CCAAT/enhancer‐binding protein β overexpression alleviates myocardial remodelling by regulating angiotensin‐converting enzyme‐2 expression in diabetes

Abstract Diabetic cardiomyopathy, a major cardiac complication, contributes to heart remodelling and heart failure. Our previous study discovered that CCAAT/enhancer‐binding protein β (C/EBPβ), a transcription factor that belongs to a family of basic leucine zipper transcription factors, interacts with the angiotensin‐converting enzyme 2 (ACE2) promoter sequence in other disease models. Here, we aimed to determine the role of C/EBPβ in diabetes and whether ACE2 expression is regulated by C/EBPβ. A type 1 diabetic mouse model was generated by an intraperitoneal injection of streptozotocin. Diabetic mice were injected with a lentivirus expressing either C/EBPβ or sh‐C/EBPβ or treated with valsartan after 12 weeks to observe the effects of C/EBPβ. In vitro, cardiac fibroblasts and cardiomyocytes were treated with high glucose (HG) to investigate the anti‐fibrosis, anti‐apoptosis and regulatory mechanisms of C/EBPβ. C/EBPβ expression was down‐regulated in diabetic mice and HG‐induced cardiac neonatal cells. C/EBPβ overexpression significantly attenuated collagen deposition and cardiomyocyte apoptosis by up‐regulating ACE2 expression. The molecular mechanism involved the binding of C/EBPβ to the ACE2 promoter sequence. Although valsartan, a classic angiotensin receptor blocker, relieved diabetic complications, the up‐regulation of ACE2 expression by C/EBPβ overexpression may exert greater beneficial effects on patients with diabetic cardiomyopathy.


Introduction
Diabetic cardiomyopathy (DCM), which is characterized by left ventricular (LV) dilatation and systolic dysfunction, occurs independently of recognized causes, such as coronary artery disease, valve disease, arterial hypertension or other cardiovascular diseases [1][2][3]. LV remodelling is one of the major pathological mechanisms that ultimately lead to congestive heart failure. Numerous mechanisms are involved in the formation and development of LV remodelling in patients with DCM, including myocardial fibrosis, cardiac hypertrophy, mitochondrial damage, inflammatory, apoptosis and activation of the renin-angiotensin system (RAS) [4].
Homeostasis of the RAS is based on a balance between the angiotensin-converting enzyme (ACE)-angiotensin (Ang) II-angiotensin II receptor type 1 (AT1R) axis and the ACE2-Ang(1-7)-Ang1-7 receptor (MasR) axis [5]. ACE2, a homologue of ACE, has been shown to exert anti-fibrosis and anti-hypertrophy effects and to reduce LV remodelling in patients with type 1 diabetes [6,7]. ACE2 catalyses the cleavage of Ang II to Ang(1-7) and then counteracts endogenous ACE by activating MasR [8,9]. Based on the results of clinical trials and experimental studies, the activation of the RAS is associated with the development of LV remodelling in patients with DCM [10,11]. However, the exact mechanism between RAS and DCM remains poorly understood.
CCAAT/enhancer-binding protein b (C/EBPb), a transcription factor that belongs to a family of basic leucine zipper transcription factors, affects cell growth and differentiation [12,13]. Reduced C/EBPb expression up-regulates the expression of a GATA-binding protein 4, T-box transcription factor 5 (Tbx5), NK 2 homeobox 5 (Nkx2.5), a-myosin heavy chain (a-MHC), troponin I (TnI) and troponin T (TnT), all of which are hypertrophy-related genes [14]. According to our previous study, C/EBPb binds the ACE2 promoter sequence and decreases ACE2 expression in Ang II-treated cells, indicating that C/ EBPb might also regulate ACE2 expression in DCM. We suggested that C/EBPb overexpression may attenuate collagen accumulation, apoptosis and LV remodelling by regulating ACE2 synthesis and other RAS members in the mouse model of type 1 diabetes.

