Nmol Ca2 Research Articles

Study of the activation of the RISK and SAFE signaling pathways by cyclosporin A pre- and post-conditioning in a mouse model of cerebral infarction

Cyclosporin A (CsA) has been shown to be useful in preventing ischemia-reperfusion injury in basic models of myocardial or cerebral infarction. CsA limits the opening of the mitochondrial transition pore (mPTP) through its direct interaction with cyclophilin D. However, recent studies in cardioprotection and neuroprotection suggest a role of CsA on the classic RISK and SAFE pathways, upstream of mitochondria. We aim to study the effect of CsA in ischemic pre- and post-conditioning on the RISK and SAFE pathways in a mouse model of stroke. Mice underwent 60 min ischemia and 30 min or 24 h reperfusion and received either no treatment (IR), a bolus of CsA (10 mg/kg) 10 min before the ischemia (pre-CsA) or 10 min after the reperfusion (post-CsA). Infarction of the middle cerebral artery was performed with a microfilament placed intra-arterially for the desired duration. Surgery was performed on Sham mice, but the filament was not occlusive. mPTP opening was evaluated by calcium retention capacity (CRC) on isolated mitochondria from ischemic right hemisphere and non-ischemic left hemisphere. Activation of the RISK and SAFE pathways was evaluated by determining the phosphorylation state of key proteins by Western blots: STAT3, GSK3-β, Akt and Erk. As expected, CRC was significantly reduced in the mitochondria from the right hemisphere in the IR group (Sham, 103 ± 21 vs. IR, 57 ± 18 nmol Ca2+/mg, n = 12/gp, P < 0.05). There was a slight increase in PTP resistance in the ischemic mitochondria from the pre-CsA and post-CsA groups. Experiments are ongoing to measure infarct size after 24 h of reperfusion. There was no significant change in protein phosphorylation in the left hemisphere. In the right ischemic hemisphere, there was a trend of activation of GSK3-β in post-CsA mice and STAT3 in pre-CsA mice. Our results suggest an effect of CsA upstream of the mPTP during stroke, which could correspond to a positive feedback loop.

P0536HYPOTHERMIA DURING RENAL ISCHEMIA-REPERFUSION IN MICE: A PROTECTIVE EFFECT ON RENAL AND MITOCHONDRIAL FUNCTIONS

Abstract Background and Aims Renal ischemia reperfusion (RIR) can induce mitochondrial stress triggering cell death and eventually leading to acute kidney injury (AKI). It has been suggested that mild hypothermia could be protective in RIR without clear underlying mechanisms. We aimed to show that mild hypothermia (34°C) during RIR protects renal mitochondrial function and prevents AKI. Method Male C57BL6 mice were assigned to 4 groups: normothermic ischemic (RIR-37°C) group (n=14) and hypothermic ischemic (RIR-34°C) group (n=14) with body temperature maintained at respectively 37°C or 34°C during 20 minutes of renal ischemia by bilateral renal clamping under general anesthesia; normothermic sham (Sham-37°C) group (n=10) and hypothermic sham (Sham-34°C) group (n=10) with only anesthesia and laparotomy at 37°C or 34°C respectively. Renal function (serum urea concentration) and isolated renal mitochondria function (capacity of mitochondria to retain calcium i.e. calcic retention capacity (CRC), and oxidative phosphorylation capacity of electron transport chain complexes (complex I, II and IV)) were assessed 2 hours and 24 hours after reperfusion. All animal procedures were approved by local Ethics Committee. Data are presented as median with IQR. Results All the parameters monitored were not modified by the temperature in the sham groups, and there was no mortality in those 2 groups. Mortality was 33% in the RIR-37°C group and 11% in the RIR-34°C group 24 hours after reperfusion (p=0.58). Renal ischemia was responsible for a significant increase of serum urea level 2 hours after reperfusion at 37°C [18.7 (17.3–19.0) mmol/L] compared to sham groups (p=0.02), whereas no significant increase was observed in the RIR-34°C group. After 24 hours of reperfusion serum urea level was improved in the RIR-34°C group [22.7 (11.5–42.0) mmol/L] compared to RIR-37°C [60.8 (58.0–69.7) mmol/L, p=0.001]. CRC was not modified by RIR after 2 hours of reperfusion in both groups. CRC was preserved 24 hours after reperfusion in the RIR-34°C group [260 (210–320) nmol Ca2+/mg protein] with no difference compared to Sham-37°C [320 (280–360) nmol Ca2+/mg protein p=0.18] whereas CRC was significantly decreased in the RIR-37°C group compared to Sham-37°C [120 (0–130) vs 320 (280–360) nmol Ca2+/mg protein p=0.004). Complexes I, II and IV were lowered after 2 hours of reperfusion in the RIR-37°C group (p&amp;lt;0.05), and complexes II and IV activities remained altered 24 hours after reperfusion, compared to Sham-37°C (p=0.009 and p=0.02 respectively). In the RIR-34°C group, complexes I, II and IV activities were preserved 2 hours after reperfusion but complex I activity decreased 24 hours after reperfusion. We found significant difference between complexes II and IV activities between IRI-34°C and RIR-37°C. Conclusion Mild hypothermia (34°C) during RIR significantly protected renal mitochondrial respiration and mitochondrial stress, associated with a preserved renal function after 2 hours of reperfusion and an improved renal function 24 hours after reperfusion compared to normothermic mice (37°C).

