Journal of Molecular and Cellular Cardiology, 2018
In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including ... more In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca 2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca 2+ ] o (0.5 mM), no changes at 1 mM [Ca 2+ ] o and a negative inotropic effect when [Ca 2+ ] o was increased to 1.8 mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca 2+ content. At all [Ca 2+ ] o studied, 8-CPT induced an increase in Ca 2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca 2+ ] o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca 2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca 2+ handling (uptake vs. leak) in the different situations.
Journal of Molecular and Cellular Cardiology, 2013
Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) loa... more Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca(2+) channel (I(CaL)), Na(+)-Ca(2+) exchanger, Ca(2+) release through the SR ryanodine receptor (RyR2) (I(rel)), Ca(2+) reuptake by the SR Ca(2+) ATPase2a (I(up)), Na(+)-K(+) pump, K(+) efflux through the inward rectifier K(+) channel and the transient outward K(+) flow (I(to)) together with increased activity of the Na(+)-H(+) exchanger (I(NHE)). Simulated CaMKII regulation affecting I(rel), I(up), I(CaL), I(NHE) and I(to) was introduced in the model to partially compensate the acidosis outcome. Late Na(+) current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca(2+) leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca(2+)], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6 min acidosis. The model showed that DADs depend on SR Ca(2+) load and on increased Ca(2+) leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on I(CaL) and I(up), since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca(2+) load at the end of acidosis. Thereafter, DADs are maintained by SR Ca(2+) reloading by Ca(2+) influx through the reverse NCX mode during the time period in which [Na(+)]i is elevated.
American Journal of Physiology-Heart and Circulatory Physiology, 2007
The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxatio... more The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentrati...
We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein ... more We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca 2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [ 3 H]-ryanodine binding and fast Ca 2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca 2+ . Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βA...
Myocardial ischemia/reperfusion (I/R) produces structural and functional alterations depending on... more Myocardial ischemia/reperfusion (I/R) produces structural and functional alterations depending on the duration of ischemia. Brief ischemia followed by reperfusion causes reversible contractile dysfunction (stunned heart) but long-lasting ischemia followed by reperfusion can result in irreversible injury with cell death. Events during I/R can alter endoplasmic reticulum (ER) function leading to the accumulation of unfolded/misfolded proteins. The resulting ER stress induces activation of several signal transduction pathways, known as unfolded protein response (UPR). Experimental evidence shows that UPR contributes to cell death in irreversible I/R injury, however there is still uncertainty for its occurrence in the stunned myocardium. This study investigated the ER Accepted Article This article is protected by copyright. All rights reserved. stress response and its functional impact on the post-ischemic cardiac performance of the stunned heart. Methods Perfused rat hearts were subjected to 20 min of ischemia followed by 30 min of reperfusion. UPR markers were evaluated by qRT-PCR and Western blot. Post-ischemic mechanical recovery was measured in absence and presence of two chemical chaperones: tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). Results Analysis of mRNA and protein levels of various ER stress effectors demonstrated that different UPR signaling cascades, involving both pro-survival and pro-apoptotic pathways, are activated. Inhibition of the UPR with chemical chaperones improved the post-ischemic recovery of cardiac mechanical function without affecting the I/R-induced increase in oxidative stress. Conclusion Our results suggest that prevention of ER stress by chemical chaperones could be a therapeutic tool to limit deterioration of the contractile function in clinical settings in which the phenomenon of myocardial stunning is present.
International journal of molecular sciences, Jan 10, 2018
Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is highe... more Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HF...
American Journal of Physiology-Heart and Circulatory Physiology, 2016
Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) b... more Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca2+ calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide s...
... Injury: Mechanisms and Functional Implications Alicia Mattiazzi, Cecilia Mundina-Weilenmann, ... more ... Injury: Mechanisms and Functional Implications Alicia Mattiazzi, Cecilia Mundina-Weilenmann, Leticia Vittone, Matilde Said, and Paola Ferrero Centro de ... and Western Blot analysis SDS-PAGE was performed using 10% acrylamide slab gels according to Porzio and Pearson as ...
