Lithium blocks the PKB and GSK3 dephosphorylation induced by ceramide through protein phosphatase-2A

The biochemical mechanism of apoptosis induced by ceramide remains still unclear, although it has been reported that dephosphorylation of PKB at Ser-473 may be a key event. In this article, we show that C(2)-ceramide (N-acetyl-sphingosine) induces the dephosphorylation of both protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) in cerebellar granule cells (CGC). We also show that lithium protects against the apoptosis induced by C(2)-ceramide by blocking the dephosphorylation of both kinases. Since lithium inhibits in vivo the observed protein phosphatase-2A (PP2A) activation induced by ceramide, we hypothesise that the neuroprotective action of lithium may be due to the inhibition of the PP2A activation by apoptotic stimuli.     

Lithium protects ethanol-induced neuronal apoptosis

Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3beta, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3beta (ser9). In addition, the selective GSK-3beta inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.     

PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells

Lithium is a well‐established non‐competitive inhibitor of glycogen synthase kinase‐3β (GSK‐3β), a kinase that is involved in several cellular processes related to cancer progression. GSK‐3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK‐3β‐independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment. J. Cell. Biochem. 117: 458–469, 2016. © 2015 Wiley Periodicals, Inc.     

Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons

In the present study we focused in the PI3K/Akt pathway which plays a key role in neuronal survival. Here we show that inhibition of PI3K/Akt by means of LY294002 induces apoptosis via a caspase-dependent and calpain-independent pathway in cerebellar granule neurons (CGNs). This finding was confirmed using zVAD-fmk, a widely caspase inhibitor that prevents apoptosis. For this purpose, we compared two models of apoptosis in CGNs, namely inhibition of PI3K/Akt, and serum potassium deprivation (S/K deprivation). In contrast to the S/K deprivation model, caspase-3 was not activated when PI3K is inhibited. Likewise, CDK5 activation was not involved in this apoptotic process, because calpain activation is responsible for the formation of CDK5/p25 neurotoxic form. However, S/K deprivation activated calpain, as it is shown by α-spectrin breakdown, and favoured the formation of CDK5/p25. Moreover, although PI3K/Akt inhibition enhanced pRbser780 phosphorylation, no increase in the expression of cell-cycle proteins, namely: cyclin D, cyclin E, CDK2 or CDK4, was detected. Furthermore, BrdU incorporation assay did not shown any increase in DNA synthesis. Likewise, PI3K/Akt inhibition increased GSK3β activity and c-Jun phosphorylation, which implicates these two pathways in this apoptotic route. Although previous reports suggest that apoptosis induced in CGNs by LY294002 and S/K deprivation causes PI3K inhibition and increases GSK3β activity and c-Jun phosphorylation activation, our results demonstrate substantial differences between them and point to a key role of GSK3β in the apoptosis induced in CGNs in the two models tested.     

Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.     

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.     

Inhibition of taurolithocholate 3-sulfate-induced apoptosis by cyclic AMP in rat hepatocytes involves protein kinase A-dependent and -independent mechanisms

The mechanisms underlying the inhibition of bile acid-induced apoptosis by cyclic AMP (cAMP) were studied in 24-h-cultured rat hepatocytes. Taurolithocholate 3-sulfate (TLCS, 100 micromol/l) led to a sustained activation of mitogen activated protein (MAP) kinases (JNK, p38(MAPK), and ERKs), dephosphorylation of protein kinase B (PKB), activation of caspases 3 and 8, and hepatocyte apoptosis. cAMP prevented TLCS-induced apoptosis, shifted the persistent TLCS-induced MAP kinase response to a transient pattern, and prevented PKB dephosphorylation. TLCS-induced CD95 and TRAIL receptor-2 trafficking to the plasma membrane were significantly inhibited. Blockade of protein kinase A (PKA) abolished the inhibitory effect of cAMP on TLCS-induced CD95 membrane targeting, but not TRAIL receptor-2 membrane targeting, PKB and MAP kinase responses. H89, an inhibitor of PKA, had no effect on cAMP-induced inhibition of TLCS-triggered poly(ADP) ribose polymerase (PARP) cleavage and caspase activation, but abolished the cAMP-induced inhibition of TLCS-triggered TUNEL- and Annexin V staining. It is concluded that cAMP inhibits bile acid-induced apoptosis via PKA-dependent and -independent mechanisms.     

