The Studies Of PGF2α-FP Receptor On Myocardial Fibrosis Diabetic Cardiomyopathy

BackgroundIn the last30years, diabetic cardiomyopathy (DCM) has attracted people’s attention for the increased morbidity and mortality in diabetes population. Emerging data now reveal that, the initial change of DCM is considered to be myocardial fibrosis. Fibrosis, in general, is a process which is characterized by cardiac fibroblasts accumulation and excess extracellular matrix (ECM) deposition. ECM is composed of a complex fibrillar collagen network, comprising mainly collagen types I and III secreted by cardiac fibroblasts, and acts as a scaffold for the myocytes. However, the mechanism mediating collagen deposition in DCM remains incompletely understood. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM. In an insulin-resistant state, the IRS/PI3K/AKT pathway was drastically blunted, whereas MAPK pathway was fully activated. This situation is known as "pathway-selective insulin resistance". Activated MAPK could induce overproduction of transforming growth factor-β(TGF-β, which has been implicated in the fibrosis. Previous studies have shown that antagonizing TGF-βdoes not prevent fibrosis completely; indicating an additional pathway also has a key role in fibrogenesis. Recently, Oga has confirmed that PGF2α-FP signaling facilitates pulmonary fibrosis independently of TGF-β. However, the role of PGF2α-FP in the development of myocardial fibrosis has not been investigated.PGF2αexerts its biological effect via activation of FP receptor. Apart from leading to a decrease of PI3K activity, activated FP receptor also leads to the activation of the PKC and Rho signaling pathways. Furthermore, inhibition of PI3K increases membrane association of FP receptor by blocking constitutive internaization of FP receptor. In turn, accumulation of the FP receptor on the cell surface membrane binds to PGF2a, thereby establishing a positive feedback loop. In an insulin-resistant state, it has not been explored whether the positive feedback loop still exists and whether PGF2a/FP receptor/PKC/Rho signaling pathway can implicate in the regulating of collagen synthesis in cardiac fibroblasts.In light of the relationship between PGF2a-FP receptor, PKC and Rho, we hypothesized that PGF2a regulates collagen expression through an FP receptor/PKC/Rho signaling pathway in cardiac fibroblasts.Objective1.To determine the effect of PGF2a on FP receptor and collagen expressions in normal and insulin-resistant cellular model2. To determine the effect of PGF2a on signaling pathways in normal and insulin-resistant cellular model3.To determine the effects of FP/PKC/Rho inhibitors on collagen synthesis induced by PGF2αin normal and insulin-resistant cellular model4.To determine the effects of FP receptor inhibitor and TGF-β inhibitor on collagen synthesis induced by PGF2a or TGF-β in normal and insulin-resistant cellular modelMethods1.Cardiac fibroblasts were separated into:normal groups, LG+PGF2a (5.5mM glucose + PGF2a) group, IR+PGF2a (insulin-resistant cellular model + PGF2α group. After treatmenting with PGF2a for1h, 4h and24h, cells were collected, and both the mRNA and protein level of FP receptor, collagen types I and III by real time PCR and enzyme linked immunosorbent assay (ELISA).2.After the cardiac fibroblasts were incubated in normal and insulin-resistant cellular model for Omin, 30min, 60min and120min, the activity of PI3K/Akt and FP receptor/PKC/Rho cascades were measured respectively.3.According to the effect of PGF2α on FP receptor/PKC/Rho cascades, specific inhibitors of FP/PKC/Rho signal pathway were used to detect the collagen types I and ⅢmRNA expression and protein secretion.4.To confirmed the TGF-βindependent, FP receptor-mediated collagen production by PGF2a, specific inhibitors of FP receptor and TGF-βwere used to detect the collagen types I and III mRNA expression.Results1.As shown, after treatment with PGF2α for1h, FP receptor mRNA expression and protein content were significantly elevated in IR+PGF2α compared with normal and LG+PGF2α. The same to stimulation with PGF2αfor4h. In comparison with normal and LG+PGF2α, the stimulation of PGF2αfor4h in IR+PGF2αdisplayed remarkably increases in collagen types I and III mRNA expression. After treatment with PGF2α for24h, collagen types I and III protein secretion were notably up-regulated in IR+PGF2α compared with normal and LG+PGF2α.2.PGF2a induced time-dependent decreases in phosphorylation of PI3K and Akt and time-dependent increases in FP receptor, PKCβ and p-MYPT-1in normal and insulin-resistant cellular model.3.1n normal, pretreatment of cardiac fibroblasts with AL8810, LY-333531, Y27632, respectively, reduced the PGF2α-induced increases in mRNA level of collagen type I by47.59%,49.11%,49.00% and type III by21.50%,27.25%,21.76% at4h; which is also decreased PGF2a-induced effects on protein level of collagen type I by14.83%,19.31%,14.11% and type III by9.5%,13.45%,17.89% at24h. In insulin-resistant cellular model, pretreatment of cardiac fibroblasts