Abstract 175: Cerebral and Myocardial Mitochondrial Injury Differ in a Rat Model of Cardiac Arrest

Introduction: Neurologic and myocardial dysfunction after successful resuscitation is prominent and mitochondrial dysfunction is predicted to be a key determinant of poor outcomes. Mitochondria contribute a critical role as effectors and targets of reperfusion injury. However, the onset and severity of mitochondrial dysfunction during cardiac arrest (CA) is not fully understood. The present study was done to explore whether changes in cerebral and myocardial mitochondria differ after cardiac arrest and cardiopulmonary resuscitation (CPR). Hypothesis: Mitochondrial injury is more severe in the brain compared to the heart during and following cardiac arrest and CPR. Methods: Sprague-Dawley rats weighing between 450 - 550 g were randomized into 4 groups (n=6): 1) sham (surgery, no ventricular fibrillation (VF) or CPR), 2) VF (VF 8 mins, no CPR); 3) VF and CPR (VF 8 mins and CPR 8 mins, no defibrillation); ROSC 1 h (VF 8 mins, CPR 8 mins, defibrillation, and observe 1 h after ROSC). VF was induced through a guide wire advanced from the right jugular vein into the right ventricle. Brain and heart mitochondria were extracted by differential centrifugation and used to measure oxidative phosphorylation and calcium retention capacity (CRC). Results: Compared with sham, brain mitochondrial CRC in VF, VF+CPR and ROSC 1 h were decreased (110±11 vs. 70±13, 62±24, 50±6 nmol Ca 2+ /mg protein, p&lt;0.05). However, the CRC of the heart mitochondria was decreased only 1 h after ROSC compared to sham (1000±150 vs. 1000±84, 993±78, 600±76 nmol Ca 2+ /mg protein). Brain mitochondrial oxidative phosphorylation with complex I substrate glutamate in VF, VF+CPR and ROSC 1 h were all decreased compared to sham (127 + 8 vs 92±17, 84±12, and 92±15, p&lt;0.05). This contrasted myocardial mitochondria oxidative phosphorylation which had no impairment (324±3 vs 338±46, 379±29, and 323±45). Conclusions: Mitochondria in the brain are more sensitive to injury during CA and CPR compared to heart mitochondria. With markedly decreased CRC, mitochondria are likely to contribute to cerebral reperfusion injury during CPR and ROSC. Preservation of cerebral mitochondrial activity and mitochondrial function during cardiac arrest may improve post-resuscitation neurological function.

Diabetes confers invitro calcific potential on serum which associates with invivo vascular calcification.