Animal protocol
All animal experiments were conducted in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals. The protocol was approved by the Animal Care and Use Committee of Shandong University Qilu Hospital. After 1 week of acclimation, 8-week-old male C57BL mice (Beijing Hua Fu Kang Biological Polytron Technologies Inc, Beijing, China) were randomized into the control group (n = 20) and the treatment group (n = 80). Type 1 diabetes was induced in the treatment group by the intraperitoneal injection of 50 mg/kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA) for five consecutive days. Meanwhile, the mice in the control group received intraperitoneal injections of a solvent (0.1 mol/l sodium citrate, pH 4.5). Random blood glucose measurements greater than 16.7 mmol/l in the treatment group for 3 days indicated the successful induction of type 1 diabetes (ACCU-CHEK Active; Roche, Indianapolis, IN, USA). After 12 weeks, the diabetic mice were randomized into the following four groups: (i) DM + shRNA negative control (N.C.) (n = 20), (ii) DM + C/EBPb (n = 20), (iii) DM + sh-C/EBPb (n = 20) and (iv) DM + valsartan (n = 20). Mice in the indicated groups were injected with 1 9 10 7 UT/30 ll of lentivector containing sh-N.C., C/ EBPb or sh-C/EBPb (GENECHEM, Shanghai, China) through the caudal vein. Valsartan (30 mg/kg; Novartis, Beijing, China) dissolved in normal saline was administered by gavage to the mice in the valsartan group [15]. Sixteen weeks after the first STZ injection, all the mice were killed.

Echocardiography
The heart function and dimension parameters were measured using a standard protocol after 16 weeks by transthoracic parasternal echocardiography using the VEVO770 imaging system (VisualSonics, Toronto, ON, Canada). LV parameters, including the left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall thickness (LVPWd), left ventricular ejection fraction (LVEF) and fractional shortening (FS), were measured in M-mode via the long/short axis view. The ratio of the early peak (E, mm/sec.) to the late peak (A, mm/sec.) mitral flow velocities was determined using pulsed-wave Doppler echocardiography.

Histology and immunohistochemistry
After fixation with 4% paraformaldehyde, dehydration with an alcohol gradient and embedding in paraffin, the heart tissues were cut into 4.5 lm sections. Sections were stained with haematoxylin and eosin (H&E) to measure the cardiomyocyte width and with Masson's trichrome to assess the collagen content. Immunohistochemical staining was performed on sections using a previously described method [16]. Sections were incubated with the following primary antibodies at the appropriate concentrations overnight at 4°C: anti-C/EBPb, anti-ACE2, anti-ACE, anti-transforming growth factor-b1 (TGF-b1), anti-collagen I and anti-collagen III (all from Abcam, Cambridge, MA, USA). The secondary antibodies were used according to the manufacturer's specifications. Images of the LV sections were obtained at 4009 magnification and measured using the computer software ImagePro Plus 6.0.2 (Media Cybernetics, Houston, TX, USA).

Statistical analysis
All data from at least three independent experiments were expressed as mean AE SD, and intergroup differences were analysed using a one-way ANOVA via SPSS software 18.0 (SPSS, Chicago, IL, USA). P < 0.05 was regarded as statistically significant.

Fasting blood glucose concentrations and morphometric profiles
As expected, 1 week after STZ injection, fasting blood glucose concentrations in diabetic mice showed a marked elevation that persisted until the end of the experiment (Table 1). Excessive water intake, excessive food intake and polyuria were observed in the diabetic mice, particularly in the DM + sh-N.C. and DM + sh-C/EBPb groups. Meanwhile, differences in body weight, heart weight and the ratio of heart weight to body weight were statistically significant among the five groups (Table 1). Thus, C/EBPb overexpression might reverse cardiac remodelling.

C/EBPb overexpression and the valsartan treatment ameliorated myocardial remodelling
Echocardiography was employed to evaluate cardiac function at the end of the experiment. LVEF, FS and the E/A ratio were substantially decreased, and LVEDd and LVPWd were increased in the DM + sh-N.C. group compared with those in the controls. Compared with the DM + sh-N.C. group, the DM + C/EBPb and DM + valsartan groups exhibited improvements in LVEF, FS and the E/A ratio and decreases in LVEDd and LVPWd, but the DM + sh-C/ EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 1A-F). The increased heart size and cardiomyocyte width were reduced by C/EBPb overexpression and valsartan (P < 0.05; Fig. 1G-J). Although the valsartan treatment improved cardiac function indices and attenuated heart size and cardiomyocyte width, C/EBPb overexpression had a much better effect on myocardial remodelling.

