Prohibitin Overexpression Improves Myocardia Function In Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) refers to the disease that happens in diabetic patients, which can’t explain with high blood pressure, coronary atherosclerosis, heart disease and other heart disease. Precious studies have found that the incidence of cardiovascular disease and mortality in patients with diabetes are both the highest in the world. Even if there is no known risk factors such as coronary artery disease and high blood pressure, diabetes is associated with increased risk of suffering from the development of cardiomyopathy. Non-invasive study showed that left ventricular systolic, diastolic dysfunction and left ventricular hypertrophy can be observed in diabetic cardiomyopathy, especially in the patients suffer from high blood pressure and coronary microcirculation disorder. Left ventricular diastolic dysfunction of patients with diabetes could be secondary to the change of collagen structure, especially the collagen cross-linking and the increase of advanced glycation end products.Myocardial cell apoptosis is another important symbol which can lead to myocardial hypertrophy and fibrosis. Inhibiting the apoptosis of myocardial cells can effectively restrain the progress of diabetic cardiomyopathy. Oxidative stress is considered to be the main factor that lead to the development of diabetes complications and is closely related to insulin resistance as well as impaired insulin secretion. Under the environment of high blood glucose, myocardial cells could release a series of cytokines and reactive oxygen species, which triggers the myocardial cell death signaling pathways.Prohibitin (PHB) is a kind of highly conservative pleiotropic protein, it is widely expressed in eukaryotic cells, such as mitochondria, nucleus and the cell membrane. It involves of a variety of cellular functions, including proliferation, apoptosis, tumor suppressor, transcription, and mitochondrial protein folding, it’s most distinctive feature is its function as mitochondrial chaperone. Previous studies have shown that PHB deficiency can lead to mitochondrial membrane depolarization and promote the generation of reactive oxygen species, which in turn induce apoptosis whereas PHB expression can alleviate the damage caused by oxidative stress and reduce the myocardial cell mitochondrial apoptosis. In addition, Supale etl have reported that PHB overexpression is negatively related to the level of the fibrosis of liver and kidney and improves the survival rate of newborn rat myocardial primary cell underthe stimulus of H2O2.The occurrence of diabetic cardiomyopathy involves a variety of molecular mechanisms, including the classic mitogen-activated protein kinase(MAPK) and Akt signaling pathway, They are closely related to the PHB, and can be activated by high glucose, what’s more, they participate in myocardial hypertrophy, cells apoptosis and cytokines mediated inflammation.The above analysis suggests that PHB is likely to play an important role in the process of diabetic cardiomyopathy. Therefore, this research will study the function of PHB gene in diabetic cardiomyopathy and its potential mechanism, which is on the basis of the rat model of type 2 diabetes.Objective1. Study the role of PHB in diabetic cardiomyopathy of rats and the underlying mechanisms.2. Study the role of PHB in abnormal cardiofibroblast proliferation and collagen secretion induced by high glucose.3. Study the role of PHB in H9C2 cardiomyoblasts apoptosis induced by high glucose. MethodsPartⅠ1. Lentivirus designBuild Lentivirus that earring rats PHB gene on the virus vector (Lv-PHB+), design green fluorescent protein on the Lentivirus (Lv-vehicle) as a negative control at the same time.2. Animal model60 healthy male Sprague-Dawley (SD) rats (120-140 g) were purchased from Beijing Weitong Lihua experimental animal technical limited company, feed the rats adaptability for a week, then the glucose tolerance test (IPGTT) were carried out for the four experimental groups which were randomly assigned into:(1) The normal control group (Con group);(2) Diabetes group (DM);(3) Diabetes+negative control group (DM+Lv-vehicle group);(4) Diabetes+PHB overexpression group (DM+Lv-phb+).Normal control group rats were fed with a regular basic feed, its composition mainly is:crude protein 20%, crude fat 3%, crude fiber 3%, other 74%. The rest of the three groups of diabetic rats was fed with high fat feed, its composition is mainly as follows:34.5% fat,17.5% protein,48% carbohydrates. Four weeks later, the glucose tolerance test (IPGTT) and insulin tolerance test (IPITT) were performed for DM group. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; Sigma, St. Louis, MO; 27.5 mg/kg in 0.1 moL/L citrate buffer, pH 4.5) in rats with insulin resistance, the same dose of sodium citrate buffer was intraperitoneal injected for control group rats. A week later, detect the fasting glucose of DM group rats, Fasting blood glucose(FBG) Levels> 11.1 mmoL/L for two consecutive analysis were considered DM rats.12 weeks after diabetes induction, two groups of diabetic rats respectively injected 5 X 107 UT/50 uL lentiviral vector carrying PHB cDNA-GFP (GenePharma, Shanghai, China) or the same volume of vehicle lentiviral vector (GenePharma, Shanghai, China) via the jugular vein.16 weeks after diabetes induction, IPGTT and IPITT were performed again, and the virus transfection efficiency in myocardial tissue was confirmed with GFP positive analysis under microscope. Rats were sacrificed at 16 weeks after diabetes induction, the triglyceride levels, serum cholesterol and FBG were analyzed by the Bayer 1650 blood chemistry analyzer (Bayer, Tarrytown, NY).3. The determination of biochemical indicatorsEvery 4 weeks, after fasting for 12 hours, angular vein blood was collected and centrifuged for supernatant. Serum triglycerides (TG), Total cholesterol (TC) and blood glucose levels was detected.4. Cardiac function measurement16 weeks after diabetes induction, the BP-98 smart rat tail noninvasive blood pressure testing instrument was used for the blood pressure levels for each group of rats; the small animals two-dimensional ultrasound detector (Vevo770) imaging system was used to measure M-mode echocardiography, pulse doppler and tissue doppler to evaluate the left ventricular systolic and diastolic function in rats.5. Histological stainingThe rat heart tissue was fixed in 4% paraformaldehyde, paraffin section was used for HE, Mason and Sirius staining to observe the heart shape as well as collagen deposition. The apoptosis of myocardial cell were determined by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining. Immunohistochemical staining was used to observe collagen I (collagen I) and collagen III (collagen III) content in the myocardial tissue.6. Western blottingExtract the protein of rat myocardial tissue to detect COL I, COL III, transforming growth factor & I (TGF-βI), PI3K, Akt/p-Akt, ERK/p-ERK, JNK/p-JNK and p38/p-P38 protein content of myocardial tissue. Using caspase 3 activity detection kits to detect the caspase 3 activity in myocardial tissue.Part II1. Cell culturePrimary rat heart tissue fibroblasts (cardiac fibroblasts; CFs) was extracted from newborn SD rats within three days. The CFs were divided into the following four groups:normal control group (Con group; 5.5 mM); high glucose group (30 mm HG group); high glucose+Lv-PHB+group (HG+Lv-PHB+group); high glucose+Lv-vehicle group (HG+Lv-vehicle group).2. ImmunofluorescenceThe laser confocal microscope was used to detect the distribution of PHB proteins in the CFs under normal conditions or high glucose stimulates.3. Cell proliferation testCFs proliferation was detected with EDU and CCK8 kits.4. Gelatin enzyme spectrumGelatin enzyme spectrum was used to detect the activity of MMP-2 and MMP-9 in CFs and culture supernatant.5. Western blottingExtracted the CFs protein, using western blot detection to analysis the content of of myocardial tissue COL I, COL Ⅲ, as well as the TGF-βⅠPart III1. Cell cultureSame as the mentioned above, H9C2 cardiomyoblasts can be divided into Con group, HG group, HG+Lv PHB+group and HG+Lv-vehicle group.2. The oxidative stress level detection in the H9c2 cardiomyoblastsUsing fluorescent probe DHE kits to detect the oxidative stress levels in the H9C2 cardiomyoblasts,3. Myocardial apoptosis detection7-AAD/Annexin V-PE kits was used to detect the H9C2 cardiomyocytes apoptosis levels.4. Western blottingH9C2 cardiomyoblasts and mitochondrial proteins were used to detect the protein expression level of bax/Bel-2 and cytochrome c. Using caspase 3 activity detection kits to analysis the caspase 3 activity of H9C2 cardiomyoblasts.Statistical analysisSPSS vl6.0 (SPSS Inc., Chicago, IL) was used for analysis. Results were compared by oneway ANOVA followed by the Tukey’s t test (2-tailed) for multiple groups and 2-tailed Student’s t test for 2 groups. Data are reported as the mean ± SEM. Differences were considered statistically significant at P< 0.05.ResultsPartⅠ1. The general properties of ratsAfter 4 weeks of high-fat diet, diabetic rats was performed with intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test. The area under curve (AUC) in the diabetic rats were higher than in normal diet group. After 20 weeks, the FBG, TC and TG level of diabetic rats were significantly higher than that of normal control group rats. But PHB expression does not decrease TC, TG and fasting blood glucose levels. However, after 20 weeks, the area under the curve for insulin tolerance test for PHB