| BackgroundDiabetic cardiomyopathy(DCM)is initialed by cardiac metabolic disorders,characterized by cardiac fibrosis,cardiac hypertrophy,and coronary artery injury,among which cardiac fibrosis is the key abnormality.Clinical trials demonstrated that glucose levels are positively correlated with cardiac fibrosis in diabetic patients,however,evidence confirming the protective role of glycemic control on cardiac fibrosis and adverse cardiovascular events is still lacking.Therefore,further studies on exploring novel targets of DCM are highly warranted.The activation of neurohumoral factors plays an important role in the initiation and progression of heart failure,among which the most widely studied factors include the renin-angiotensin-aldosterone system(RAAS)and the natriuretic peptide(NPs)family.In the early stages of heart failure,hemodynamic abnormalities lead to RAAS activation as a compensatory mechanism to maintain cardiac output.However,continued activation of RAAS leads to ventricular remodeling and exacerbates the symptoms of heart failure.NPs,as endogenous factors that can antagonize the activation of RAAS and decrease cardiac load,have attracted increasing attention in recent years.NP family consists of three kinds of NPs,atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),C-type natriuretic peptide(CNP),and their three kinds of receptors(NPRs),NPRA,NPRB,and NPRC.NPs bind to NPRs on various tissues and exert physiological effects including diuresis,natriuresis,vasodilation,and inhibiting cardiac fibrosis and remodeling to antagonize the activation of RAAS.However,few studies have thoroughly explored the function of NPRC.Previous studies found that NPRC knockout increased atrial fibrosis under angiotensin Ⅱ treatment,but attenuated atrial fibrosis under TGF-β1 treatment and TAC in mice on the contrary.However,no studies explored the relation of NPRC with ventricular fibrosis.Our recent research has found that NPRC deficiency inhibited the inflammation,and enhanced the browning and lipolysis in white adipose tissue,suggesting the potential protective role of NPRC on adipose metabolic diseases.Considering that DCM is also related to metabolic disorders,whether NPRC deficiency plays a protective role in the progression of left ventricular fibrosis in DCM remains to be explored,which may be of great significance for the development of NP-based therapy for DCM.For decades,a more promising strategy in the treatment of heart failure is to enhance the effects of NPs.To date,sacubitril/valsartan(sac/val)is the first-in-class product that is recommended by the European and American guidelines for heart failure.Sacubitril is an inhibitor of neprilysin(NEP),which is responsible for the enzymatic degradation of NPs.Treatment with sacubitril/valsartan increases the serum level of NPs in patients and inhibits the activation of RAAS simultaneously.Preclinical studies have confirmed that sacubitril/valsartan can attenuate cardiac fibrosis,inhibit cardiomyocyte apoptosis,and improve cardiac function in DCM mice.However,in addition to the NEP-mediated enzymatic degradation pathway,the NPRC-mediated receptor degradation pathway is also one of the clearance pathways of NPs in vivo.So far,there is no study comparing the therapeutic effects of the inhibition of NPRC and NEP on cardiac fibrosis in DCM mice.Therefore,we constructed NPRC-/-diabetic mice and sac/val-treated diabetic mice to compare their effects on cardiac fibrosis in DCM.Dissertation Ⅰ The role and mechanism of natriuretic peptide receptor C on cardiac fibrosis in diabetic cardiomyopathyObjectives1.To investigate the expression of NPRC in the heart of diabetic mice.2.To investigate whether NPRC is involved in the regulation of cardiac fibrosis in DCM.3.To investigate the potential mechanism in the regulation of cardiac fibrosis by NPRC in DCM.Methods1.Experimental animalsNPRC knockout mice were constructed by CRISPR/Cas9 and backcrossed with C57BL/6J mice to obtain NPRC knockout mice(NPRC-/-)and wild-type mice(WT)with the same genetic background.2.Groups of experimental animals(1)Eight-week-old male C57BL/6J mice were randomly divided into the following 2 groups:control and DM