Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury

Figures

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  FIGURE 1. ADK inhibition prevents myocardial I/R injury and improves cardiac function. A, The expression of ADK from I/R‐injured hearts was immunoblotted and quantitatively analysed. (n = 5). B, Infarct size was visualized by Evans blue/TTC staining and was quantitatively analysed. (n = 10). C, LDH was determined in the serum from mice exposed to myocardial 30‐min ischaemia/2‐h reperfusion. (n = 5). D and E, Cardiac function was evaluated via echocardiography, as indicated by LVEF and LVFS at the end of the 24‐h reperfusion. (n = 8). F, Myocardial ultrastructure changes were observed by electron microscope. (n = 5). Scale bar = 0.5 μm. G and H, Mouse heart rates and blood pressures were measured by tail‐cuff plethysmography (MRBP, IITC Life Science). (n = 6). *P < .05 vs DMSO group

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  FIGURE 2. Cell apoptosis and necroptosis are decreased by ADK inhibition in I/R‐injured hearts. A and B, Apoptotic cells in the area at risk were determined by TUNEL staining and quantitatively analysed. (n = 5). Scale bar = 20 μm. C and D, Protein lysates from I/R‐injured hearts (30‐min ischaemia/4‐h reperfusion) were immunoblotted for cleaved caspase‐12, cleaved caspase‐9, cleaved caspase‐8, cleaved caspase‐3, Bax, Bcl‐2, phosphor‐P38 and P38. (n = 5). E and F, Myocardial necrosis were evaluated by double staining with CaV3 (viable cardiomyocytes) and EBD (necrotic cells). (n = 5). Scale bar = 50 μm. G and H, Protein lysates from I/R‐injured hearts (30‐min ischaemia/4‐h reperfusion) were immunoblotted for RIP1, phosphor‐RIP1, RIP3, phosphor‐RIP3, MLKL, phosphor‐MLKL, CaMKII and phosphor‐CaMKII (n = 5)

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  FIGURE 3. Cell apoptosis and necroptosis are decreased by ADK inhibition in vitro. H9c2 cells were incubated in hypoxia chamber for 12 h and exposed to reoxygenation for 4 h with/without ABT‐702 (1 μmol/L) pre‐treatment. A and B, Apoptotic cells were stained by TUNEL method and quantitatively analysed. (n = 5). Scale bar = 20 μm. C and D, Representative immunoblots of cleaved caspase‐12, cleaved caspase‐9, cleaved caspase‐8, cleaved caspase‐3, Bax, Bcl‐2, phosphor‐P38 and P38 in H/R‐stimulated H9c2 cells. (n = 5). E and F, Necroptotic cells were analysed by Annexin V/PI staining using flow cytometry. (n = 5). G and H, Representative immunoblots of RIP1, phosphor‐RIP1, RIP3, phosphor‐RIP3, MLKL, phosphor‐MLKL, CaMKII and phosphor‐CaMKII in H/R‐stimulated H9c2 cells (n = 5)

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  FIGURE 4. ADK inhibition prevents H/R‐induced cell apoptosis and necroptosis via Akt/XIAP pathway. A, H9c2 cells were incubated in hypoxia chamber for 12 h and exposed to reoxygenation for 4 h. cIAP1, cIAP2, XIAP and phosphor‐XIAP were immunoblotted in H/R‐stimulated H9c2 cells with/without ABT‐702 pre‐treatment. (n = 5). B, H9c2 cells were incubated in hypoxia chamber for 12 h and exposed to reoxygenation for 1 h with/without ADK pre‐treatment. Lysates were separated and immunoblotted for Akt and phosphor‐Akt. (n = 5). C, H9c2 cells were pre‐treated with MK‐2206 (10 μmol/L) before H/R and ADK inhibition. Lysates were immunoblotted for XIAP and phosphor‐XIAP. (n = 5). D, H9c2 cells were transfected with XIAP‐siRNA or scrambled siRNA. Apoptotic cells were stained by TUNEL method and quantitatively analysed. (n = 6). E, Cleaved caspase‐9, cleaved caspase‐8 and cleaved caspase‐3 were immunoblotted. (n = 5). F, Necroptotic cells were analysed by Annexin V/PI staining using flow cytometry. (n = 6). G, RIP3, phosphor‐RIP3, MLKL, phosphor‐MLKL, CaMKII and phosphor‐CaMKII were immunoblotted. (n = 5)

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  FIGURE 5. Adenosine receptor A2b mediated the protective effect of ADK inhibition on H/R‐induced cell death. A, H9c2 cells were exposed to H/R injury and pre‐treated with ABT‐702 and/or 8‐SPT (100 μmol/L). Lysates were immunoblotted for XIAP and phosphor‐XIAP. (n = 5). B and C, mRNA expression of adenosine receptors in I/R‐injured mouse hearts, H/R‐treated H9c2 cells and their control were quantitatively analysed. (n = 6). D, Adenosine receptor A2B was immunoblotted in H/R‐treated H9c2 cells. (n = 5). E, H9c2 cells were exposed to H/R injury and pre‐treated with ABT‐702 and/or MRS 1754 (10 nmol/L). Lysates were immunoblotted for Akt, phosphor‐Akt, XIAP and phosphor‐XIAP. (n = 5). F, Apoptotic cells were stained by TUNEL method and quantitatively analysed. (n = 6). G, Cleaved caspase‐9, cleaved caspase‐8 and cleaved caspase‐3 were immunoblotted. (n = 5). H, Necroptotic cells were analysed by Annexin V/PI staining using flow cytometry. (n = 6). I, RIP3, phosphor‐RIP3, MLKL, phosphor‐MLKL, CaMKII and phosphor‐CaMKII were immunoblotted. (n = 5)

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  FIGURE 6. ADK inhibition improves mitochondrial function. H9c2 cells were pre‐treated with/without ABT‐702 and exposed to H/R injury. A and B, ΔΨm was detected by JC‐1 staining. (n = 5). Scale bar = 20 μm. C and D, Opening of mPTP was determined by calcein‐AM staining. (n = 5). Scale bar = 20 μm. E and F, Mitochondrial superoxide production was determined by MitoSox staining (n = 5). Scale bar = 100 μm. G, ATP levels were tested by ATP Assay Kit. (n = 5). H, Morphology of mitochondria was observed with transmission electron microscopy. (n = 5). Scale bar = 0.5 μm