Pharmacological inhibition of translocon is sufficient to alleviate endoplasmic reticulum stress and improve Ca2+ handling and contractile recovery of stunned myocardium.

INTRODUCTION: The function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. AIMS: In this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. METHODS: Mechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 µM), comparing its effects with those of the chaperones TUDCA (30 µM) and 4-PBA (3 mM). RESULTS: Emetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. CONCLUSION: Blocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.

Chemical chaperones improve the functional recovery of stunned myocardium by attenuating the endoplasmic reticulum stress.

AIM: Myocardial ischaemia/reperfusion (I/R) produces structural and functional alterations depending on the duration of ischaemia. Brief ischaemia followed by reperfusion causes reversible contractile dysfunction (stunned heart) but long-lasting ischaemia 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 stress response and its functional impact on the post-ischaemic cardiac performance of the stunned heart. METHODS: Perfused rat hearts were subjected to 20 minutes of ischaemia followed by 30 minutes of reperfusion. UPR markers were evaluated by qRT-PCR and western blot. Post-ischaemic 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 signalling cascades, involving both pro-survival and pro-apoptotic pathways, are activated. Inhibition of the UPR with chemical chaperones improved the post-ischaemic 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.

Reversible redox modifications of ryanodine receptor ameliorate ventricular arrhythmias in the ischemic-reperfused heart.

Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca(2+) 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 synthase. Left ventricular contractile parameters and monophasic action potentials were recorded. Oxidation and phosphorylation of CaMKII and RyR2 were assessed. Increased oxidation of CaMKII during reperfusion had no consequences on the level of RyR2 phosphorylation. Avoiding the reperfusion-induced thiol oxidation of RyR2 with MPG produced a reduction in the number of arrhythmias and did not modify the contractile recovery. Conversely, selective prevention of S-nitrosylation and S-glutathionylation of RyR2 was associated with higher numbers of arrhythmias and impaired contractility. In S2814A mice, treatment with MPG further reduced the incidence of arrhythmias. Taken together, the results suggest that redox modification of RyR2 synergistically with CaMKII phosphorylation modulates reperfusion arrhythmias.

Increased Na⁺/Ca²âº exchanger expression/activity constitutes a point of inflection in the progression to heart failure of hypertensive rats.

UNLABELLED: Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca²âº-handling alterations are known to occur in SHR. However, the putative modifications of Ca²âº-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 Ca²âº, Ca²âº-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 Ca²âº 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-Ca²âº transient decay and an increase in Na⁺/Ca²âº exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca²âº-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 Ca²âº efflux from the cell, constitutes a primary alteration of Ca²âº-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.

Stimulation of NOX2 in isolated hearts reversibly sensitizes RyR2 channels to activation by cytoplasmic calcium.

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.

Role of CaMKII in post acidosis arrhythmias: a simulation study using a human myocyte model.

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.

Subchronic microcystin-LR exposure increased hepatic apoptosis and induced compensatory mechanisms in mice.

Acute lethal cytotoxicity of microcystin-LR (MC-LR), a toxin produced by fresh-water cyanobacteria, has been attributed to protein phosphatases type 1 and type 2A (PP1/PP2A) inhibition and reactive oxygen species (ROS) generation. However, the effects and molecular mechanisms of prolonged, sublethal MC-LR exposure are less known. We studied mice intraperitonealy injected with saline or 25 µg MC-LR/kg for 28 days (every 2 days). MC-LR induced apoptosis in liver and not in kidneys or heart of treated animals. Liver also showed decreased α-tubulin levels (45.56% ± 7.65% of controls) and activation of p38-MAPK and CaMKII pathways (137.93% ± 11.64% and 419.35% ± 67.83% of the control group, respectively). PP1/PP2A activity decreased from 1.82 ± 0.23 (controls) to 0.91 ± 0.98 mU/mg (MC-LR-treated mice); however, no difference in total Ser/Thr phosphatase activity was found between both the groups. The results demonstrated that apoptosis and cytoskeleton disruption contributed to the hepatic cytotoxic effects of subchronic MC-LR administration. These effects occurred in association with sustained activation of signaling cascades and development of compensatory mechanisms to maintain total Ser/Thr phosphatase activity.

Phospholamban phosphorylation by CaMKII under pathophysiological conditions.

Sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) transports Ca2+ into the SR, decreasing the cytosolic Ca2+ during relaxation and increasing the SR Ca2+ available for contraction. SERCA2a activity is regulated by phosphorylation of another SR protein: Phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a. Phosphorylation of PLN by either cAMP or cGMP-dependent protein kinase at Ser16 or the Ca2+-calmodulin-dependent protein kinase (CaMKII), at Thr17, relieves this inhibition, increasing SR Ca2+ uptake and SR Ca2+ load. Thus, PLN is a major player in the regulation of myocardial relaxation and contractility. This review will examine the main aspects of the role of CaMKII and Thr17 site of PLN, on different pathophysiological conditions: acidosis, ischemia/reperfusion (I/R) and heart failure (HF). Whereas CaMKII-activation and PLN phosphorylation contribute to the functional recovery during acidosis and stunning, CaMKII results detrimental in the irreversible I/R injury, producing apoptosis and necrosis. Phosphorylation of Thr17 residue of PLN and CaMKII activity vary in the different models of HF. The possible role of these changes in the depressed cardiac function of HF will be discussed.

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation.

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 beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-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 betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced 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 beta-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 betaAR stimulation.

Ca2+/calmodulin-dependent protein kinase: a key component in the contractile recovery from acidosis.

Intracellular acidosis exerts substantial effects on the contractile performance of the heart. Soon after the onset of acidosis, contractility diminishes, largely due to a decrease in myofilament Ca(2+) responsiveness. This decrease in contractility is followed by a progressive recovery that occurs despite the persistent acidosis. This recovery is the result of different mechanisms that converge to increase diastolic Ca(2+) levels and Ca(2+) transient amplitude. Recent experimental evidence indicates that activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an essential step in the sequence of events that increases the Ca(2+) transient amplitude and produces contractile recovery. CaMKII may act as an amplifier, providing compensatory pathways to offset the inhibitory effects of acidosis on many of the Ca(2+) handling proteins. CaMKII-induced phosphorylation of the SERCA2a regulatory protein phospholamban (PLN) has the potential to promote an increase in sarcoplasmic reticulum (SR) Ca(2+) uptake and SR Ca(2+) load, and is a likely candidate to mediate the mechanical recovery from acidosis. In addition, CaMKII-dependent phosphorylation of proteins other than PLN may also contribute to this recovery.

beta 2-Adrenergic stimulation is involved in the contractile dysfunction of the stunned heart.

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 alpha- and beta-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 alpha 1- and beta-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 (alpha 1-blocker), atenolol (beta 1-blocker), ICI 118,551 (beta 2-blocker) and selective inhibitors of Gi-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 beta 2-AR-Gi-PI3K-pathway, which was counteracted by a beneficial effect, triggered by the stimulation of alpha 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.

Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions.

The sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) is under the control of a closely associated SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits the SR Ca(2+) pump, whereas phosphorylation of PLN, at either Ser(16) by PKA or Thr(17) by calmodulin-dependent protein kinase II (CaMKII), reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca(2+) uptake by the SR. This would in turn lead to an increase in the velocity of relaxation, SR Ca(2+) load, and myocardial contractility. Thus, PLN is a major determinant of cardiac contractility and relaxation. Although in the intact heart, beta-adrenoceptor stimulation results in phosphorylation of PLN at both Ser(16) and Thr(17) residues, the role of Thr(17) site has long remained equivocal. In this review, we attempt to highlight the signaling cascade and the physiological relevance of the phosphorylation of this residue in the heart under both physiological and pathological situations.

Role of phosphorylation of Thr(17) residue of phospholamban in mechanical recovery during hypercapnic acidosis.

OBJECTIVES: To assess the time course of phosphorylation of phospholamban residues, the underlying mechanisms determining these phosphorylations, and their functional impact on the mechanical recovery during acidosis. METHODS: Langendorff perfused rat hearts were submitted to 30 min of hypercapnic acidosis. Contractility, relaxation, and phosphorylation of phospholamban residues, immunodetected by specific antibodies, were determined. RESULTS: Acidosis produced a mechanical impairment followed by a spontaneous recovery, most of which occurred within the first 3 min of acidosis (early recovery). During this period, contractility and relaxation recovered by 67+/-9% and 77+/-11%, respectively, from its maximal depression, together with an increase in the Ca(2+)-calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of Thr(17). The CaMKII inhibitor KN-93, at 1, 5 and 10 microM, decreased Thr(17) phosphorylation to basal levels and produced a similar impairment of the early relaxation recovery (50%). However, only 5 and 10 microM KN-93 inhibited the early contractile recovery and completely blunted the late mechanical recovery. Inhibition of the reverse mode of the Na(+)/Ca(2+) exchanger by KB-R7943 decreased Thr(17) phosphorylation but accelerated the early contractile recovery. CONCLUSIONS: CaMKII-dependent Thr(17) phosphorylation significantly increased at the beginning of acidosis, is responsible for 50% of the early relaxation recovery, and is linked to the activation of the reverse Na(+)/Ca(2+) mode. The early contractile recovery and the late mechanical recovery are dependent on CaMKII but independent of the phosphorylation of the Thr(17) residue of phospholamban. The reverse Na(+)/Ca(2+) mode has an additional negative effect that opposes the early mechanical recovery.