C/EBPb overexpression ameliorates extracellular matrix deposition in vitro and in vivo
Masson's trichome staining of cardiac sections revealed the elevated expression of the extracellular matrix (ECM) in the interstitial areas of diabetic mice compared to the expression in the control mice. C/EBPb overexpression and the valsartan treatment dramatically reduced collagen deposition in the intramyocardial and perivascular regions compared to diabetic mice that were transfected with sh-N.C. Additionally, the expression was much lower in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/ EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 2A).
The induction of diabetes increased the accumulation of the fibrotic markers collagen I, collagen III and TGF-b1 compared to that in healthy controls. C/EBPb overexpression and the valsartan treatment reduced the levels of collagen and TGF-b1 compared to those in the DM + sh-N.C. group, and collagen was expressed at much lower levels in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group. The effects of all groups were confirmed by immunohistochemistry and Western blotting (P < 0.05; Figs 2B,C and D and 3A,B and D).
According to the Western blot results, MMP-9 expression was not significantly different from all groups (P < 0.05; Fig. 3C). But C/EBPb overexpression and the valsartan treatment ameliorated the diabetesinduced reduction in MMP-2 levels, and MMP-2 was expressed at much higher levels in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 3E). Consistent with the Western blot results, the ELISA showed that the serum MMP-9 levels were not significantly different from all groups, whereas C/EBPb overexpression and the valsartan treatment increased the serum MMP-2 levels compared to those in the DM + sh-N.C. group, and MMP-2 levels were much higher in the C/ EBPb overexpression group than in the valsartan group. The sh-C/ EBPb treatment decreased MMP-2 levels, but the difference was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 3F and G). Consistent with the alterations observed in diabetic mice, the expression of the collagen I and III and TGF-b1 proteins was  increased in high glucose-treated CFs. Both C/EBPb overexpression and the valsartan treatment attenuated the up-regulation of the collagen I and III and TGF-b1 compared to expression in the HG + sh-N.C. group, and collagen was expressed at lower levels in the C/EBPb overexpression group than in the valsartan-treated group. The HG + sh-C/EBPb group was not significantly different from the HG + sh-N.C. group (P < 0.05; Fig. 3H-J). Likewise, the results of MMPs proteins expression in tissue were verified by exposing CFs to HG (P < 0.05; Fig. 3K and L). Additionally, significant differences in activity of MMP-9 were not observed among the groups, but C/EBPb overexpression and the valsartan treatment both remarkably ameliorated the HG-induced decrease in MMP-2 activity (P < 0.05; Fig. 3M and N).

C/EBPb overexpression suppresses apoptosis in the diabetic myocardium and H9C2 cardiomyocytes and decreases inflammatory factors expression in serum
The Bax/Bcl-2 ratio and AT2R expression were increased in vehicle-treated diabetic mice compared with expression in the healthy controls. C/EBPb overexpression effectively reduced the Bax/Bcl-2 ratio and AT2R expression compared to that in the DM + sh-N.C. group, whereas the valsartan treatment only exerted a significant effect on the Bax/Bcl-2 ratio. The apoptosis indices of the DM + sh-C/EBPb group were not significantly different from that of the DM + sh-N.C. group (P < 0.05; Fig. 4A and B). The results were verified by exposing H9C2 cardiomyocytes to HG (P < 0.05; Fig. 4C and D).
Serum IL-6 and MCP-1 levels were increased in the diabetic mice, but they were alleviated by C/EBPb overexpression and the valsartan treatment compared to the levels in the DM + sh-N.C. group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 4E and F).

Diabetes and the HG treatment decrease C/EBPb expression, and C/EBPb overexpression up-regulates ACE2 expression
The C/EBPb protein was expressed at lower levels in the sh-N.C. group than in the controls, but it was up-regulated in the C/EBPb overexpression group. The sh-C/EBPb and valsartan-treated groups showed no significant differences compared with the vehicle-treated group (P < 0.05; Fig. 5A). Meanwhile, the ACE2 protein was expressed at lower levels in the sh-N.C. group than in the control group. C/EBPb overexpression increased ACE2 protein expression compared to that with the sh-N.C. treatment. The sh-C/EBPb and valsartan-treated groups were not significantly different from the vehicle group in vivo (P < 0.05; Fig. 5B). Similar effects of C/ EBPb and ACE2 expressions were verified in CFs treated with HG and in the myocardium by immunohistochemistry (P < 0.05; Fig. 5D-G).

C/EBPb overexpression decreases Ang II and increases Ang(1-7) levels in DCM
The sh-N.C. treatment, C/EBPb silencing and valsartan treatment markedly increased myocardium Ang II levels in diabetic mice compared to the levels in the controls. C/EBPb overexpression alleviated the increased Ang II content compared to the sh-N.C. treatment. The levels of Ang (1-7) were significantly increased in the C/EBPb overexpression group and slightly increased in the valsartan-treated groups compared to the sh-N.C. group. The sh-N.C. treatment and C/EBPb silencing decreased the myocardium Ang(1-7) levels, but the difference was not statistically significant (P < 0.05; Fig. 6A and B). Serum Ang II and Ang(1-7) levels are consistent with those in tissue (P < 0.05; Fig. 6C and D).