groups;(2)Eight-week-old male NPRC-/-mice and littermate WT mice were randomly divided into the following 4 groups:WT+Control,NPRC-/-+Control,WT+DM,and NPRC-/-+DM.3.Construction of mice with DCMEight-week-old male mice were given an intraperitoneal injection of streptozotocin solution(STZ,55mg/kg/d for 5 consecutive days).The mice in the control groups were injected with the buffer of the same volume.After 2 weeks,fasting blood glucose levels were measured.Mice with fasting blood glucose levels>16.7mmol/L were considered diabetic.After 16 weeks,the mice were euthanized.4.EchocardiographyThe left ventricular systolic and diastolic function were measured using the Vevo2100 imaging system.The following parameters were measured:the left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD),the left ventricular fractional shortening(LVFS)and left ventricular ejection fraction(LVEF),the peak mitral flow velocities in early diastole(E)and late diastole(A),the mitral annular velocities in early diastole(e’).5.Collection of mouse tissues and pathological examinationAfter 16 weeks of diabetes inducement,mice were euthanized,and mouse serum was collected,centrifuged,and frozen at-80℃.ANP,BNP,and CNP levels in the serum of mice were measured using ELISA kits.Heart tissues were either stored in liquid nitrogen and transferred to-80℃ or fixed in 4%paraformaldehyde for subsequent histopathological examination.After that,the heart tissues were embedded in paraffin,and cut into cross-sections for HE staining,Masson staining,immunohistochemical(IHC)detection of Collagen Ⅰ,Collagen Ⅲ,p-VASP,p-CREB,p-PKA substrates,ANP,BNP,CNP and immunofluorescence staining for NPRC.6.Primary cell isolation and treatmentPrimary cardiomyocytes(CM)and cardiac fibroblasts(CF)were isolated from neonatal rats by differential attachment technique.The isolated primary CMs were cultured in a low glucose DMEM medium containing 8%equine serum and 5%newborn calf serum.The isolated primary CFs were cultured in a low glucose DMEM medium containing 10%fetal bovine serum.All cells were cultured in a 3 7℃ incubator containing 5%CO2.Cells were grouped as follows:(1)CFs were treated with the normal concentration of glucose(NG),high concentration of glucose(HG,33.3mM),and high osmotic solution(HO,33.3mM).(2)CFs were transfected with si-NC or si-NPRC,followed by NG or HG treatment.(3)CMs were transfected with si-NC or si-NPRC,followed by NG or HG treatment.The supernatant of CMs was collected and added to CFs.(4)CFs were transfected with si-NC or si-NPRC,or si-NPRC combined with si-TGIF1,followed by HG treatment.(5)CFs were transfected with adenovirus overexpressing ANP,BNP,CNP(Ad-ANP,Ad-BNP,and Ad-CNP),and control adenovirus(Ad-GFP).(6)CFs were treated with forskolin,the agonist of cAMP,and H89,the antagonist of PKA.(7)CFs were treated with 8-br-cGMP,the agonist of cGMP,and KT5823,the antagonist of PKG.7.Immunofluorescence staining of cellsAfter treatment,the cells were fixed with 4%paraformaldehyde and permeabilized with 0.1%Triton X-1 00.Then the cells were blocked with 5%BSA and incubated with primary antibodies overnight at 4℃.On the next day,the cells were washed and incubated with secondary antibodies.The nuclei were stained with DAPI and the fluorescent signal was detected by a fluorescence microscope.8.Cell proliferation assayAfter cell samples were prepared,the EdU labeling reagent was added to the culture medium(1:2500),followed by culture for 12 h before cell collection.Next,the cells were fixed with 4%paraformaldehyde,incubated with Apollo staining solution,and counterstained with Hoechst 33342 for cell nuclei.Finally,fluorescence images were captured by a microscope.In addition,the CCK-8 reagent was added to the cell medium when cell samples were prepared,and after incubation for 4 h,the absorbance at 450 nm was measured by a microplate reader.9.Co-immunoprecipitation assayCFs were cultured in a 100 mm cell dish and transfected with si-NC or si-NPRC.The CFs were lysed with lysis buffer.Then cPML antibody and mouse IgG were added to the lysis followed by incubation for 1 h.Protein A/G-agarose beads were then incubated with the mixture at 4℃ overnight.The next day,the beads were washed and