Journal of Molecular and Cellular Cardiology, 2014
Ca 2+-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/... more Ca 2+-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120 min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24 h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knockin mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca 2+ current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.
Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural... more Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca 2+ -handling alterations are known to occur in SHR. However, the putative modifications of Ca 2+ -handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca 2+ , Ca 2+ -handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca 2+ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca 2+ transient decay and an increase in Na + /Ca 2+ exchanger (NCX) abundance (p, 0.05) with no changes in SERCA2a expression/activity. An increased Ca 2+ -calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca 2+ efflux from the cell, constitutes a primary alteration of Ca 2+ -handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.
En la célula miocárdica el aumento del Ca 2+ citoplasmático desencadena la contracción, y su dism... more En la célula miocárdica el aumento del Ca 2+ citoplasmático desencadena la contracción, y su disminución, la relajación. En el miocardio humano la remoción de Ca 2+ del citosol ocurre principalmente a través del secuestro de este ion hacia el retículo sarcoplasmático (RS) por la bomba de Ca 2+ del RS (SERCA2). La phospholamban (PLB) es una proteína del RS que, en su forma desfosforilada, disminuye la afinidad de la SERCA2 por el Ca 2+ e inhibe el secuestro de Ca 2+ por el RS. La fosforilación de la PLB, como ocurre durante la estimulación betaadrenérgica, libera esta inhibición aumentando el secuestro y la carga de Ca 2+ del RS. En constitutive viral versus an endothelin-1-responsive cardiac promoter.
Naunyn-Schmiedeberg's Archives of Pharmacology, 2006
Endogenous catecholamines released during myocardial ischemia have been considered both to aggrav... more Endogenous catecholamines released during myocardial ischemia have been considered both to aggravate cell injury and exacerbate arrhythmias and to exert a protective action on the post-ischemic contractile function. The present work was addressed to look for evidence to explain this controversy. The effects of cardiac catecholamine depletion and of αand β-adrenoceptor (AR) blockade on the post-ischemic contractile dysfunction, as well as its possible relationship with cardiac oxidative stress, were studied in isolated and perfused rat hearts submitted to 20 min of ischemia and 30 min of reperfusion (stunning). Catecholamine depletion improves the contractile recovery in the stunned heart. This mechanical effect was associated with decreased levels of lipid peroxidation. A similar enhancement of the contractile function during reperfusion was detected after the simultaneous blockade of α 1-and β-ARs with prazosin plus propranolol. To ascertain which specific AR pathway was involved in the effects of catecholamines on the stunned heart, selective AR blockers, prazosin (α 1-blocker), atenolol (β 1-blocker), ICI 118,551 (β 2-blocker) and selective inhibitors of G i-PI3K pathway (pertussis toxin and wortmannin) were alternatively combined. The results indicate that catecholamines released during ischemia exert a dual action on the contractile behavior of the stunned heart: a deleterious effect, related to the activation of the β 2-AR-G i-PI3K-pathway, which was counteracted by a beneficial effect, triggered by the stimulation of α 1-AR. Neither the depression nor the enhancement of the post-ischemic contractile recovery were related with the increase in ROS formation induced by endogenous catecholamines.
Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser 16 b... more Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser 16 by PKA and/or at Thr 17 by CaMKII increases the affinity of the SR Ca 2+ pump for Ca 2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr 17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser 16 and Thr 17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr 17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr 17 phosphorylation was dependent on Ca 2+ entry to the cardiac cell. This Ca 2+ influx would mainly occur by the coupled activation of the Na + /H + exchanger and the Na + /Ca 2+ exchanger working in the reverse mode, since phosphorylation of Thr 17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca 2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr 17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr 17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131-136, 2004)
Journal of Molecular and Cellular Cardiology, 2007
We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein ... more We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca 2+ influx to the cell by low [Ca] o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [ 3 H]-ryanodine binding and fast Ca 2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca 2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulation by PKA and CaMKII, respectively. The isoproterenolinduced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the β-induced increase in the channel activity as indicated by the enhancement of the [ 3 H]-ryanodine binding and the velocity of fast SR Ca 2+ release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca 2+ release during βAR stimulation.