Although vascular calcification (VC) is prevalent in Type 2 diabetes mellitus (T2DM), underlying mechanisms remain unclear. Neither is it known whether T2DM confers calcific potential (CP) on serum, enabling it to induce VC outside the disease milieu. We, therefore, investigated the CP of serum from controls and subjects with T2DM with and without in vivo VC. Samples from 20 healthy controls and 44 age- and sex-matched patients with T2DM with modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) > 60 ml·min−1 were analysed for CP using rat aortic smooth muscle cells in vitro. CT scans of femoral arteries identified individuals with in vivo calcification. Serum from subjects with T2DM revealed significantly greater CP than controls. This was further enhanced in the presence of in vivo VC. Addition of β-glycerophosphate (β-GP) plus CaCl2 increased the CP of T2DM serum but not of controls. Along with age, CP was an independent predictor of the presence of VC. In receiver operator curve (ROC) analysis, CP was a significant predictor of femoral arterial VC (C-statistic 0.70: P=0.009). The distribution of CP was bimodal around a cutoff of 100 nmoles of Ca2+ protein mg−1, with a higher proportion of Type 2 diabetes subjects with in vivo calcification (T2DM+) sera above the cutoff value. This group also showed elevated levels of osteoprotegerin (OPG) and matrix Gla protein (MGP). Diabetes confers CP on the serum which is enhanced by the presence of in vivo VC. The CP acquired may be dependent on levels of OPG and MGP. These findings may be clinically relevant for early identification of individuals at risk of VC and for informing therapeutic strategies.

Infarct size reduction by cyclosporine A at reperfusion involves inhibition of the mitochondrial permeability transition pore but does not improve mitochondrial respiration

IntroductionIschemic postconditioning (PoCo) and cyclosporine A (CysA) given prior to reperfusion reduce myocardial infarct size after ischemia/reperfusion. Ischemic postconditioning's protection is characterized by better preservation of mitochondrial respiration and calcium retention capacity. Protection by CysA is not entirely clear. Cyclosporine A inhibits not only mitochondrial permeability transition pore (mPTP) opening but also the phosphatase calcineurin. We have investigated whether CysA mediates protection not only by mPTP inhibition but also through a more upstream inhibition of calcineurin with subsequently better preserved mitochondrial respiration.Material and methodsAnesthetized pigs were subjected to 90 min ischemia and 10 min reperfusion initiated with either PoCo (6 × 20 s reperfusion/re-occlusion; n = 9), CysA infusion (5 mg/kg i.v.; 5 min before reperfusion; n = 4), or immediate full reperfusion (IFR; n = 8). Mitochondria were isolated from myocardial tissue for measurement of respiration and calcium retention capacity.ResultsIn mitochondria from ischemic/reperfused myocardium, ADP-stimulated complex I respiration was similar between CysA (116 ±11 nmol O2/min/mg protein) and IFR (117 ±8), but better preserved with PoCo (160 ±9; p < 0.05). Calcium retention capacity was greater with both PoCo and CysA (1096 ±45 and 1287 ±128 nmol Ca2+/mg protein) than with IFR (756 ±103; p < 0.05).ConclusionsCyclosporine A's protection is not associated with improved mitochondrial respiration. Protection is unlikely related to an upstream calcineurin inhibition, but is indeed secondary to mPTP inhibition.

Cardiac Subsarcolemmal and Interfibrillar Mitochondria Display Distinct Responsiveness to Protection by Diazoxide

ObjectiveCardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.MethodsMitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca2+-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca2+ and TPP+ ion-selective electrodes.ResultsCompared to IFM, isolated SSM exhibited a higher sensitivity to Ca2+ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca2+-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca2+/mg protein in SSM and IFM, respectively), suggestive of increased Ca2+ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca2+ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).ConclusionThese results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca2+-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Evaluation of serum nitrite, nitrate and malonyldialdehyde levels in preeclampsia

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca++ uptake by inside–out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49±3% in the preeclamptic women as compared to the gestational controls (8.06±0.11 nmol Ca++/mg protein min, gestational controls; 4.08±0.1 nmol Ca++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05±0.96 nmol Pi/mg protein min, gestational controls; 8.85±0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.

