BMP7 ameliorated viral myocardial fibrosis by inhibiting endothelial-to-mesenchymal transition

: 1 Background: Endothelial-to-mesenchymal transition (Endo-MT) is associated with myocardial 2 fibrosis in dilated cardiomyopathy (DCM). Endothelial-to-mesenchymal transition (Endo-MT) is 3 induced by coxsackievirus B3 (CVB3) in cardiac microvascular endothelial cells (CMVECs). Bone 4 morphogenetic protein 7 (BMP7) significantly inhibits Endo-MT and the progression of cardiac 5 fibrosis. The study was aimed to investigate the effect and the underlying mechanism of BMP7 on 6 Endo-MT in myocardial fibrosis induce by CVB3 infection in vivo. 7 Methods: BALB/c mice were intraperitoneally injected by CVB3 to induce viral myocarditis (VMC). 8 Mice were treated with BMP7 after CVB3 infection. Subsequently, all groups of mice were 9 determined by echocardiography, histopathologic and molecular detection. 10 Results: We found that the ratio of BMP7/TGF-β1 in mRNA levels was decreased obviously at 11 different time points after CVB3 injection. Double immunofluorescence staining indicated that Endo-MT was implicated in was BMP7. The protein levels of pSmad3 and Smad4 were significantly upregulated in VMC group, as well as Wnt/β-catenin and the transcription factor snail. BMP7 treatment reversed the changes of these protein levels. Moreover, CO-IP demonstrated the crosstalk between β-catenin and Smad3 in VMC mice, which was downregulated by BMP7 treatment. Conclusions: These results indicated that BMP7 obviously ameliorated myocardial fibrosis in CVB3-infected mice via Endo-MT, which was involved in the TGF-β/Smad and Wnt/β-catenin pathway. β-Catenin/Smad3 interaction may be associated with Endo-MT in the development of viral 22 myocardial fibrosis.


Background 27
Myocarditis is an inflammatory disease associated with a large variety of triggers, including 28 infectious agents, autoimmune diseases and drugs. Coxsackievirus B3 (CVB3) is still considered to 29 represent the dominant etiological agent in viral myocarditis (VMC). One-third of patients with 1 VMC evolve into a chronic stage of disease and progress to dilated cardiomyopathy (DCM) and 31 heart failure, a frequent reason for heart transplantation [1]. Accumulating studies have demonstrated 32 that myocardial fibrosis orchestrates predominantly the development from VMC to DCM [2][3][4]. The 33 emergence of cardiac fibroblasts with an endothelial cell origin, referred to as endothelial-to- (CMVECs) in vitro [11]. Therefore, we assume that Endo-MT may contribute to the myocardial 42 fibrosis in CVB3-infected mice. 43 Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β (TGF-44 β) superfamily，has opposing effects on many pathophysiological processes [12]. BMP7 counteracts 45 TGF-β-induced accumulation of myofibroblasts and extracellular matrix (ECM), referred to as an 46 antifibrotic factor [13,14]. Of note, BMP7 alleviates myocardial fibrosis via inhibition of Endo-MT 47 3 assessed semi-quantitatively using image-Pro Plus 6.0 analyzing software. 91

Double immunofluorescence staining 92
Confocal fluorescence microscopy was used to explicit the co-location of endothelial markers 93 and mesenchymal markers. 5μm-thick frozen heart sections were treated with two mixed primary 94 antibodies at 4°C overnight. The primary antibodies groups were respectively αSMA (ab5694, rabbit 95 polyclonal, 1:100) and CD31 antibody (ab24950, mouse polyclonal, 1:100), as well as FSP-1 96 (ab27957, rabbit polyclonal, 1:50) and VE-cadherin antibody (ab7047, mouse polyclonal; 1:50). 97 After washing for at least 3 times, the sections were continually incubated with a compound of two 98 secondary antibodies from different species with conjugation to two distinct fluochromes 99 (respectively goat-anti-rabbit conjugation with rhod-red and goat-anti-mouse conjugation with 100 FITC, Jackson). After slides were stained with DAPI, confocal images were taken (Leica confocal 101 microscope) and the expression of double-labelled picture was analyzed (LAS software). 102

Western blot analysis 103
Western Blot Analysis was performed according to standard procedures. In brief, heart tissues 104 were extracted with RIPA buffer. Protein lysate was separated and transferred to polyvinylidene 105 fluoride membrane, which were incubated with primary antibodies as follows: p-Smad3 (SC11769,

Co-immunoprecipitation (Co-IP) assay 113
Co-IP was performed following standard procedure. Supernatants obtained from heart samples 114 were incubated with selective antibodies and protein A/G agarose beads overnight. Beads were 115 washed at least five times and the cleared supernatants were subjected to SDS-PAGE followed by 116 western blot analysis, which was performed with antibodies as mentioned above.

