Changes Of Cardiac Collagen Metablism And Interventional Effects Of Benazepril On Cardiac Interstitial Remodeling In Rats With Type 1 Diabetic Mellitus

Backgroud and objectivesType 1 diabetic mellitus (DM1), that is insulin-dependent diabetic mellitus (IDDM) in children has an increasing morbidity in recent years. Diabetic cardiomyopathy (DC) is one of the independent complications of diabetes mellitus. It is caused by metabolism disturbance of blood glucose, subsequenced by cardiac dysfunction and cellular changes, and then accompanied with small vascular lesions, microcirculation disturbances and autonomic neuropathy, finally leading to cardiac insufficiency. The clinic manifestation of DC mainly includes left ventricular hypertrophy and impairment of diastolic function, and systolic malfunction appears in the late course of DC. Early signs of diastolic malfunction are first observed in children and adolescents with DM1, continued with left ventricular remodeling and finally congestive cardiac failure in the progression of disease. Therefore, early diagnosis and suitable intervention are very important in patients with DC.Diabetic cardiomyopathy is expressed histomorphologically mainly through proliferation and hypertrophy of cardiocytes and interstitial fibrosis. Changes of cardiocyte have been regarded as the focus of ventricular remodeling of DC in the past. However, more and more experts are putting an emphasis on non-cardocytes (mainly fibroblasts) and excellular matrix (mainly collagen) recently. Cardiac interstitial fibrosis might cause ventricular hypertrophy, impairment of cardiac function, and lead to heart failure in the end. So the pathological mechanisms and preventions and treatments of interstitial remodeling in DC become the key target in the fields of diabetic complications.Accumulation of cardiac interstitial collagen is caused by either excess production or weakened degradation of collagens. If collagen synthesis exceeds its degradation, interstitial fibrosis occurs. Collagen proteins are synthesized and secreted mainly in cardiac fibroblasts. Type I procollagen carboxyterminal propeptide (PICP) and type III procollagen aminoterminal propeptide (PIIINP) are released into blood during the synthesis of collagen. Serum PICP and PIIINP have been regarded as indirect indexes of oversynthesis of collagen in vivo. And they have been found to play an important role in cardiovascular diseases such as hypertension, heart failure and acute myocardial infarction. Some studies reported that there were fibroblast proliferation, oversynthesis of collagen and interstitial fibrosis in DC. However, changes of serum PICP and PIIICP and their relationship with cardiac fibrosis in diabetic patients remain uncertain.Collagens are degraded mainly under the control of the family of zincdependent, redox sensitive, endopeptidases: MMPs. Generally metabolism of extracellular matrix and tissue remodeling are maintained under the balance of MMPs and tissue inhibitors of metalloproteinases (TIMPs). There are many members in the MMP family. They are classified into subgroups according to substrate specificity and structure, including the collagenase, the gelatinase, the stromelysin and the membrane-type MMPs. Gelatinases MMP-2 is involved in degrading gelatins (collagen fragments) and cleaving basement membrane matrix proteins. MMPs and TIMPs have been found to take part in the genesis and development of many cardiovascular diseases. However, the molecular changes and the roles of MMPs/TIMPs in diabetic cardiomyopathy remain unclear yet.The present study was designed to investigate the changes of cardiac collagen metabolism by monitoring serum PIIINP levels, cardiac collagen type III (III-C) protein expression and collagen vollume fraction (CVF) , and determine their relationships with MMPs/TIMPs system in a rat model of DM1 induced by streptozocin (STZ), in order to probe what roles MMPs/TIMPs system might play in the pathophysiolgic course of DC.Studies have demonstrated that renin-angiotensin system (RAS) plays an important role in diabetic angiocardiopathy, and angiotensin-converting enzyme inhibitors (ACEIs), which reduce the production of Ang-II by inhibiting ACE, could prevent the progression of cardiovascular complications in DM. However, what role RAS plays in ECM remodeling and how it regulates the expressions of MMPs/TIMPs in DC remains unknown. Benazapril is a potent ACEI with prolonged action and hence used in our study to observe its therapeutic effects in cardiac remodeling in diabetes. The present study was designed to explore the effects of ACEIs on myocardial collagen metabolism and MMPs/TIMPs to gain insight into the potential molecular mechanism of ACEIs therapy in delaying cardiac interstitial fibrosis in streptozotocin-induced typel diabetic rats.Methods1. The rat diabetic model: 46 healthy male Wistar rats (220-250g) were divided randomly into control group (NC, n=16) and diabetes group (n=30). Diabetic models were induced by a single intraperitoneal injection of STZ 60 mg/kg and in the following 72 hours blood glucose level was measured. Rats with blood glucose≥16.7 mmol/L and urine glucose≥3+ were indicated as diabetes. And then diabetic rats were subdivided into 4-week diabetes group (DM4W) , 12-week diabetes group (DM12W) and 12-week diabetes group with benazepril treatment (DB12W) . DB12W rats were treated with benazepril (10mgkg-1·d-1, qd by intragastric administration). The whole study last 12 weeks. Eight NC and DM rats were killed on week 4 and 12 respectively; and DB12W rats on week 12.2. The level of serum PIIINP was measured by radioimmunity methods.3. The levels of serum MMP-2 and TIMP-2 were detected quantitatively by ELISA method.4. Histopathologic evaluation: (l)Myocardial ultrastructure were observed by transmission electron microscopy. (2)Areas integra of collagen fiber were calculated by Van Gieson staining.5. Cardiac MMP-2 activity was determined by zymography.6. The protein abundence