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.

CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.

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 (45/120min), 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 (1/24h). 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, knock-in 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.

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.

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury.

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr(17) site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na(+)/Ca(2+)exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca(2+)-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca(2+) loading and release (thapsigargin/dantrolene), mitochondrial Ca(2+) uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr(17) of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.

Angiotensin II-induced oxidative stress resets the Ca2+ dependence of Ca2+-calmodulin protein kinase II and promotes a death pathway conserved across different species.

RATIONALE: Angiotensin (Ang) II-induced apoptosis was reported to be mediated by different signaling molecules. Whether these molecules are either interconnected in a single pathway or constitute different and alternative cascades by which Ang II exerts its apoptotic action, is not known. OBJECTIVE: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II-induced apoptosis. METHODS AND RESULTS: Ang II (1 micromol/L) reduced cat/rat myocytes viability by approximately 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca(2+)/calmodulin-dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [autocamtide 2-related inhibitory peptide]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II-induced cell mortality. Moreover, although KN-93 did not affect Ang II-induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H(2)O(2), even in the presence of the Ca(2+) chelator BAPTA-AM, in myocytes and in EGTA-Ca(2+)-free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments. CONCLUSIONS: (1) The Ang II-induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca(2+) concentrations, suggesting a new mechanism by which ROS reset the Ca(2+) dependence of CaMKII to extremely low Ca(2+) levels.

CaMKII inhibition protects against necrosis and apoptosis in irreversible ischemia-reperfusion injury.

OBJECTIVES: Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of cardiac excitation-contraction coupling (ECC) as well as in apoptotic signaling and adverse remodeling. The goal of the present study is to investigate the role of CaMKII in irreversible ischemia and reperfusion (I/R) injury. METHODS: Isovolumic Langendorff perfused rat hearts were subjected to global no-flow I/R (45 min/120 min), and isolated myocytes were subjected to a protocol of simulated I/R (45 min simulated ischemia/60 min reoxygenation) either in the absence or presence of CaMKII inhibition [KN-93 (KN) or the CaMKII inhibitory peptide (AIP)]. RESULTS: In I/R hearts, an increase in CaMKII activity at the beginning of reperfusion was confirmed by the significantly increased phosphorylation of the Thr(17) site of phospholamban. In the presence of KN, contractile recovery at the end of reperfusion was almost double that of I/R hearts. This recovery was associated with a significant decrease in the extent of infarction, lactate dehydrogenase release (necrosis), TUNEL-positive cells, caspase-3 activity, and an increase in the Bcl-2/Bax ratio (apoptosis). In isolated myocytes, both KN and AIP prevented simulated I/R-induced spontaneous contractile activity and cell mortality. Similar results were obtained when inhibiting the reverse mode Na(+)/Ca(2+) exchanger (NCX) with KB-R7943, sarcoplasmic reticulum (SR) function with ryanodine and thapsigargin, or SR Ca(2+) release with tetracaine. In contrast, overexpression of CaMKII decreased cell viability from 52+/-3% to 26+/-2%. CONCLUSIONS: Taken together, the present findings are the first to establish CaMKII as a fundamental component of a cascade of events integrating the NCX, the SR, and mitochondria that promote cellular apoptosis and necrosis in irreversible I/R injury.

Contractile recovery from acidosis in toad ventricle is independent of intracellular pH and relies upon Ca2+ influx.

Hypercapnic acidosis produces a negative inotropic effect on myocardial contractility followed by a partial recovery that occurs in spite of the persistent extracellular acidosis. The underlying mechanisms of this recovery are far from understood, especially in those species in which excitation-contraction coupling differs from that of the mammalian heart. The main goal of the present experiments was to obtain a better understanding of these mechanisms in the toad heart. Hypercapnic acidosis, induced by switching from a bicarbonate-buffered solution equilibrated with 5% CO2 to the same solution equilibrated with 12% CO2, evoked a decrease in contractility followed by a recovery that reached values higher than controls after 30 min of continued acidosis. This contractile pattern was associated with an initial decrease in intracellular pH (pHi) that recovered to control values in spite of the persistent extracellular acidosis. Blockade of the Na+/H+ exchanger (NHE) with cariporide (5 micromol l-1) produced a complete inhibition of pHi restitution, without affecting the mechanical recovery. Hypercapnic acidosis also produced a gradual increase of diastolic and peak Ca2+i transient values, which occurred immediately after the acidosis was settled and persisted during the mechanical recovery phase. Inhibition of Ca2+ influx through the reverse mode of the Na+/Ca2+ exchanger (NCX) by KB-R (1 micromol l-1 for myocytes and 20 micromol l-1 for ventricular strips), or of L-type Ca2+ channels by nifedipine (0.5 micromol l-1), completely abolished the mechanical recovery. Acidosis also produced an increase in the action potential duration. This prolongation persisted throughout the acidosis period. Our results show that in toad ventricular myocardium, acidosis produces a decrease in contractility, due to a decrease in Ca2+ myofilament responsiveness, followed by a contractile recovery, which is independent of pHi recovery and relies on an increase in the influx of Ca2+. The results further indicate that both the reverse mode NCX and the L-type Ca2+ channels, appear to be involved in the increase in intracellular Ca2+ concentration that mediates the contractile recovery from acidosis.

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.