Abstract 3563: Postconditioning Inhibits Mitochondrial Permeability Transition Pore Opening Independently of any Modification of the Oxidative Phosphorylation and of the Inner Membrane Potential at Reperfusion

Mitochondrial permeability transition pore (mPTP) inhibition plays a crucial role in postconditioning (PostC). We sought to determine whether oxidative phosphorylation and mitochondrial membrane potential (ΔΨ m ), which both modulate mPTP opening, are involved in the inhibition of mPTP opening in the postconditioned heart. Anesthetized rabbits underwent 30 minutes of ischemia followed by 10 minutes of reperfusion. At the onset of reperfusion, they received either no intervention (Control, C), 4 cycles of 1 min ischemia followed by 1 min reperfusion (PostC), or an IV injection of 5mg/kg of the powerful inhibitor of mPTP opening, i.e. cyclosporine A (CsA). Sham rabbits underwent no ischemic insult throughout the 40 minute experiment. At the end of the 10 minute reperfusion period, the myocardial area at risk was excised, and mitochondria were isolated by differential centrifugations. Calcium retention capacity, an index of mPTP inhibition (CRC: nmol Ca 2+ /mg prot) and ΔΨ m (at state 4: % of FCCP-evoked maximum) were assessed by spectrofluorimetry in isolated subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation (at states 3 and 4: nmol O 2 /min/mg) was assessed using a Clark-type electrode (RCR: state 3 / state 4). As expected, PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of ΔΨ m , together with a comparable significant decrease of RCR in both SSM and IFM. In all three groups, this latter effect was related to a concomitant significant decrease in state 3 and to an increase in state 4 respiration. These data suggest that during the early minutes of reperfusion, postconditioning inhibits mPTP opening, independent of any specific modification of the oxidative phosphorylation or of ΔΨ m . Summarized data:

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.

Cardioprotective Effect of Morphine and a Blocker of Glycogen Synthase Kinase 3β, SB216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via Inhibition of the Mitochondrial Permeability Transition Pore

Morphine has been shown to protect the myocardium against ischemia-reperfusion injury through inhibition of glycogen synthase kinase-3beta (GSK-3beta). Given that GSK-3beta is known to modulate the mitochondrial permeability transition pore (mPTP), we investigated the role of mPTP in the cardioprotective effect of morphine and the GSK-3beta inhibitor SB216763 [SB; 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] during ischemia-reperfusion. Both morphine (0.3 mg/kg) and SB (0.6 mg/kg) reduced infarct size in a model of regional myocardial ischemia-reperfusion in rats (13 +/- 1 and 14 +/- 3% of the area at risk versus 33 +/- 4% in controls; p < 0.05). Morphine and SB protected the ischemic myocardium against Ca(2+)-induced mPTP opening as demonstrated by the increased capacity of mitochondria to retain Ca(2+) when they were isolated from the ischemic zone 10 min after the onset of reperfusion (59 +/- 8 and 66 +/- 3 versus 29.5 +/- 6 nmol Ca(2+)/mg x protein, respectively; p < 0.05). This was associated with a restoration of mitochondrial oxidative phosphorylation parameters. In isolated adult rat cardiomyocytes subjected to anoxia-reoxygenation, morphine (2 microM), SB (3 microM), and the direct mPTP inhibitor cyclosporine A (3 microM) delayed mPTP opening as assessed by the calcein loading Co(2+)-quenching technique. This was accompanied by an increase in cell survival as measured by nuclear staining with propidium iodide. These in vitro effects of morphine on inhibition of mPTP opening during anoxia-reoxygenation were suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin (0.1 microM). These data indicate that the infarct-limiting effect of morphine and SB is linked by a cause-effect relationship, which leads to an increased mitochondrial resistance and inhibition of mPTP opening through the PI3-kinase pathway and subsequent inactivation of GSK-3beta.

