Patricia Holemans

Ultrastructural and Functional Remodeling of the Coupling Between Ca2+ Influx and Sarcoplasmic Reticulum Ca2+ Release in Right Atrial Myocytes From Experimental Persistent Atrial Fibrillation

Rationale: Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function. Objective: To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca2+ release in persistent AF. Methods: We studied cell shortening, membrane currents, and [Ca2+]i in right atrial myocytes isolated from sheep with persistent AF (duration 129±39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca2+]i transients were examined in confocal imaging. Results: Myocyte shortening and underlying [Ca2+]i transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca2+]i. SR Ca2+ content was not different. Calculated fractional SR Ca2+ release was reduced in AF (by 20.6%, P<0.05). Peak Ca2+ current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca2+ release in AF was significantly reduced with increased areas of delayed Ca2+ release. Propagation between RyR was unaffected but Ca2+ release at subsarcolemmal sites was reduced. Rate of Ca2+ extrusion by Na+/Ca2+ exchanger was increased. Conclusions: In persistent AF, reduced SR Ca2+ release despite preserved SR Ca2+ content is a major factor in contractile dysfunction. Fewer Ca2+ channel–RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na+/Ca2+ exchanger, underlie the reduction in Ca2+ release.

Pharmacological Inhibition of Na/Ca Exchange Results in Increased Cellular Ca2+ Load Attributable to the Predominance of Forward Mode Block

Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca2+ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 &mgr;mol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP−/−). During voltage clamp ramps with [Ca2+]i buffering, block of reverse mode block was slightly larger than of forward mode (by 25±5%, P<0.05). In the absence of [Ca2+]i buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca2+ influx and Ca2+ efflux were reduced to the same extent (to 36±6% and 32±4%, respectively). With normal SR function the reduction of inward NCX current (INCX) was 57±10% (n=10); during large caffeine-induced Ca2+ transients, it was larger (82±3%). [Ca2+]i transients evoked during depolarizing steps increased (from 424±27 to 994±127 nmol/L at +10mV, P<0.05), despite a reduction of ICaL by 27%. Resting [Ca2+]i increased; there was a small decrease in the rate of decline of [Ca2+]i. SR Ca2+ content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP−/− mice, in which SR Ca2+ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca2+. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of INCX reduction against effects on Ca2+ handling by refining mode dependence and/or including additional targets.

Different Patterns of Angiotensin II and Atrial Natriuretic Peptide Secretion in a Sheep Model of Atrial Fibrillation

Angiotensin II and Atrial Natriuretic Peptide in AF. Introduction: It is well established that rapid atrial rates, as in atrial fibrillation (AF), cause atrial electrical and structural remodeling leading to the maintenance of AF. The role of neurohumoral changes in this pathophysiologic vicious circle remains unclear.

Exercise training does not improve cardiac function in compensated or decompensated left ventricular hypertrophy induced by aortic stenosis

There is ample evidence that regular exercise exerts beneficial effects on left ventricular (LV) hypertrophy, remodeling and dysfunction produced by ischemic heart disease or systemic hypertension. In contrast, the effects of exercise on pathological LV hypertrophy and dysfunction produced by LV outflow obstruction have not been studied to date. Consequently, we evaluated the effects of 8 weeks of voluntary wheel running in mice (which mitigates post-infarct LV dysfunction) on LV hypertrophy and dysfunction produced by mild (mTAC) and severe (sTAC) transverse aortic constriction. mTAC produced ~40% LV hypertrophy and increased myocardial expression of hypertrophy marker genes but did not affect LV function, SERCA2a protein levels, apoptosis or capillary density. Exercise had no effect on global LV hypertrophy and function in mTAC but increased interstitial collagen, and ANP expression. sTAC produced ~80% LV hypertrophy and further increased ANP expression and interstitial fibrosis and, in contrast with mTAC, also produced LV dilation, systolic as well as diastolic dysfunction, pulmonary congestion, apoptosis and capillary rarefaction and decreased SERCA2a and ryanodine receptor (RyR) protein levels. LV diastolic dysfunction was likely aggravated by elevated passive isometric force and Ca(2+)-sensitivity of myofilaments. Exercise training failed to mitigate the sTAC-induced LV hypertrophy and capillary rarefaction or the decreases in SERCA2a and RyR. Exercise attenuated the sTAC-induced increase in passive isometric force but did not affect myofilament Ca(2+)-sensitivity and tended to aggravate interstitial fibrosis. In conclusion, exercise had no effect on LV function in compensated and decompensated cardiac hypertrophy produced by LV outflow obstruction, suggesting that the effect of exercise on pathologic LV hypertrophy and dysfunction depends critically on the underlying cause.

Role of nitric oxide and oxidative stress in a sheep model of persistent atrial fibrillation