overexpression group rats is lower than the control group rats, suggesting the abnormal insulin resistance levels in diabetic rats.2. PHB overexpression improve the heart shape and cardiac functionIn the diabetic group, PHB protein expression level in myocardial tissue was significantly lower than the control group, and PHB expression was obviously increased after overexpression of PHB in myocardial tissue. After 20 weeks, the phenotype of eccentric ventricular hypertrophy was shown in the DM group showed.At the end of the experiment, left ventricular ejection fraction (LVEF), fractional shortening(FS), early to late mitral inflow velocity (E/A) and E’/A’ were all lower than the normal rats. In contrast, the left ventricular end-diastolic diameter (LVEDd) is higher than the normal diet group rats. Compared with the empty vector control, PHB over expression can significantly improve the LVEF, FS, E/A, E’/A’ and lower LVEDd. Diabetic rats blood pressure is slightly increased, but no statistical significance was found.3. PHB overexpression could improve myocardial fibrosis and apoptosis in DCMAccording to Masson and Sirius staining, diabetic rats myocardial interstitial collagen accumulation is significantly higher than normal rats. Compared with the negative control, PHB overexpression reduced the interstitial and perivascular collagen deposition. Similarly, TUNEL staining showed that PHB can alleviate diabetic rats myocardial apoptosis level of the myocardial tissue.4. Molecular biology level detectionResults show that diabetes increases the expression of fibrosis markers COL I, COL III, as well as the expression of TGF-βI, which is consistent with immunohistochemical results, PHB significantly reduced the expression level of these markers. In addition, the caspase-3 activity of diabetic myocardial was increased significantly and the ratio of Bax/Bcl-2 was higher than control, consistent with this, TUNEL staining showed that PHB can reduce the expression of apoptosis related indicators.We further found that compared with normal rats myocardial tissue, diabetes can reduce the protein level of PI3K, p-Akt and p-P38 in myocardial tissue, while the PHB overexpression can improve the levels of these proteins whereas reduce p-ERK protein level in the myocardial tissue of rats caused by hyperglycemia.Part Ⅱ1. The expression and localization of PHB in CFsAccording to the results of the western blot, under the stimulation of high glucose, PHB protein level was increased at the first 12h then decreased after then. Compared with the normal environment, the PHB expression in the nucleus was higher than control under the environment of high glucose.2. PHB overexpression alleviates CFs proliferationEDU and CCK8 kit test showed that high glucose could promote the proliferation of CFs, and PHB expression can reduce the proliferation of CFs.3. Molecular biological detectionSimilar to the results of the experiment in vivo, western blot showed that in CFs under high glucose stimulates, collagen Ⅰ, collagen Ⅲ, as well as the expression of TGF-βI were significantly increased. According to the results of Gelatin zymography, high glucose stimulation increased the content of MMP-2 and MMP-9 and activity at the same time. PHB overexpression in vitro could reduced the expression of fibrosis markers, as well as the expression and activity of MMP-2.Part Ⅲ1. PHB expression in H9C2 cardiomyocytesSimilar to fibroblasts, the expression of PHB in H9C2 cardiomyocytes also showed the trend of first increase then reduce, but the peak point was different.2. Oxidative stress level detection in H9C2 cardiomyocytesDHE staining showed that compared with normal control group, high glucose stimulation could increase the generation of reactive oxygen species in H9C2 cardiomyocytes, and PHB overexpression effectively reduced the generation of reactive oxygen species in the cells.3. Apoptosis detection in H9C2 cardiomyocytesFlow cytometry results indicated that, PHB overexpression could reduce the 7-AAD staining positive rate in H9C2 cardiomyocytes induced by high glucose, namely early cell apoptosis rate. At the molecular level, PHB overexpression can prevent the release of cytochrome c from mitochondria into the cytoplasm of cells and the ratio of Bax/Bcl-2 as well as the activity of caspase 3 in H9C2 cardiomyocytes stimulated by high glucose.Conclusion1. PHB overexpression effectively improve diabetic rats cardiac function and fibrosis in vivo.2. PHB overexpression alleviates abnormal fibroblasts proliferation, collagen secretion in vitro.3. PHB overexpression suppresses the H9C2 cardiomyocytes apoptosis induced by high glucose, improves the oxidative stress state in H9C2 cardiomyocytes.4. The role of PHB in diabetic cardiomyopathy is associated with PI3K/Akt and MAPK signal pathway.