Phenotype‐Specific Permeability Transition Pore Regulation in Rat Skeletal Muscle

The aim of this study was to determine whether the susceptibility of mitochondria to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the soleus (SOL: 95% type I fibers) and superficial white gastrocnemius (WG: 92% type IIb fibers). Sensitivity to Ca2+‐induced PTP opening was determined with a novel approach using permeabilized myofibers devoid of their contractile apparatus (phantom fibers). Ca2+ uptake in this preparation is specific to mitochondria and calcium retention capacity (CRC) is a good indicator of PTP sensitivity. Mitochondrial volume density was ~ 2 fold greater in SOL vs. WG, as judged by respiratory capacity per mg fiber weight and activity of the marker enzyme citrate synthase (CS). CRC values were 2.2 fold lower in phantom fibers from SOL compared to WG when expressed per unit of CS (CRC: 36.6±9.9 vs. 81.0±26.2 pmol Ca2+/mIU CS; p&lt;0,001; n=4), indicating a pronounced vulnerability to PTP opening in type I vs. IIb fibers. A similar phenomenon was also observed in isolated mitochondria (CRC: &lt;83 and 121 ± 60 nmol Ca2+/mg prot. in SOL and WG). This phenomenon was partly due to a 2‐fold increase in endogenous matrix Ca2+ levels in mitochondria from SOL compared to WG, but not to enhanced ROS production. Expression of the PTP regulatory protein cyclophilin‐D was similar in mitochondria from both muscles. However, expression of ANT, a protein reported to be involved in pore formation and which contributes to Ca2+‐sensitivity of the PTP, was significantly 2.3 fold higher in SOL compared to WG. The present study provides evidence that regulation of the PTP varies according to the phenotype of muscle fibers, which may impact on muscle cell death under conditions of stress.Funded by NSERC.

Presence of Secretogranin II and High-Capacity, Low-Affinity Ca2+ Storage Role in Nucleoplasmic Ca2+ Store Vesicles

Chromogranins and secretogranins have traditionally been known as marker proteins of secretory granules that contain the highest concentrations of cellular calcium, reaching approximately 40 mM. In addition, chromogranin B was also shown to exist in the nucleus, localizing in the putative inositol 1,4,5-trisphosphate (IP3)-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranins A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+ binding proteins, binding 30-90 mol of Ca2+/mol with dissociation constants (Kd) of 1.5-4 mM. Yet the Ca2+-binding property of secretogranins has not been studied. Here, we show the localization of secretogranin II (SgII) in the nucleus, more specifically, in the IP3-sensitive nucleoplasmic Ca2+ store vesicles along with CGB and the IP3 receptors. We have also determined the Ca2+-binding property of SgII and found that SgII binds 61 mol of Ca2+/mol (910 nmol Ca2+/mg) with a Kd of 3.0 mM at the intragranular pH 5.5 and 30 mol of Ca2+/mol (440 nmol Ca2+/mg) with a Kd of 2.2 mM at a near-physiological pH 7.5. Chromogranin B also bound 50 mol of Ca2+/mol (670 nmol Ca2+/mg) with a Kd of 3.1 mM at pH 7.5. Given the high-capacity, low-affinity Ca2+-binding property of SgII and its presence in the IP3-sensitive nucleoplasmic Ca2+ store vesicles, these results suggest that SgII may function in the storage and control of Ca2+ in the nucleus through its interaction with CGB in the nucleoplasmic vesicles.

Abstract 846: Gsk3β Inhibition Is Required For Postconditioning To Modulate Mitochondrial Permeability Transition Pore Opening

