivity of both dyes was approximately 10% to 20%. Signal decay half-life due to dye internalization was 80 to 210 minutes, which is 5 to 7 times slower than for di-4-ANEPPS. In transillumination mode, DeltaF/F was as high as 20%. In blood-perfused tissues, DeltaF/F reached 5.5% (1.8 times higher than for di-4-ANEPPS). CONCLUSION: We have synthesized and characterized two new near-infrared dyes with excitation/emission wavelengths shifted >100 nm to the red. They provide both high voltage sensitivity and 5 to 7 times slower internalization rate compared to conventional dyes. The dyes are optimized for deeper tissue probing and optical mapping of blood-perfused tissue, but they also can be used for conventional applications.

Mechanisms of atrial fibrillation termination by pure sodium channel blockade in an ionically-realistic mathematical model.

The mechanisms by which Na+-channel blocking antiarrhythmic drugs terminate atrial fibrillation (AF) remain unclear. Classical "leading-circle" theory suggests that Na+-channel blockade should, if anything, promote re-entry. We used an ionically-based mathematical model of vagotonic AF to evaluate the effects of applying pure Na+-current (I(Na)) inhibition during sustained arrhythmia. Under control conditions, AF was maintained by 1 or 2 dominant spiral waves, with fibrillatory propagation at critical levels of action potential duration (APD) dispersion. I(Na) inhibition terminated AF increasingly with increasing block, terminating all AF at 65% block. During 1:1 conduction, I(Na) inhibition reduced APD (by 13% at 4 Hz and 60% block), conduction velocity (by 37%), and re-entry wavelength (by 24%). During AF, I(Na) inhibition increased the size of primary rotors and reduced re-entry rate (eg, dominant frequency decreased by 33% at 60% I(Na) inhibition) while decreasing generation of secondary wavelets by wavebreak. Three mechanisms contributed to I(Na) block-induced AF termination in the model: (1) enlargement of the center of rotation beyond the capacity of the computational substrate; (2) decreased anchoring to functional obstacles, increasing meander and extinction at boundaries; and (3) reduction in the number of secondary wavelets that could provide new primary rotors. Optical mapping in isolated sheep hearts confirmed that tetrodotoxin dose-dependently terminates AF while producing effects qualitatively like those of I(Na) inhibition in the mathematical model. We conclude that pure INa inhibition terminates AF, producing activation changes consistent with previous clinical and experimental observations. These results provide insights into previously enigmatic mechanisms of class I antiarrhythmic drug-induced AF termination. The full text of this article is available online at http://circres.ahajournals.org

Frequency-dependent breakdown of wave propagation into fibrillatory conduction across the pectinate muscle network in the isolated sheep right atrium.

Atrial fibrillation (AF) may result from stationary reentry in the left atrium (LA), with fibrillatory conduction toward the right atrium (RA). We hypothesize that periodic input to the RA at an exceedingly high frequency results in disorganized wave propagation, compatible with fibrillatory conduction. Simultaneous endocardial and epicardial optical mapping (di-4-ANEPPS) was performed in isolated, coronary-perfused sheep RA. Rhythmic pacing of Bachmann's bundle allowed well-controlled and realistic conditions for LA-driven RA. Pacing at increasingly higher frequencies (2.0 to 6.0 Hz) led to increasing delays in activation distal to major branching sites of the crista terminalis and pectinate bundles, culminating in spatially distributed intermittent blockade at or above approximately 6.5 Hz. At this "breakdown frequency," the direction of RA propagation became completely variable from beat to beat and thus transformed into fibrillatory conduction. Such frequency-dependent changes were independent of action potential duration. Rather, the spatial boundaries between proximal and distal frequencies correlated well with branch sites of the pectinate musculature. Thus, there exists a breakdown frequency in the sheep RA below which activity is periodic throughout the atrium and above which it is fibrillation-like. The data are consistent with the ideas that during AF, high-frequency activation initiated in the LA undergoes fibrillatory conduction toward the RA, and that sink-to-source effect at branch points of the crista terminalis and pectinate muscles is important in determining the complexity of the arrhythmia.