Ionic basis of ventricular action potentials

Dr. Escobar will talk about his cutting-edge approach to understanding molecular mechanisms underlying electrical activity in the heart

Calcium Regulation of Single Ryanodine Receptor Channel Gating Analyzed Using HMM/MCMC Statistical Methods

Type-II ryanodine receptor channels (RYRs) play a fundamental role in intracellular Ca2+ dynamics in heart. The processes of activation, inactivation, and regulation of these channels have been the subject of intensive research and the focus of recent debates. Typically, approaches to understand these processes involve statistical analysis of single RYRs, involving signal restoration, model estimation, and selection. These tasks are usually performed by following rather phenomenological criteria that turn models into self-fulfilling prophecies. Here, a thorough statistical treatment is applied by modeling single RYRs using aggregated hidden Markov models. Inferences are made using Bayesian statistics and stochastic search methods known as Markov chain Monte Carlo. These methods allow extension of the temporal resolution of the analysis far beyond the limits of previous approaches and provide a direct measure of the uncertainties associated with every estimation step, together with a direct assessment of why and where a particular model fails. Analyses of single RYRs at several Ca2+ concentrations are made by considering 16 models, some of them previously reported in the literature. Results clearly show that single RYRs have Ca2+-dependent gating modes. Moreover, our results demonstrate that single RYRs responding to a sudden change in Ca2+ display adaptation kinetics. Interestingly, best ranked models predict microscopic reversibility when monovalent cations are used as the main permeating species. Finally, the extended bandwidth revealed the existence of novel fast buzz-mode at low Ca2+ concentrations

Phase 1 repolarization rate defines Ca2+ dynamics and contractility on intact mouse hearts.

In the heart, Ca2+ influx through L-type Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum. In most mammals, this influx occurs during the ventricular action potential (AP) plateau phase 2. However, in murine models, the influx through L-type Ca2+ channels happens in early repolarizing phase 1. The aim of this work is to assess if changes in the open probability of 4-aminopyridine (4-AP)-sensitive Kv channels defining the outward K+ current during phase 1 can modulate Ca2+ currents, Ca2+ transients, and systolic pressure during the cardiac cycle in intact perfused beating hearts. Pulsed local-field fluorescence microscopy and loose-patch photolysis were used to test the hypothesis that a decrease in a transient K+ current (Ito) will enhance Ca2+ influx and promote a larger Ca2+ transient. Simultaneous recordings of Ca2+ transients and APs by pulsed local-field fluorescence microscopy and loose-patch photolysis showed that a reduction in the phase 1 repolarization rate increases the amplitude of Ca2+ transients due to an increase in Ca2+ influx through L-type Ca2+ channels. Moreover, 4-AP induced an increase in the time required for AP to reach 30% repolarization, and the amplitude of Ca2+ transients was larger in epicardium than endocardium. On the other hand, the activation of Ito with NS5806 resulted in a reduction of Ca2+ current amplitude that led to a reduction of the amplitude of Ca2+ transients. Finally, the 4-AP effect on AP phase 1 was significantly smaller when the L-type Ca2+ current was partially blocked with nifedipine, indicating that the phase 1 rate of repolarization is defined by the competition between an outward K+ current and an inward Ca2+ current

Thermodynamics of myoplasmic Ca²⁺ alternans reveal the molecular mechanisms involved in its genesis

Myoplasmic Ca²⁺ alternans commonly occur during conditions such as tachycardia, ischemia, or hypothermia. This is a serious condition that can lead to sudden cardiac death. Myoplasmic Ca²⁺ alternans are alternating beat-to-beat changes in the amplitude of the Ca²⁺ transient. They typically arise from a variation in the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variability in the release of Ca²⁺ has previously been attributed to a delay in the recovery of the ryanodine receptor (RyR2), an incomplete Ca²⁺ refilling of the SR, or a change in the duration of the action potential. In each case, the RyR2 will mobilize Ca²⁺ from the SR in an alternating manner, thus generating Ca²⁺ alternans. To investigate the myoplasmic Ca²⁺ alternans in more depth, we utilized a novel experimental approach, Fluorescence Local Field Optical Mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. These recordings were collected in conjunction with a local cold finger, where a temperature gradient was locally imposed on the tissue. In the colder regions, Ca²⁺ alternans were larger and occurred without changes in the duration of the action potential duration. Upon analyzing changes in the Q10 of several kinetic processes defining the intracellular Ca²⁺ dynamics, we found the imposed temperature gradient to have a significant effect on the relaxation of intracellular Ca²⁺ transients. The precipitous temperature dependency of Ca²⁺ alternans observed suggests they arise from an insufficient Ca²⁺ uptake into the SR by the ATPase of SR (SERCA2a). Interestingly, we found Ca2+ alternans to be heavily dependent on the SR Ca²⁺ and could be fostered with increased heart rate, which decreased the time for SERCA2a reuptake into SR beat to beat. Similarly, the partial pharmacological inhibition of SERCA2a with Thapsigargin increased the amplitude of myoplasmic Ca²⁺ alternans. Finally, the FLOM experimental approach is a valuable technique that can shed light on how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.Las alternancias de Ca²⁺ en el mioplasma suelen ocurrir durante una taquicardia, isquemia o hipotermia. Esta condición fisiopatológica puede desencadenar una muerte súbita. Las alternancias de Ca²⁺ mioplásmicas se expresan como un cambio latido a latido en la amplitud del Ca²⁺ sistólico. Estos cambios cíclicos en la amplitud del Ca²⁺ sistólico provienen de variaciones en la liberación de Ca²⁺ desde el retículo sarcoplasmático (RS) entre dos latidos consecutivos. Esta variabilidad en la liberación de Ca²⁺ desde el RS ha sido asociados con una incompleta recuperación del estado inactivado del receptor a rianodina tipo 2 (RyR2), un rellenado incompleto del RS o cambios en la duración del potencial de acción. En cualquier caso, el RyR2 moviliza Ca²⁺ de manera alternadas, generando alternancias mioplásmicas de Ca²⁺. Con la intención de estudiar las alternancias mioplásmicas de Ca²⁺ con mayor profundidad, hemos desarrollado una nueva aproximación experimental, el Mapeo Óptico Fluorescente de Campo Local (FLOM, por sus siglas en ingles). Esta técnica nos ha permitido registrar en la capa epicárdica del ventrículo izquierdo imágenes fluorescentes en un corazón intacto con resolución subcelular. Estas imágenes han sido obtenidas en conjunción con un dedo frio, que permite generar un gradiente de temperatura en el tejido. En las regiones más frías las alternancias mioplásmicas de Ca²⁺ son mayores y ocurren en ausencia de cambios en la duración del potencial de acción. Analizando el Q10 de algunos procesos cinéticos que determinan la dinámica de Ca²⁺ intracelular, encontramos que los gradientes de temperatura afectan la relajación de los transitorios de Ca²⁺. Interesantemente, encontramos que la dependencia de temperatura de las alternancias mioplásmicas de Ca²⁺ provienen de una recaptura insuficiente de Ca2+ hacia el SR mediada por la bomba de Ca2+ SERCa2. Aún más, la inhibición de la bomba de Ca²⁺ con Thapsigargina ha mostrado que si la capacidad de transporte de Ca²⁺ hacia el RS es menor, la amplitud de las alternancias mioplásmicas de Ca²⁺ se incrementan. Finalmente, el desarrollo de FLOM como una nueva aproximación experimental nos ha permitido dilucidad como la arritmogénesis está correlacionada con la distribución espacial de disfunciones metabólicas en miocitos en el miocardio.Sociedad Argentina de Fisiologí

