Bruno D. Stuyvers

Wide long lasting perinuclear Ca2+ release events generated by an interaction between ryanodine and IP3 receptors in canine Purkinje cells.

The purpose of this study was to determine whether IP(3)Rs contribute to the generation of wide long lasting perinuclear Ca(2+) release events in canine Purkinje cells. Spontaneous Ca(2+) release events (elevations of basal [Ca(2+)] equivalent to F/F(0) 3.4SD over F(0)) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca(2+) waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 microm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035+/-0.0007 events (ev)/microm(2)/s, N=34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022+/-0.00005 ev/microm(2)/s, N=41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0+/-3.9 vs. TE 9.0+/-0.3 microm(2), P<0.01) and higher amplitude (F/F(0) 1.38+/-0.02 vs. TE 1.20+/-0.003, P<0.01). WLE event rate was increased by phenylephrine (10 microM, P<0.01), inhibited by 2APB and U73122 (P<0.05), and abolished by tetracaine (1 mM) and ryanodine (100 microM). While SSL WLEs were scattered randomly, Core WLEs (n=69 events) were predominantly distributed longitudinally 18.2+/-1.6 microm from the center of nuclei. Immunocytochemistry showed that IP(3)R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca(2+) release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP(3)R1s evoked Ca(2+) release and may play a role in Ca(2+) dependent nuclear processes.

Ventricular arrhythmias and the His-Purkinje system

Ventricular arrhythmias are a major cause of sudden death, which accounts for approximately half of cardiac mortality. The His–Purkinje system is composed of specialized cells responsible for the synchronous activation of the ventricles. However, experimental studies show that the Purkinje system can be arrhythmogenic during electrolyte imbalance, after exposure to various drugs, and in myocardial ischaemia, during which Purkinje cells can survive in anaerobic conditions. Purkinje cells can generate both automatic and triggered focal rhythms, and their network configuration can accommodate re-entrant circuits. In humans, a variety of monomorphic ventricular tachycardias can be sustained within the architecture of the Purkinje branches. Furthermore, discrete Purkinje sources can serve as critical triggers of ventricular fibrillation in a wide spectrum of patients with structural heart disease or with an apparently normal heart. In drug-resistant cases of monomorphic and polymorphic Purkinje-related ventricular tachycardias, catheter ablation is a very effective treatment. The specific transcriptional signatures and functional properties of Purkinje cells, including their intracellular calcium dynamics, underlie their extreme arrhythmogenicity. However, the identification of vulnerable individuals remains challenging, and the molecular mechanisms of Purkinje-related arrhythmias have to be characterized further to enable the development of interventions to prevent lethal cardiac arrhythmias.

Modeling our understanding of the His-Purkinje system.

The His-Purkinje System (HPS) is responsible for the rapid electric conduction in the ventricles. It relays electrical impulses from the atrioventricular node to the muscle cells and, thus, coordinates the contraction of ventricles in order to ensure proper cardiac pump function. The HPS has been implicated in the genesis of ventricular tachycardia and fibrillation as a source of ectopic beats, as well as forming distinct portions of reentry circuitry. Despite its importance, it remains much less well characterized, structurally and functionally, than the myocardium. Notably, important differences exist with regard to cell structure and electrophysiology, including ion channels, intracellular calcium handling, and gap junctions. Very few computational models address the HPS, and the majority of organ level modeling studies omit it. This review will provide an overview of our current knowledge of structure and function (including electrophysiology) of the HPS. We will review the most recent advances in modeling of the system from the single cell to the organ level, with considerations for relevant interspecies distinctions.

Release kinetics of circulating cardiac myosin binding protein-C following cardiac injury

Diagnosis of myocardial infarction (MI) is based on ST-segment elevation on electrocardiographic evaluation and/or elevated plasma cardiac troponin (cTn) levels. However, troponins lack the sensitivity required to detect the onset of MI at its earliest stages. Therefore, to confirm its viability as an ultra-early biomarker of MI, this study investigates the release kinetics of cardiac myosin binding protein-C (cMyBP-C) in a porcine model of MI and in two human cohorts. Release kinetics of cMyBP-C were determined in a porcine model of MI (n = 6, pigs, either sex) by measuring plasma cMyBP-C level serially from 30 min to 14 days after coronary occlusion, with use of a custom-made immunoassay. cMyBP-C plasma levels were increased from baseline (76 ± 68 ng/l) at 3 h (767 ± 211 ng/l) and peaked at 6 h (2,418 ± 780 ng/l) after coronary ligation. Plasma cTnI, cTnT, and myosin light chain-3 levels were all increased 6 h after ligation. In a cohort of patients (n = 12) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy, cMyBP-C was significantly increased from baseline (49 ± 23 ng/l) in a time-dependent manner, peaking at 4 h (560 ± 273 ng/l). In a cohort of patients with non-ST segment elevation MI (n = 176) from the SYNERGY trial, cMyBP-C serum levels were significantly higher (7,615 ± 4,514 ng/l) than those in a control cohort (416 ± 104 ng/l; n = 153). cMyBP-C is released in the blood rapidly after cardiac damage and therefore has the potential to positively mark the onset of MI.