subjected to Western blot.10.Extraction of cytoplasmic and nuclear proteinsCytoplasmic and nuclear proteins were separated using Cytoplasmic&Nuclear Extraction Kits.Briefly,cytoplasmic extraction buffer and nuclear extraction buffer was added to obtain the cytoplasmic and nuclear proteins which were further subjected to Western blot analysis.11.Measurement of intracellular cAMP and cGMPAfter cell treatment,the cyclic AMP/GMP chemiluminescence detection kit was used to detect the levels of intracellular cAMP and cGMP,and the OD value at 450nm was read on a microplate reader.12.Transcriptome RNA sequencingAfter cell treatment,total RNA was lysed with Trizol solution and stored at-80℃.Then the samples were shipped to Shanghai Whale Boat Biotechnology Co.,LTD(Shanghai,China)for RNA sequencing and analysis.13.RNA extraction,reverse transcription,and real-time PCRTotal RNA in cells was extracted and mRNA was reversed-transcribed to cDNA.Then the Ct values of NPRA,NPRB,NPRC,and TGIF1 were obtained by real-time PCR,and the relative gene expressions were calculated by the 2-ΔΔCt method.14.Western blotThe total protein of cardiac tissues was extracted using the protein extraction kit,and the total cell protein was extracted using a lysis buffer.The protein concentration was determined by the BCA method.SDS-polyacrylamide gel electrophoresis was performed,followed by the transfer of protein to the PVDF membranes by the semidry transfer method.The membranes were blocked and incubated with primary antibodies at 4℃ overnight.The next day,the membranes were washed and incubated with secondary antibodies,followed by detection using a chemiluminescent substrate.15.Statistical analysisAll data were presented as mean ± SEM.All analyses were performed with GraphPad Prism 8 software.The normality assumption of the data distribution was assessed.Unpaired two-tailed Student’s t-tests were used to determine the statistical difference between two groups.One-way ANOVA followed by Turkey’s post-hoc test was performed to determine the statistical difference between multiple groups with one variable.Two-way ANOVA followed by Tukey’s post-hoc test was used to compare multiple groups with more than one variable.For all statistical comparisons,p<0.05 was considered statistically significant.Results1.NPRC expression increased in the hearts of diabetic miceImmunofluorescent staining and Western blot showed increased NPRC expression in the hearts of the mice in the DM group relative to the control group.2.Comparison of NPRC expression in major cardiac cellsWe examined the expression of NPRC in CM and CF.PCR results showed that the mRNA level of NPRC in CF was higher than that in CM.In addition,immunofluorescence and PCR results showed that the distribution of NPRC on CF was much higher than that of NPRA and NPRB.3.HG treatment increased the expression of NPRC in CFsWestern blot and PCR showed that compared with the NG group,the expression of NPRC in HG-treated CFs was significantly increased,but the expression level of NPRC in the HO group was not significantly changed.4.DCM was successfully induced in NPRC-/-mice(1)NPRC-/-mice were constructed by CRISPR/Cas9 technology and the deletion of NPRC was confirmed by Western blot of cardiac tissues.(2)Diabetes was induced in eight-week-old male NPRC-/-and littermate WT mice by injection of STZ.Measurement of body weight and fasting blood glucose in 4 groups of mice(WT+Control,NPRC-/-+ Control,WT+DM,and NPRC-/-+DM)showed that NPRC knockout did not affect the body weight and fasting blood glucose in mice.5.NPRC deficiency improved cardiac function and remodeling in diabetic miceEchocardiography showed that compared with the mice in WT+Control group,mice in the WT+DM group developed cardiac systolic and diastolic dysfunction,as indicated by a significant decrease in LVEF,LVFS,and E/A,and an increase in E/e’.NPRC deletion improved cardiac function induced by diabetes in NPRC-/-+DM group,as demonstrated by restored LVEF,LVFS,E/A,and E/e’.Furthermore,DM-induced enlargement in LVEDD and LVESD in WT+ DM group was also restored by NPRC deletion in NPRC-/-+DM group,indicating an improvement in cardiac remodeling.6.NPRC deficiency alleviated