Journal of Molecular and Cellular Cardiology, 2014
The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca ... more The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca 2+ ]) is redox sensitive. Here, we report the effects of a mild oxidative stress on cardiac RyR (RyR2) channels in Langendorff perfused rat hearts. Single RyR2 channels from control ventricles displayed the same three responses to Ca 2+ reported in other mammalian tissues, characterized by low, moderate, or high maximal activation. A single episode of 5 min of global ischemia, followed by 1 min of reperfusion, enhanced 2.3-fold the activity of NOX2 compared to controls and changed the frequency distribution of the different responses of RyR2 channels to calcium, favoring the more active ones: high activity response increased and low activity response decreased with respect to controls. This change was fully prevented by perfusion with apocynin or VAS 2870 before ischemia and totally reversed by the extension of the reperfusion period to 15 min. In vitro activation of NOX2 in control SR vesicles mimicked the effect of the ischemia/reperfusion episode on the frequencies of emergence of single RyR2 channel responses to [Ca 2+ ] and increased 2.2-fold the rate of calcium release in Ca 2+-loaded SR vesicles. In vitro changes were reversed at the single channel level by DTT and in isolated SR vesicles by glutaredoxin. Our results indicate that in whole hearts a mild oxidative stress enhances the response of cardiac RyR2 channels to calcium via NOX2 activation, probably by S-glutathionylation of RyR2 protein. This change is transitory and fully reversible, suggesting a possible role of redox modification in the physiological response of cardiac RyR2 to cellular calcium influx.
Journal of Molecular and Cellular Cardiology, 2018
In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including ... more In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca 2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca 2+ ] o (0.5 mM), no changes at 1 mM [Ca 2+ ] o and a negative inotropic effect when [Ca 2+ ] o was increased to 1.8 mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca 2+ content. At all [Ca 2+ ] o studied, 8-CPT induced an increase in Ca 2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca 2+ ] o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca 2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca 2+ handling (uptake vs. leak) in the different situations.
Journal of Molecular and Cellular Cardiology, 2013
Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) loa... more Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca(2+) channel (I(CaL)), Na(+)-Ca(2+) exchanger, Ca(2+) release through the SR ryanodine receptor (RyR2) (I(rel)), Ca(2+) reuptake by the SR Ca(2+) ATPase2a (I(up)), Na(+)-K(+) pump, K(+) efflux through the inward rectifier K(+) channel and the transient outward K(+) flow (I(to)) together with increased activity of the Na(+)-H(+) exchanger (I(NHE)). Simulated CaMKII regulation affecting I(rel), I(up), I(CaL), I(NHE) and I(to) was introduced in the model to partially compensate the acidosis outcome. Late Na(+) current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca(2+) leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca(2+)], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6 min acidosis. The model showed that DADs depend on SR Ca(2+) load and on increased Ca(2+) leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on I(CaL) and I(up), since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca(2+) load at the end of acidosis. Thereafter, DADs are maintained by SR Ca(2+) reloading by Ca(2+) influx through the reverse NCX mode during the time period in which [Na(+)]i is elevated.
American Journal of Physiology-Heart and Circulatory Physiology, 2007
The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxatio... more The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentrati...
We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein ... more We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca 2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [ 3 H]-ryanodine binding and fast Ca 2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca 2+ . Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βA...
