| Background:Ischemic heart disease (IHD) is a serious threat to global public health. Establishment of coronary collateral circulation and improvement of myocardial microcirculation is the key link in the process of reducing myocardial ischemia; however, there is no effective therapy in clinic now. The previous results of our study have indicated that low-energy pulse waves produced by Cardiac Shock Wave Therapy could induce cavitation effect, which could exert a mechanical shear stress on the myocardial tissue and cells. The CSWT was able to promote angiogenesis in ischemic myocardium by up regulating many growth factors and improve myocardial perfusion and metabolism in patients with IHD. Shear stress of CSWT also could promote the establishment of the coronary collateral circulation and improve coronary microcirculation in animal models. But angiogenesis has palyed only a limited role in restoring effective blood perfusion of ischemic tissues. The transformation of microvascular into markedly larger vessels during arteriogenesis, forming a functional collateral circulation, can effectively improve the ischemic myocardial tissue perfusion. However, whether CSWT could promote arteriogenesis of coronary microvascular and improve collateral circulation and the molecular mechanism underlying this biological remain poorly understood.Objectives:The purpose of this study were to establish SD rats acute myocardial infarction models and give CSWT treatment; to investigate whether CSWT shear force could promotes arteriogenesis of coronary microvascular in the infarction border zone and its possible molecular mechanisms. The study will explore further mechanism of CSWT, and provide a scientific basis for new treatment strategies and methods of coronary heart disease.Methods:① 55 adult male SD rats were selected randomly. The SD rats’acute myocardial infarction models were built by ligation of the left anterior descending artery (LAD) in thoracotomy surgery. High-frequency echocardiography was used to evaluate the rat cardiac function after surgery. In order to indentify the size of myocardial infarction area, the myocardium of rats after surgery was stained with TTC. The success rate and stability of rat AMI model were also evaluated.② Another 70 adult male SD rats were randomly divided into sham group (n=10), AMI group (n=30) and CSWT group (n=30). Both AMI group and CSWT group had been built into acute myocardial infarction models. The sham group received the same procedure as the other groups without surgery. Then, the rats in CSWT group were subjected to CSWT therapy. A total of 200 shots (60 shots per minute.) were delivered at a total energy flux density of 0.24mJ/mm2 in every treatment session during 4 weeks, with three sessions in 1 week.③ After 4 weeks, the heart function of all rats were identified with high-frequency echocardiography again. Left ventricular muscle was sliced for pathological observation. Immunofluorescence confocal microscopy was used to observe the arteriogenesis situation of coronary microvascular. The apoptosis and apoptosis-related proteins were evaluated by TUNEL and western blot. The myocardium fibrosis in the infarction border zone in the three experimental groups was showed by Masson’s trichrome staining.④ Using label-free quantitative proteomic technology, we compared the protein profiles in AMI group and CSWT group at weeks 1,2,4 after operation. Bioinformatics analysis method was applied for GO functional annotations, differentially expressed proteins in KEGG pathways annotation and analysis of the enrichment. To identify differentially expressed proteins significantly enriched GO and KEGG entries. In the biomechanics process of coronary microvascular arteriogenesis induced by CSWT, the nuclear regulatory pathway and related interest proteins had been screened and analyzed. Using Western blot analysis, we detected the expression of related target protein in myocardial tissue for validation.Results:①We completed 55 SD rats with LAD ligation surgery. The surgical success rate was 100%.1 week postoperative survival rate was 90.9%. After surgery, high-frequency echocardiography parameters including the left ventricular end-systolic diameter (LVESD), the left ventricular end-diastolic diameter (LVEDD), fractional shortening (FS) were detected. The results showed that ventricular septum motion significantly diminished or without movement and right ventricular apical formed a balloon-like sac. The LVESD (3.09±0.40mm versus 5.77±0.48mm, P< 0.01) and LVEDD(4.92±0.37mm versus 8.06±0.47mm, P<0.01)were significantly enlarged. And FS (37.07±8.01% versus 28.37±6.79%, P<0.01) was significantly decreased. The left ventricular wall got thinner, collapsed and gray which could be seen by the naked eyes. TTC staining after measuring the infarcted myocardium area percentage measured after TTC staining was 35.66±3.77%.