Ariel L. Escobar

Transient Ca2+ depletion of the sarcoplasmic reticulum at the onset of reperfusion

AIMS Myocardial stunning is a contractile dysfunction that occurs after a brief ischaemic insult. Substantial evidence supports that this dysfunction is triggered by Ca2+ overload during reperfusion. The aim of the present manuscript is to define the origin of this Ca2+ increase in the intact heart. METHODS AND RESULTS To address this issue, Langendorff-perfused mouse hearts positioned on a pulsed local field fluorescence microscope and loaded with fluorescent dyes Rhod-2, Mag-fluo-4, and Di-8-ANEPPS, to assess cytosolic Ca2+, sarcoplasmic reticulum (SR) Ca2+, and transmembrane action potentials (AP), respectively, in the epicardial layer of the hearts, were submitted to 12 min of global ischaemia followed by reperfusion. Ischaemia increased cytosolic Ca2+ in association with a decrease in intracellular Ca2+ transients and a depression of Ca2+ transient kinetics, i.e. the rise time and decay time constant of Ca2+ transients were significantly prolonged. Reperfusion produced a transient increase in cytosolic Ca2+ (Ca2+ bump), which was temporally associated with a decrease in SR-Ca2+ content, as a mirror-like image. Caffeine pulses (20 mM) confirmed that SR-Ca2+ content was greatly diminished at the onset of reflow. The SR-Ca2+ decrease was associated with a decrease in Ca2+ transient amplitude and a shortening of AP duration mainly due to a decrease in phase 2. CONCLUSION To the best of our knowledge, this is the first study in which SR-Ca2+ transients are recorded in the intact heart, revealing a previously unknown participation of SR on cytosolic Ca2+ overload upon reperfusion in the intact beating heart. Additionally, the associated shortening of phase 2 of the AP may provide a clue to explain early reperfusion arrhythmias.

Calsequestrin 2 deletion shortens the refractoriness of Ca2+ release and reduces rate-dependent Ca2+-alternans in intact mouse hearts

Calsequestrin (Casq2) is a low affinity Ca(2+)-binding protein located in sarcoplasmic reticulum (SR) of cardiac myocytes. Casq2 acts as a Ca(2+) buffer regulating free Ca(2+) concentration in the SR lumen and plays a significant role in the regulation of Ca(2+) release from this intracellular organelle. In addition, there is experimental evidence supporting the hypothesis that Casq2 also modulates the activity of the cardiac Ca(2+) release channels, ryanodine receptors (RyR2). In this study, Casq2 knockout mice (Casq2-/-) were used as a model to evaluate the effects of the Casq2 on the cytosolic and intra-SR Ca(2+) dynamics, and the electrical activity in the ventricular epicardial layer of intact beating hearts. Casq2-/- mice have accelerated intra-SR Ca(2+) refilling kinetics (76 ± 22 vs. 136.5 ± 15 ms) and a reduced refractoriness of Ca(2+) release (182 ± 32 ms Casq2+/+ and 111 ± 22 ms Casq2-/- ). In addition, mice display reduced Ca(2+) alternans (67% decline in the amplitude of Ca(2+) alternans at 7 Hz, 21oC) and less T-wave alternans at the electrocardiographic level. The results presented in this paper support the idea of Casq2 acting both as a buffer and a direct regulator of the Ca(2+) release process. Finally, we propose that alterations in Ca(2+) release refractoriness shown here could explain the relationship between Casq2 function and an increase in the risk for ventricular arrhythmias.

