Role of sarcoplasmic reticulum in mitochondrial permeability transition and cardiomyocyte death during reperfusion.

There is solid evidence that a sudden change in mitochondrial membrane permeability (mitochondrial permeability transition, MPT) plays a critical role in reperfusion-induced myocardial necrosis. We hypothesized that sarcoplasmic reticulum (SR) Ca(2+) cycling may induce partial MPT in microdomains of close anatomic proximity between mitochondria and SR, resulting in hypercontracture and cell death. MPT (mitochondrial calcein release), cell length, and sarcolemmal rupture (Trypan blue and lactate dehydrogenase release) were measured in adult rat cardiomyocytes submitted to simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) and reperfusion. On simulated reperfusion, 83 +/- 2% of myocytes developed hypercontracture. In 22 +/- 6% of cases, hypercontracture was associated with sarcolemmal disruption [Trypan blue(+)]. During simulated reperfusion there was a 25% release of cyclosporin A-sensitive mitochondrial calcein (with respect to total mitochondrial calcein content). Simultaneous blockade of SR Ca(2+) uptake and release with thapsigargin and ryanodine, respectively, significantly reduced mitochondrial calcein release, hypercontracture, and cell death during simulated reperfusion. SR Ca(2+) blockers delayed mitochondrial Ca(2+) uptake in digitonin-permeabilized cardiomyocytes but did not have any effect on isolated mitochondria. Pretreatment with colchicine to disrupt microtubule network reduced the degree of fluorescent overlap between SR and mitochondria and abolished the protective effect of SR Ca(2+) blockers on MPT, hypercontracture, and cell death during reperfusion. We conclude that SR Ca(2+) cycling during reperfusion facilitates partial mitochondrial permeabilization due to the close anatomic proximity between both organelles, favoring hypercontracture and cell death.

Effect of acidic reperfusion on prolongation of intracellular acidosis and myocardial salvage.

AIMS: It has been proposed that intracellular acidosis may be the basis of the cardioprotection of different interventions, including postconditioning. However, contradictory reports exist on the effects of acidic reperfusion on myocardial salvage. Here we characterized the effect of lowering the pH of the reperfusion media (pHo) on intracellular pH (pHi) and cell death. METHODS AND RESULTS: The effect of acidic perfusion on reperfusion injury was studied in isolated rat hearts submitted to 40 min of ischaemia and 30 min of reperfusion, and its effect on the Na(+)/Ca(2+)-exchanger (NCX) was analysed in isolated myocytes. pHi and phosphocreatine (PCr) were monitored by nuclear magnetic resonance spectroscopy. Lowering pHo to 6.4 during the initial 3 min of reperfusion delayed pHi normalization, improved PCr recovery, and markedly reduced (P < 0.001) lactate dehydrogenase release and infarct size (tetrazolium reaction). This cardioprotection was attenuated as pHo was increased, and was lost at pH0 7.0. Extending acidic reperfusion to the first 15 or 30 min of reflow did not result in further delay of pHi normalization and abolished the protection afforded by the initial 3 min of acidic reperfusion unless the Na(+)/H(+)-exchanger (NHE) blocker cariporide was added to the acidic perfusate and HCO(3)(-) substituted for N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid]. In experiments performed in fura-2-loaded myocytes exposed to low Na(+) buffer adjusted to pH 6.4, the lower Ca(2+) uptake indicated an inhibitory effect of acidosis on NCX. CONCLUSION: Acidic reperfusion for 3 min delays normalization of pHi and enhances myocardial salvage, but extending it beyond this period fails to further delay pHi recovery. This is probably due to persisting NHE and Na(+)/HCO(3)(-)-cotransporter activities, and it is detrimental, possibly through prolonged NCX inhibition.

Opening of mitochondrial permeability transition pore induces hypercontracture in Ca2+ overloaded cardiac myocytes.

UNLABELLED: After myocardial ischemia, necrotic cell death occurs mainly during the first minutes of reperfusion through ATP-dependent hypercontracture leading to sarcolemmal rupture. Recent studies indicate that opening of a mitochondrial permeability transition pore (mPTP) is a critical event in reperfusion-induced necrosis. OBJECTIVE: We investigated the hypothesis that mPTP can induce hypercontracture. METHODS: Both intact and digitonin-permeabilized rat cardiac myocytes were loaded with TMRE and submitted to oxidative damage (intermittent 568 nm laser illumination) to promote mPTP, detected as mitochondrial depolarization. The effect of cytosolic Ca(2+) overload (5 mmol/L extracellular Ca(2+)) and ATP availability on mPTP-induced cell shortening were analyzed, and changes in cytosolic and mitochondrial Ca(2+) were simultaneously monitored by confocal microscopy (Fluo-4 and Rhod-2). RESULTS: In the absence of Ca(2+) overload, induction of mPTP was consistently followed by mitochondrial depolarization and rigor shortening that, in permeabilized cells, was prevented by ATP. Exposure of intact cardiac myocytes to 5 mmol/L Ca(2+) induced an increase in cytosolic and mitochondrial Ca(2+) content. In Ca(2+) overloaded myocytes, induction of mPTP resulted in a further increase in cytosolic Ca(2+) and hypercontracture (> 50% reduction in length with distortion of cell geometry) that started before depolarization involved all mitochondria within the cell and could be prevented by the mPTP inhibitor cyclosporin A. In permeabilized myocytes, mPTP could promote hypercontracture when cytosolic Ca(2+) overload was mimicked in the presence of ATP, and was prevented when ATP was removed from the intracellular-like medium. CONCLUSIONS: mPTP opening may induce ATP-dependent hypercontracture in Ca(2+) overloaded myocytes. This phenomenon could reconcile the apparently contradictory hypotheses of hypercontracture and mPTP opening as main determinants of necrosis during the first minutes of reperfusion.