with AL8810, LY-333531, Y27632, respectively, remarkably down-regulated the PGF2a-induced effects on mRNA level of collagen type I by80.48%,82.03%,83.50% and type III by26.49%,29.60%,36.08%at4h; which also reversed the PGF2α-induced increases in protein level of collagen type I by41.62%,35.07%,41.71% and type III by22.33%,18.13%,18.20%at24h.4.The effect of PGF2a was inhibited by AL8810but not by SB-50124alone in normal and insulin-resistant cellular model; the effect of TGF-βwas inhibited by SB-50124but not by AL8810alone in normal and insulin-resistant cellular model.Conclusions １.PGF2a induces higher levels of FP receptor, collagen types I and III in insulin-resistant cellular model2.PGF2a not only activates FP receptor/PKC/Rho cascades, but also further blunts PI3K/Akt pathways3.These data revealed that FP receptor/PKC/Rho pathway mediates the role of PGF2a in regulating collagen types I and III mRNA expression and protein secretion in normal and insulin-resistant cellular model4.PGF2a facilities synthesis of collagen types I and III in cardiac fibroblasts via FP receptor signaling independently of TGF-β BackgroundDiabetic cardiomyopathy (DCM) is described as the structural and functional changes in the myocardium that are associated with diabetes, which is one of the leading causes of morbidity and mortality worldwide. Several mechanisms have been implicated in the pathophysiology of diabeticcardiomyopathy. Of these, metabolic disturbances, myocardial fibrosis, small vessel disease, and cardiac autonomic neuropathy are the major players in the pathophysiology of diabeticcardiomyopathy.One major pathophysiology player of DCM is myocardial fibrosis. In this regard, myocardial fibrosis is multifactorial and can be caused by several processes including insulin resistance, inflammation, and various hormones. Many studies revealed the relationship of the above processes with myocardial fibrosis respectively; however, the crosstalk between insulin resistance and hormones is unclear. Hence, there is a need to understand the detailed mechanisms and factors associated with myocardial fibrosis in DCM.PGF2a, a metabolism of arachidonic acid, attracts people’s attention as a new fibrosis hormone. Ogo have found that PGF2α/FP receptor signaling facilitates pulmonary fibrosis. What’s more, PGF2α-FP receptor could mediate protein kinase C (PKC) or Rho kinase signaling molecules resulting in a series of biological activity. Furthermore, PKC and Rho kinase molecules can participate in the progression of fibrosis respectively. However, whether PGF2a-FP receptor/PKC/Rho kinase pathway could play an important role in the development of fibrosis has been little known. In our previous study, we have demonstrated that PGF2α regulated collagen I and III expression by an FP receptor/PKC/Rho kinase pathway in insulin-resistant cardiac fibroblasts, but the extrapolation of these finding to myocardial fibrosis in the diabetic animal model warrants further investigation.Objective1.To established the type2diabetic’s model.2.To explore the mechanisms of PGF2a-FP receptor involved in DCM by FP-receptor gene silencing in vivo.MethodsThis study enrolled forty male Sprague-Dawley (SD) rats at4weeks. All the rats were randomly assigned to control group (n =8) and diabetic group (n =32). The control group received normal chow; the diabetic groups were fed an HF diet. After four weeks, IPGTT and IPITT were performed again. The control group received citrate buffer (intraperitoneally) alone. The rats with insulin resistance in diabetic group were rendered with an intraperitoneal injection of a single dose of STZ (Sigma, St. Louis, MO;27.5mg/kg i.p. in0.1mol/L citrate buffer, pH4.5). Tail vein blood glucose was measured every3days in the first week and those with plasma glucose levels≥11.1mmol/L were considered to be diabetic. The diabetic group (n =32) was redivided into three groups: diabetes + vehicle group (n =8), diabetes + FP receptor-shRNA group (n =8) and diabetes + FP receptor-shRNA+PGF2αgroup (n8). After14weeks of diabetes, the last two groups were injected with2.5 *1010plaque-forming units of an adenovirus harboring FP receptor gene (FP receptor-shRNA) and the vehicle group was injected with a control empty virus (vehicle) by the jugular vein. Adenoviru transfer was repeated in2weeks. The FP receptor-shRNA + PGF2α group not only repeated adenovirus transfer but also injected with PGF2a (0.1mg/kg) for two weeks. Four weeks after the first adenovirus injection, rats were sacrificed. The heart was excised and weighed. During the experiments, we carried out:①Measurement of body weight and biochemical indexes.②fter rats deprived of food overnight, we collected blood sample through the jugular vein. Total cholesterol, triglyceride levels, FBG, PGF2α and fasting insulin levels were measured.③IPGTT was performed to access glucose tolerance; IPITT was carried out to evaluate insulin sensitivity.④Changes in the left ventricular end diastolic diameter pressure (LVEDP) were measured.