Biologia de las proteinas del shock térmico / Biology of heat shock proteins

Las proteínal del shock térmico (Hsp) constituyen una família que se encuentra en forma constitutiva en todas las células pro y eucariotas. Cumplen diversas funciones fisiológicas: colaboran en la adquisición de la estructura terciarua de las proteínas en formación, interviniendo en su ensamble, translocación y secreción así como también en la degradación o reparación de proteínas anormales, actuando como chaperonas moleculares. Cuando las células son sometidas a distintos estímulos como el estrés del shock calórico, radiaciones, diversas drogas, infecciones virales, etc, las Hsp se sobreexpresan. De esta manera confieren protección a las células, volviéndolas resistentes a la apoptosis. Esta familia de proteínas comprende numerosos miembros que se agrupan según su peso molecular. En los seres humanos, las Hsp se expresan también en tejidos neoplásicos de ovario, endometrio, mama, aparato digestivo, etc. En algunos casos, la sobreexpresión está asociada a mal pronóstico de la enfermedad debido a que podría favorecer el proceso metastásico. Algunos autores las correlacionan tanto con la proliferación como con la diferenciación de los tejidos neoplásicos. Recientes estudios muestran su influencia en el desarrollo de la resistencia a drogas quimioterapéuticas. En enfermedades autoimunes, como artritis reumatoidea, las Hsp pueden suprimir la respuesta inflamatoria. En otras enfermedades pueden resultar inmunógenas por sí mismas. Por consiguiente su papel en el sistema inmune aún no está bien definido.

Biologia de las proteinas del shock térmico / Biology of heat shock proteins

Las proteínal del shock térmico (Hsp) constituyen una família que se encuentra en forma constitutiva en todas las células pro y eucariotas. Cumplen diversas funciones fisiológicas: colaboran en la adquisición de la estructura terciarua de las proteínas en formación, interviniendo en su ensamble, translocación y secreción así como también en la degradación o reparación de proteínas anormales, actuando como chaperonas moleculares. Cuando las células son sometidas a distintos estímulos como el estrés del shock calórico, radiaciones, diversas drogas, infecciones virales, etc, las Hsp se sobreexpresan. De esta manera confieren protección a las células, volviéndolas resistentes a la apoptosis. Esta familia de proteínas comprende numerosos miembros que se agrupan según su peso molecular. En los seres humanos, las Hsp se expresan también en tejidos neoplásicos de ovario, endometrio, mama, aparato digestivo, etc. En algunos casos, la sobreexpresión está asociada a mal pronóstico de la enfermedad debido a que podría favorecer el proceso metastásico. Algunos autores las correlacionan tanto con la proliferación como con la diferenciación de los tejidos neoplásicos. Recientes estudios muestran su influencia en el desarrollo de la resistencia a drogas quimioterapéuticas. En enfermedades autoimunes, como artritis reumatoidea, las Hsp pueden suprimir la respuesta inflamatoria. En otras enfermedades pueden resultar inmunógenas por sí mismas. Por consiguiente su papel en el sistema inmune aún no está bien definido. (AU)

Celulas dendriticas y su papel en patologia / Dendritic cells: their role in pathology

La finalidad de esta revisión es aportar un conocimiento general sobre las células dendríticas (CD), células accesorias de la respuesta inmune. Se las reconoce como las células presentadoras de antígenos por excelencia (APC) y por lo tanto expresan antígenos clase II del complejo mayor de histocompatibilidad (MHC). Los diversos tipos de CD tienen un origen común en la médula ósea diferenciándose luego bajo la influencia de variados estímulos y distribuyéndose en órganos linfoideos y no linfoideos. Desde los tejidos periféricos migran a los ganglios linfáticos donde presentan el antígeno a los linfocitos T. Dependiendo del microambiente expresan diversos marcadores de superfície siendo capaces de la secreción de citoquinas como IL-12, IL-1 y TNFalpha. Como APC cumplen un importante papel en la patogenia de enfermedades autoinmunes y virales destacándose su participación en la infección por HIV. Se las encuentran en el infiltrado de numerosos cánceres humanos donde actuando como APC podrían incluir una respuesta inmune antitumoral. En esta propiedad se basa su utilización para el tratamiento de linfomas y melanomas llevada a cabo actualmente en diversos laboratorios.

Celulas dendriticas y su papel en patologia / Dendritic cells: their role in pathology

La finalidad de esta revisión es aportar un conocimiento general sobre las células dendríticas (CD), células accesorias de la respuesta inmune. Se las reconoce como las células presentadoras de antígenos por excelencia (APC) y por lo tanto expresan antígenos clase II del complejo mayor de histocompatibilidad (MHC). Los diversos tipos de CD tienen un origen común en la médula ósea diferenciándose luego bajo la influencia de variados estímulos y distribuyéndose en órganos linfoideos y no linfoideos. Desde los tejidos periféricos migran a los ganglios linfáticos donde presentan el antígeno a los linfocitos T. Dependiendo del microambiente expresan diversos marcadores de superfície siendo capaces de la secreción de citoquinas como IL-12, IL-1 y TNFalpha. Como APC cumplen un importante papel en la patogenia de enfermedades autoinmunes y virales destacándose su participación en la infección por HIV. Se las encuentran en el infiltrado de numerosos cánceres humanos donde actuando como APC podrían incluir una respuesta inmune antitumoral. En esta propiedad se basa su utilización para el tratamiento de linfomas y melanomas llevada a cabo actualmente en diversos laboratorios. (AU)