Pharmacological Inhibition Of Focal Adhesion Kinase Attenuates Cardiac Fibrosis In Mice Post-Myocardial Infarction Models And Preliminary Research On Transparence Of Cardiac Tissue

Myocardial infarction is a very common and severe disease in clinic, to some extent, post-infarction cardiac fibrosis may affect the prognosis and outcome of the disease. Although there are some theories trying to explain the mechanism, and uncover the signaling pathways which are involved in the cardiac fibrosis, such as the changes in myocardial tension/pressure, activation of the transforming growth factor-beta(TGF-β) signaling pathway, and the improper activation from fibroblasts to myofibroblasts, and so on. However, all of these points could not completely expound the underlying mechanism of cardiac fibrosis, which is still needed to be studied.According to the researches of our team and others both domestic and abroad, focal adhesion kinase (FAK) is thought to play an important role in fibrotic diseases (such as skin fibrosis, pulmonary fibrosis, liver fibrosis and so on). Also, FAK participates in some inflammatory pathways to mediate fibrotic procedures; on the other hand, FAK could directly mediate the differentiation of fibroblasts into myofibroblasts. Therefore, FAK may be used as a promising target for the treatment of fibrotic diseases. However, there were few researches and reports on the function and mechanism of FAK in the hypoxic-ischemic heart diseases. Based on our previous study, which is the role of FAK in atrial fibrillation and myocardial fibrosis, we planned to further inquiry that:firstly, the differentiation of cardiac fibroblasts could be induced under the treatment of hypoxia, with the mediating function of FAK, in addition, when targeted inhibit the activation of FAK, the differentiation of cardiac fibroblasts could be ameliorated; secondly, in the in vivo experiment, a myocardial infarction model of mice induced by the coronary artery ligation would be established, with the pharmacological inhibition of FAK, its effect on cardiac fibrosis post-infarction will be verified.At the same time, based on the newly published "passive clarity technique" (PACT), we made some exploration and improvement of this technique on the thick heart tissue. First of all, the structure of heart tissue was fully preserved, and this transparent technique was optimized, furthermore, the location and expression of FAK and fibrosis-related collagen protein was measured with the method of immunofluorescence staining on a spatial three-dimensional structure. And this optimized technique and way of thinking should be applied to detect tissues or proteins on other pathological conditions.Based on the above description, the function and mechanism of FAK on cardiac fibrosis under the condition of myocardial infarction (or the environment of hypoxia and ischemia) could be more fully explained, which would provide a new idea and method for the treatment of myocardial fibrosis, and provide a promising technical support for a more accurate detection of myocardial fibrosis on a three-dimensional mode.The first part:Focal adhesion kinase (FAK) regulates the hypoxia-induced differentiation of cardiac fibroblasts.Objective:To study the regulation of FAK on the hypoxia -induced differentiation of cardiac fibroblasts.Methods:Primary cardiac fibroblasts (CFs) were isolated and cultured from two-to four-day-old neonatal C57BL/6 mice, the third-generation cells at nearly 70% concentration were used for experiments. The normal CFs were treated under the condition of hypoxia/serum-free with or without PP2 (FAK inhibitor) as the control group, the experimental group and the intervention group, respectively. At the end point of 24 hours, changes in cell morphology were observed, and the inhibition of FAK activity, as well as its downstream AKT signaling activity were detected by western blot, the expression of CFs differentiation-related protein were analyzed by immunofluorescence, Real-time PCR were used to detect the expression of FAK and CFs differentiation gene expression.Results:CFs differentiation was stimulated under the hypoxia/serum-free conditions, with the activation of FAK. However, given the treatment of PP2, the activation of FAK was inhibited, which can be detected by western blot (p<0.05), Real-time PCR revealed the gene expression levels of FAK, α-SMA and type I collagen, were all increased in the experimental group comparing with the control group, but significantly decreased after the PP2 inhibition (p<0.001, p<0.05, and p<0.05, respectively). The expressions of α-SMA and viraentin protein, as markers of CFs differentiation, also showed the tendency of higher in the experimental group and downregulated by PP2 intervention, in which AKT signaling pathway was involved.Conclusions:CFs differentiation could be induced under the conditions of hypoxia/serum-free. Giving PP2 intervention to inhibit the activation of FAK, the level of CFs differentiation can be significantly suppressed via AKT signaling pathway.The second part:Focal