Evoked centripetal Ca2+ mobilization in cardiac Purkinje cells: insight from a model of three Ca2+ release regions

•u2002 Abnormal oscillations of calcium (Ca2+) concentration in cardiac Purkinje cells (P‐cells) have been associated with life‐threatening arrhythmias, but the mechanism by which these cells control their Ca2+ level in normal conditions remains unknown. •u2002 We modelled our previous hypothesis that the principal intracellular Ca2+ compartment (endoplasmic reticulum; ER) which governs intracellular Ca2+ concentration, formed, in P‐cells, three concentric and adjacent layers, each including a distinct Ca2+ release channel. We then tested the model against typical Ca2+ variations observed in stimulated P‐cells. •u2002 We found in swine P‐cells, as in the rabbit and dog, that stimulation evokes an elevation of Ca2+ concentration first under the membrane, which then propagates to the interior of the cell. •u2002 Our mathematical model could reproduce accurately this typical ‘centripetal’ Ca2+ spread, hence supporting (1) the existence of the ‘3 layered’ Ca2+ compartment, and (2) its central role in the regulation of Ca2+ concentration in P‐cells. •u2002 To model the ‘centripetal’ Ca2+ spread, local variations of Ca2+ concentration were calculated for a virtual cell environment encompassing three different regions that mimicked the three layers of ER in P‐cells. Various tests of the model revealed that the second intermediate layer was essential for ‘forwarding’ the Ca2+ elevation from the periphery to the cell centre. •u2002 This novel finding suggests that a thin intermediate layer of specific ER Ca2+ channels controls the entire Ca2+ signalling of P‐cells. Because Ca2+ plays a role in the electric properties of P‐cells, any abnormality affecting this intermediate region is likely to be pro‐arrhythmic and could explain the origin of serious cardiac arrhythmias known to start in the Purkinje fibres.

Small caliber arterial endothelial cells calcium signals elicited by PAR2 are preserved from endothelial dysfunction

Endothelial cell (EC)‐dependent vasodilation by proteinase‐activated receptor 2 (PAR2) is preserved in small caliber arteries in disease states where vasodilation by muscarinic receptors is decreased. In this study, we identified and characterized the PAR2‐mediated intracellular calcium (Ca2+)‐release mechanisms in EC from small caliber arteries in healthy and diseased states. Mesenteric arterial EC were isolated from PAR2 wild‐type (WT) and null mice, after saline (controls) or angiotensin II (AngII) infusion, for imaging intracellular calcium and characterizing the calcium‐release system by immunofluorescence. EC Ca2+ signals comprised two forms of Ca2+‐release events that had distinct spatial‐temporal properties and occurred near either the plasmalemma (peripheral) or center of EC. In healthy EC, PAR2‐dependent increases in the densities and firing rates of both forms of Ca2+‐release were abolished by inositol 1,4,5‐ trisphosphate receptor (IP3R) inhibitor, but partially reduced by transient potential vanilloid channels inhibitor ruthenium red (RR). Acetylcholine (ACh)‐induced less overall Ca2+‐release than PAR2 activation, but enhanced selectively the incidence of central events. PAR2‐dependent Ca2+‐activity, inhibitors sensitivities, IP3R, small‐ and intermediate‐conductance Ca2+‐activated potassium channels expressions were unchanged in EC from AngII WT. However, the same cells exhibited decreases in ACh‐induced Ca2+‐release, RR sensitivity, and endothelial nitric oxide synthase expression, indicating AngII‐induced dysfunction was differentiated by receptor, Ca2+‐release, and downstream targets of EC activation. We conclude that PAR2 and muscarinic receptors selectively elicit two elementary Ca2+ signals in single EC. PAR2‐selective IP3R‐dependent peripheral Ca2+‐release mechanisms are identical between healthy and diseased states. Further study of PAR2‐selective Ca2+‐release for eliciting pathological and/or normal EC functions is warranted.

Role of reactive oxygen species and Ca2+ dissociation from the myofilaments in determination of Ca2+ wave propagation in rat cardiac muscle

Ca(2+) waves are initiated not only by Ca(2+) leak from the sarcoplasmic reticulum (SR), but also by Ca(2+) dissociation from the myofilaments in the myocardium with nonuniform contraction. We investigated whether contractile properties and the production of reactive oxygen species (ROS) affect Ca(2+) wave propagation. Trabeculae were obtained from 76 rat hearts. Force was measured with a strain gauge, sarcomere length with a laser diffraction technique, and [Ca(2+)](i) with fura-2 and a CCD camera (24°C, 2.0mmol/L [Ca(2+)](o)). ROS production was estimated from 2,7-dichlorofluorescein (DCF) fluorescence. Trabeculae were regionally exposed to a jet of solution containing 1) 10mmol/L Ca(2+) to initiate Ca(2+) waves by SR Ca(2+) leak due to Ca(2+) overload within the jet-exposed region, and 2) 0.2mmol/L Ca(2+) or 5mmol/L caffeine to initiate such waves by Ca(2+) dissociation from the myofilaments due to nonuniform contraction. Ca(2+) waves were induced by stimulus trains for 7.5s. Ten-percent muscle stretch increased DCF fluorescence and accelerated Ca(2+) waves initiated due to both Ca(2+) overload and nonuniform contraction. Preincubation with 3μmol/L diphenyleneiodonium or 10μmol/L colchicine suppressed the increase in DCF fluorescence but suppressed acceleration of Ca(2+) waves initiated only due to Ca(2+) overload. Irrespective of preincubation with colchicine, reduction of force after the addition of 10μmol/L blebbistatin did not decelerate Ca(2+) waves initiated due to Ca(2+) overload, while it did decelerate waves initiated due to nonuniform contraction. These results suggest that Ca(2+) wave propagation is modulated by ROS production through an intact microtubule network only during stretch and may be additionally modulated by Ca(2+) dissociated from the myofilaments in the case of nonuniform contraction.

