The Significance Of MIF And AMPK In Myocardial Adaptation To Chronic Hypoxia

Background and Objective:Congenital heart disease, especially cyanotic cardiac defects which result in chronic hypoxia in the neonate until operative correction is achieved, remains a major cause of death in infancy. The effects of chronic hypoxia on neonatal myocardial metabolic adaptation have not been well defined. In the heart, AMP-activated protein kinase (AMPK) orchestrates the regulation of energy generating and consuming pathways by regulating many metabolic pathways, and it's the key kinase under stress conditions such as hypoxia, ischemia et al. However, the activator of AMPK under chronic hypoxia need to be identified.Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient and highly conserved cytokine. It was originally described as a product of T-cells that inhibits the migration of macrophages in vitro and promotes macrophage accumulation in the skin in delayed-type hypersensitivity reactions. Now, the pleiotropic cytokine MIF has emerged as a key player in cardiovascular disease, and a recent study has demonstrated that MIF modulates the activation of AMPK during ischemia, functionally linking inflammation and metabolism in the heart. So, MIF might play an important role in the adaptive responses of cardiomyocytes during chronic hypoxia by activating the AMPK signal pathway. Meanwhile, the expression of MIF in cardiomyocytes under chronic hypoxia might increase because the stability of hypoxia-inducible factor -1α(HIF-1α) is increased under chronic hypoxia, and HIF-1 can stimulate upregulation the expression of MIF. Thus, the aim of this study is to provide an insight into the involvement of MIF in the metabolic adaptation of cardiomyocytes during chronic hypoxia both in vivo and in vitro.Methods:Samples from the right ventricular outflow tract were collected from patients with cyanotic(n = 21) or acyanotic (n = 14) congenital heart disease. The expression of HIF-1α and MIF were examined by immunohistochemistry, while mRNA and protein production of HIF-1αand MIF were evaluated by RT-PCR and western blot, respectively. Meanwhile, the phosphorylated AMPK was measured by western blot.To evaluated the effect of chronic hypoxia on the expression of MIF by cardiomyocytes in vitro, primary rat cardiomyocytes and embryonic rat-heart-derived H9c2 cells were cultivated and exposed to 1.0% O2, 5.0% CO2 for different durations to establish the chronic hypoxic cell model. Control cells were cultivated in the same conditions except for 21% O2 concentration. The growth of cells was tested by MTT cell proliferation assays, and after different duration of hypoxic exposure, cells were collected and subjected to RT-PCR and western blot to detecting the mRNA and protein expression of MIF, and the phosphorylated AMPK.Results:1. Immunohistochemistry revealed that HIF-1αwas expressed both in the cytoplasm and nucleus. The protein levels of HIF-1αwere significantly elevated (p < 0.001) in patients with cyanotic compared to acyanotic congenital heart disease and positively correlated with the degree of hypoxemia. The expression of HIF-1αmRNA has no significant difference between the two groups (p > 0.05).2. Immunoreactivity for MIF was identified in the cytoplasm of cardiomyocytes. The protein level of MIF was significantly higher in the cyanotic group than acyanotic group (p < 0.001), and inversely correlated with SaO2 (Spearman correlation coefficient:–0.74, p < 0.01). Meanwhile, MIF mRNA is constitutively expressed in the cardiomyocytes and significantly increased in infants with cyanotic cardiac defects (p < 0.001).3. Western blot analysis revealed that phosphorylation of AMPK at Thr 172 was significantly increased in cyanotic hearts compared with their acyanotic counterparts (p < 0.001).4. In both normal and hypoxia-exposed cells, the MIF mRNA and protein were expressed constitutively at low levels by the cardiomyocytes initially. By contrast, the expression of MIF mRNA and protein in hypoxia-exposed cells were significantly increased in a duration dependent manner.5. Hypoxia stimulated AMPK activation in a time-dependent manner. The phosphorylation of AMPK at Thr 172 gradually increased in the first 3 d (compared with control: p < 0.001) and remained high throughout the study period. Moreover, the activity of AMPK was correspondingly increased.6. When comparing the growth of cells under hypoxia and normoxia, we found out that the growth rate under hypoxia was dramatically low in the first 24 h (p < 0.01), however, there was no significant difference between them after 24 h.Conclusions:Upregulation of endogenous HIF-1αand MIF expression and increased activation of AMPK in cardiomyocytes under chronic hypoxia has been observed both in vivo and in vitro. Taken together, the findings here suggested that HIF-1αcould stimulate MIF expression, and MIF might be a key activator of AMPK under chronic hypoxia. So, MIF might be the key modulator of the metabolic adaptation of cardiomyocytes to chronic hypoxia.