Levels of the ACE, AT1R and MasR proteins in the diabetic myocardium and CFs
Diabetes significantly elevated the expression of the ACE and AT1R proteins and decreased the expression of the MasR protein. C/EBPb overexpression remarkably reduced levels of the ACE and AT1R proteins and increased levels of the MasR protein, but the valsartan and C/EBPb silenced groups were not significantly different from the sh-N.C. group (P < 0.05; Fig. 6E and F). The results were verified in HG-treated CFs (P < 0.05; Fig. 6G and H). The expression of ACE was tested consistently by Western blot in vivo and in vitro and verified by immunohistochemistry analysis (P < 0.05; Figs 5C and 6I and J).   C/EBPb overexpression alleviates fibrosis and apoptosis by up-regulating ACE2 levels Consistent with the changes described above, ACE2 was expressed at significantly higher levels in CFs in the si-N.C + C/EBPb group than in the sh-N.C. + si-N.C. group and decreased in the C/EBPb + si-ACE2 group compared to the levels in the si-N.C. + C/EBPb group. Additionally, the expression of collagen III and TGF-b1 was not reduced in the C/EBPb + si-ACE2 group compared to expression in the si-N.C + C/EBPb group, which corroborated our hypothesis regarding the C/EBPb-ACE2 pathway (P < 0.05; Fig. 7A-D).
ChIP assays were used to obtain further insights into the molecular interactions between C/EBPb and ACE2 following the HG treatment. The ACE2 promoter sequence was obviously enriched in the DNA immunoprecipitated with the anti-C/EBPb antibody, indicating that C/EBPb directly binds to the ACE2 promoter. Furthermore, the results verified that the binding of C/EBPb to the ACE2 promoter decreased following the HG treatment, further indicating that hyperglycaemia-induced ACE2 down-regulation may be directly caused by the weaker binding force between C/EBPb and the ACE2 promoter (P < 0.05; Fig. 7E). Based on these findings, C/EBPb regulates ACE2 expression by directly binding to its promoter.