cardiac fibrosis induced by diabetes in vivoMasson’s trichrome staining showed that diabetes increased collagen accumulation in the cardiac perivascular and interstitial spaces in WT-/-+DM mice,compared with the NPRC-/-+DM mice.IHC and Western blot for Collagen Ⅰ and Collagen Ⅲ showed similar results.7.NPRC deficiency inhibited collagen synthesis and proliferation of CFsWestern blot,EdU assay,and CCK-8 assay showed that HG treatment increased the protein levels of Collagen Ⅰ,Collagen Ⅲ,PCNA,and the proliferation of CFs,which were inhibited by NPRC knockdown.8.The supernatant of NPRC-deficient CMs inhibited collagen synthesis and proliferation of CFsWe treated CFs with the supernatant of CMs that were transfected with si-NC or si-NPRC and treated with NG or HG.Western blot,EdU assay,and CCK-8 assay showed that the protein levels of Collagen Ⅰ,Collagen Ⅲ,PCNA,and the proliferation of CFs were decreased in CFs treated with NPRC-deficient CM supernatant.9.NPRC deficiency inhibited TGF-β1/Smad signaling in vivoWestern blot of cardiac tissues in 4 groups of mice(WT+Control,NPRC-/-+Control,WT+DM,and NPRC-/-+DM)showed that the protein levels of TGF-β1,pSmad2,and p-Smad3 were decreased in NPRC-/-+ DM mice,compared with that of WT+DM mice.10.NPRC deficiency inhibited TGF-β1/Smad signaling in vitro(1)Western blot showed that NPRC deficiency decreased the protein levels of TGFβ1,p-Smad2,and p-Smad3 of CFs in the si-NPRC+HG group,compared with that in the si-NC+HG group.(2)We treated CFs with the supernatant of CMs that were transfected with si-NC or si-NPRC and treated with NG or HG.Western blot showed that TGF-β1 expression was not changed by treatment with NPRC-deficient CM supernatant.However,the expression of p-Smad2 and p-Smad3 were still reduced in CFs treated with the CM supernatant from the si-NPRC+HG group than from si-NC+HG group.11.The effect of NPRC deficiency on the mRNA expression profile in CFsTranscriptome sequencing of CFs showed that TGIF1 was the most prominent fibrosis-related gene in the highly enriched biological processes after NPRC knockdown.RT-PCR and Western blot showed that NRPC deficiency increased the mRNA and protein level of TGIF1 in CFs.Meanwhile,the protein level of TGIF1 was also increased in CFs treated with NPRC-deficient CM supernatant.Western blot of cardiac tissues also showed that TGIF1 protein expression was increased in mice of NPRC-/-+control group and NPRC-/-+DM group relative to the WT+control and WT+DM group,respectively.12.NPRC regulated collagen synthesis and proliferation of CFs by targeting TGIF1We knocked down both NPRC and TGIF1 through the transfection of siRNAs.Compared with CFs transfected with si-NC,si-NPRC transfection decreased the protein expression of collagen Ⅰ and collagen Ⅲ and increased the protein expression of TGIF1 in the si-NPRC group.However,when the elevated levels of TGIF1 were knocked down by si-TGIF1,the protein expression of collagen Ⅰ and collagen Ⅲ was partially reversed in the si-NPRC+si-TGIF group,suggesting that TGIF1 played a crucial role in the mechanism through which NPRC deficiency regulated collagen synthesis and proliferation in CFs.13.TGIF1 sequestered cPML in the nucleus as the mechanism by which NPRC regulated the collagen synthesis and proliferation of CFsTGIF1 is a repressor of the TGF-β1/Smad pathway and one of the mechanisms is to sequester cPML in the nucleus and inhibit the phosphorylation of Smad2 and Smad3.Immunofluorescence staining showed that the nuclear localization of cPML was markedly enhanced in si-NPRC+NG and si-NPRC+HG groups,relative to the si-NC+NG and si-NC+HG groups,respectively.In addition,we separated nuclear and cytoplasmic proteins and found that the ratio of nuclear to cytoplasmic cPML levels was significantly higher in the si-NPRC+NG and si-NPRC+ HG groups than in the si-NC+NG and si-NC+HG.The co-IP assay showed that NPRC knockdown in CFs resulted in increased binding of cPML with TGIF1.14.NPRC deficiency activated cAMP/PKA signaling in vivo and in vitroELISA and Western blot showed that the level of intracellular cAMP and the protein levels of p-PKA substrates and p-CREB were increased in NPRC-deficient CFs.IHC and Western blot of cardiac