Introduction: Activation of the PI3kinase pathway and subsequent Inhibition of GSK3β by phosphorylation at serine-9 residue has recently been shown to protect against myocardial lethal reperfusion injury. Hypothesis: We hypothesized that GSK3β may play a key role in protection afforded by postconditioning. We used transgenic mice (Tg) that express a constitutively active (i.e. that cannot be inactivated by phosphorylation at serine 9) form of GSK-3β. Methods: Anesthetized WT and Tg mice underwent 60 minutes of coronary artery occlusion followed by 24 hrs of reperfusion (n=9/gr). At reflow, mice received either no intervention (controls: C-WT and C-Tg), postconditioning (postC) by 3 cycles of [1 min ischemia / 1 min reperfusion], or an IV bolus injection (1 minute before reflow) of either 10 mg/kg of cyclosporine A (CsA), a potent inhibitor of the mitochondrial permeability transition pore (mPTP) opening, or 70μg/kg of SB 216763 (SB), a specific inhibitor of GSK3β. Infarct size (%of area at risk) was measured by TTC staining. CRC, an index of Ca 2+ -induced mPTP opening in isolated mitochondria was measured by a fluorimetric approach. Results: PostC, CsA and SB significantly reduced infarct to 29%, 36% and 34% respectively (p&lt;0.05 vs. C: 56±5%) in WT mice. In contrast, postC failed to significantly reduce infarct size in Tg mice (51±5%), while as expected CsA limited necrosis (36±6%). In WT, CRC was significantly decreased in C versus sham (75±14 nmol Ca 2+ /mg), but improved in postC, CsA and SB mice, averaging 155±31, 267±11 and 183±37 nmol Ca 2+ /mg respectively (p&lt;0.05). In Tg mice, CRC was not significantly changed in postC versus control (139±44 nmol Ca 2+ /mg), but improved in CsA. Conclusion: These results suggest that inhibition of GSK3β is involved in postconditioning’s protection against infarction, likely by preventing mPTP opening during the first minutes of reperfusion.

Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice

Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca(2+) retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca(2+) retention capacity (132 +/- 13 and 153 +/- 31 vs. 53 +/- 16 nmol Ca(2+)/mg protein in control) and reduced infarct size to 35 +/- 4 and 32 +/- 7% of area at risk vs. 61 +/- 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 +/- 6 and 77 +/- 6% in postconditioned and Debio-025 groups, respectively, vs. 62 +/- 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.

Elevated Ca2+ ATPase (SERCA2) activity in tuna hearts: Comparative aspects of temperature dependence

Tunas have an extraordinary physiology including elevated metabolic rates and high cardiac performance. In some species, retention of metabolic heat warms the slow oxidative swimming muscles and visceral tissues. In all tunas, the heart functions at ambient temperature. Enhanced rates of calcium transport in tuna myocytes are associated with increased expression of proteins involved in the contraction-relaxation cycle. The cardiac SR Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling. Measurements of oxalate-supported Ca2+-uptake in atrial SR vesicles isolated from four species of tunas indicate that bluefin have at least two fold higher Ca2+-uptake than all other tunas examined between 5 and 30 degrees C. The highest atrial Ca2+-uptake was measured in bluefin tuna at 30 degrees C (23.32+/-1.58 nmol Ca2+/mg/min). Differences among tunas in the temperature dependency of Ca2+-uptake were similar for ATP hydrolysis. Western blot analysis revealed a significant increase in SERCA2 content associated with higher Ca2+ uptake rates in the atrial tissues of bluefin tuna and similar RyR expression across species. We propose that the expression of EC coupling proteins in cardiac myocytes, and the higher rates of SERCA2 activity are an important evolutionary step for the maintenance of higher heart rates and endothermy in bluefin tuna.

Ca2+Storage Capacity of Rat Brain Mitochondria Declines During the Postnatal Development Without Change in ROS Production Capacity

Ca2+ overload of mitochondria and oxidants are considered as crucial factors inducing the opening of the permeability transition pore (PTP) in mitochondria. The interdependence between permeability transition (PT), calcium retention capacity (CRC), and reactive oxygen species (ROS) generation was studied in mitochondria from immature and mature rat brain. Brain mitochondria isolated from 1-day- and 1-week-old rats are much more resistant to Ca2+-triggered PT in phosphate-containing incubation medium than mitochondria from adult brain, since the CRC decreases with development. CRC of mitochondria from 1-week-old rat brain was higher than for adult rat brain (450 +/- 112 vs. 175 +/- 35 nmol Ca2+ per mg of protein). In contrast, for ROS generation there was no age difference. In immature and mature mitochondria, basal, respiratory chain-inhibited or glutathione-depleted ROS generations were similar. In addition, the extent of the Ca2+ load was without effect on the basal ROS generation before mitochondria underwent PT. In summary, ROS generation does not crucially affect the ability of immature mitochondria to buffer high levels of extramitochondrial Ca2+ without undergoing PT. However, we hypothesize that the high resistance of immature mitochondria is related to the low content of some PTP complex constituents, such as creatine kinase.