Adaptation of Single Cardiac Ryanodine Receptor Channels

Single cardiac ryanodine receptor (RyR) channel adaptation was previously defined with Ca2+ stimuli produced by flash photolysis of DM-nitrophen (caged-Ca+2). Photolysis of DM-nitrophen induced a very fast Ca+2 overshoot (Ca+2 spike) at the leading edge of the Ca+2 stimuli. It has been suggested that adaptation (tau approximately 1.3 s) may reflect Ca+2 slowly coming off the RyR Ca+2 activation sites following the faster Ca+2 spike (tau approximately 1 ms). This concern was addressed by defining the Ca2+ deactivation kinetics of single RyR channels in response to a rapid reduction in free Ca2+ concentration ([Ca2+]FREE). The [Ca2+]FREE was lowered by photolysis of Diazo-2. Single RyR channels deactivated (tau approximately 5.3 ms) quickly in response to the photolytically induced [Ca2+]FREE reduction. Improved estimates of the Ca2+ spike time course indicate that the Ca2+ spike is considerably faster (10-100-fold) than previously thought. Our data suggest that single RyRs are not significantly activated by fast Ca2+ spikes and that RyR adaptation is not due to deactivation following the fast Ca2+ spike. Thus, RyR adaptation may have an important impact on Ca2+ signaling in heart.

Chasing cardiac physiology and pathology down the CaMKII cascade

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such as Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation but also in cell death, transcriptional activation of hypertrophy, inflammation, and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation, and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.

Beat to beat Ca2+-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca(2+) regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca(2+) regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca(2+) buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca(2+). Prior to introduction of the caged Ca(2+) buffer, spontaneous local Ca(2+) releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r²=0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca(2+) transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca(2+) was acutely released from the caged compound by flash photolysis, intracellular Ca(2+) dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca(2+) dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca(2+) regulates normal SANC automaticity on a beat-to-beat basis.

Luminal Ca2+ content regulates intracellular Ca2+ release in subepicardial myocytes of intact beating mouse hearts: effect of exogenous buffers

Ca(+)-induced Ca(2+) release tightly controls the function of ventricular cardiac myocytes under normal and pathological conditions. Two major factors contributing to the regulation of Ca(2+) release are the cytosolic free Ca(2+) concentration and sarcoplasmic reticulum (SR) Ca(2+) content. We hypothesized that the amount of Ca(2+) released from the SR during each heart beat strongly defines the refractoriness of Ca(2+) release. To test this hypothesis, EGTA AM, a high-affinity, slow-association rate Ca(2+) chelator, was used as a tool to modify luminal SR Ca(2+) content. An analysis of the cytosolic and luminal SR Ca(2+) dynamics recorded from the epicardial layer of intact mouse hearts indicated that the presence of EGTA reduced the diastolic SR free Ca(2+) concentration and fraction of SR Ca(2+) depletion during each beat. In addition, this maneuver shortened the refractory period and accelerated the restitution of Ca(2+) release. As a consequence of the accelerated restitution, the frequency dependence of Ca(2+) alternans was significantly shifted toward higher heart rates, suggesting a role of luminal SR Ca(2+) in the genesis of this highly arrhythmogenic phenomenon. Thus, intra-SR Ca(2+) dynamics set the refractoriness and frequency dependence of Ca(2+) transients in subepicardial ventricular myocytes.

Role of inositol 1, 4, 5-trisphosphate in the regulation of ventricular Ca2+ signaling in intact mouse heart

Inositol 1,4,5-trisphosphate (InsP(3)R)-mediated Ca(2+) signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP(3)-mediated Ca(2+) signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP(3)-sensitive intracellular Ca(2+) signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP(3)] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP(3). Our results indicate that the basal [Ca(2+)] increased after the flash photolysis of caged InsP(3) without affecting the action potential (AP)-induced Ca(2+) transients. The amplitude of the basal [Ca(2+)] elevation depended on the intracellular [InsP(3)] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP(3)-induced Ca(2+) release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca(2+) release from both RyR- and InsP(3)R-containing Ca(2+) stores, suggesting that these pools have similar Ca(2+) reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP(3) was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitation-contraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.

Ca2+ Sparks and Ca2+ waves are the subcellular events underlying Ca2+ overload during ischemia and reperfusion in perfused intact hearts

Abnormal intracellular Ca(2+) cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca(2+). At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca(2++), which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca(2+) increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses: (1) An increase diastolic Ca(2+) sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca(2+) increase; (2) an increase in cytosolic Ca(2+) pro-arrhythmogenic events (Ca(2+) waves), mediates the abrupt diastolic Ca(2+) rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca(2+) sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07±0.33 vs. 1.13±0.20 sp/s/100 μm, n=29/34, 7 hearts). Reperfusion significantly changed Ca(2+) sparks kinetics, by prolonging Ca(2+) sparks rise time and decreased cspf. However, it significantly increased Ca(2+) wave frequency relative to ischemia (0.71±0.14 vs. 0.38±0.06 w/s/100 μm, n=32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca(2+) sparks and provides direct evidence of increased Ca(2+) sparks in ischemia that transform into Ca(2+) waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.