tissues also showed that the protein levels of p-PKA substrates and p-CREB were increased in NPRC-/-mice,suggesting that NPRC deficiency activated cAMP/PKA signaling in vivo and in vitro.15.NPRC deficiency activated cGMP/PKG signaling in vivo through elevated NPsIHC and Western blot of cardiac tissues in 4 groups of mice(WT+Control,NPRC/-+Control,WT+DM,and NPRC-/-+DM)showed that the levels of p-VASP increased in NPRC-/-mice.Meanwhile,IHC and ELISA showed that the levels of ANP,BNP,and CNP increased in the cardiac tissues and serum of NPRC-/-mice,suggesting that NPRC deficiency activated cGMP/PKG signaling in vivo through elevated NPs.16.NPRC knockdown activated cGMP/PKG signaling in CFsELISA and Western blot showed that the cGMP level and p-VASP expression were increased in NPRC-deficient CFs and the levels of three NPs in the cell supernatant were also increased,indicating that NPRC knockdown activated cGMP/PKG signaling through the increase of NP levels in an autocrine manner.17.NPRC knockdown in CMs activated cGMP/PKG signaling in CFsELISA and Western blot results showed that the cGMP level and p-VASP expression,were increased in CFs treated with NPRC-deficient CM supernatant due to the elevated levels of the three NPs in the CM supernatant.The results suggested that NRPC knockdown of CMs activated cGMP/PKG signaling in CFs in a paracrine manner.18.Activation of cAMP/PKA signaling increased TGIF expression and inhibited TGF-β1 expressionWestern blot showed that forskolin,a cAMP agonist,increased the protein expression of TGIF1 while decreasing that of p-Smad2/3,both of which were counteracted by treatment with H89,an antagonist of PKA.Moreover,forskolin decreased the expression level of TGF-β1,which was abrogated by H89,suggesting that the activation of PKA inhibited TGF-β1/Smad signaling by inhibiting TGF-β1 and increasing TGIF1 expression simultaneously.19.Activation of cGMP/PKG signaling increased TGIF expressionRT-PCR and Western blot showed that the mRNA and protein level of TGIF1 were increased in ANP,BNP,or CNP-overexpressed CFs,but there were no significant differences in TGF-β1 expression.Meanwhile,8-br-cGMP,a cGMP agonist,increased the protein expression of TGIF1 while decreasing that of p-Smad2/3,both of which were counteracted by treatment with KT5823,an antagonist of PKG.Conclusions1.NPRC expression increased in the hearts of diabetic mice;2.NPRC deficiency attenuated cardiac fibrosis and improved cardiac remodeling and function in diabetic mice;3.NPRC deficiency decreased collagen synthesis and proliferation in CFs;4.NPRC deficiency inhibited TGF-β1/Smad signaling by increasing TGIF1 and decreasing TGF-β1 expression through the activation of cAMP/PKA and cGMP/PKG pathways.Dissertation Ⅱ Comparison of the role and mechanism of NPRC deficiency and sacubitril/valsartan in diabetic cardiomyopathyObjectives1.To compare the effects of NPRC knockout and sac/val gavage on cardiac fibrosis and cardiac function in DCM mice.2.To compare the effects of NPRC knockdown and sac/val on collagen synthesis and proliferation of cardiac fibroblasts.3.To investigate the different mechanisms of NPRC knockdown and sac/val treatment on cardiac fibroblasts.Methods1.Groups of experimental animalsEight-week-old male NPRC-/-mice and littermate WT mice were randomly divided into the following 6 groups:Con,NPRC-/-+Con,Sac/val+Con,DM,NPRC/-+DM,Sac/val+DM.2.Construction of mice with DCMThe same as Dissertation Ⅰ.3.Method of drug deliveryThe sac/val tablets were ground and dissolved in 0.5%methylcellulose,and the mice in the Sac/val+Con and Sac/val+ DM groups were given an intragastric administration of 60mg/kg/d sac/val.The mice in other groups were given 0.5%methylcellulose solution of the same volume.The intragastric administration started from the day that diabetes was successfully induced to the day of euthanasia.4.EchocardiographyThe same as Dissertation Ⅰ.5.Collection of mouse tissues and pathological examinationAfter 16 weeks of diabetes inducement,mice were euthanized,and mouse serum was collected,centrifuged,and frozen at-80℃.Heart tissues were either stored in liquid nitrogen and transferred to-80℃ or fixed in 4%paraformaldehyde for subsequent