The modulatory effects of connexin 43 on cell death/survival beyond cell coupling.

Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.

Mitochondrial Ca2+ uptake during simulated ischemia does not affect permeability transition pore opening upon simulated reperfusion.

OBJECTIVE: Reenergization of ischemic cardiomyocytes may be associated with acute necrotic cell death due in part to cytosolic Ca2+ overload and opening of a permeability transition pore (PTP) in mitochondria. It has been suggested that Ca2+ overload during ischemia primes mitochondria for PTP opening during reperfusion. We investigated the ability of mitochondria to uptake Ca2+ during simulated ischemia (SI) and whether this uptake determines PTP opening and cell death upon simulated reperfusion (SR). METHODS: Rat heart mitochondria were submitted to either hypoxia (anoxic chamber) or to SI (respiratory inhibition, substrate depletion and acidosis) and subsequent SR. Mitochondrial Ca2+ uptake was monitored using Ca2+ microelectrodes after exposure to different [Ca2+] up to 25 microM during SI, and PTP opening was assessed by quantification of mitochondrial swelling (changes in absorbance rate at 540 nm) and calcein release. Mitochondrial Ca2+ uptake (Rhod-2 fluorescence) and cytosolic Ca2+ rise (Fura-2 ratio fluorescence) were further investigated in HL-1 cardiac myocytes submitted to SI/SR, and the effect of reducing mitochondrial Ca2+ load (with 25 microM ruthenium red) or blocking PTP opening (with 0.5 microM cyclosporin A) on the rate of cell death was investigated in adult cardiomyocytes exposed to SI/SR. RESULTS: SI induced a progressive dissipation of mitochondrial membrane potential (TMRE fluorescence); however, prior to the completion of depolarization, high levels of Ca2+ uptake were observed in mitochondria. SR induced PTP opening but this phenomenon was not influenced by the magnitude of mitochondrial Ca2+ uptake during previous SI. Blockade of the mitochondrial Ca2+ uniporter during SI in cardiomyocytes attenuated mitochondrial Ca2+ uptake but increased cytosolic Ca2+ overload and cell death upon subsequent SR. CONCLUSION: Mitochondrial Ca2+ uptake during SI buffers cytosolic Ca2+ overload but its magnitude appears not to be an important determinant of PTP opening upon subsequent SR.

Effects of hypoxia, glucose deprivation and acidosis on phosphatidylcholine synthesis in HL-1 cardiomyocytes. CTP:phosphocholine cytidylyltransferase activity correlates with sarcolemmal disruption.

A decrease in [3H]Cho (choline) incorporation in to PtdCho (phos-phatidylcholine) preceded the onset of LDH (lactate dehydrogenase) release in HL-1 cardiomyocytes submitted to simulated ischaemia. This observation led us to examine the role of PtdCho synthesis in sarcolemmal disruption in HL-1 cardiomyocytes. To address this objective we analysed the individual effects of hypoxia, glucose deprivation and acidosis, three prominent components of ischaemia, on the different steps of the Kennedy pathway for the synthesis of PtdCho. Pulse and pulse-chase experiments with [3H]Cho, performed in whole HL-1 cells submitted to hypoxia or normoxia, in the presence or absence of glucose at different pHs indicated first, that CK (choline kinase) was inhibited by hypoxia and acidosis, whereas glucose deprivation exacerbated the inhibition caused by hypoxia. Second, the rate-limiting reaction in PtdCho synthesis, catalysed by CCT (CTP:phosphocholine cytidylyltransferase), was inhibited by hypoxia and glucose deprivation, but unexpectedly activated by acidosis. In cellfree system assays, acidosis inhibited both CK and CCT. In experiments performed in whole cells, the effect of acidosis was likely to be direct on CK, but indirect or intact-cell-dependent on CCT. Since hypoxia and glucose deprivation favoured membrane disruption, but acidosis prevented it, we hypothesized that the modulation of CCT could be an important determinant of cell survival. Supporting this hypothesis, we show that CCT activity in whole-cell experiments clearly correlated with LDH release, but not with ATP concentration. Altogether our results suggest a significant role for CCT activity in sarcolemmal disruption during ischaemia.