Discussion
The major finding in our study was that C/EBPb overexpression ameliorated diabetes-induced myocardial remodelling by up-regulating ACE2 expression and modulating the expression of other members of the RAS.
The most important effect of C/EBPb on the successful model was the amelioration of DCM-induced fibrosis and remodelling. Enhanced fibrosis in cardiac tissue is a vital hallmark of cardiac dysfunction. Ang II, which is converted from Ang I by ACE, is one of the most important factors that contribute to the up-regulation of collagen expression in patients with diabetes [19,20]. Ang II stimulates collagen synthesis in fibroblasts and myofibroblasts via AT1R [21]. Candesartan inhibited the increased collagen I expression in HG-treated fibroblasts in a previous study, indicating that Ang II regulates HG-induced collagen deposition in CFs [22]. The up-regulation of ACE2 and Ang(1-7) expression alleviates the fibrosis and cardiac dysfunction in subjects with DCM [7,23]. In the present study, C/EBPb overexpression decreased Ang II levels and reduced collagen production and ECM deposition in HG-treated fibroblasts and diabetic mice. As shown in our previous study, diabetic mice and CFs both expressed increased levels of TGF-b1, which acts as a major mediator of cardiac remodelling by altering collagen metabolism and inducing cardiomyocyte hypertrophy [24,25]. TGF-b1 expression is induced by Ang II in cardiac cells [26,27]. Ang II inhibition has been reported to reduce ECM synthesis by modulating TGF-b1 expression in CFs and diabetic rats [28,29]. In the present study, levels of the TGF-b1 protein were significantly suppressed by C/EBPb overexpression or the valsartan treatment, whereas diabetes and sh-C/EBPb exacerbated fibrosis in vitro and in vivo by up-regulating TGF-b1 expression, indicating that C/EBPb overexpression reduces ECM deposition by decreasing Ang II-induced TGF-b1 expression.
An imbalance between ECM deposition and degradation in the heart plays a major role in fibrosis in DCM. The expression and activity of MMP-2 mediate collagen degradation and are down-regulated in diabetic mice and in CFs treated with HG or Ang II [7,25,30,31]. ACE2 overexpression reduces ECM deposition by increasing MMP-2 activity and expression [7]. MMP-2 primarily degrades collagen, which consists of fibrillary peptides and newly generated fibres. However, MMP-9, which is similar to MMP-2, also degrades collagen, although with lower proteolytic activity [25]. The sh-N.C. and sh-C/ EBPb treatment enhanced the reduced MMP-2 levels, whereas C/ EBPb overexpression increased MMP-2 expression and activity by up-regulating ACE2 expression, thus increasing the degradation of the ECM in mice with DCM. However, MMP-9 activity was not significantly altered among the groups in our study.
An increase in cardiomyocyte loss and hypertrophy underlies cardiac fibrosis and dysfunction in diabetes [32]. As expected, the Bax/ Bcl-2 ratio, which reflects the rate of apoptosis, was higher in diabetic mice and in HG-induced cardiomyocytes, consistent with previous findings [18]. Ang II triggers apoptosis and exacerbates cell growth, both of which are vital pathological features in DCM [33,34]. AT2R, which has controversial functions, was expressed at high levels in the diabetic mice and HG-induced CFs in our study. However, according to some studies, AT2R, like MasR, opposes AT1R activity and counteracts the negative effects induced by AT1R [35]. An increase in AT2R levels accompanied by a reduction in Bcl-2 levels was previously observed in DCM. Specific stimulation of AT2R after serum starvation exacerbates apoptosis [36]. In addition, AT2R exerts a proapoptotic effect on neonatal cardiomyocytes and R3T3 mouse fibroblasts [37]. Based on our results, AT2R promoted apoptosis in diabetes. C/EBPb overexpression not only decreased the Bax/Bcl-2 ratio but also reduced AT2R expression in DCM. Although the exact mechanism by which C/EBPb regulated AT2R was not investigated in the present study, our findings suggest a new method for studying apoptosis in DCM.
Based on our study and previous research, the mechanisms by which C/EBPb alleviated fibrosis and apoptosis were mainly due to the up-regulation of ACE2 expression and reduction of Ang II levels. In a previous study, ACE2 was recognized as an important modulator of DCM [38]. However, as an enzyme, the development of ACE2 as a clinical drug is very difficult due to its instability and ease of degradation. Therefore, future studies should focus on identifying a factor that induces ACE2 expression or activity. Using ChIP assays, we revealed a new regulatory mechanism in which C/EBPb binds to the ACE2 promoter sequence and enhances ACE2 production as a transcription factor. The HG treatment reduced the expression of C/ EBPb and its binding to the ACE2 promoter. Meanwhile, the expression of fibrosis markers was not reduced following treatment with C/ EBPb + ACE2-siRNA. Therefore, following up-regulation by C/EBPb, ACE2 catalyses the cleavage of Ang II to Ang(1-7) and inhibits ACE function. Ang(1-7) alleviates fibrosis and cardiac dysfunction by activating MasR, leading to reductions in ACE, AT1R and AT2R levels, as verified in previous studies [7,23]. However, we did not comprehensively examine whether the effects of C/EBPb suppression on fibrosis and apoptosis in DCM depended on ACE2 completely, and the new mechanism by which C/EBPb regulates DCM warrants further investigation.
Valsartan and other ARBs have been shown to prevent DCM. We also believe that the effects of valsartan and other ARBs on relieving DM are undeniable. However, ARBs have been shown to relieve diabetic complications by specifically blocking rather than circulating intracellular Ang II. Furthermore, intracellular Ang II is more relevant to fibroblasts in diabetic hearts, and several research studies have indicated that some effects of intracellular Ang II are not inhibited by ARBs [7,39]. In our study, C/EBPb up-regulated ACE2 expression, ACE2 catalysed the cleavage of Ang II to Ang(1-7) and decreased intracellular Ang II level and counteracted the effects of ACE on CFs and cardiomyocytes in ameliorating diabetic fibrosis and cardiac dysfunction, but valsartan had no effect on ACE2 expression [7]. As AT1R antagonists, valsartan only effects on AT1R and has no effect on AT2R and MasR according to its pharmacological action. C/EBPb up-regulated Ang(1-7), which decreased ACE, AT1R and AT2R levels, and up-regulated MasR expression levels [7,23], which ultimately prevented DCM progression, whereas valsartan increased Ang(1-7) levels much lower than C/EBPb. Overall, C/EBPb decreased intracellular Ang II level by up-regulating ACE2 and Ang (1-7) levels, indicating that C/EBPb may be more effective in treating DCM than ARBs.
In conclusion, we first investigated the role of C/EBPb as a transcription factor that promotes ACE2 expression and C/EBPb overexpression as a protective factor against fibrosis and apoptosis by upregulating ACE2 expression in DCM. Additionally, the underlying mechanisms involve the down-regulation of the ACE-Ang II-AT1R axis and the up-regulation of the ACE2-Ang(1-7)-MasR axis that directly suppressed collagen deposition and apoptosis. Furthermore, C/EBPb-induced ACE2 up-regulation is more efficacious in treating DCM than ARBs. Our study revealed a novel potential therapeutic target for the amelioration of cardiac dysfunction and remodelling in patients with DCM.