Myocardial infarction is one of the most important causes of mortality worldwide. Timely recovery myocardial perfusion remains the major treatment for acute MI to salvage the ischemic myocardium, but reperfusion result in the excess formation of reactive oxygen species, mitochondrial dysfunction, intracellular calcium overload, uncoordinated excess contractile activity and activation of intracellular proteolysis caused irreversible myocardial injury [33, 34]. IPO, a phenomenon involving brief repetitive episodes of I/R at the directed onset of reperfusion, can effectively limit MI/RI in both experimental and clinical situations. Endogenous adenosine release to activate adenosine receptors during reperfusion plays a key role in the IPO-mediating cardioprotective effect. Subsequently, abundant experimental and clinical studies have demonstrated that adenosine applied directly before the onset of reperfusion also alleviates I/R-induced myocardial injury. However, numerous studies have reported that the cardioprotective effect of IPO is considerably suppressed in chronic diabetic myocardium due to the development of metabolic abnormalities And T2MD is associated with enhanced uptake of interstitial adenosine by cardiac fibroblasts and reduced ability of these cells to release adenosine during ATP deprivation.
Previous studies have confirmed that adenosine limits infarct size only with massive adenosine receptor activation by a prolonged high-dose infusion of adenosine but not a short-acting bolus injection[5, 34]. In the present study, we increased extracellular adenosine concentrations in diabetic hearts during reperfusion by continuous intravenous administration of NECA. a non-selective adenosine receptor agonist (Ki = 14 nM, 20 nM, and 6.2 nM for human A1R, A2AR, and A3R, respectively, and EC50 = 2,400 nM for A2BR). Our study showed for the first time that administration of NECA (2µg/kg) infused 5 min before the onset of reperfusion followed by 0.2µg/kg/min infusion for 60 min exerted a cardioprotective effect in vivo in T2MD rat hearts, as demonstrated by the improved post-MI/R cardiac functional recovery, reduced myocardial infarct size and cTnI release and attenuated myocardial apoptosis (Fig. 3–5). These findings indicated that maintaining high extracellular adenosine concentrations in cardiac tissue can alleviate MI/RI even in the diabetic state. A similar phenomenon was observed in non-diabetic rabbits and mice[25, 26].
Adenosine exerts cardioprotective effects by binding to any of four G protein-coupled receptors( A1R, A2AR, A2BR and A3R)[5, 6], and abundant studies have revealed that A1R and A3R agonists protect cardiomyocytes against ischemic injury, while A2AR and A2BR agonists function primarily by attenuating reperfusion injury to the heart[5, 11, 37, 38]. However, A2BR is a low-affinity receptor that requires high adenosine concentrations to be significantly activated, and normal adenosine concentrations fail to activate this receptor[6, 34]. Therefore, NECA postconditioning alleviate I/R-induced myocardial injury predominantly by activating A2AR[39, 40]. Our study found that the anti-MI/RI effect of NECA was reversed by the selective A2A antagonist ZM241385(222-fold more potent for A2A than A2B) and mimicked by CGS21680, a selective A2A agonist (Ki = 290 nM, 27 nM, and 67 nM for A1R, A2AR, and A3R, EC50 = 88,800 nM for A2BR) (Fig. 3–5) suggested that A2AR are primarily involved in the action of NECA during reperfusion. Similarly, the anti-infarction effect of NECA was abolished by the selective A2AR antagonist SCH58261 in vitro in a non-diabetic rat heart.
Previous studies have demonstrated that adenosine can increase PKC activity by binding with adenosine receptors[5, 41], and activation of PKC was also confirmed to protect the myocardium against I/R. Therefore, it is logical that adenosine alleviates MI/RI by activating PKC, but the underlying mechanisms and the specific PKC isoforms involved in this protective cascade are unclear and have been widely investigated. PKC isozymes as a family of serine/threonine kinases including classical PKCs (α, β1, β2, γ), novel PKCs (δ, ε, η, θ), and atypical PKCs (ζ, λ), were confirmed to be expressed in cardiac tissues from various mammalian species including mice, rats, rabbits, dogs and pigs. phosphorylation or translocation is the activation state of PKC isoforms. Wang et al. found that PKCα, PKCδ and PKCε were translocated from the soluble to the particulate fraction in response to H/R(120 min/30 min) in rat primary cultured cardiomyocytes, and inhibition of the translocation of PKCα reduced I/R-induced apoptosis and myocardial injury. Moreover, Hsu et al.reported that decreased PKCα/ε activity (reduction in the phosphorylation of PKCα/ε by Go6976/εV1–2) also provided cardioprotection against I/R-induced heart injury. However, Zatta et al.reported that IPO limited infarct size by increasing the translocation of PKCε to sites outside the mitochondrial outer membrane but limited translocation of PKCδ to the mitochondria. Additionally, Lu et al. showed that IPC increasing the translocation of PKCα and PKCδ from the cytosol to the sarcolemma in rat heart, if inhibited the translocation of PKCα will abolish cardioprotection induced by IPC[30, 44]. Furthermore, PKCα was shown to be activated in the heart and associated with sevoflurane-, fibroblast growth factor-2- and sildenafil-induced cardioprotection[2, 31]. Therefore, different PKC isoforms may play opposite roles in regulating MI/RI.
