| Background:Heart failure is associated with a high rate of mortality and morbidity,and ventricular remodeling is an integral aspect of heart failure progression.Ventricular remodeling is a mechanotransduction process that is regulated by the nervous system and immune system.However,it remains unknown which key molecular factors govern the neuro/immune/cardio axis that underlies mechanotransduction during ventricular remodeling.Piezo1 is a novel protein involved in mechanotransduction.Piezo1 can regulate inflammation,both Piezo1 and inflammation especially interleukin-6（IL-6）in heart play key roles in ventricular remodeling,but the therapeutic effect against IL6 in heart on ventricular remodeling is controversial.However,the cell bodies of afferent nerves innervating the heart are mainly located in the thoracic dorsal root ganglion（TDRG）of T2-T6,the role of Piezo1 and IL6 on ventricular remodeling focused on TDRG stays unknown.Therefore,we focuse on the role and mechanism of neurogenic inflammation mediated by TDRG Piezo1 channel in ventricular remodeling,aiming to understand the neuroimmune network underlying mechanotransduction and explore new targets for treatment or prevention of ventricular remodeling.Methods:1.To observe the effect of ventricular remodeling post-MI on Piezo1/Piezo2 in the TDRG,SD rats were randomly divided into six groups of six rats each:sham,3 days,1week,2 weeks,4 weeks and 6 weeks post-MI,which subsequently are denoted as sham,3d MI,1WMI,2WMI,4WMI and 6WMI,respectively.The severity of ventricular remodeling was monitored by echocardiography,hematoxylin and eosin staining,intermuscular fibrosis,cardiomyocyte hypertrophy,apoptosis and necroptosis.The expression of Piezo1 and Piezo2 in the TDRG was analyzed by western blotting,quantitative real-time PCR,and immunostaining.In order to further validate the results of rats and lay the foundation for subsequent mouse experiments,WT mice were randomly divided into two groups of six mice each:sham and 4WMI.The gene expression of Piezo1 in TDRG was analyzed by in situ sequencing（ISS）experiments.2.To explore whether downregulation of Piezo1 in the TDRG could help remediate ventricular remodeling after MI,Piezo1 m RNA was targeted in the TDRG by delivering a LV-Piezo1-RNAi into the TDRG of rats via spinal nerve injection 1 week before MI（LV-Piezo1-RNAi）;a negative control lentivirus was delivered to control rats（LV-NC）.Successful transfection was confirmed by fluorescence microscopy,western blotting,and quantitative real-time PCR analysis of Piezo1 in the TDRG.SD rats were randomly divided into two groups of six rats each,namely LV-NC and LV-Piezo1-RNAi,and ventricular remodeling was tested 4 weeks post-MI.3.To investigate the mechanism of Piezo1 in the TDRG and its potential involvement in promoting ventricular remodeling after 4WMI.（1）SD rats and WT mice were respectively randomly divided into two groups of six rats each,namely sham and 4WMI.IL6 in the TDRG and components of the IL-6/IL-6R/STAT3 cascade in the infarcted area of each heart were quantified by western blotting,enzyme-linked immunosorbent assay（ELISA）,or quantitative real-time PCR in rats.Expression and localization of IL-6,and neuronal excitement in TDRG were analyzed by ISS method in mice.（2）SD rats were randomly divided into two groups of six rats each,namely LV-NC and LV-Piezo1-RNAi.c-Fos immunostaining and delta-Fos B western blotting were performed to assess neuronal activation in the TDRG.IL-6 in the TDRG and components of the IL-6/IL-6R/STAT3 cascade in the infarcted area of each heart were detected by western blotting,ELISA,or quantitative real-time PCR.