We explored the possible mechanism of pathological myocardial remodeling induced by miR-1929-3p in mice after MCMV infection on the cellular and molecular level. Here, MCMV tended to infect MCFs in myocardial tissue, whereafter, up-regulated the expression of ETAR through the endogenous low expression of miR-1929-3p in MCFs. Subsequently, it caused myocardial remodeling by promoting the proliferation, activation and autocrine of MCFs and paracrine of MCFs on MCMs. The specific mechanism was related to the activation of NLRP3 inflammasome as well as the mature and release of IL-18 induced by high expression of ETAR. HCMV is a kind of double-stranded DNA virus in Herpesviridae, which has species-specific infection to the body. HCMV has a high latent infection rate in the population, which is a conditional pathogenic pathogen. It is related to a variety of cardiovascular diseases including essential hypertension, atherosclerosis, and coronary heart disease . Our previous studies have shown that MCMV infection leads to hypertensive ventricular remodeling in mice, which is closely related to the low expression of cardiovascular miR-1929-3p. urthermore, MCFs are the most abundant cell types in the heart and play a key role in regulating normal myocardial function and adverse myocardial remodeling caused by hypertension, myocardial infarction, and heart failure . HCMV can widespread attack human tissues and cells while MCMV specifically infects mice and is not pathogenic to humans. In the laboratory, MCMV strain can be obtained in large quantities by infection with primary mouse embryonic fibroblasts or homologous cell lines . However, there is few research on MCMV-infected myocardial tissue cells in mice. To explore the cellular and molecular mechanism of myocardial remodeling caused by MCMV infection, MCMs and MCFs were selected as the research objects in the experimental design, and it was found that MCMV was more likely to infect the latter. Moreover, the expression of miR-1929-3p in MCMs after the same MOI intervention at the same time did not show a change consistent with the previous overall level, further indicating that MCMs were not the host cells of MCMV. It is well known that stable cardiac fibroblasts undergo phenotypic transformation during myocardial remodeling, and their proliferation ability is significantly enhanced. The MCMV Smith strain used in this study is a non-toxic pathogen, which can reduce cell viability. In our vivo studies, MCMV infection caused myocardial remodeling in mice with hypertension, which theoretically promoted the proliferation of MCFs. When MCFs were infected with MOI = 0.1 for 72 hours, its proliferation activity was obviously decreased, and the cell morphology was significantly abnormal or even dead. The CCK8 cell proliferation assay showed that the proliferation rate of MCFs was the highest under the conditions of MOI = 0.01 and time = 48 hours, which was chosen as the optimal condition for infection in subsequent experiments in this study.
Myocardial remodeling is the main mechanism of disability and death caused by cardiovascular diseases such as hypertension, heart failure and myocardial infarction. This involves the overall structure and morphology of the heart, as well as changes in CMs and non-CMs cells and subcellular levels, which adversely affect cardiac function. Our previous studies have shown that MCMV infection can induce hypertensive myocardial remodeling in mice. This study suggests that MCFs are the key cells to induce this effect. On the one hand, it was found that MCMV did not successfully infect MCMs, and the expression of miR-1929-3p in cells did not change significantly after intervention. Namely, MCMV may not induce myocardial hypertrophy by directly inducing the expression changes of related regulatory factors in MCMs. On the other hand, although CMs are essential for cardiac pumping, the main cell type of the heart is CFs. The main role of CFs is to secrete collagen to cause ECM deposition, which maintains the integrity of cardiac structure and function. Excessive collagen deposition or pathological fibrosis is an important cause of left ventricular dysfunction and adverse outcomes in patients with hypertension, myocardial infarction, and heart failure. In fact, in our study, MCMV showed a tendency to infect MCFs, and miR-1929-3p expression was significantly down-regulated. CFs are the major regulator of ECM metabolism, maintaining the balance between the synthesis and degradation of ECM components. Specifically, the key aspects of CFs involved in cardiac remodeling-related functions include proliferation, migration, differentiation, ECM conversion and secretion of growth factors and cytokines . In addition, CFs mediate many biological processes through differentiation into myofibroblasts, which often show high sensitivity to pro-inflammatory cytokines (including IL-1, IL-6, TNF-α) and vasoactive peptides (including Ang II, ET-1) that cause adverse myocardial remodeling . We found that MCFs proliferated actively after MCMV infection and showed phenotypic transformation to myofibroblasts. The outcome of these changes was the synthesis of type I and type III collagen. In other studies, it has been found that there is ‘crosstalk’between CFs and CMs. In a rat model of aortic coarctation, hypoxia-induced mitogens induce CFs migration, proliferation and myofibroblast differentiation through CMs-CFs paracrine . We added the supernatant of MCFs, which was cultured for 48 hours after infection, into MCMs medium and measured cell size of MCMs after 24 hours. It is well known that CFs can secrete various types of cytokines. The above phenomena suggested that MCFs may release some cytokines or gene products to induce myocardial hypertrophy through paracrine effect. MCMV infection could cause myocardial remodeling directly and indirectly. Of course, the specific mechanisms of paracrine mediators and occurrence need further exploration, but the leading role of MCFs in myocardial remodeling induced by MCMV infection in mice with hypertension was beyond doubt.
