Calreticulin-mediated Endoplasmic Reticulum Stress-related Apoptosis Is Involved In Hypoxia/Reoxygenation Injury Of Cardiomyocytes

Ischemic heart diseases seriously endanger human health. Restoration of blood flow as soon as possible is the key to reduce ischemic myocardial injury. Reperfusion therapies such as thrombolysis, percutaneous transluminal coronary angioplasty and coronary artery bypass surgery, are the most effective treatments to salvage myocardium following ischemic injury. However, the effect of reperfusion therapy is restricted by vascular recanalization time and reperfusion injury. To interpret the pathogenesis of ischemia/reperfusion injury is the major issue need to be resolved urgently in the medical field.Endoplasmic reticulum (ER) is the most important organelle in eukaryotic cell, adjusting protein folding, Ca²⁺ homeostasis and stress response. ER is very sensitive to stress stimulation. Ischemia/hypoxia, glucose/nutrient deprivation, ATP depletion, a large number of free radicals production or disturbance of Ca²⁺ homeostasis can cause dysfunction of endoplasmic reticulum and trigger endoplasmic reticulum stress. A certain degree of ERS improves the restoration of ER function, but excessive and prolonged ERS, as one of the mechanisms of ischemia/reperfusion injury, destroy Ca²⁺ homeostasis and induce ER associated cell death, and result in tissue injury. ERS induces upregulation of ER molecular chaperones such as glucose-regulated proteins(GRPs), calreticulin(CRT), folding enzymes, and activation of pro-apoptotic factors such as caspase-12 and CHOP/GADD153. Among these factors, calreticulin(CRT) is an essential Ca²⁺-binding /storage chaperone resident protein of endoplasmic reticulum or sarcoplasmic reticulum found across a diverse range of species. The protein is involved in the regulation of intracellular Ca²⁺ homeostasis and endoplasmic reticulum Ca²⁺ storage capacity, and is also an important molecular chaperone involved in "quality control" within secretory pathways. CRT plays an important role in apoptosis, cell adhesion, gene expression and autoimmunity. It is focus on the effect of calreticulin in the pathogenesis of ischemia/reperfusion injury. Our previous work has confirmed that ischemia/reperfusion obviously upregulate CRT expression and ischemia preconditioning protects cardiomyocyte through moderately upregulating CRT expression. It is reported that CRT has a dual role in I/R injury. It is very important to investigate the effect and molecular mechanism of CRT in I/R injury.The present study used neonatal rat cardiomyocyte hypoxia/reoxygenation model to simulate in vivo cardiac ischemia/reperfusion injury. At first, we confirmed that H/R triggered severer ERS and induced cardiomyocyte apoptosis. ERS inducer tunicamycin(TM) simulated H/R injury. In order to investigate whether CRT is the key factor to induce ERS-related apoptosis in H/R injury and its signaling pathway, pCDB-CRT plasmid transfection or siRNA interference were used to interfere with CRT expression. Then the effect of hypoxic postconditioning(H-postC) on CRT-mediated H/R injury was investigated. Major methods and results were as follows:1. H/R triggers severer ERS and induces cardiomyocyte apoptosis.Neonatal rat cardiomyocytes were prepared from Sprague-Dawley rats aged24 hours. H/R was induced by 4 hours' hypoxia(H) followed by 24 hours' reoxygenation(R). Different concentrations of TM were used to induce ERS directly. Morphological studies, lactate dehydrogenase (LDH) leakage, and flow cytometry were employed to assess cell apoptosis and necrosis. ER specific resident fluorochrome Dapoxyl was used to observe ER morphological changes. RT-PCR and/or Western blot were used to detect expression of ERS molecules such as ATF4, p-PERK, GRP78, CRT, CHOP and apoptosis-related molecules such as caspase-12, caspase-3, Bax and Bcl-2. It was suggested that H/R as the same as Tm up-regulate expression of CRT, CHOP, caspase-12, caspase-3 and Bax, and increase phosphorylation of PERK, and induce cardiomyocyte apoptosis.2. CRT is the key factor to induce ERS-related apoptosis in H/R injuryIn the modols of TM-induced ERS and cardiomyocyte H/R, we showed that pCDB-CRT plasmid transfection aggratated ERS, activated ERS-related apoptosis pathway and led to excessive cell injury, while siRNA interference attanuated cell injury and inhibited the ERS-related apoptosis pathway.3. Signalling pathway of CRT-mediated H/R injuryCRT overexpression upregulated the expression of CHOP both in mRNA (by RT-PCR) and protein (by Western blot), and the phosphorylation of PERK. Correlation analysis indicated that PERK phosphorylation and CHOP expression was positive con-elation with CRT expression. Activities of sarco(endo)plasmic reticulum Ca²⁺-ATPase and calcineurin, and intracellular Ca²⁺ concentration ([Ca²⁺]i) were dectected, which showed a negative correlation between CRT expression and SERCA activity and a positive correlation between CRT expression with [Ca²⁺]i and CaN activity.4. H-postC suppresses CRT-mediated ERS-related apoptosis induced by H/RH-postC suppressed the up-regulation of CRT, CHOP, and phosphorylation of PERK and inhibited ERS-related apoptosis. H-postC also up-regulated SERCA activity, down-regulated [Ca²⁺]i and CaN activity, and attenuated cell injury. Extraneous over-expression of CRT decreased the cardioprotective effect of H-postC, suggesting that suppression of CRT-mediated ERS-related apoptosis was invlolved in cardioprotection of H-postC.Conclusion: CRT overexpression mediates H/R injury im cardiomyocytes through the pathways as follws: (1) CRT overexpression induces ERS-related apoptosis through phosphorylating PERK and up-regulating CHOP expression. (2) CRT overexpression down-regulates SERCA activity, up-regulates [Ca²⁺]i and CaN activity. H-postC attenuates CRT-mediated ERS-related apoptosis during H/R injury.