BMP7 improved Cardiac function and reduced fibrosis in viral myocarditis 123
In the study, we analyzed the effects of BMP7 administration in a mouse model of CVB3-124 induced myocarditis. Inbred male BALB/c mice were injected intraperitoneally with 10 3 TCID50 of 125 CVB3 to establish animal model of viral myocarditis. As shown in table 1, BMP7 administration 126 significantly decreased the mortality rate after CVB3 injection (40% vs. 25%, p<0.05). 127 Additionally, the ratio of BMP7/TGF-β1 in mRNA levels was demonstrated to decline obviously at 128 different time points after CVB3 injection (Figure 1), denoting that CVB3 infection may contribute 129 to the imbalance of BMP7/TGF-β1 in the heart. 130 To assess the myocardial function, echocardiography was performed. The data revealed that 131 However, BMP7 treatment significantly improved cardiac function, as characterized by the 134 alteration of the echocardiographic functional parameters (Table 1, Figure 2). 135 To evaluate the extent of myocardial damage, the hearts were examined using HE and SR 136 staining. The analysis revealed extensive myocardial damage and interstitial fibrosis in the hearts of 137 VMC group, whereas BMP7 ameliorated myocardial necrosis and fibrosis ( Figure 3A). In addition, 138 murine heart weight to body weight ratio (HW/BW) was markely elevated in VMC group. However, 139 BMP7 treatment decreased the HW/BW ratio compared with VMC group ( Figure 3B). More than 140 that, virus RNA replication was obviously decreased in CVB3-infected hearts by BMP7 141 administration ( Figure 3C). 142

BMP7 inhibited Endo-MT in viral myocarditis 143
Compared with control group, immunohistochemistry staining showed that the expression of 144 CD31, the marker of endothelial cells, was significantly decreased in VMC mice, which was 145 attenuated by BMP7 (Figure 4, P<0.05). It was also found that FSP-1, a mesenchymal marker, was 146 markedly increased in VMC mice, which was inhibited by BMP7 intervention (Figure 4, p<0.01). 147 Those results suggested that Endo-MT was involved in CVB3-induced VMC. 148 To further delineate Endo-MT, co-expression of endothelial markers (CD31 and VE-cadherin, 149 green) and mesenchymal markers (FSP-1-and αSMA, red) were analyzed using double 150 5 immunofluorescence staining. Confocal microscopy revealed double-labeled of αSMA/CD31 and 151 FSP-1/VE-cadherin after CVB3 intrusion, whereas the number of co-expression cells was greatly 152 decreased with BMP7 intervention (Figure5). Based on these results, it was indicated that BMP7 153 inhibited Endo-MT in viral myocarditis, which might contribute to a switch to cardiac fibrosis 154 ( Figure 3A). and Smad4 were significantly upregulated in VMC group, as well as Wnt/β-catenin and the 168 transcription factor snail. BMP7 treatment reversed the changes of these protein levels, indicating 169 that BMP7 may inhibit Endo-MT via TGF-β/Smad and Wnt/β-catenin pathway ( Figure 6). 170 Furthermore, the crosstalk between Smad3 and β-catenin was determined. Co-IP assay demonstrated 171 that co-localization of β-catenin and Smad3 was increased in CVB3-infected mice. However, BMP7 172 treatment inhibited the protein complex between Smad3 and β-catenin (Figure 7). These results 173 indicated that BMP7 hampered the activation of TGF-β/Smad and Wnt/β-catenin signaling after 174 virus intrusion, which is associated with the interaction of Smad3/β-catenin and upregulation of 175 snail (Figure 8). In the study, it was shown that BMP7/TGF-β1 ratio in mRNA levels was significantly declined 196 followed by CVB3 invasion. Altogether, these results give rise to the possibility that the imbalance 197 of BMP7/TGF-β1 may be the initiating mechanism of Endo-MT and its subsequent fibrosis elicited 198 by CVB3.

81300096) 246
Availability of data and materials 247 All data generated or analyzed during this study are included in this article. 248

Ethics approval and consent to participate 249
This study was approved by Animal Experimentation Ethics Committee of Zhongshan Hospital, 250 Fudan University, and all procedures were performed in accordance with the Guide for the Care and 251 Use of Laboratory Animals and the Regulation of Animal Protection Committee. 252

Consent for publication 253
Not applicable. 254