Regulatory Mechanism Of NO Pathway In Cardiac Mitochondrial Biogenesis During Chronic Hypoxia

BackgroundMyocardial chronic hypoxia is a common clinical pathophysiological process and leads to a series of myocardial metabolic adaptive changes. Mitochondrial biogenesis which controls the quantity and quality of mitochondria may plays an important role in myocardial metabolic adaptation. However, the effect of chronic hypoxia in myocardial mitochondrial biogenesis has not been fully understood. Nitric oxide (NO) may be involved in the regulation of mitochondria biogenesis in a variety of mammalian cell lines, but its role in cardiac mitochondrial biogenesis during chronic hypoxia remains unknown. Research on the relationship between NO and myocardial mitochondrial biogenetic adaptation during chronic hypoxia may help to clarify the mechanism of chronic hypoxic adaptation and provide some new ideas to enhance myocardial protection in health and patients. To better clarify the regulatory mechanisms of NO pathway in cardiac mitochondrial biogenesis during chronic hypoxia, we performed a detailed morphologic and molecular analysis on heart tissue samples from patients with congenital heart disease and cultured cardiomyocytes exposed to chronic hypoxia.MethodsIn the first part of this study, samples from the right ventricular outflow tract myocardium were taken from patients with cyanotic(n=10) and acyanotic(n=10) congenital heart diseases. The ultrastructure of samples was analyzed by transmission electron microscope. MtDNA copy number, cytochrome c oxidase I (COX I), peroxisome proliferators activated receptorγcoactivator-1α(PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam) and three nitric oxide synthase(NOS) isoforms transcript and(or) protein levels were detected by real-time PCR , RT-PCR and Western blot respectively. The activity levels of NOS were also analyzed.In the second part, cultured neonatal rat cardiomyocytes were treated with SNAP (NO donor), oxygenated hemoglobin (OxyHb, NO scavenger) or ODQ (soluble guanylate cyclase inhibitor). Mitochondrial specific fluorescence probe was used for detecting the mitochondrial mass, real-time PCR, RT-PCR and Western blot were used for detecting the expression of related gene and protein.In the last part, neonatal rat cardiomyocytes were cultured in 1%O2 for 48h and treated with L-NAME(NOS inhibitor),OxyHb or ODQ. The NO production in cardiomyocytes, mitochondrial mass, related gene and protein expression were also detected.Results1. Mitochondrial volume density (Vv) and numerical density (Nv) were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease (p<0.05, p<0.01). Elevated mtDNA and up-regulated COXI, PGC-1α, NRF1 and Tfam mRNA levels were observed in cyanotic patients (p<0.01). Levels of COXI and eNOS protein were significantly higher in the myocardium of cyanotic patients than those of acyanotic (p<0.05, p<0.05). Levels of PGC-1αtranscript and eNOS protein inversely correlated with SaO2(r=-0.75, p<0.01 and r=-0.64, p<0.01, respectively). Levels of PGC-1αtranscript correlated with the levels of eNOS(r=0.58, p<0.01).2. (1) In SNAP-treated cardiomyocytes, the mitochondria fluorescence intensity, copy number of mtDNA, levels of COXI mRNA and protein were significantly higher than those of control group (p <0.01, p <0.01, p <0.01, p <0.01). Mitochondrial biogenesis related signaling molecules PGC-1α, and NRF1 Tfam mRNA levels were also elevated (p <0.01, p <0.01, p <0.05).(2) Comparing with the SNAP group, the mitochondria fluorescence intensity, copy number of mtDNA and COXI mRNA decreased in SNAP + OxyHb group (p <0.01, p <0.01, p <0.05), and levels of PGC-1αand NRF1 mRNA had also decreased (p <0.01, p <0.05).(3) Comparing with the SNAP group, the mitochondria fluorescence intensity, mtDNA copy number and COXI mRNA levels decreased in the SNAP + ODQ group (p <0.05, p <0.01, p <0.05), and PGC-1αmRNA levels have also decreased (p < 0.05).3. (1) Comparing with the control group, NO production, eNOS and iNOS protein levels were significantly higher in cardiomyocytes exposed to chronic hypoxia (p <0.01, p <0.05, p <0.05). The mitochondria fluorescence intensity, mtDNA copy number and COXI mRNA levels were significantly higher (p <0.01, p <0.01, p <0.01). Mitochondrial biogenesis related signaling molecules PGC-1α, and NRF1 Tfam mRNA levels were also elevated in cardiomyocytes exposed to chronic hypoxia (p <0.01, p <0.05, p <0.01).(2) Comparing with the chronic hypoxia group, the mitochondria fluorescence intensity, COXI mRNA and protein levels decreased (p <0.01, p <0.01, p <0.05), and PGC-1αand Tfam mRNA levels have also decreased (p <0.05, p <0.01) in chronic hypoxia + L-NAME group.(3) Comparing with the chronic hypoxia group, levels of PGC-1αmRNA decreased in chronic hypoxia + OxyHb group (p <0.05). No significant changes were observed in other parameters.(4) Comparing with the chronic hypoxia group, the mitochondria fluorescence intensity, mtDNA copy number, COXI mRNA and protein levels decreased (p <0.01, p <0.05, p <0.01, p <0.05), levels of PGC-1αand Tfam mRNA were also decreased (p <0.01, p <0.05) in chronic hypoxia + ODQ group.ConclusionsThis study based on a detailed morphologic and molecular analysis on heart tissue samples from congenital heart disease patients and cultured cardiomyocytes exposed to chronic hypoxia. The results indicated that:1. The mitochondrial mass, mtDNA copy number, mitochondrial encoded COXI mRNA and protein levels were increased in the right ventricular outflow tract myocardium of congenital heart disease with cyanosis, suggesting that mitochondrial biogenesis is activated in myocardium in congenital heart disease with cyanosis. Meanwhile, levels of eNOS protein were also elevated in cyanotic group, and were significantly positive correlated with mitochondrial biogenesis, which implied that eNOS may be involved in the regulation of mitochondrial biogenesis.2. NO donor can promote the mitochondrial biogenesis in cultured cardiomyocytes, possibly mediated by sGC-cGMP pathway.3. NO production, eNOS and iNOS protein expression as well as mitochondrial biogenesis were up-regulated in cardiomyocytes exposed to chronic hypoxia. NO may regulate mitochondrial biogenesis through NOS-NO-sGC-cGMP pathway in cardiomyocytes exposed to chronic hypoxia.