AIMS Oxidative stress can modulate nitric oxide (NO) signalling pathways. Both pathways have been shown to be involved in the pathophysiology of atrial fibrillation (AF), but data are conflicting. We aimed to characterize the NO-pathway and its relation to oxidative stress in persistent AF in a sheep model. METHODS AND RESULTS Persistent AF was induced by rapid atrial pacing for a mean of 136.5 ± 21.7 days. Non-stimulated sheep served as controls. Nicotine adenine dinucleotide phosphate (NADPH) oxidase-stimulated superoxide production was significantly increased in the AF group (+51.3 ± 23.2%, P < 0.01). Although there were no changes in mRNA expression of the different NADPH oxidase subunits, the increased activity was associated with markedly increased protein expression of the NADPH oxidase activator, Rac1 (+26 ± 4.6%, P < 0.05). No differences were seen in superoxide dismutase activity, but glutathione peroxidase activity was lower in the AF group. There was a marked accumulation of 3-nitrotyrosine, a biomarker for peroxynitrite, in atrial tissue of AF animals, as demonstrated by immunohistochemical staining and dot blot analysis (+15.6 ± 1.8%, P < 0.05). Expression of atrial NOS3 mRNA was 24.9 ± 4.4% lower in the AF group vs. control (P < 0.05), while NOS1 and 2 were unchanged. Immunoblot analysis revealed no changes in protein expression. Nitrite/nitrate levels were significantly lower during AF (-24.8 ± 5.8%, P < 0.05). CONCLUSION In a sheep model of persistent AF, NOS3 transcript levels are attenuated and circulating NOx levels decreased. Persistent AF is associated with increased oxidative stress, probably resulting from increased NADPH oxidase activity, without major changes in anti-oxidant capacity of the atrial tissue.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

Aims In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and results Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release.

Early exercise training after myocardial infarction prevents contractile but not electrical remodelling or hypertrophy

AIMS Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca(2+). We investigated whether this represents partial or complete reversal of cellular remodelling. METHODS AND RESULTS Mice with MI following left coronary ligation were given free access to a running wheel (MI(EXE), N = 22) or housed sedentary (MI(SED), N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 +/- 3 microm in MI(EXE) vs. 148 +/- 3 microm in MI(SED) and 136 +/- 2 microm in SHAM; P < 0.05, mean +/- SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MI(SED) (at 1 Hz, L/L(0)=4.4 +/- 0.3% vs. 6.7 +/- 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MI(EXE), L/L(0)=6.4 +/- 0.5%). Membrane currents and [Ca(2+)](i) were measured via whole-cell patch clamping, with Fluo-3 as Ca(2+) indicator, all at 30 degrees C. Ca(2+) transient amplitude, I(CaL) and sarcoplasmic reticulum Ca(2+) content were not different between the three groups. Diastolic Ca(2+) levels at 4 Hz were significantly elevated in MI(SED) only, with a trend to increased spontaneous Ca(2+) release events (sparks). Action potential duration was increased and transient outward K(+) currents significantly reduced after MI; this was unaffected by exercise. CONCLUSIONS Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca(2+) response and has a beneficial effect on diastolic Ca(2+) handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K(+) currents are not affected.

Simultaneous Creation and Evaluation of Linear Radiofrequency Lesions

AbstractBackground: Catheter based “maze” procedures for atrial fibrillation have been hampered by difficult creation and evaluation of continuous and transmural linear lesions. Our aim was to develop an online evaluation method for effective lesion creation based on conventional techniques and using the multipolar ablation catheter, already in place. Methods and Results: We created 15 linear lines in right atria of 13 anesthetized sheep using three multipolar catheter designs (8 × 4 mm 7 Fr, 4 × 6 mm 7 Fr, 8 × 4 mm 3.7 Fr). The lesions were placed on the right posterolateral wall between the orifices of the superior and inferior vena cava. Radiofrequency energy was applied in the temperature-controlled mode to prespecified endpoints (electrogram amplitude decrease to ≤50%; pacing threshold increase by ≥100%; split potentials indicating conduction block). Macroscopically transmural and continuous lesions were achieved in only 3 experiments (29 ± 12 mm × 5 ± 1 mm), all created by 3.7 Fr octapolar catheters inserted through long sheaths. Preset temperature was reached in 96% of the electrodes (vs. 64% in the non-effective experiments; p < 0.01). Electrogram amplitude decrease (to ≤50%) and pacing threshold increase (by ≥100%) did not predict effectiveness. The only criterion that could reliably predict transmural continuous necrosis at histology was the development of split potentials (p ≤ 0.05). Conclusions: Effective creation of linear lesions is difficult. Pliable catheters that conform to the endocardial contour give the best results. The only endpoint that reliably predicted histological transmural continuous necrosis was development of split potentials indicating conduction block.

Paradoxical Undersensing at a High Sensitivity in Dual Chamber Pacemakers

WILLEMS, R., et al.: Paradoxical Undersensing at a High Sensitivity in Dual Chamber Pacemakers. In view of the evolving indications for device therapy in atrial arrhythmia the accurate detection of high rate atrial events is a necessity. In a sheep model of atrial fibrillation (AF) we observed a contradictory behavior of the Thera DR pacemaker. The pacemakers were programmed to deliver burst pacing on detection of sinus rhythm (SR). Paradoxically, progressively more inappropriate bursts were delivered during AF, at a higher sensitivity. This implied that the pacemaker interpreted AF as SR. We assessed the atrial detection of the Thera DR, Diamond, Saphir, and Marathon pacemakers during AF, in a sheep model and in vitro using a waveform generator. By counting the annotated atrial‐sensed events reported by the pacemaker we charted the behavior of the pacemakers at different sensitivities. At a higher sensitivity both the Thera DR and the Diamond paradoxically reported fewer atrial events during AF. This behavior led to inappropriate mode switching and incorrect diagnostic data collection. It could be reproduced in vitro. The Marathon did not show this paradoxical undersensing in vivo or in vitro. This paradoxical undersensing at a high sensitivity is dependent on the amplitude of the input signal and could not be explained by an overlap of programmable timing parameters. It is caused by ringing of the atrial sensing amplifier. At high atrial rates and a high sensitivity the ringing of the repetitive atrial signal input results in blanking of the atrial amplifier. This observation may be relevant in the device management of patients with paroxysmal AF.

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca2+/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation. Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca2+ transient are primarily protein kinase A‐dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events.