Mathematical model of mitochondrial ionic homeostasis: Three modes of Ca 2+ transport

Mitochondria play an important role in regulation of Ca 2+ homeostasis in a cell. Here we present a mathematical model of mitochondrial ion transport and use this model to analyse different modes of Ca 2+ uptake by mitochondria. The model includes transport of H +, Ca 2+, K +, inorganic phosphate and oxidative substrates across the inner mitochondrial membrane harboring permeability transition pore (PTP). The detailed description of ion fluxes is based on the experimental ion kinetics in isolated mitochondria. Using the model we show that the kinetics of Ca 2+ uptake by mitochondria is regulated by the total amount of Ca 2+ in the system and the rate of Ca 2+ infusion. Varying these parameters we find three different modes of ion transport. When the total amount of Ca 2+ is below 140 nmol Ca 2+/mg protein, all available Ca 2+ is accumulated in the matrix without activation of the PTP. Between 140 and 160 nmol Ca 2+/mg protein, accumulation of Ca 2+ generates periodic opening and closure of the PTP and oscillations of ion fluxes. Higher levels of Ca 2+ (>160 nmol Ca 2+/mg protein) result in a permanently open PTP, membrane depolarization and loss of small ions from the matrix. We show that in the intermediate range of Ca 2+ concentrations the rate of Ca 2+ infusion regulates the PTP state, so that slow Ca 2+ infusion does not lead to PTP opening, while fast Ca 2+ infusion results in an oscillatory state.

Exercise training induces respiratory substrate-specific decrease in Ca2+-induced permeability transition pore opening in heart mitochondria

The purpose of this study was to determine whether regular exercise (treadmill running, 10 wk) alters the susceptibility of rat isolated heart mitochondria to Ca(2+)-induced permeability transition pore (PTP) opening and whether this could be associated with changes in the modulation of PTP opening by selected physiological effectors. Basal leak-driven and ADP-stimulated respiration in the presence of substrates for complex I, II, and IV were not affected by training. Fluorimetric studies revealed that in the control and exercise-trained groups, the amount of Ca(2+) required to trigger PTP opening was greater in the presence of complex II vs. I substrates (230 +/- 12 vs. 134 +/- 7 nmol Ca(2+)/mg protein, P < 0.01; pooled average of control and trained groups). In addition, with a substrate feeding the complex II, training increased by 45% (P < 0.01) the amount of Ca(2+) required to trigger PTP opening both in the presence and absence of the PTP inhibitor cyclosporin A. However, membrane potential, reactive oxygen species production, NAD(P)H ratio, and Ca(2+) uptake kinetics were not different in mitochondria from both groups. Together, these results suggest the existence of a substrate-specific regulation of the PTP in heart mitochondria and suggest that regular exercise results in a reduced sensitivity to Ca(2+)-induced PTP opening in presence of complex II substrates.

Ca2+-binding proteins in nuclei.

Nuclei isolated from skeletal muscle of 15-day-old chick embryos, adult chickens, rabbits and from rat liver contain on the average 8-18 nmol Ca2+/mg protein. Digestion of nuclei with DNAase I and RNAase at 37 degrees C for 8--12 h reduced the Ca2+ binding by more than 90%. After nuclease treatment, Ca2+-binding proteins were identified in the nonhistone chromosomal protein fractions and in the insoluble residue by equilibrium dialysis and centrifuge transport, in media of 0.1 M KCl and 1 mM MgCl2. The interaction of Ca2+-binding proteins with chromatin may be of importance in the regulation of the gene expression in response to changes in cytoplasmic and nucleoplasmic free-Ca2+ concentration.