Myocardial ischemia/reperfusion (I/R) produces structural and functional alterations depending on... more Myocardial ischemia/reperfusion (I/R) produces structural and functional alterations depending on the duration of ischemia. Brief ischemia followed by reperfusion causes reversible contractile dysfunction (stunned heart) but long-lasting ischemia followed by reperfusion can result in irreversible injury with cell death. Events during I/R can alter endoplasmic reticulum (ER) function leading to the accumulation of unfolded/misfolded proteins. The resulting ER stress induces activation of several signal transduction pathways, known as unfolded protein response (UPR). Experimental evidence shows that UPR contributes to cell death in irreversible I/R injury, however there is still uncertainty for its occurrence in the stunned myocardium. This study investigated the ER Accepted Article This article is protected by copyright. All rights reserved. stress response and its functional impact on the post-ischemic cardiac performance of the stunned heart. Methods Perfused rat hearts were subjected to 20 min of ischemia followed by 30 min of reperfusion. UPR markers were evaluated by qRT-PCR and Western blot. Post-ischemic mechanical recovery was measured in absence and presence of two chemical chaperones: tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). Results Analysis of mRNA and protein levels of various ER stress effectors demonstrated that different UPR signaling cascades, involving both pro-survival and pro-apoptotic pathways, are activated. Inhibition of the UPR with chemical chaperones improved the post-ischemic recovery of cardiac mechanical function without affecting the I/R-induced increase in oxidative stress. Conclusion Our results suggest that prevention of ER stress by chemical chaperones could be a therapeutic tool to limit deterioration of the contractile function in clinical settings in which the phenomenon of myocardial stunning is present.
International journal of molecular sciences, Jan 10, 2018
Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is highe... more Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HF...
American Journal of Physiology-Heart and Circulatory Physiology, 2016
Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) b... more Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca2+ calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide s...
... Injury: Mechanisms and Functional Implications Alicia Mattiazzi, Cecilia Mundina-Weilenmann, ... more ... Injury: Mechanisms and Functional Implications Alicia Mattiazzi, Cecilia Mundina-Weilenmann, Leticia Vittone, Matilde Said, and Paola Ferrero Centro de ... and Western Blot analysis SDS-PAGE was performed using 10% acrylamide slab gels according to Porzio and Pearson as ...
Journal of Molecular and Cellular Cardiology, 2014
Ca 2+-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/... more Ca 2+-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120 min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24 h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knockin mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca 2+ current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.
Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural... more Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca 2+ -handling alterations are known to occur in SHR. However, the putative modifications of Ca 2+ -handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca 2+ , Ca 2+ -handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca 2+ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca 2+ transient decay and an increase in Na + /Ca 2+ exchanger (NCX) abundance (p, 0.05) with no changes in SERCA2a expression/activity. An increased Ca 2+ -calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca 2+ efflux from the cell, constitutes a primary alteration of Ca 2+ -handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.
En la célula miocárdica el aumento del Ca 2+ citoplasmático desencadena la contracción, y su dism... more En la célula miocárdica el aumento del Ca 2+ citoplasmático desencadena la contracción, y su disminución, la relajación. En el miocardio humano la remoción de Ca 2+ del citosol ocurre principalmente a través del secuestro de este ion hacia el retículo sarcoplasmático (RS) por la bomba de Ca 2+ del RS (SERCA2). La phospholamban (PLB) es una proteína del RS que, en su forma desfosforilada, disminuye la afinidad de la SERCA2 por el Ca 2+ e inhibe el secuestro de Ca 2+ por el RS. La fosforilación de la PLB, como ocurre durante la estimulación betaadrenérgica, libera esta inhibición aumentando el secuestro y la carga de Ca 2+ del RS. En constitutive viral versus an endothelin-1-responsive cardiac promoter.
