Endoplasmic Reticulum Stress/Ca2+-Calmodulin-Dependent Protein Kinase/Signal Transducer and Activator of Transcription 3 Pathway Plays a Role in the Regulation of Cellular Zinc Deficiency in Myocardial Ischemia/Reperfusion Injury

Figures

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  FIGURE 1. Zinc deficiency can induce ER Stress and Ca2+ leak. (A) Cells were exposed to TPEN (10 μM, n = 3) for 4 h. (B) TPEN was applied 1 h before exposure to ZnCl2 (10 μM, n = 3) and ionophore pyrithione (4 μM) for 1 h. (C) ER and free Ca2+ concentrations in cells were labeled with Mag-fluo4 (5 μM, n = 3) and Fluo-4 (5 μM, n = 3), respectively, and detected with confocal microscopy. (D) Mouse hearts were ischemic for 30 min and then reperfused for 30 min. Cardiomyocytes were labeled with Mag-fluo4 (5 μM) and Fluo-4 (5 μM), respectively (n = 10). *p < 0.05, **p < 0.01, and ***p < 0.001. ns, no significant difference P > 0.05.

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  FIGURE 2. Calcium is responsible for zinc deficiency-induced STAT3 activation. BAPTA-AM (10 μM) (A,B) or EGTA-AM (10 μM) (C,D) was applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 3. Zinc deficiency activates Ca2+ leak from ER via RyR2. (A,B) H89 (PKA inhibitor,10 μM, n = 5) was applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation. (C) Mouse hearts were ischemic for 30 min and then reperfused for 30 min. H89 (10 mg/kg) was injected 5 min before reperfusion and continued for 30 min through the tail vein (n = 7). (D) H89 (10 μM, n = 6) was applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation and labeled with Mag-fluo-4 (5 μM, n = 6) and Fluo-4 (5 μM, n = 6), respectively. (E) H89 (10 μM, n = 5) and 2APB (100 μM, n = 5) were applied 2 h before exposure to TPEN for 2 h. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, no significant difference P > 0.05.

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  FIGURE 4. P-CaMKII is involved in zinc deficiency-induced STAT3 activation. KN93, KN92 (A,B), H89 (C), or BAPTA-AM (D) (10 μM, n = 5) were applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation. *p < 0.05, **p < 0.01, and ***p < 0.001. ns, no significant difference P > 0.05.

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  FIGURE 5. Zinc deficiency-induced STAT3 activation promotes ZIP9 expression. (A,B) Stattic (10 μM, n = 5) was applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation. (C) Mouse hearts were ischemic for 30 min and then reperfused for 30 min. Stattic (3 mg/kg, n = 5) was injected 5 min before reperfusion and continued for 30 min through the tail vein. *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 6. ER Stress/CaMKII is involved in the regulation of ZIP9 by STAT3 (A,B) H89, KN93, or BAPTA-AM (10 μM, n = 5) were applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation. (C) Mouse hearts were ischemic for 30 min and then reperfused for 30 min. H89, KN93 or BAPTA-AM (10 mg/kg) were injected 5 min before reperfusion and continued for 30 min through the tail vein (n = 7). (D) Zn2+ levels were monitored with inductively coupled plasma optical emission spectroscopy (ICPOES, n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 7. Activated STAT3 can reduce myocardial reperfusion injury. (A) KN93 (10 μM, n = 4) was applied 2 h before exposure to TPEN for 2 h or 30 min before the onset of reoxygenation. (B) Mouse hearts were ischemic for 30 min and then reperfused for 30 min. After I/R, the release of these myocardial injury markers were detected (n = 9). (C) Myocardial infarct size was assessed by TTC staining. Mouse hearts were subjected to 30 min ischemia followed by 2 h of reperfusion (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 8. Cellular endogenous protective mechanism induced by zinc deficiency.