Cardiac Alternans and Ventricular Fibrillation: A Bad Case of Ryanodine Receptors Reneging on Their Duty

Since the first description in 1872 of cardiac alternans by Traube1 in a patient with alcoholic cardiomyopathy, there has been significant progress in understanding the significance of this clinical sign of heart disease. Traube reported this phenomenon before Einthoven invented the ECG in 1903. Thus, he actually described pulsus alternans in his patient, a strong–weak arterial pulse alternation perceptible by unaided finger tact. The patient died shortly after diagnosis, but because he had severe cardiomyopathy, cardiac alternans was not recognized as an ancillary index of disease severity or as a harbinger of mortality. Later, the introduction of ECGs in the routine examination of patients made it clear that cardiac alternans, in the form of T-wave alternans, was a common sign in several cardiomyopathies, such as heart failure, coronary artery disease, genetic and acquired channelopathies, and even in electrolyte disturbances of the body. However, an unequivocal association between cardiac alternans and arrhythmia risk was not recognized until the 1990s,2 and only recently a multicenter clinical trial established that patients with moderate cardiac dysfunction but lacking T-wave alternans may not need an implantable cardiac defibrillator to improve their odds of avoiding sudden cardiac death.3 Thus, from the peculiar weak–strong arterial stroke oscillation detected by Traube to a critical risk stratification factor for sudden cardiac death, cardiac alternans have come a long way as diagnostic and prognostic manifestation of cardiac disease. Article see p 1410 If the pathway of cardiac alternans in the clinical arena has been a smoothly ascending line, explaining its precise cellular mechanisms has resulted in a more tortuous process, reflecting the complexity of the phenomenon. The ability to induce mechanical alternans by rapidly stimulating the heart was recognized early as an inherent ability of all mammalian ventricular muscles.4 Initially, researchers relied on traditional whole heart …

Intact Heart Loose Patch Photolysis Reveals Ionic Current Kinetics During Ventricular Action Potentials

RATIONALE Assessing the underlying ionic currents during a triggered action potential (AP) in intact perfused hearts offers the opportunity to link molecular mechanisms with pathophysiological problems in cardiovascular research. The developed loose patch photolysis technique can provide striking new insights into cardiac function at the whole heart level during health and disease. OBJECTIVE To measure transmembrane ionic currents during an AP to determine how and when surface Ca(2+) influx that triggers Ca(2+)-induced Ca(2+) release occurs and how Ca(2+)-activated conductances can contribute to the genesis of AP phase 2. METHODS AND RESULTS Loose patch photolysis allows the measurement of transmembrane ionic currents in intact hearts. During a triggered AP, a voltage-dependent Ca(2+) conductance was fractionally activated (dis-inhibited) by rapidly photo-degrading nifedipine, the Ca(2+) channel blocker. The ionic currents during a mouse ventricular AP showed a fast early component and a slower late component. Pharmacological studies established that the molecular basis underlying the early component was driven by an influx of Ca(2+) through the L-type channel, CaV 1.2. The late component was identified as an Na(+)-Ca(2+) exchanger current mediated by Ca(2+) released from the sarcoplasmic reticulum. CONCLUSIONS The novel loose patch photolysis technique allowed the dissection of transmembrane ionic currents in the intact heart. We were able to determine that during an AP, L-type Ca(2+) current contributes to phase 1, whereas Na(+)-Ca(2+) exchanger contributes to phase 2. In addition, loose patch photolysis revealed that the influx of Ca(2+) through L-type Ca(2+) channels terminates because of voltage-dependent deactivation and not by Ca(2+)-dependent inactivation, as commonly believed.