Naunyn-Schmiedeberg's Archives of Pharmacology, 2006
Endogenous catecholamines released during myocardial ischemia have been considered both to aggrav... more Endogenous catecholamines released during myocardial ischemia have been considered both to aggravate cell injury and exacerbate arrhythmias and to exert a protective action on the post-ischemic contractile function. The present work was addressed to look for evidence to explain this controversy. The effects of cardiac catecholamine depletion and of αand β-adrenoceptor (AR) blockade on the post-ischemic contractile dysfunction, as well as its possible relationship with cardiac oxidative stress, were studied in isolated and perfused rat hearts submitted to 20 min of ischemia and 30 min of reperfusion (stunning). Catecholamine depletion improves the contractile recovery in the stunned heart. This mechanical effect was associated with decreased levels of lipid peroxidation. A similar enhancement of the contractile function during reperfusion was detected after the simultaneous blockade of α 1-and β-ARs with prazosin plus propranolol. To ascertain which specific AR pathway was involved in the effects of catecholamines on the stunned heart, selective AR blockers, prazosin (α 1-blocker), atenolol (β 1-blocker), ICI 118,551 (β 2-blocker) and selective inhibitors of G i-PI3K pathway (pertussis toxin and wortmannin) were alternatively combined. The results indicate that catecholamines released during ischemia exert a dual action on the contractile behavior of the stunned heart: a deleterious effect, related to the activation of the β 2-AR-G i-PI3K-pathway, which was counteracted by a beneficial effect, triggered by the stimulation of α 1-AR. Neither the depression nor the enhancement of the post-ischemic contractile recovery were related with the increase in ROS formation induced by endogenous catecholamines.
Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser 16 b... more Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser 16 by PKA and/or at Thr 17 by CaMKII increases the affinity of the SR Ca 2+ pump for Ca 2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr 17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser 16 and Thr 17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr 17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr 17 phosphorylation was dependent on Ca 2+ entry to the cardiac cell. This Ca 2+ influx would mainly occur by the coupled activation of the Na + /H + exchanger and the Na + /Ca 2+ exchanger working in the reverse mode, since phosphorylation of Thr 17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca 2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr 17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr 17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131-136, 2004)
Journal of Molecular and Cellular Cardiology, 2007
We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein ... more We aimed to define the relative contribution of both PKA and Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca 2+ influx to the cell by low [Ca] o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [ 3 H]-ryanodine binding and fast Ca 2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca 2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulation by PKA and CaMKII, respectively. The isoproterenolinduced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the β-induced increase in the channel activity as indicated by the enhancement of the [ 3 H]-ryanodine binding and the velocity of fast SR Ca 2+ release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca 2+ release during βAR stimulation.
Journal of Molecular and Cellular Cardiology, 2014
The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca ... more The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca 2+ ]) is redox sensitive. Here, we report the effects of a mild oxidative stress on cardiac RyR (RyR2) channels in Langendorff perfused rat hearts. Single RyR2 channels from control ventricles displayed the same three responses to Ca 2+ reported in other mammalian tissues, characterized by low, moderate, or high maximal activation. A single episode of 5 min of global ischemia, followed by 1 min of reperfusion, enhanced 2.3-fold the activity of NOX2 compared to controls and changed the frequency distribution of the different responses of RyR2 channels to calcium, favoring the more active ones: high activity response increased and low activity response decreased with respect to controls. This change was fully prevented by perfusion with apocynin or VAS 2870 before ischemia and totally reversed by the extension of the reperfusion period to 15 min. In vitro activation of NOX2 in control SR vesicles mimicked the effect of the ischemia/reperfusion episode on the frequencies of emergence of single RyR2 channel responses to [Ca 2+ ] and increased 2.2-fold the rate of calcium release in Ca 2+-loaded SR vesicles. In vitro changes were reversed at the single channel level by DTT and in isolated SR vesicles by glutaredoxin. Our results indicate that in whole hearts a mild oxidative stress enhances the response of cardiac RyR2 channels to calcium via NOX2 activation, probably by S-glutathionylation of RyR2 protein. This change is transitory and fully reversible, suggesting a possible role of redox modification in the physiological response of cardiac RyR2 to cellular calcium influx.
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Papers by Matilde Said