Phospholamban phosphorylation sites enhance the recovery of intracellular Ca2+ after perfusion arrest in isolated, perfused mouse heart

Objective: To investigate the importance of the phosphorylation of Ser16 and Thr17 sites of phospholamban (PLN) on intracellular Ca2+ (Cai2+) handling and contractile recovery of the stunned myocardium. Methods: Cai2+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. Results: Pre-ischemic values of Cai2+ and LVDP were similar in both groups. In PLN-WT, a transient increase in Thr17 phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9 ± 7.6% of pre-ischemic values, n = 14. In contrast, in PLN-DM, there was a poor recovery of Cai2+ transient amplitude and LVDP was significantly lower (28.3 ± 6.7%, n = 11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Cai2+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. Conclusions: PLN phosphorylation appears to be crucial for the mechanical and Cai2+ recovery during stunning and protective against the mechanical abnormalities typical of Cai2+ overload. The importance of PLN phosphorylation would primarily reside in the Thr17 residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations.Facultad de Ciencias Médica

Reperfusion after Ichemia Causes Cytosolic Calcium Overload Due to Rapid Calcium Release from the Sarcoplasmic Reticulum

After a brief ischemic insult, a sustained contractile dysfunction occurs manifested as a sluggish recovery of pump function (myocardial stunning). Substantial evidence supports that myocardial dysfunction is triggered by Ca2+ overload during reperfusion (R). Previous results from different laboratories including our own, describe a cascade of events triggered by R that involves the activation of Na+/H+ and Na+/Ca2+ (NCX) exchangers, with enhanced Ca2+ influx. Whether this Ca2+ influx directly produces the increase in cytosolic Ca2+ or this increase occurs as a consequence of sarcoplasmic reticulum (SR) Ca2+ release triggered in turn by the Ca2+ influx, is not known. To address this issue, we performed 12 min of global no-flow ischemia followed by R in the isovolumic Langendorff perfused mouse heart positioned on a Pulsed Local Field Fluorescence microscope and loaded with fluorescent dyes (Rhod-2 or Mag-Fluo-4 to assess cytosolic or SR Ca2+, respectively). The results indicated an initial increase in diastolic Ca2+ during early R that gradually returned to pre-ischemic levels. This increase was associated with a decrease in SR Ca2+ content that recovered within 10 min, as a mirror image of the diastolic Ca2+ profile. Additional experiments in which caffeine pulses (20 mM) were applied, confirmed that SR Ca2+ content was greatly diminished at the onset of R and gradually recovered within 10 min of R. The present findings indicate that the increase in diastolic Ca2+ that occurs upon R is due to a SR Ca2+ release and not just because of the Ca2+ entry through the reverse NCX mode, as has been previously thought.Facultad de Ciencias Médica

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.Facultad de Ciencias Médica

Phospholamban phosphorylation sites enhance the recovery of intracellular Ca2+ after perfusion arrest in isolated, perfused mouse heart

Objective: To investigate the importance of the phosphorylation of Ser16 and Thr17 sites of phospholamban (PLN) on intracellular Ca2+ (Cai2+) handling and contractile recovery of the stunned myocardium. Methods: Cai2+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. Results: Pre-ischemic values of Cai2+ and LVDP were similar in both groups. In PLN-WT, a transient increase in Thr17 phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9 ± 7.6% of pre-ischemic values, n = 14. In contrast, in PLN-DM, there was a poor recovery of Cai2+ transient amplitude and LVDP was significantly lower (28.3 ± 6.7%, n = 11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Cai2+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. Conclusions: PLN phosphorylation appears to be crucial for the mechanical and Cai2+ recovery during stunning and protective against the mechanical abnormalities typical of Cai2+ overload. The importance of PLN phosphorylation would primarily reside in the Thr17 residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations.Facultad de Ciencias Médica