adhesion kinase (FAK) regulates the ventricular myocardial fibrosis post-infarction in mice.Objective:To study the regulation of FAK on ventricular fibrosis in mice after myocardial infarction.Methods:Eight- to ten-week-old C57BL/6 wild-type mice (male only) were used, and myocardial infarction was induced by permanent left anterior descending (LAD) coronary artery ligation (CAL).30 mice were randomly divided into sham group, myocardial infarction (MI) and drug intervention (PF) group. At the 7th day post-MI, an employed dosage of PF-562,271 (15 mg/kg body weight in 0.2 mL of 0.5% methylcellulose) was orally given to experimental animals daily over a period of 21 days. The postoperative status was monitored. At the end point of total 4 weeks, the heart function was detected by echocardiography, heart tissue biopsy with Masson trichrome and Sirius red staining were used to discover collagen levels, protein expressions of FAK and type I collagen were detected by immunohistochemistry analysis, FAK protein levels, and the activity and expression levels of FAK signaling pathway proteins such as mTOR, ERK1/2, AKT, P70S6K were analyzed by western blot.Results:MI model were established, only 4 mice in MI group died of cardiac rupture or heart failure. The left ventricular anterior wall, left ventricular internal dimension, and left ventricular volume were significantly changed in MI and PF group, but no obvious difference of the heart function (percent fractional shortening) could be seen between the former two groups. The measurements of heart, lung mass, and heart weight/body weight in MI and PF groups were increased compared with the sham group. Expression of FAK was significantly increased due to the myocardial infarction, but pharmacologic administration could suppress its activation, which was also supported by imraunohistochemistry, and the expression of type I collagen showed the same tendency. Collagen levels were increased at the border zone of infarction in MI group by Masson trichrome and Sirius red staining, and decreased in PF group for pharmacologic inhibition. CAL treatment significantly increased activated FAK, phosphorylated mTOR, AKT (ser473), P70S6K and ERK1/2 expressions assessed by Western Blot (Figure 4). However, FAK (p<0.01), AKT and P70S6K (p<0.01 and p<0.001, respectively), and ERK1/2 (p<0.05) expressions could be suppressed at the 28th day of post-MI pharmacological intervention with PF-562,271.Conclusions:During the 4 weeks’myocardial infarction, an increased activation and expression of FAK could be seen, heart function deteriorated, and the level of myocardial fibrosis was also increased. After the administration of drug intervention, FAK activity was inhibited, ventricular fibrosis was significantly improved. Meanwhile, the FAK downstream related protein expression levels were appropriate changed. All these results support the notion that FAK inhibition may represent a useful therapeutic target in treating fibrotic diseases.The third part:Preliminary research on transparent method of mice heart tissue.Objective:To preliminary explore the heart tissue transparent method and its use of study on myocardial fibrosis.Methods:Eight-to ten-week-old C57BL/6 wild-type mice (male only) were used, PBS flush was perfused to cleaning tissues through the inferior vena cava, heart tissues were harvested and fixed with 4% paraformaldehyde, then cut into 1 mm slices perpendicular to the longitudinal direction of the heart. The slices were immersed in Acrylamide + paraformaldehyde (set to PBS group, A2P0 group, A4P0 group, A4P4 group, AOP4 group, respectively) and 0.25% VA-044 solution to prepare for tissue-hydrogel hybridization. After deoxidization, all slices were gently washed in different concentrations of SDS solutions (PBS group,4%,8%,12%,16%, and 20% SDS groups, respectively). Then the visibility of heart tissue transparency could be gradually recorded. The changes of tissue weight, the total protein loss after rinsing, and photographs of all slices were recorded. After antibody incubation, the expression levels of interested proteins (type I collagen and phosphorylation of FAK) were detected with a laser confocal microscopy in 3D stereoscopic images.Results:The 1 mm thick heart tissue slices in A4P0 group could form favorable tissue-hydrogel hybridization, and after 3 days of washing in 8% or 16% SDS solutions, the heart tissue slices showed visible transparency. The increase of tissue weight could be seen during the rinsing. The 8% SDS group showed a less protein loss than other groups, and the analyzed proteins of phosphorylated FAK and type I collagen were fully demonstrated in 3D stereoscopic images after the antibodies incubation.Conclusions:Heart tissue slices of 1 mm thickness can be transparent by rinsing with A4P0 combined with 8% SDS solutions, and the transparent tissue slices could be used to detect protein expression level in situ space by the immunofluorescence method. This provides a novel method to analyze myocardial fibrosis in a more accurate 3D stereoscopic mode.