② 4 weeks after CSWT therapy, high-frequency echocardiography parameters were detected. LVESD and LVEDD in CSWT group were improved significantly than the AMI group (4.32±0.20mm versus 5.71 ±0.33mm AND 6.43±0.32mm versus 7.73±0.55mm, P<0.01). In addition to this, FS in CSWT group were improved greatly than the AMI group (32.54±5.43% versus 25.80±7.34%, P<0.05).③ We performed collateral morphometry by immunofluorescence confocal microscopy to define whether CSWT contributed to arteriogenesis of coronary microvascular in AMI rats. In the infarction border zone of the CSWT group, both the collateral diameter of coronary microvascular and smooth muscle layer thickness were increased more obviously than the AMI group. As expected, the dedifferentiated state of vascular smooth muscle cells (VSMCs) is characterized by an increase in G-actin.④The myocardium Bcl-2 levels were elevated in CSWT group and the Sham group than in the AMI group (1.08±0.03 and 1.02±0.02 versus 0.10±0.02, P<0.01). On the contrary, The myocardium Bax and Caspase-3 levels were declined in CSWT group than in the AMI group (0.65±0.05 and 0.30±0.02 versus 0.10±0.10 and 1.31±0.12, P<0.01). The apoptotic index of CSWT group was significantly lower than AMI group (29.91±2.96 versus 15.42±1.33, P<0.05).⑤Masson’s trichrome staining showed markedly increased fibrotic area in AMI group compared with that in CSWT group and Sham group. In AMI group, myocardial fibers were arranged irregularly and collagen was deposited obviously. Myocardium fibrosis CVF in CSWT group (18.70±13.06%) was significantly higher than in Sham group (0.31±0.17%) but lower than in AMI group (70.70±12.34%), P<0.05.⑥We applied label-free quantitative proteomic approach to compare the protein of myocardium tissue in CSWT group and in AMI group. A total of 1320 protein groups were quantified under highly stringent criteria with a false discovery rate of<1% for peptide and protein groups. Among these proteins, differentially expressed proteins between the two experiment groups were identified with statistically significant differences. Compared to the AMI group, there were 50 proteins up-regulated and 29 proteins down-regulated in the CSWT group at 1 week after CSWT therapy. At 2 weeks, there were 60 proteins up-regulated and 22 proteins down-regulated. And there were 65 proteins up-regulated and 24 proteins down-regulated at 4 weeks after CSWT therapy. Bioinformatics analysis revealed that these differentially expressed proteins were largely enriched in focal adhesion signaling pathway.9 proteins changed dynamically during the CSWT therapy process. The Integrin-linked protein kinase (ILK), Collagen α-3(Ⅵ) chain (Co16a3), laminin α5 (Lama5) and Calpain-2 catalytic subunit (Capn2) levels showed a trend of increase gradually, while the Ras-related protein Rap-1b (Rap1b), Vitronectin (Vtn), Integrin (33 (Itgb3), a-actinin-1 (Actnl) and Myosin light chain 7 (My17) levels showed a trend of decrease gradually. The dynamic changes in the expression of ILK, Co16a3, Capn2, Vtn, Itgb3 and Actnl were tested by the Western blot assay, and the results were consistent with the label-free quantitative proteomic results.⑦ Direct protein-protein interaction network analyses of these significantly changed proteins showed that the up-regulation of intergrin linked kinase (ILK) during CSWT therapy may be closely related to the arteriogenesis of coronary microvascular in AMI rats.Conclusions:①The rat model of AMI established by the modified surgical LAD ligation has higher successful rate, and the model stability and reproducibility are good. It could be an ideal animal model for proteomics and genomics research in cardiovascular academic field.② The CSWT with shear force could promote coronary microvascular arteriogenesis in the infarction border zone of AMI rats. The VSMCs may dedifferentiate and proliferate that could increase the collateral diameter of coronary microvascular and smooth muscle layer thickness, thereby improving coronary collateral circulation, inhibiting myocardial apoptosis and fibrosis, alleviating left ventricular remodeling and improving cardiac function in AMI rats.③During the process of the CSWT mechanical signal transforming into biological effect in heart of AMI rats, focal adhesion signaling pathway may have a central role in the related signal network.④ ILK and other sequential differentially expressed proteins were closely related to the phenotype of coronary microvascular arteriogenesis with CSWT therapy. These proteins would be the key factors that account for series of biomechanical effects and expected to become the new breakthrough point for biomechanical mechanism research of CSWT. This innovative strategy allowed novel insights for detecting the molecular mechanism of coronary microvascular arteriogenesis promoted by CSWT. |
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