Akt/PKB controls the activity-dependent bulk endocytosis of synaptic vesicles

Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode during intense neuronal activity. The dephosphorylation of Ser774 on dynamin I is essential for triggering of ADBE, as is its subsequent rephosphorylation by glycogen synthase kinase 3 (GSK3). We show that in primary cultures of cerebellar granule neurons the protein kinase Akt phosphorylates GSK3 during intense neuronal activity, ensuring that GSK3 is inactive during intense stimulation to aid dynamin I dephosphorylation. Furthermore, when a constitutively active form of Akt was overexpressed in primary neuronal cultures, ADBE was inhibited with no effect on clathrin-mediated endocytosis. Thus Akt has two major regulatory roles (i) to ensure efficient dynamin I dephosphorylation via acute activity-dependent inhibition of GSK3 and (ii) to negatively regulate ADBE when activated in the longer term. This is the first demonstration of a role for Akt in SV recycling and suggests a key role for this protein kinase in modulating synaptic strength during elevated neuronal activity.     

Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.     

Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways

We used cultured cerebellar granule cells to examine whether native group-III metabotropic glutamate (mGlu) receptors are coupled to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways. Cultured granule cells responded to the group-III mGlu receptor agonist, L-2-amino-4-phosphonobutanoate (l-AP4), with an increased phosphorylation and activity of MAPKs (ERK-1 and -2) and an increased phosphorylation of the PI-3-K target, protein kinase B (PKB/AKT). These effects were attenuated by the group-III antagonists, alpha-methyl-serine-O -phosphate (MSOP) and (R,S )-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l-AP4 also induced the nuclear translocation of beta-catenin, a downstream effector of the PI-3-K pathway. To assess the functional relevance of these mechanisms we examined the ability of l-AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K(+) from 25 to 10 mm. Neuroprotection by l-AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI-3-K pathway. Taken together, these results show for the first time that native group-III mGlu receptors are coupled to MAPK and PI-3-K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.     

Bone Morphogenetic Protein-6 Promotes Cerebellar Granule Neurons Survival by Activation of the MEK/ERK/CREB Pathway

Bone morphogenetic proteins (BMPs) have been implicated in the generation and postnatal differentiation of cerebellar granule cells (CGCs). Here, we examined the eventual role of BMPs on the survival of these neurons. Lack of depolarization causes CGC death by apoptosis in vivo, a phenomenon that is mimicked in vitro by deprivation of high potassium in cultured CGCs. We have found that BMP-6, but not BMP-7, is able to block low potassium–mediated apoptosis in CGCs. The neuroprotective effect of BMP-6 is not accompanied by an increase of Smad translocation to the nucleus, suggesting that the canonical pathway is not involved. By contrast, activation of the MEK/ERK/CREB pathway by BMP-6 is necessary for its neuroprotective effect, which involves inhibition of caspase activity and an increase in Bcl-2 protein levels. Other pathways involved in the regulation of CGC survival, such as the c-Jun terminal kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt/PKB, were not affected by BMP-6. Moreover, failure of BMP-7 to activate the MEK/ERK/CREB pathway could explain its inability to protect CGCs from low potassium–mediated apoptosis. Thus, this study demonstrates that BMP-6 acting through the noncanonical MEK/ERK/CREB pathway plays a crucial role on CGC survival.     

High fluence low‐power laser irradiation induces apoptosis via inactivation of Akt/GSK3β signaling pathway