Cardiac expression of ryanodine receptor subtype 3; a strategic component in the intracellular Ca2+ release system of Purkinje fibers in large mammalian heart

BACKGROUNDnThree distinct Ca2+ release channels were identified in dog P-cells: the ryanodine receptor subtype 2 (RyR2) was detected throughout the cell, while the ryanodine receptor subtype 3 (RyR3) and inositol phosphate sensitive Ca2+ release channel (InsP3R) were found in the cell periphery. How each of these channels contributes to the Ca2+ cycling of P-cells is unclear. Recent modeling of Ca2+ mobilization in P-cells suggested that Ca2+ sensitivity of Ca2+induced Ca2+release (CICR) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca2+ release exists in live P-cells. We compared the regional Ca2+ dynamics with the arrangement of intracellular Ca2+ release (CR) channels.nnnMETHODSnGene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca2+ activity and electrically-evoked Ca2+ mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca2+ events. We used the Ca2+ propagation velocities as indicators of channel Ca2+ sensitivity.nnnRESULTSnRyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RyR3 and InsP3R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca2+ release channels revealed an intermediate layer of RyR3 expression between a near-membrane InsP3R-region and a central RyR2-region. We found that cell periphery produced two distinct forms of spontaneous Ca2+-transients: (1) large asymmetrical Ca2+ sparks under the membrane, and (2) typical Ca2+-wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca2+ waves (CWWs) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3μM) and xestospongin C (XeC; 50μM) dramatically reduced their frequency. The Ca2+ wavelets propagated in a 5-10μm thick layered space which matched the intermediate zone of RyR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RyR3 antagonist dantrolene (10μM). The velocity of wavelets was two times larger (86±16μm/s; n=14) compared to CWWs (46±10μm/s; n=11; P<0.05). Electric stimulation triggered a uniform and large elevation of Ca2+ concentration under the membrane which preceded the propagation of Ca2+ into the interior of the cell. Elevated Cai propagated at 150μm/s (147±34μm/s; n=5) through the region equivalent to the zone of RyR3 expression. This velocity dropped by 50% (75±24μm/s; n=5) in the central region wherein predominant RyR2 expression was detected.nnnCONCLUSIONnWe identified two layers of distinct Ca2+ release channels in the periphery of Pcell: an outer layer of InsP3Rs under the membrane and an inner layer of RyR3s. The propagation of Ca2+ events in these layers revealed that Ca2+ sensitivity of Ca2+ release was larger in the RyR3 layer compared to that of other sub-cellular regions. We propose that RyR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

Effect of Carbenoxolone on Arrhythmogenesis in Rat Ventricular Muscle.

BACKGROUNDnConnexin43 (Cx43) is a major connexin that forms gap junction (GJ) channels in the heart and is also present in the cell membrane as unopposed/non-junctional hemichannels and in the inner mitochondrial membrane. By using carbenoxolone (CBX), a blocker of Cx43, the effect of the blockade of Cx43 on Ca(2+)waves and triggered arrhythmias in the myocardium with non-uniform contraction was examined.nnnMETHODS AND RESULTSnTrabeculae were obtained from rat hearts. Force, [Ca(2+)]i, and the diffusion coefficient were measured. Non-uniform contraction was produced with a 2,3-butanedione monoxime jet. Ca(2+)waves were induced by electrical stimulation. Inducibility of arrhythmias was estimated based on the minimal [Ca(2+)]oat which arrhythmias were induced. The Ca(2+)spark rate was measured in isolated single rat ventricular myocytes. CBX reduced the GJ permeability, whereas it did not change force and [Ca(2+)]itransients. CBX increased the Ca(2+)leak from the sarcoplasmic reticulum in trabeculae and increased the Ca(2+)spark rate in isolated single myocytes. CBX increased the velocity of Ca(2+)waves and further increased the inducibility of arrhythmias. Modulation of mitochondrial KATPchannels by diazoxide, cromakalim and 5-hydroxydecanoic acid affected the inducibility of arrhythmias increased by CBX.nnnCONCLUSIONSnThese results suggest that in diseased hearts, Cx43 plays an important role in the occurrence of triggered arrhythmias, probably under the modulation of mitochondrial KATPchannels.