for myocardial MMP-2, TIMP-2 and collagen III (III-C) were observed by immunohistochemical stain.7. Determination of the mRNA expression of MMP-2 and TIMP-2 by RT-PCR method.Results1. Blood glucose, heart weight, body weight and the ratio of heart weight/body weight: At 4 and 12 weeks, diabetic rats exhibited decreased heart weight (HW) and body weight (BW) but increased HW/BW ratio compared with those of NC. However, the indexes mentioned above had no statistical difference between DM12W and DB12W groups.2. Myocardial ultrastructure: Obviously focal degeneration and rupture of myofibrils and increased mitochondria appeared in the myocardium at 4 weeks. Degeneration and necrosis of mitochondria, accumulation of cardiac fibers were significant at 12 weeks. These changes were decreased significantly in ACEI-treated group.3. Collagen metabolism:(1)Myocardial collagen contents: Considerable fibrosis was seen mainly between cardiomyocytes and around vessels in diabetic hearts at 4 weeks compared with the values in NC group. Much more fibrosis appeared in diabetic hearts at 12 weeks. These structural changes were reduced significantly in DB12W group.(2)Protein expressions of III-C in left ventricular: In DM group, the cardiac III-C protein expression was higher than that in NC group at 4 weeks, and kept increasing with the longer course. The indicator values in DB groups were markedly lower than those in DM group.(3)Serum PIIINP levels: PIIINP level in plasma was markedly higher from DM rats than in NC group at 4 weeks, ,and kept increasing with the longer course. Treatment with benazepril significantly reduced the levels of PIIINP in diabetic group.4.The level of serum MMP-2 and TIMP-2: compared with that of control group, serum MMP-2 level was slightly elevated but not statistically significantly in diabetic group at 4 weeks, and markedly increased at 12 weeks . Increased concentrations of TIMP-2 in blood were evident in diabetic rats at 4 and 12 weeks.5. MMP-2 activity of left ventricular tissue: MMP-2 activity assessed by gelatin zymography was weaker obviously in the hearts of diabetic rats compared to normal values at 4 and 12 weeks. MMP-2 activity was mildly but not significantly higher in DB group than that in DM group.6. MMP-2 and TIMP-2 mRNA and protein expression of left ventricular tissue: MMP-2 mRNA and protein expression was down-regulated and TIMP-2 mRNA expression were up-regulated in diabetic hearts compared with NC group at 4 and 12 weeks. TIMP-2 protein expression was slightly elevated but not statistically significantly at 4 weeks, but markedly increased at 12 weeks in diabetic group. The above changes were prevented with benazepril treatment in DB rats .7. Correlative relationship analysis: serum MMP-2 were significantly negatively correlated with cardiac activity and gene expression of MMP-2 and positively correlated with CVF, III-C and serum PIIINP. Serum TIMP-2 were significantly positively correlated with CVF, III-C, but not correlated with TIMP-2 mRNA and protein expression and serum PIIINP.8. Correlative relationship analysis: CVF and III-C were significantly negatively correlated with cardiac activity, gene and protein expression of MMP-2 and positively correlated with gene and protein expression of TIMP-2.Conclusions 1. Cardiac hypertrophy and the imbalance of collagen metabolism occurred in the early stages of diabetes at least in a rat animal model, with degeneration and necrosis of cardiocytes and accumulation of interstitial collagens.2. Serum PIIINP, cardiac III-C protein expression and CVF increased gradually with the DM course prolonging, indicating that collagen dysmetabolism may be a possible pathway to accumulate ECM in DC.3. Serum PIIINP increased gradually with the DM course prolonging and correlated positively with cardiac III-C protein expression and CVF respectively. It meaned that serum PIIINP may be a possible index of collagen fibrosis in DC.4. MMP-2 activity, mRNA and protein expression was downregulated and TIMP-2 mRNA and protein expression were upregulated in diabetic hearts. CVF and III-C were significantly negatively correlated with cardiac activity, gene and protein expression of MMP-2 and positively correlated with gene and protein expression of TIMP-2. These indicated that a decrease in MMP-2/TIMP-2 complex might contribute to an impairment in ECM degradation, eventually resulting in cardiac fibrosis in diabetic myocardiopathy.5. Serum MMP-2 and TIMP-2 levels were significantly positively correlated with CVF, III-C, which indicated that both serum MMP-2 and TIMP-2 might be used to predict the extent of collagen fibrosis in DC.6. Benazepril treatment helps to the recovery of cardiac substantial and interstitial remodeling by ameliorating hypertrophy and necrosis of cardiocytes and accumulation and fibrosis of collagen fibers.7. Recovery of diabetic changes of cardiac MMP-2 and TIMP-2 with benazepril treatment indicated that the drug may promote collagen degradation and inhibit its accumulation by improving MMP-2/TIMP-2 imbalance.Innovations and meanings1. This study investigated the relationships between cardiac collagen metabolism and MMP-2/TIMP-2 imbalance in the same animal model of DM1, disclosed the roles of MMP-2/TIMP-2 in interstitial remodeling of DC. Thus potent theoretical evidences were offered for MMPs as a new therapeutic target in DC.2. This study firstly viewed serum MMP-2, TIMP-2 and PIIINP were significantly correlated with cardiac collagen accumulation in the same diabetic models, therefore these indexes could be used to predict the extent of collagen fibrosis and contribute to early discovery and early intervention of DC.3. This study explored the effects of benazepril on in vivo collagen metabolism and MMP-2/TIMP-2 in diabetic animals. ACEIs were confirmed to inhibit diabetic collagen accumulation and cardiac remodeling by regulating the expressions of MMPs and TIMPs system. It offered new theoretic evidence for ACEIs in the treatment of DC in clinic.