Attenuation Of Cardiac Fibrosis Consequent On Inhibition Of Periostin By Using Ramipril Or A Traditional Chinese Medicine In A Heart Failure Rat Model

Post-myocardial infarction left ventricular remodeling should be considered an important therapeutic target to postpone refractory heart failure. Cardiac healing process after acute myocardial infarction (AMI) is a common cause of left ventricular (LV) remodeling and frequently leads to the development of chronic heart failure (CHF), which is characterized by cardiomyocyte hypertrophy, an increase in extracellular matrix (ECM) fibrillar collagen content and the development of abnormal diastolic function. Cardiac healing process after AMI can be divided into two successive phases: the inflammatory phase and the scar formation phase. All the surviving people after myocardial infarction (MI) must suffer the scar phase. It is of great importance to study the mechanism of heart failure and establish a therapy to prevent left ventricular after MI by supporting of healing.Renin - angiotensin system (RAS) plays an important role in the scar stage healing after myocardial infarction. Angiotensin II (Ang II) stimulates progrowth responses, such as TGF-β1, and causes cardiac remodeling which were characters of cardiomyocyte hypertrophy, fibroblast proliferation, and interstitial fibrosis. Periostin, which is a TGF-β1 responding factor, is important to recruit cardiac fibroblasts and create a durable collagen network caused by collagen synthesis and cross-linking. Indeed, collagen increased not only in the scar but also in the viable tissues remote from the infarct after MI. Modulation of collagen deposition may be an effective therapeutic strategy to inhibit the deleterious process of left ventricular fibrosis, which plays a role in the development of congestive heart failure in the post- MI.An enormous amount of research and clinical experience has confirmed the ability of ACE inhibitors to reduce morbidity and mortality in patients with MI and heart failure.Qiliqiangxin is a kind of traditional Chinese medicine.So in our study, we observed the effects of ramipril and qiliqiangxin capsule on attenuation of periostin and regulation of collagen synthesis in the scar and the surviving myocardium after the solid scar developed. Here we report a novel mechanism that can explain the cardiac antifibrotic effect of ACE inhibitors or Qiliqiangxin capsule and subsequence upon cardiac protection of left ventricular remodeling.We randomized 72 rats surviving left anterior descending coronary artery ligation to placebo (MI-control), Ramipril (MI-ramipril) therapy, qiliqiangxin large dosage (MI-large) therapy and qiliqiangxin small dosage (MI-small) therapy with additional eight other rats being sham. At 8th weeks, cardiac hemodynamic parameters and cardiac morphological parameters such as heart weight (HW), left ventricular weight (LVW), and infarct zone weight (IZW) were determined. The myocardial fibrosis was detected by photomicrography.The level of plasma angiotensin II (Ang II) was detected using ELISA. The expressions of TGF-β1, periostin, and collagen types I (collagen I) were measured by reverse transcription-polymerase chain reaction (RT-PCR) in the regional infarct zone (IZ) and non-infarct zone (NIZ). Hydroxyproline assay was selected for total collagen concentration in the IZ and NIZ tissues.Results: Compared with sham rats, body weight-matched MI-control group rats showed significant increase in morphological parameters (P<0.05), while hemodynamic parameters were impaired (P<0.05). This increase was significantly inhibited by ramipril (P<0.01) and qiliqiangxin large dosage (P<0.05), while their hemodynamic parameters were clearly improved (P<0.05). But the morphological parameters and the hemodynamic parameters were not improved in the small dosage qiliqiangxin admission group. Hematoxilin and eosin (HE) staining for fibrosis was increased in MI-control group, while decreased signigicantly in both MI-ramipril and MI-large. It was decreased slightly in MI-small. Plasma Ang II concentration evidently dropped in the MI- ramipril rats(111.3±12.8 pg/ml), MI- large (89.0±13.5 pg/ml)and MI-small rats(112.7±19.6 pg/ml), compared with the control rats(143.1±17.9pg/ml). Compared with MI- ramipril rats, plasma Ang II concentration was significantly attenuated in