（3）To investigate the Piezo1 function,calcium imaging experiment was carried out to TDRG primary neurons in vitro.When the primary neurons of TDRG were cultured to the 3rd day,the calcium imaging experiment was performed.TDRG neurons were divided into four groups:sham+Yoda1（TDRG neurons extracted from sham mice,30μM Yoda1 was continuously perfused for30s）,4WMI+Yoda1（TDRG neurons extracted from 4WMI mice,30μM Yoda1 was continuously perfused for 30s）,sham+Gs MTx4+Yoda1（TDRG neurons extracted from sham mice,pre-perfusion of Piezo1 specific inhibitor 2.5μM Gs MTx4 for 60s,and then30μM Yoda1 continuous perfusion for 30s）,4WMI+Gs MTx4+Yoda1（TDRG neurons extracted from 4WMI mice,pre-perfusion 2.5μM Gs MTx4 for 60s,and then 30μM Yoda1 was continuously perfused for 30s）,and the ratio of F340/380 in each group was recorded.TDRG neurons in each group were from 6 mice and n=20 neurons.Next,the cell culture medium of neurons in each group was collected for IL-6 detection by ELISA.They were divided into 6 groups:sham（TDRG neurons extracted from sham mice,no treatment）,4WMI（TDRG neurons extracted from 4WMI mice,no treatment）,sham+Yoda1（TDRG neurons extracted from sham mice,30μM Yoda1 co-cultured for18 hours）,4WMI+Yoda1（TDRG neurons extracted from 4WMI mice,30μM Yoda1co-cultured for 18 hours）,Sham+Gs MTx4+Yoda1（TDRG neurons extracted from Sham mice,after 2.5μM Gs MTx4 pretreatment for 18 hours,30μM Yoda1 co-cultured for 18 hours）,4WMI+Gs MTx4+Yoda1（TDRG neurons extracted from 4WMI mice,after 2.5μM Gs MTx4 pretreatment for 18 hours,30μM Yoda1 co-cultured for 18hours）.TDRG neurons in each group were from 6 mice,n=6.（4）Downregulation of IL-6 in the TDRG was achieved by intrathecal injection of anti-rat IL-6 diluted in artificial cerebral spinal fluid（ACSF）2 h before MI（anti-IL-6）,and intrathecal injection of ACSF served as the control.SD rats were randomly divided into two groups of six rats each,namely anti-IL-6 and ACSF.Ventricular remodeling was tested 4 weeks post-MI,and IL-6 as well as Piezo1 in the TDRG and components of the IL-6/IL-6R/STAT3 axis in the infarcted area of each heart were detected by western blotting,ELISA,or quantitative real-time PCR.To study how IL-6 in TDRG affects IL-6 in the heart,After treatment of TDRG neurons with 20μM colchicine（axoplasmic transport inhibitor）for 18 hours,ELISA was used to determine whether it could reduce the release of IL-6 caused by the neurons activation after co-culture of 30μM Yoda1 for 18 hours.（5）Mice were randomly divided into two groups of six mice each,namely WT and IL-6Rα–/–.Piezo1 and IL-6 in the TDRG and components of the IL-6/IL-6R/STAT3cascade in the heart,along with ventricular remodeling,were tested at 4WMI.Results:1.Piezo1 expression was the highest at 4W after MI.2.Echocardiographic analysis demonstrated that the LV-Piezo1-RNAi rats had significantly improved heart pump function as reflected by EF,FS,LVIDd and LVIDs.Histological analysis revealed that LV-Piezo1-RNAi treatment moderately reversed the dilation of the left ventricles,improved the organization of myofibers,and increased the thickness of the thinned walls.Masson’s trichrome staining of longitudinal heart sections revealed significant alleviation of intermuscular fibrosis.Accordingly,western blotting and quantitative real-time PCR analyses of the infarcted area of LV-Piezo1-RNAi heart tissues verified a significant decrease in the levels of the fibrosis markers collagen I,collagen III and CTGF and necrosis markers p-MLKL and RIPK3as well as m RNA levels of the hypertrophy marker genes ANP,BNP andβ-MHC.3.