histopathological examination.After that,the heart tissues were embedded in paraffin,and cut into cross-sections for HE staining,Masson staining,immunohistochemical(IHC)detection of Collagen Ⅰ,Collagen Ⅲ,p-VASP,p-CREB,p-PKA substrates.6.Primary cell isolation and cultureNeonatal C57BL/6J mice were used for the isolation of cardiac fibroblasts by differential attachment technique.The isolated primary cells were cultured in the low glucose DMEM medium containing 10%fetal bovine serum.All cells were cultured in a 37℃ incubator containing 5%CO2.Cells were grouped as follows:(1)Si-NC+NG group:cells were transfected with negative control siRNAs,followed by DMSO and normal concentration of glucose(NG)treatment.(2)Si-NPRC+NG group:cells were transfected with NPRC siRNAs,followed by DMSO and NG treatment.(3)LCZ696+NG group:cells were transfected with negative control siRNAs,followed by LCZ696(10μM)and NG treatment.(4)Si-NC+HG group:cells were transfected with negative control siRNAs,followed by DMSO and high concentration of glucose(HG,final concentration was 33.3mM)treatment.(5)Si-NPRC+HG group:cells were transfected with NPRC siRNAs,followed by DMSO and HG treatment.(6)LCZ696+HG group:cells were transfected with negative control siRNAs,followed by LCZ696(10μM)and HG treatment.7.Cell proliferation assayThe same as Dissertation Ⅰ.8-Western blotTotal cell protein was extracted using lysis buffer and the protein concentration was determined by the BCA method.SDS-polyacrylamide gel electrophoresis was performed,followed by the transfer of protein to the PVDF membranes by the semidry transfer method.The membranes were blocked and incubated with primary antibodies at 4℃ overnight.The next day,the membranes were washed and incubated with secondary antibodies,followed by detection using a chemiluminescent substrate.9.Statistical analysisThe normality assumption of the data distribution was assessed.Continuous variables with normal distribution were presented as mean ± SEM.Two-way ANOVA followed by Tukey’s post-hoc test was used to compare multiple groups with more than one variable.For all statistical comparisons,p<0.05 was considered statistically significant.All analyses were performed with GraphPad Prism 8 software.Results1.General conditions of experimental animalsAfter 16 weeks of diabetes inducement,we measured the body weight and fasting blood glucose levels of the mice in 6 groups(Con group,NPRC-/-+Con group,sac/val+Con group,DM group,NPRC-/-+DM group,Sac/val+DM group).The results showed that compared with the Con group,the diabetic mice had decreased body weight and increased blood glucose levels,but neither NPRC deficiency nor sac/val gavage affected the body weight and blood glucose levels of the mice.The systolic blood pressure,diastolic blood pressure,and heart rate of the mice in 6 groups were measured.The systolic blood pressure of NPRC-/-mice was lower than that of WT mice(Con vs NPRC-/-+Con;DM vs NPRC-/-+DM).And the systolic blood pressure of sac/val-treated mice in the control group and DM group was also lower than that of WT mice in the control group and DM group(Con vs Sac/val+Con;DM vs Sac/val+DM).2.NPRC deficiency more significantly improved cardiac function and remodeling in diabetic mice than sac/val treatmentEchocardiography of the mice in 6 groups(Con group,NPRC-/-+Con group,Sac/val+Con group,DM group,NPRC-/-+DM group,Sac/val+DM group)showed that LVEF,LVFS,and E/A of the diabetic mice were significantly decreased than that of the mice in Con group while LVEDD and LVESD were significantly increased.NPRC deficiency and sac/val treatment both improved the above parameters,suggesting that both exerted protective effects on cardiac function and remodeling in diabetic mice.However,compared with the mice in Sac/val+DM group,the LVFS of the mice in the NPRC-/-+DM group was higher while the LVEDD and LVESD were lower,suggesting that the systolic function and remodeling of the NPRC-/-diabetic mice were more significantly improved than sac/val-treated diabetic mice.3.NPRC deficiency more significantly improved heart size and morphology in diabetic mice than sac/val treatmentPhotographs of mouse hearts and analyses of heart weight/body weight