MI- large rats(P<0.01).There was no evident difference between ramipril rats and MI-small rats. The expressions of TGF-β1, periostin, collagen I and total collagen concentration in IZ were as follow: Compared with sham rats, the expressions of TGF-β1, periostin, collagen I and total collagen concentration of MI-control rats and the other three admission groups were evidently increased. Compared with MI-control rats, the expressions of TGF-β1, periostin, collagen I and total collage concentration were attenuated in MI-ramipril rats and MI-large rats. The treatment with small dosage qiliqiangxin significantly attenuated the expression of these proteins ,but not as effective as the treatment with large dosage qiliqiangxin (P﹤0.05). And the expressions of TGF-β1, periostin, collagen I and total collagen concentration in NIZ were as follow: Compared with sham rats, the expressions of TGF-β1, periostin, collagen I and total collagen concentration of MI-control rats and the other three admission groups were evidently increased(P﹤0.05). Compared with MI-control rats, the proteins were down-regulated in MI-ramipril rats and MI-large rats. There were no significant differences between MI-ramipril rats and MI-large rats(P﹥0.05). And also there were no differences between MI-control rats and MI-small rats(P﹥0.05).Conclusions: Myocardial infarction triggers a local inflammatory response that ultimately results in the replacement of dead cardiomyocytes with fibrous tissue and in the formation of a collagen-based scar. Increased deposition of total collagen proteins exists not only in the scar but also in the left ventricle remote from the infarct regions, resulting in the impairment of heart function due to morphological and functional separation of myocytes with subsequent inhibition of electrical coupling and increase of oxygen diffusion distance which leads to hypoxia. Increased deposition of collagen contributes to increased cardiac muscle stiffness and therefore may affect diastolic function, which is one of the characters of cardiovascular remodeling.Periostin is a novel secreted and soluble extracellular matrix protein of the fasciclin family and is known to be expressed in bone and to a lesser extent in lung, kidney, and heart valves. However, it is not detected in the normal adult myocardium, except in the case of various heart diseases. Periostin, a protein induced by TGF-β1, coclusted with several isoforms of collagen, laminin, and fibronectin within the adult rat heart, is essential for left ventricular remodeling. In this study, we tested the hypothesis that inhibition of periostin contributes to the antifibrotic effect of ACE inhibitors on cardiac remodeling.To confirm the periostin expression in the fibrous area in heart failure rats after AMI, we performed RT-PCR analysis on both the infarct and noninfarct regions. Periostin mRNA was not only highly expressed in the infarct regions bur also expressed in noninfarct regions. To assess the importance of ramipril and qiliqiangxin in the improvement of left ventricular remodeling, we examined the expression of periostin protein in IZ and NIZ myocardium of the left ventricle (LV) with the treatment of ramipril, qiliqiangxin or placebo. The increase in periostin expression was significantly inhibited in IZ and NIZ in the heart failure rats by the treatment of ramipril or qiliqiangxin.We also confirmed the increase in Ang II in plasma after MI and this increment was greatly inhibited by ramipril. Compared with ramipril, large dosage qiliqiangxin treatments exerted more powerful effect on attenuation of plasma Ang II. However, no significant difference was found between the ramipril and small dosage qiliqiangxin treatment. Ang II is a chemoattractant to inflammatory cells and induces leukocyte-endothelial cell interactions via Angiotensin II type 1 receptor (AT1) and Angiotensin II type 2 receptor (AT2). ACE inhibitors reduce the inflammatory infiltrates into the IZ in the early stage and prevent accumulation of type I myocardial collagen in the IZ secondary to activation of TGF-β. Ramipril and qiliqiangxin in our experiment, significantly decreased the concentration of plasma Ang II and subsequently decreased the expression of TGF-β1 not only in the scar but also in the remote surviving myocardium in heart failure rats after MI.TGF-β1 is a locally