（1）The levels of IL-6 protein and m RNA in the TDRG,the levels of components of the IL-6/IL-6R/STAT3 cascade,and m RNA levels of IL-6 and IL-6R in the infarcted area were all significantly increased in 4WMI rats compared with sham rats.The upregulation of Piezo1 protein in the TDRG correlated positively with IL-6 level in the TDRG and IL-6/IL-6R/STAT3 cascade components in the infarcted area of the heart of both sham and 4WMI rats.The gene expression of IL-6,c-Fos,and delta-Fos B in TDRG were also significantly increased in 4WMI mice compared with sham mice.（2）Compared with LV-NC rats,the percentage of c-Fos-positive cells and delta-Fos B protein expression both decreased in LV-Piezo1-RNAi rats;The levels of IL-6 protein and m RNA in the TDRG,the levels of IL-6/IL-6R/STAT3 cascade components,and the levels of IL-6 and IL-6R m RNAs in the infarcted area were all significantly decreased in LV-Piezo1-RNAi rats.（3）Calcium imaging showed that compared with sham+Yoda1 group,F340/380 in4WMI+Yoda1 group was significantly higher;After pre-perfusion of Gs MTx4,the respective F340/380 ratio decreased significantly,but compared with sham+Gs MTx4+Yoda1 group,the F340/380 ratio of 4WMI+Gs MTx4+Yoda1 group still increased significantly.ELISA results showed that compared with sham+Yoda1group,the release of IL-6 in 4WMI+Yoda1 group increased;Compared with sham+Gs MTx4+Yoda1 group,the release of IL-6 in 4WMI+Gs MTx4+Yoda1 group also increased significantly.（4）Piezo1 expression（m RNA and protein）in the TDRG did not differ significantly between the anti-IL-6 and ACSF groups,indicating that IL-6 level did not affect Piezo1level in the TDRG.Intrathecal injection of the neutralizing anti-IL-6 decreased the levels of IL-6,IL-6R,and phosphorylated STAT3（p STAT3）as well as the levels of m RNAs encoding IL-6 and IL-6R in the infarcted area of the heart.The ELISA results howed that the release of IL-6 in the 4WMI+Yoda1+Colchicine group was significantly reduced compared with the 4WMI+Yoda1 group.There was a significant improvement in heart pump function—as assessed by measuring EF,FS,LIDd and LVIDs—and alleviation of the dilated left ventricles that had thin walls and disorganized myofibers.Anti-IL-6 also protected rats from cardiac fibrosis as reflected by Masson’s trichrome staining and expression of collagen I,collagen III and CTGF.Finally,there was a concomitant decrease in both necrosis（as reflected by p-MLKL and RIPK3 levels）and hypertrophy（as reflected by expression of m RNAs encoding ANP,BNP andβ-MHC）.（5）IL-6Rα–/–mice had a decreased p STAT3/STAT3 ratio,but there was no change in the protein and m RNA levels of Piezo1 and IL-6 in the TDRG and IL-6 in the infarcted area of the heart.In addition,IL-6Rα–/–mice exhibited better heart pump function and relatively improved dilated left ventricles with thicker walls and fewer disorganized myofibers.Finally,the IL-6Rα–/–mice had reduced cardiac fibrosis,necrosis,and hypertrophy.Conclusions:In response to ventricular remodeling post-MI,TDRG-derived Piezo1 was up-regulated and its function was enhanced,which led to the activation of neurons in TDRG and the release of neurogenic inflammatory factor IL-6.IL-6 in TDRG is transported to the myocardial infarction area through the mechanism of axoplasmic transport,thus activating the IL-6/IL-6R/STAT3 pathway in the myocardial infarction area and aggravating the ventricular remodeling.The revelation of a Piezo1-mediated neurogenic inflammatory cascade uncovers an as-yet unknown facet of the neuronal immune signaling axis underlying mechanotransduction during ventricular remodeling. |
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