Frequency-dependent acceleration of relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a phosphorylation.

An increase in stimulation frequency causes an acceleration of myocardial relaxation (FDAR). Several mechanisms have been postulated to explain this effect, among which is the Ca(2+)-calmodulin-dependent protein kinase (CaMKII)-dependent phosphorylation of the Thr(17) site of phospholamban (PLN). To gain further insights into the mechanisms of FDAR, we studied the FDAR and the phosphorylation of PLN residues in perfused rat hearts, cat papillary muscles and isolated cat myocytes. This allowed us to sweep over a wide range of frequencies, in species with either positive or negative force-frequency relationships, as well as to explore the FDAR under isometric (or isovolumic) and isotonic conditions. Results were compared with those produced by isoprenaline, an intervention known to accelerate relaxation (IDAR) via PLN phosphorylation. While IDAR occurs tightly associated with a significant increase in the phosphorylation of Ser(16) and Thr(17) of PLN, FDAR occurs without significant changes in the phosphorylation of PLN residues in the intact heart and cat papillary muscles. Moreover, in intact hearts, FDAR was not associated with any significant change in the CaMKII-dependent phosphorylation of sarcoplasmic/endoplasmic Ca(2+) ATPase (SERCA2a), and was not affected by the presence of the CaMKII inhibitor, KN-93. In isolated myocytes, FDAR occurred associated with an increase in Thr(17) phosphorylation. However, for a similar relaxant effect produced by isoprenaline, the phosphorylation of PLN (Ser(16) and Thr(17)) was significantly higher in the presence of the beta-agonist. Moreover, the time course of Thr(17) phosphorylation was significantly delayed with respect to the onset of FDAR. In contrast, the time course of Ser(16) phosphorylation, the first residue that becomes phosphorylated with isoprenaline, was temporally associated with IDAR. Furthermore, KN-93 significantly decreased the phosphorylation of Thr(17) that was evoked by increasing the stimulation frequency, but failed to affect FDAR. Taken together, the results provide direct evidence indicating that CaMKII phosphorylation pathways are not involved in FDAR and that FDAR and IDAR do not share a common underlying mechanism. More likely, a CaMKII-independent mechanism could be involved, whereby increasing stimulation frequency would disrupt the SERCA2a-PLN interaction, leading to an increase in SR Ca(2+) uptake and myocardial relaxation.

Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a beta-adrenergic challenge.

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser16, PKA site, and Thr17, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal beta-adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser16 of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and beta-blockade. Thr17 site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr17 was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr17 phosphorylation was similar in P and NP hearts and was significantly diminished by the Na+/Ca2+ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to beta-adrenergic stimulation was significantly decreased both in P and NP hearts.

The relative relevance of phosphorylation of the Thr(17) residue of phospholamban is different at different levels of beta-adrenergic stimulation.

Contractility and relaxation measurements were combined with the determination of total phospholamban (PLB) phosphorylation and the immunodetection of PLB-phosphorylation sites in the intact, beating rat heart to identify the contributions of PLB phosphorylation at the Thr(17) and Ser(16) residues at different levels of beta-adrenoceptor stimulation. Whereas with 30-300 nM isoproterenol, phosphorylation of Thr(17), the Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) site and Ser(16), the protein kinase A (PKA) site, contributed approximately 50% each to PLB phosphorylation, and both participated in the relaxant action of isoproterenol, at lower a level of beta-adrenoceptor stimulation (isoproterenol 0.3-3 nM), both effects were exclusively due to Ser(16) phosphorylation. Increasing [Ca](o) at 3 nM isoproterenol, to obtain an increase in contractility comparable to that produced by 30 nM isoproterenol, significantly increased Thr(17) phosphorylation and the relaxant effect produced by 3 nM isoproterenol. An increase in Thr(17) phosphorylation and in the relaxant effect of 3 nM isoproterenol was also obtained by phosphatase inhibition (okadaic acid). In this case, Ser(16) phosphorylation was also increased. Moreover, perfusion with 30 nM isoproterenol in the presence of the PKA inhibitor H-89 decreased phosphorylation at both PLB residues and diminished the inotropic and relaxant responses to the beta-agonist. The relative contribution of Thr(17) phosphorylation to the isoproterenol-induced phosphorylation of PLB and relaxation thus increased with the level of beta-adrenoceptor stimulation and the consequent increase in PKA activity. The lack of Thr(17) phosphorylation at low isoproterenol concentrations might therefore be attributed to a level of PKA activity insufficient to increase [Ca](i) to activate the CaMKII system and/or to inhibit the phosphatase that dephosphorylates PLB

Time course and mechanisms of phosphorylation of phospholamban residues in ischemia-reperfused rat hearts. Dissociation of phospholamban phosphorylation pathways.

Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that occurs in ischemia-reperfused hearts. Because SR function is regulated by phosphorylation of phospholamban (PLB), a SR protein phosphorylated by cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser(16)phosphorylation increased significantly after 20 min of ischemia from 2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific phosphorylation and decreased to control values immediately after reperfusion. Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in Ser(16)phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after beta-blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with Ca(2+)overload, transiently favoring the recovery of relaxation during early reperfusion.

Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo Arenarum Hensel) ventricle

En tiritas intactas de ventrículo de sapo, se estudió el efecto relajante o lusitrópico positivo de diferentes intervenciones qu aumentan el AMPc intracelular. El isoproterenol aumentó la tensión desarrollada (DT), la velocidad máxima de contracción (+T), y la velocidad máxima de relajación (-T aumentó proporcionalmente más que +T a concentraciones de isoproterenol desde 10-8 a 10-4M, por lo que la relación +T/-T disminuyó significativamente. Una dosis única de isoproterenol (3x10**-8M) aumentó significativamente los niveles de AMPc desde 0.174 ñ 0.022 a 0.329 ñ pmoles/mg peso húmedo y produjo un aumento en la contractilidad de 69 ñ 13% y una disminución de +T/-T de 18.5 ñ 4.55%. La administración de 10**-3M de dibutiril AMPc(dAMPc) aumentó significativamente DT y +T y disminuyó significativamente la relación +T/-T. Efectos similares produjo la administración de milrinona, un inhibidor específico de la fosfodiesterasa de AMPc. La papaverina, un inhibidor inespecífico de fosfodiesterasas, no produjo aumentos en +T, pero aumentó significativamente -T. En trabéculas desprovistas químicamente de membrana, la sensibilidad al calcio de las proteínas contráctiles aumentó significativamente por la administración de 10**-5M del inhibidor de fosfodiesterasa 3-isobutil-1-metil-xantina (IBMX). La administración de 10**-3M de dAMPc no afectó la sensibilidad al calcio de las trabéculas desprovistas de membrana. Sin embargo la misma concentración de dAMPc produjo una disminución en la sensibilidad al calcio de las proteínas contráctiles cuando se administró en presencia de IBMX o de papavarina. Los resultados indicarían que el efecto relajante del isoproterenol es mediado en el ventrículo de sapo por un aumento en los niveles de AMPc intracelular. Estos resultados sugieren además que la disminución de la sensibilidad al calcio de los miofilamentos podría ser un mecanismo por el que el AMPc produce su efecto relajante

Positive lusitropic effect and diminished myofibrillar sensitivity to calcium produced by cAMP on toad (Bufo Arenarum Hensel) ventricle

En tiritas intactas de ventrículo de sapo, se estudió el efecto relajante o lusitrópico positivo de diferentes intervenciones qu aumentan el AMPc intracelular. El isoproterenol aumentó la tensión desarrollada (DT), la velocidad máxima de contracción (+T), y la velocidad máxima de relajación (-T aumentó proporcionalmente más que +T a concentraciones de isoproterenol desde 10-8 a 10-4M, por lo que la relación +T/-T disminuyó significativamente. Una dosis única de isoproterenol (3x10**-8M) aumentó significativamente los niveles de AMPc desde 0.174 ñ 0.022 a 0.329 ñ pmoles/mg peso húmedo y produjo un aumento en la contractilidad de 69 ñ 13% y una disminución de +T/-T de 18.5 ñ 4.55%. La administración de 10**-3M de dibutiril AMPc(dAMPc) aumentó significativamente DT y +T y disminuyó significativamente la relación +T/-T. Efectos similares produjo la administración de milrinona, un inhibidor específico de la fosfodiesterasa de AMPc. La papaverina, un inhibidor inespecífico de fosfodiesterasas, no produjo aumentos en +T, pero aumentó significativamente -T. En trabéculas desprovistas químicamente de membrana, la sensibilidad al calcio de las proteínas contráctiles aumentó significativamente por la administración de 10**-5M del inhibidor de fosfodiesterasa 3-isobutil-1-metil-xantina (IBMX). La administración de 10**-3M de dAMPc no afectó la sensibilidad al calcio de las trabéculas desprovistas de membrana. Sin embargo la misma concentración de dAMPc produjo una disminución en la sensibilidad al calcio de las proteínas contráctiles cuando se administró en presencia de IBMX o de papavarina. Los resultados indicarían que el efecto relajante del isoproterenol es mediado en el ventrículo de sapo por un aumento en los niveles de AMPc intracelular. Estos resultados sugieren además que la disminución de la sensibilidad al calcio de los miofilamentos podría ser un mecanismo por el que el AMPc produce su efecto relajante (AU)