⑤The tissue sections underwent HE staining, Masson’s trichrome staining and Picrosirius red staining. The myocyte cross-sectional area, the collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were analyzed.⑥The mRNA expression of FP receptor, the protein expression of FP receptor, PKC and Rho were measured.⑦Tissue sections (4μm) were subjected to immunohistochemistry.Results1.Animal characteristicsThe heart weight, water intake, food intake and urine volume were significantly higher in diabetic group than the control group (P<0.05). In FP receptor-shRNA group and FP receptor-shRNA + PGF2α group, the water intake and urine volume were significantly reduced (P<0.05) compared to vehicle group, thus the heart weight and food intake were slightly reduced with no significantly differences.2.Total cholesterol, triglyceride, FBG and PGF2α concentrationsAfter4weeks of a HF diet, serum cholesterol, PGF2α and insulin began to slightly increase in the diabetic group (P>0.05). The level of total triglyceride and FBG were significantly elevated in the diabetic group; the ISI was also decreased in the diabetic group (P<.05). One week after STZ injection, the levels of serum total cholesterol, triglyceride, FBG, PGF2α and insulin were remarkably elevated in the diabetic group and remained elevated until the end of the experiment. With a4-week transfection of FP receptor shRNA, the elevated serum levels of total cholesterol, triglyceride, FBG, and insulin were greatly reduced (P<0.05).3. Glucose and insulin toleranceIPGTT revealed that, the levels of blood glucose were significantly higher at week4in diabetic group at0,30min compared to control group. The AUC across the time for glucose level was higher at week4than at baseline. Similarly, IPITT showed impaired insulin sensitivity.At the end of the experiment, the diabetic group showed elevated levels of blood glucose and higher AUC on both IPGTT and IPITT compared with the control (P<0.05). In FP receptor shRNA group and FP receptor shRNA + PGF2a group, the IPGTT and IPITT showed the levels of blood glucose were significantly lower to vehicle group; the AUC for glucose and insulin were also lower to vehicle group.4. LV function assessed by catheterizationWe found that left ventricular end diastolic diameter pressure (LVEDP) was higher in the diabetic group than control group. With FP receptor gene silencing, LVEDP was lower in FP receptor shRNA group and FP receptor shRNA + PGF2a group than the vehicle group.5. Pathology characteristicsThe diabetic group showed cardiac fibrosis, with a scattered, small, inordinate pattern, as well as damaged and irregular collagen network structure in the interstitial and perivascular area. CVF and PVCA/LA were higher in the diabetic group than control group. With FP receptor shRNA treatment, CVF and PVCA/LA were decreased in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared to vehicle group.6. FP receptor/PKC/Rho kinase and Akt pathwaysThe relative mRNA and protein expression of FP receptor was significantly elevated in diabetic group. Accompanied by FP receptor overexpression, the phosphorylation of PKCβ2and MYPT-1were markedly increased. With FP receptor shRNA treatment, the relative mRNA and protein expression of FP receptor was significantly downregulated in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared with vehicle treatment, as well as the phosphorylation of PKCβ2and MYPT-1.7. The distribution of FP receptorImmunohistochemistry revealed the protein distribution of the FP receptor was upregulated in the diabetic group. With FP receptor shRNA treatment, the FP receptor was downregulated in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared with vehicle group.8. Collagen Ⅰ and ⅢImmunohistochemistry revealed the protein expression of collagen Ⅰ and Ⅲ content was upregulated in the diabetic group. With FP receptor shRNA treatment, collagen Ⅰ and Ⅲ was downregulated in FP receptor shRNA group and FP receptor shRNA+PGF2a group compared with vehicle group.Conclusion1.Metabolite disturbance appeared at week4in rats fed a HF diet. With a4-week transfection of FP receptor shRNA, the metabolite-disturbant states were ameliorated.2.The diabetic group showed impaired glucose tolerance and insulin sensitivity; with FP receptor shRNA treatment, the impaired glucose tolerance and insulin sensitivity were ameliorated.3.Diastolic dysfunction was developed and progressed during DCM. With FP receptor gene silencing, the diastolic dysfunction was improved, which also confirmed the beneficial effect of FP receptor shRNA on improving LV diastolic dysfunction.4.Our results revealed that severe cardiac fibrosis exists in type2diabetic rats; cardiac fibrosis may ameliorate with FP receptor gene silencing.5.FP receptor/PKC/Rho kinase signaling pathway involves in the pathogenesis of DCM.6.In diabetic states, the deposition of collagen Ⅰ and Ⅲ was accelerated. With FP receptor gene silencing, the excessive deposition of collagen Ⅰ and Ⅲ was ameliorated, which reconfirmed the beneficial effect of FP receptor shRNA in DCM.