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

Corazón de ratón perfundido tipo Langendorff: isquemia global simulada versus no simulada

El corazón sometido a isquemia sufre una disfunción en el manejo del calcio (Ca2+) intracelular y en la función contráctil, que se recupera parcialmente durante la reperfusión del mismo. La magnitud de estas alteraciones se agrava en función del tiempo de duración de la isquemia. Para el estudio de esta patología se utilizan diferentes preparados, siendo el más común la retroperfusión aórtica (tipo Langendorff) del corazón aislado sometido a isquemia global o regional por cese de flujo. Algunos estudios requieren la utilización de cardiomiocitos aislados, los cuales por sus características son sometidos a isquemia simulada químicamente. Objetivos: Comparar la función mecánica, el Ca2+ citosólico y la fosforilación de proteínas clave en el manejo del Ca2+ citosólico durante la isquemia global no simulada y la simulada químicamente.Facultad de Ciencias Médica

Corazón de ratón perfundido tipo Langendorff: isquemia global simulada versus no simulada

El corazón sometido a isquemia sufre una disfunción en el manejo del calcio (Ca2+) intracelular y en la función contráctil, que se recupera parcialmente durante la reperfusión del mismo. La magnitud de estas alteraciones se agrava en función del tiempo de duración de la isquemia. Para el estudio de esta patología se utilizan diferentes preparados, siendo el más común la retroperfusión aórtica (tipo Langendorff) del corazón aislado sometido a isquemia global o regional por cese de flujo. Algunos estudios requieren la utilización de cardiomiocitos aislados, los cuales por sus características son sometidos a isquemia simulada químicamente. Objetivos: Comparar la función mecánica, el Ca2+ citosólico y la fosforilación de proteínas clave en el manejo del Ca2+ citosólico durante la isquemia global no simulada y la simulada químicamente.Facultad de Ciencias Médica

Corazón de ratón perfundido tipo Langendorff: isquemia global simulada versus no simulada

El corazón sometido a isquemia sufre una disfunción en el manejo del calcio (Ca2+) intracelular y en la función contráctil, que se recupera parcialmente durante la reperfusión del mismo. La magnitud de estas alteraciones se agrava en función del tiempo de duración de la isquemia. Para el estudio de esta patología se utilizan diferentes preparados, siendo el más común la retroperfusión aórtica (tipo Langendorff) del corazón aislado sometido a isquemia global o regional por cese de flujo. Algunos estudios requieren la utilización de cardiomiocitos aislados, los cuales por sus características son sometidos a isquemia simulada químicamente. Objetivos: Comparar la función mecánica, el Ca2+ citosólico y la fosforilación de proteínas clave en el manejo del Ca2+ citosólico durante la isquemia global no simulada y la simulada químicamente.Facultad de Ciencias Médica

The two-loop four-fermion scattering amplitude in QED

We present the analytic evaluation of the two-loop corrections to the amplitude for the scattering of four fermions in Quantum Electrodynamics, $f^- + f^+ + F^- + F^+ \to 0$, with $f$ and $F$ representing a massless and a massive lepton, respectively. Dimensional regularization is employed to evaluate the loop integrals. Ultraviolet divergences are removed by renormalizing the coupling constant in the ${\overline{\text{MS}}}$-scheme, and the lepton mass as well as the external fields in the on-shell scheme. The analytic result for the renormalized amplitude is expressed as Laurent series around $d=4$ space-time dimensions, and contains Generalized Polylogarithms with up to weight four. The structure of the residual infrared divergences of the virtual amplitude is in agreement with the prediction of the Soft Collinear Effective Theory. Our analytic results are an essential ingredient for the computation of the scattering cross section for massive fermion-pair production in massless fermion-pair annihilation, i.e. $f^- f^+ \to F^- F^+$, and crossing related processes such as the elastic scattering $f F \to f F$, with up to Next-to-Next to Leading Order accuracy.Comment: 5 pages, 2 figures, 1 table + supplemental materia

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

Two-Loop Four-Fermion Scattering Amplitude in QED

We present the first fully analytic evaluation of the transition amplitude for the scattering of a massless into a massive pair of fermions at the two-loop level in quantum electrodynamics. Our result is an essential ingredient for the determination of the electromagnetic coupling within scattering reactions, beyond the currently known accuracy, which has a crucial impact on the evaluation of the anomalous magnetic moment of the muon. It will allow, in particular, for a precise determination of the leading hadronic contribution to the (g−2)μ in the MUonE experiment at CERN, and therefore can be used to shed light on the current discrepancy between the standard model prediction and the experimental measurement for this important physical observable

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

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 Ca2+ in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca2+ signaling explains the subtle and critical control of important events of ECC and relaxation, such as Ca2+ influx and SR Ca2+ release and uptake. The multifunctional Ca21-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 Ca2+ levels. This activity can be sustained, creating molecular memory after the decline in Ca2+ 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 Ca2+ regulation and dysregulation in cardiac health and disease.Centro de Investigaciones Cardiovasculare