generated cytokine with an important function for healing and fibrosis by modulating periostin expression. In the early stage (3 days and 5 days), TGF-β1 appear to be protective against ischemic myocardial infarction by improving the mortality and ventricular remodeling. But when a solid scar had already developed, 4 weeks after coronary artery ligation, the benefit on left ventricular remodeling will not exist any longer. In the scar stage, the activation of TGF-β1 is important to induce special myofibroblasts, which expressαsmooth muscle actin (αSMA), to synthesize ECM such as collagen. Recently, Paige et al, using the Snad6, TGF-βor periostin transgenic animals, pointed that periostin is linked to TGF-βsuperfamily signaling. Increased TGF-βactivity alone is not sufficient to promote ventricular fibrosis in adult ventricle. TGF-βactivation directly correlates with periostin upregulation.Consistent with the previous study, our study comfirmed that the expressions of TGF-β1 and periostin were increased in IZ and NIZ myocardium, and attenuated with the treatment of ramipril and large dosage qiliqiangxin capsule. However, small dosage qiliqiangxin treatment exerted less powerful effects on TGF-β1 and periostin mRNA expressions in NIZ myocardium. TGF-β1 appears to cause the expression of periostin and secondary to the collagen deposition. This finding supports the hypothesis that ACEI and qiliqiangxin capsule may mediate the expression of periostin in IZ and NIZ post-MI myocardiumThe fibroblastic cells activated by Ang II–TGF-β1 signals can secrete periostin, which contributed to the amount or cross-linking of newly synthesized collagen. The decease of periostin in IZ and NIZ with the treatment of ramipril and qiliqiangxin prompted us to thoroughly investigate the change of collagen deposition and cross-link in IZ and NIZ myocardium. The expression of collagen I mRNA is significant deceased in IZ and NIZ with the treatment of ramipril and large dosage qiliqiangxin capsule. Our present study confirmed that ramipril and large dosage qiliqiangxin capsule successively attenuated the mRNA expression of TGF-β1, periostin, collagen I in IZ and NIZ after the decrease of plasma Ang II concentration. To investigate the role of ramipril and qiliqiangxin capsule in inhibition of cross-linking collagen, we examined the total collagen in IZ and NIZ. Most total collagen is the mature cross-linking collagen, while ramipril and large dosage qiliqiangxin capsule in fact decreased the total collagen in the IZ and NIZ significantly. There was a direct relationship between insoluble collagen content and myocardial stiffness. With the insoluble collagen content increased, the myocardial diastolic stiffness was also increased. Our finding is that ramipril and large dosage qiliqiangxin capsule inhibited the initial production of collagen mRNA but also the final production of cross-link collagen, while their heart function parameters were clearly improved. But the expressions of TGF-β1, periostin, collagen I and collagen concentration in NIZ were not improved in small dosage qiliqiangxin treatment group, while the heart function parameters were impaired.In summary, Ang II activated the Ang II type 1 receptor and increased the various growth factors, such as TGF-β1. Ang II and TGF-β1 increased periostin expression, and eventually contributed to the amount of cross-linking of newly synthesized collagen, which is essential for the normal mechanical properties of collagen-containing tissues after MI, while ramipril and qiliqiangxin inhibited the expressions of these proteins and mature collagen in the IZ and NIZ myocardium. Our results indicated that periostin protein, as a secreted extracellular matrix protein, involved in healing processes and tissue fibrosis in heart failure rat hearts after MI. Also, we provided evidence that the antifibrotic effects of ramipril and qiliqiangxin, a kind of traditional Chinese medicine,are mediated via the inhibition of periostin, which maybe a novel mechanism to attenuate left ventricular remodeling in heart failure rats after MI. And qiliqiangxin is in a dosage dependent manner.The treatment with Ramipril and qiliqiangxin significantly attenuated the expression of periostin and cardiac collagen deposition and cross linking, accompanied by a significant improvement in cardiac dysfunction.