Gina Sánchez

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S-Glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91phox membrane subunit and the cytoplasmic regulatory p47phox subunit co-localized in transverse tubules of adult mice fibers with the α1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91phox and p22phox, which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47phox and p67phox. NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Exercise induces early and late myocardial preconditioning in dogs

OBJECTIVE We tested the hypothesis that exercise induces myocardial preconditioning in dogs. METHODS We instrumented dogs with a snare on the anterior descending coronary artery and catheters in the root of the aorta, left ventricular cavity and coronary sinus. After recovering from surgery the dogs were trained to stay in the laboratory and run on a treadmill. Subsequently, they were randomly allocated to five groups: (1) non-preconditioned dogs: under anesthesia, the anterior descending coronary artery was occluded during 1 h and then reperfused during 4.5 h. (2) Early preconditioned dogs: procedure similar to group 1 but the dogs performed exercise on a treadmill for five periods of 5 min each before the coronary occlusion. (3) Late preconditioned dogs: procedure similar to group 2 but 24 h were allowed to elapse between the preconditioning exercise and the coronary occlusion. (4) Early preconditioned dogs plus 5-hydroxydecanoate: procedure similar to group 2 but 5-hydroxydecanoate was administered prior to exercise. (5) Non-preconditioned dogs with 5-hydroxydecanoate: procedure similar to group 1 but 5-hydroxydecanoate was administered at a time equivalent to that in group 4. RESULTS Exercise did not induce myocardial ischemia and the hemodynamics during the experiments did not differ between groups. Exercise immediately before the coronary occlusion decreased the infarct size (percent of the risk region) by 78% (P<0.05), an effect that was abolished with 5-hydroxydecanoate. Exercise 24 h prior to coronary occlusion decreased infarct size by 46% (P<0.05 vs. non-preconditioned dogs, P<0.05 vs. early preconditioned dogs). 5-Hydroxydecanoate by itself did not modify infarct size. These effects could not be explained by changes in collateral flow to the ischemic region. CONCLUSIONS Exercise prior to a coronary occlusion induces early and late preconditioning of the infarct size. The early effect is mediated through mitochondrial ATP-sensitive potassium channels.

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.

Ischemia Enhances Activation by Ca2+ and Redox Modification of Ryanodine Receptor Channels from Rat Brain Cortex

Cerebral ischemia stimulates Ca2+ influx and thus increases neuronal intracellular free [Ca2+]. Using a rat model of cerebral ischemia without recirculation, we tested whether ischemia enhances the activation by Ca2+ of ryanodine receptor (RyR) channels, a requisite feature of RyR-mediated Ca2+-induced Ca2+ release (CICR). To this aim, we evaluated how single RyR channels from endoplasmic reticulum vesicles, fused into planar lipid bilayers, responded to cytoplasmic [Ca2+] changes. Endoplasmic reticulum vesicles were isolated from the cortex of rat brains incubated without blood flow for 5 min at 37°C (ischemic) or at 4°C (control). Ischemic brains displayed increased oxidative intracellular conditions, as evidenced by a lower ratio (∼130:1) of reduced/oxidized glutathione than controls (∼200:1). Single RyR channels from ischemic or control brains displayed the same three responses to Ca2+ reported previously, characterized by low, moderate, or high maximal activity. Relative to controls, RyR channels from ischemic brains displayed with increased frequency the high activity response and with lower frequency the low activity response. Both control and ischemic cortical vesicles contained the RyR2 and RyR3 isoforms in a 3:1 proportion, with undetectable amounts of RyR1. Ischemia reduced [3H]ryanodine binding and total RyR protein content by 35%, and increased at least twofold endogenous RyR2 S-nitrosylation and S-glutathionylation without affecting the corresponding RyR3 endogenous levels. In vitro RyR S-glutathionylation but not S-nitrosylation favored the emergence of high activity channels. We propose that ischemia, by enhancing RyR2 S-glutathionylation, allows RyR2 to sustain CICR; the resulting amplification of Ca2+ entry signals may contribute to cortical neuronal death.

Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion

Type-2 ryanodine receptors (RyR2)--the calcium release channels of cardiac sarcoplasmic reticulum--have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems--calpains, the proteasome and autophagy--on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

Drp1 loss-of-function reduces cardiomyocyte oxygen dependence protecting the heart from ischemia-reperfusion injury.

Abstract: Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.

Redox regulation of calcium release in skeletal and cardiac muscle

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

AIMS Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles.

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.