High fluence low‐power laser irradiation (HF‐LPLI) is a newly discovered stimulus through generating reactive oxygen species (ROS) to trigger cell apoptosis. Activation of glycogen synthase kinase 3β (GSK3β) is proved to be involved in intrinsic apoptotic pathways under various stimuli. However, whether the proapoptotic factor GSK3β participates in HF‐LPLI‐induced apoptosis has not been elucidated. Therefore, in the present study, we investigated the involvement of GSK3β in apoptosis under HF‐LPLI treatment (120 J/cm², 633 nm). We found that GSK3β activation could promote HF‐LPLI‐induced apoptosis, which could be prevented by lithium chloride (a selective inhibitor of GSK3β) exposure or by GSK3β‐KD (a dominant‐negative GSK3β) overexpression. We also found that the activation of GSK3β by HF‐LPLI was due to the inactivation of protein kinase B (Akt), a widely reported and important upstream negative regulator of GSK3β, indicating the existence and inactivation of Akt/GSK3β signaling pathway. Moreover, the inactivation of Akt/GSK3β pathway depended on the fluence of HF‐LPLI treatment. Furthermore, vitamin c, a ROS scavenger, completely prevented the inactivation of Akt/GSK3β pathway, indicating ROS generation was crucial for the inactivation. In addition, GSK3β promoted Bax activation by down‐regulating Mcl‐1 upon HF‐LPLI treatment. Taken together, we have identified a new and important proapoptotic signaling pathway that is consisted of Akt/GSK3β inactivation for HF‐LPLI stimulation. Our research will extend the knowledge into the biological mechanisms induced by LPLI. J. Cell. Physiol. 226: 588–601, 2011. © 2010 Wiley‐Liss, Inc.     

Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase.

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB-415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB-415286 induced a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and SB-415286 all caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB-415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3-kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB-203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB-415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.     

Apoptosis-inducing and -preventing signal transduction pathways in cultured cerebellar granule neurons

1. Cultured cerebellar granule neurons maintained in medium containing 26 mM potassium (high K+ or HK+) undergo cell death when switched to medium with 5 mM potassium (low K+ or LK+). This low K(+)-induced cell death has typical features of apoptosis. The intracellular signaling pathway of low K(+)-induced apoptosis has been investigated. 2. Cerebellar granule neurons become committed to undergo apoptosis between 2 and 5 h after K+ deprivation, judging from the inability of high K+ to rescue them after this time. Although the levels of most mRNAs decrease markedly concomitant with commitment, expression of c-jun mRNA increases 2-3 h after K+ deprivation. Among the family of caspases, a caspase-3-like protease is activated within 4 h of lowering the K+ concentration. A caspase-1-like protease is also activated within 2 h of K+ deprivation. 3. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity by LY294002 or wortmannin also induces apoptosis in cerebellar granule neurons. The intracellular signaling pathway of LY294002-induced apoptosis has been investigated. The activity of c-Jun N-terminal kinase (JNK) increases 8 h after addition of LY294002 to high K+ medium or low K+ medium containing BDNF. Expression of c-Jun protein also increases almost simultaneously. 4. The low K(+)-induced apoptosis of cerebellar granule neurons is prevented by high K+ (membrane depolarization by high K+), BDNF, IGF-1, bFGF or cAMP. The intracellular signaling pathways by which these agents prevent low K(+)-induced apoptosis have been investigated. Agents other than cAMP prevent apoptosis through PI3-K and a Ser/Thr kinase, Akt/PKB. The survival-promoting effect of cAMP does not depend on the PI3-K-Akt pathway.     

GSK3beta mediates the induced expression of synaptic acetylcholinesterase during apoptosis

Besides its role in terminating acetylcholine-mediated neurotransmission, acetylcholinesterase (AChE) is found to be expressed and participate in the process of apoptosis in various cell types. However, the mechanisms underlying AChE up-regulation in neuronal cells remain elusive. Herein we demonstrated that glycogen synthase kinase-3β (GSK3β) mediates induced AChE-S expression during apoptosis. In this study, A23187 and thapsigargin (TG) were employed to induce apoptosis in neuroendocrine PC12 cells. The results showed that exposure of PC12 cells to A23187 and TG up-regulated AChE activity significantly. The same treatment also led to activation of GSK3β. Two different inhibitors of GSK3β (lithium and GSK3β-specific inhibitor VIII) could block A23187- or TG-induced up-regulation of AChE activity, AChE-S mRNA level and protein expression. However, lithium could not inhibit the induction of AChE-R mRNA and protein under similar conditions. Taken together, our results show that GSK3β is specifically involved in the induction of AChE-S expression in PC12 cells during apoptosis.     

Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.