Studies have found that ET-1 levels in myocardium and circulation are elevated in patients with heart failure and experimental models of heart failure. ET-1 mainly increases collagen synthesis by stimulating CFs from different species (including humans), thereby promoting MF. ETAR and ETBR are both involved in this process. There is evidence that ET-1 can reduce the activity of collagenase through ETAR. These two receptors can be expressed by CFs and myofibroblasts in adult and rat, and the expression of ETBR is dominant. Other studies have shown that ET-1 can promote the proliferation of CFs in neonatal rats and adult rats by activating ETAR and induce the expression of myofibroblast phenotypic marker α-SMA in neonatal rats [24, 25]. In previous study, we found that ETAR is one of the target genes of miR-1929-3p related to hypertension by miRNA microarray combined with bioinformatics analysis and MCMV infection in mice. In this study, MCFs infected with MCMV was used as the cell disease model. We found that the expression of miR-1929-3p was down-regulated after MCMV infection, while the mRNA and protein expression of its target gene ETAR was increased. Over-expression of ETAR by adenovirus vector intervention significantly weaken the beneficial effect of miR-1929-3p mimic on reducing MF, which was manifested as the increase of MCFs proliferation activity, α-SMA protein expression, as well as collagen I and III mRNA levels. However, whether ETBR has a beneficial effect on MF after MCMV infection deserves further exploration. Combined with previous in vivo studies, we believe that the down-regulated miR-1929-3p induced by MCMV infection is involved in the occurrence of hypertensive pathological myocardial remodeling through high expression of ETAR, which may be one of the important links of hypertension and target organ damage induced by MCMV infection.
miRNA plays a negative regulatory role by inhibiting mRNA translation or promoting mRNA degradation. Traditionally, fibrosis is considered as an indirect response to myocardial cell death and/or injury-induced hypertrophy. A report on the role of miR-21 in CFs demonstrated its ability to regulate MAPK pathway. MiR-21 regulated cell proliferation in the reconstructed heart and increased the release of CFs growth factor, thereby promoting cardiomyocyte hypertrophy . In another study, miR-21 negatively regulated the expression of phosphatase and tensin homolog (PTEN) deleted on chromosome 10 in CFs, resulting in an up-regulation of AKT signaling pathway and an increase in matrixmetallo proteinase-2 (MMP-2) expression . According to the results of previous studies, we proposed that MCMV caused the decrease of miR-1929-3p expression in mice by infection, which further led to the increase of ETAR expression, leading to essential hypertension and adverse myocardial remodeling . This study further proved that MCMV in myocardium mainly caused the low expression of miR-1929-3p in CFs, and MF was mainly caused by the activation of NLRP3 inflammasome mediated by ETAR. These new findings suggest that miRNAs expressed in CFs may be targets for the treatment of poor myocardial remodeling in the future. However, due to the individual differences of miRNA expression in the circulation among hypertensive patients, it is difficult to be used as a biomarker for diagnosis or screening of hypertension. Therefore, these multifunctional cells provide attractive but challenging therapeutic targets for future cardiovascular disease diagnosis and treatment. So far, many scholars have begun to develop small non-coding RNA-related therapeutic strategies. For example, miRNA has been developed for the treatment of liver fibrosis. In addition, a variety of viral and non-viral vectors have been developed for miRNA mimics .
NLRP3 inflammasome can be stimulated by a variety of signals and trigger inflammatory reactions to participate in myocardial remodeling. NLRP3 inflammasome can be stimulated by a variety of signals and trigger inflammatory reactions to participate in myocardial remodeling. In this study, the expressions of NLRP3, caspase-1 and IL-18 in CFs of MCMV infection group were increased, while miR-1929-3p inhibitor aggravated the above effect. This suggests that the mechanism of MCMV infection promoting the occurrence and development of MF may be related to the low expression of miR-1929-3p caused by MCMV infection and the activation of NLRP3 inflammasome induced by high expression of ETAR. Selective NLRP3 inflammasome MCC950 attenuated NLRP3 inflammasome activation and completely reversed the inflammatory effect induced by miR-1929-3p inhibitor, suggesting that the inflammatory response triggered by MCMV infection in MCFs was mainly mediated by down-regulated miR-1929-3p. In addition, we also compared the expression of inflammasome and their downstream effectors after MCMV infection and after the addition of ETAR antagonist BQ123. The results showed that ETAR as a target gene was indeed involved in the activation of MCMV-miR-1929-3p-ETAR-NLRP3 pathway in MCFs. Unfortunately, expression of IL-1β did not change after MCMV infection and after treatment with miR-1929-3p inhibitors, but it was reduced only by the effects of MCC950 drugs. This phenomenon is most likely to be affected by drugs themselves, but not MCMV infection and miR-1929-3p downregulation. We speculated that MCMV infection may selectively trigger the activation of IL-18 in MCFs and promote the survival and DNA replication of MCMV in host cells. At the same time, IL-18 is also the main effector molecule of NLRP3 inflammasome activation and participation in MF. From the perspective of disease treatment, inhibiting the activation of inflammatory cytokines may be more effective than blocking any of these downstream cytokines. However, this research limitation lies in has not explore the participation of other mesenchymal cells in cardiac tissue in myocardial remodeling caused by MCMV, and the specific mechanism of ETAR activating NLRP3 inflammasome in MCFs.