showed that NPRC deficiency and sac/val treatment both effectively improved the heart size in diabetic mice,but NPRC deficiency more significantly improved the heart size than sac/val treatment.4.NPRC deficiency more significantly attenuated cardiac fibrosis in diabetic mice than sac/val treatmentSirius red staining of cardiac tissues showed that diabetes increased collagen accumulation in the cardiac perivascular and interstitial spaces in the DM group,compared with the NPRC-/-+DM and sac/val+DM groups.Further comparison showed that NPRC deficiency more significantly inhibited cardiac fibrosis than sac/val treatment.IHC for collagen Ⅰ and collagen Ⅲ showed similar results.5.NPRC deficiency more significantly inhibited the collagen synthesis in cardiac fibroblasts than LCZ696 treatmentWestern blot showed that HG treatment increased the expression of collagen Ⅰ and collagen Ⅲ in cardiac fibroblasts.The expression of collagen Ⅰ and collagen Ⅲ in siNPRC+HG and LCZ696+HG groups were decreased than that in the si-NC+HG group.However,compared with LCZ696 treatment,the levels of collagen Ⅰ and collagen Ⅲ were lower in NPRC-deficient cells,suggesting that NPRC deficiency more significantly inhibited the collagen synthesis in cardiac fibroblasts than LCZ696 treatment.6.NPRC deficiency more significantly inhibited the proliferation of cardiac fibroblasts than LCZ696 treatmentThe EdU and CCK-8 assays indicated that HG treatment promoted the proliferation of cardiac fibroblasts in the si-NC+HG group,which was decreased by NPRC deficiency in the si-NPRC+HG group and LCZ696 treatment in the LCZ696+HG group.Meanwhile,the proliferation of cardiac fibroblasts in the si-NPRC+HG group was decreased than that in the LCZ696+HG group,suggesting NPRC deficiency more significantly inhibited the proliferation of cardiac fibroblasts than LCZ696 treatment.7.NPRC deficiency more significantly inhibited TGF-β1/Smad signaling in cardiac fibroblasts than LCZ696 treatmentWestern blot showed that the protein levels of TGF-β1,p-Smad2,and p-Smad3 were decreased by NPRC knockdown in the si-NPRC+HG group.Although the protein levels were also slightly decreased in the LCZ696+HG group,the extent of reduction was not as significant as that in the si-NPRC+HG group,suggesting that NPRC deficiency more significantly inhibited TGF-β1/Smad signaling in cardiac fibroblasts than LCZ696 treatment.8.NPRC deficiency more significantly activated PKG signaling in cardiac tissues and fibroblasts than sac/val treatmentIHC of cardiac tissues for p-VASP showed that the expression of p-VASP in NPRC-/-mice were increased than that of WT mice and sac/val-treated mice,regardless of the presence of diabetes.In addition,the expression of p-VASP in sac/val-treated mice was also increased than that of WT mice,suggesting that both treatments could activate PKG signaling in cardiac tissues but were more significant by NPRC deficiency.Western blot of cardiac fibroblasts showed that the protein expression of p-VASP in NPRC-deficient cells was remarkedly increased than the rest,showing similar results to the in vivo results.9.NPRC deficiency activated PKA signaling in cardiac tissues and fibroblastsIHC of cardiac tissues for p-CREB and p-PKA substrates showed that the expression of p-CREB and p-PKA substrates in NPRC-/-mice were increased than that of WT mice and sac/val-treated mice,regardless of the presence of diabetes.Meanwhile,there was no significant difference between WT mice and sac/val-treated mice,suggesting that sac/val did not affect PKA signaling in vivo.Western blot of cardiac fibroblasts showed that the protein expression of p-CREB and p-PKA substrates in NPRC-deficient cells was more remarkedly increased than the rest,showing similar results to the in vivo results.Conclusions1.NPRC deficiency more significantly attenuated cardiac fibrosis and improved cardiac function in diabetic mice than sac/val treatment.2.NPRC deficiency more significantly inhibited collagen synthesis and proliferation of cardiac fibroblasts than LCZ696 treatment.3.The reason for the greater benefit of NPRC deficiency over Sac/val treatment was related to the activation of both PKG and PKA pathways by NPRC deficiency. |