In our experimental model, we found that NECA treatment significantly increased the phosphorylation level of PKCα, and this effect was aborted by ZM241385 and mimicked by CGS21680 (Fig. 6). NECA treatment also resulted in PKCα translocation from the cytosol to the nucleus but not the membranes, and this phenomenon disappeared following pretreatment with ZM241385 and reappeared following administration of CGS21680 alone (Fig. 6). These findings indicated that NECA-induced activation of PKCα was dependent on A2AR. In addition, the cardioprotection of NECA was ablated by the PKCα selective inhibitor Go6976. PMA postconditioning also ameliorated MI/RI, as shown by the reduced infarct size, apoptotic index and cTnI release (Fig. 7A-F). These beneficial effects were weakened by co-administration of Go6976. The data in the present study demonstrated that NECA-induced cardioprotection depends on the activation of PKCα, which play essential roles in this process. However, different adenosine receptor subtypes corresponding to specific PKC isoforms may be involved. Mitsuhiro et al reported that the A1R mediated cardioprotection via activation of the PKCδ signaling pathway, and A1Rs were also reported to promote the translocation of PKCɛ and PKCδ to the plasma membrane in rat cardio myocytes. However, the A3Rs have an opposite role in PKC activation, A3R agonist IB-MECA was shown to attenuate sunitinib-induced PKCα phosphorylate; and A2BR was described as a downstream signal in the PKCs signaling pathway[25, 48]. Our data suggest that NECA can activate PKCα via A2AR.
Next, we investigated how NECA-induced PKC-α activation contributes to attenuation MI/RI. Increasing evidence suggested that apoptosis leads to the reduction of viable contractile cardiomyocytes. Persistent acute ischemia can trigger apoptosis, reperfusion boosts this process, diabetic metabolic abnormalities further aggravation. Suppressing myocardial apoptosis could reduce the loss of contractile cells attenuated cardiac injury and improve cardiac function[28, 49]. Numerous studies have shown that the miR-15 family has an effect on I/R-induced cardiomyocyte apoptosis. The expression of the miR-15 family was upregulated in the infarcted cardiac region including miR-15a, miR-15b, miR-16, miR-195, and miR-497 after MI, which increases cardiomyocyte apoptosis by negatively regulating the expression of target genes. miR-15a, a member of miR-15 family was shown to aggravate myocardial injury by promote cardiomyocyte apoptosis via targeting Bcl-2. Because miR-15a family members have the same ‘seed sequence’ for mRNA recognition (Fig. 5G), they are expected to show an overlap in targets. miR-15b, miR-16,miR-195 and miR-497 were also confirmed to promote cardiomyocyte apoptosis and exacerbate cardiac injury by targeting Bcl-2, and downregulation of miR-15family members recovered Bcl-2 protein expression and attenuated I/R-induced apoptosis[18, 19].
Interestingly, PKCα has been demonstrated to promote cell survival and suppress apoptosis by increasing the activity of the anti-apoptotic protein Bcl-2. Furthermore, Cohen et al.showed that upregulated PKCα is inversely related to decreased production of miR-15a in head and neck squamous cell carcinoma, and similarly Brandenstein et al. showed that PKCα inhibited apoptosis and promoted cell proliferation by binding directly to pri-miRNA-15a in the nucleus, which reduced mature cytoplasmic miR-15a levels[21, 22]. Therefore, we concluded that PKCα regulate Bcl-2 activity by inhibiting the expression of miR-15a in I/R hearts. In the present study we found that NECA, CGS and PMA-induced PKCα activation is associated with down-regulation miR-15a expression and upregulation Bcl-2 protein level. Furthermore, we confirmed that PKCα binding directly to pri-miRNA-15a in the nucleus by EMSA (Fig. 8B), which confirmed our hypothesis that PKCα regulates Bcl-2 activity by inhibiting the expression of miR-15a.
In summary, our findings suggested that NECA reduces MI/RI and cardiomyocytes apoptosis in T2DM rats through the A2AR/PKCα/miR-15a signal pathway (illustrated in Fig. 9). Our study suggested that NECA is a useful target candidate for the treatment of MI/RI in patient with type 2 diabetes.