KLF15 Negatively Regulates Cardiac Fibrosis and SDF-1β Attenuates Cardiac Fibrosis Through KLF15 Pathway in Type 2 Diabetic Mice

Yuanyuan Tian Jilin University First Hospital Zhenyu Wang Jilin University First Hospital Xiangyu Zheng Jilin University First Hospital Shihuan Cheng Jilin University First Hospital Wenjing Song Jilin University First Hospital Zhe Jing Jilin University First Hospital Lu Cai University of Louisville School of Medicine Madhavi Rane University of Louisville School of Medicine Yuguang Zhao (  zhaoyuguang@jlu.edu.cn ) Jilin University First Hospital

lead to DCM and heart failure (5). Therefore, identifying mechanisms regulating myocardial brosis caused by T2D, can lead to identi cation of therapies to prevent or to inhibit progression of myocardial brosis, to prevent or delay the occurrence of DCM in T2D patients.
In 2005, Balabanian et al revealed that SDF-1 can bind to the second chemokine receptor CXCR7 with a 10-fold higher a nity than CXCR4 (15). In humans, CXCR7 is expressed in heart, brain, endothelium, kidney, and tumor cells (16,17). Previous studies have shown that SDF-1/CXCR4 signaling has a protective effect on the survival of hypoxic cardiomyocytes after myocardial infarction and ischemia/reperfusion injury (18)(19)(20). However, recent studies have implicated that CXCR7 and CXCR4 mediate distinct functions and can also play an important role in mediating cell survival or the antiapoptotic effect of SDF-1 (14). In our previous study, we have demonstrated that SDF-1β protects against cardiac apoptosis via CXCR7 in T2D animal model (21). Currently, the role of SDF-1β in modulating myocardial brosis in T2D mice is not known. Therefore, research examining the role of SDF-1β in delaying or preventing myocardial brosis in T2D mice along with identifying the mechanisms underlying these effects is necessary.
Krüppel-like factors (KLFs) as a subfamily of the zinc-nger class of DNA binding transcriptional regulators (22), played diverse roles in the differentiation and development of various mammalian cells, such as regulating gene expression, growth, and differentiation (23). The transcriptional factor KLF15 is widely expressed, with abundant expression in liver, kidney, heart and skeletal muscle.
Immunohistochemistry revealed that KLF15 protein is located in interstitial broblasts of heart and skeletal muscle and in potentially brogenic cells such as the mesangial cells in the kidneys or stellate cells in the liver, therefore, it is reasonable to postulate that KLF15 may be involved in the brogenesis in these organs (24). Under pathophysiological conditions, KLF15 repressed expression of connective tissue growth factor (CTGF), tissue growth factor-β (TGF-β), and myocardin-related transcription factor-A in cardiac broblasts, leading to the alleviation of cardiac brosis and improvement of cardiac function. Accordingly, a low expression of KLF15 could promote brotic remodeling during pathological left ventricular hypertrophy (LVH) (25,26). In experimental models, Fisch et.al have demonstrated KLF15 expression in the heart and that KLF15 functioned as a transcriptional repressor in pathological cardiac hypertrophy through inhibition of the myocyte enhancer factor 2 (MEF2) and GATA binding protein 4 (GATA4) transcriptional pathway (27). Wang et.al also demonstrated that adenoviral overexpression of KLF15 signi cantly inhibited the basic expression of CTGF in neonatal rat ventricular broblasts and as well as CTGF expression induced by TGF-β1. After ligating the aorta of Klf15 knockout mice, the expression of CTGF in the heart increased and the degree of cardiac brosis increased (26). In 2017, for the rst time, a human study indicated the potential importance of KLF15 in promoting cardiopathological changes in the patients with T2D. In this study, they associated Klf15 SNP rs9838915 A allele with increased left ventricular (LV) mass in patients with T2D and these ndings were replicated in a large independent cohort (28). These studies promoted us to question whether KLF15 plays an important role in the left ventricular hypertrophy of patients with T2D (29). In our previous study, we have demonstrated that SDF-1β protects against cardiac apoptosis via CXCR7 in T2D animal model (21). Therefore, in the current study we hypothesize that SDF-1β could alleviate T2D-induced myocardial brosis in a KLF15 dependent manner. In the following research, we examined the protective effect of SDF-1β on T2D-induced myocardial brosis and systematically investigated the function of KLF15 in this process.
Leenders et.al revealed that TGF-β1 down-regulated KLF15 expression in cardiomyocytes in a p38 mitogen activated protein kinase (p38 MAPK) dependent manner and promoted ventricular hypertrophy (30). However, they did not examine the involvement of a precise p38 MAPK isoform. There are four members of the p38 MAPK family in mammals: α, β, γ and δ (31). Of the four known isoforms of p38 MAPKs, p38α and p38β are abundant in cardiomyocytes, while no expression of p38γ and p38δ isoforms was detected in the heart (32). In contrast, we have demonstrated that p38β MAPK activation was required for SDF-1β mediated cardiac protection from palmitate-induced cardiac cell apoptosis (21). In the current study, we evaluated the protective role of SDF-1β on T2D-induced myocardial brosis. Moreover, we also examined whether p38 MAPK mediated the protective effects of SDF-1β and if so, which isoform of p38 MAPK was involved in this process.
The dose of SDF-1β used in the present study was based on our previous study. The control mice were given the same volume of vehicle (1% dimethyl sulfoxide diluted with PBS). All T2D and age-matched control mice continually received their HFD or ND for an additional 3 months. After 3 months of SDF-1β or vehicle treatment, their cardiac function was measured, after which animals were euthanized and heart tissues were collected. Echocardiography Transthoracic echocardiography was measured by a Vevo 770 ultrasound system (Visualsonics, Toronto, Canada) equipped with a high frequency ultrasound probe (RMV-707B) as described previously (36). Mice were anesthetized with intraperitoneal injection of 1.2% Avertin (Sigma, St. Louis, MO) and placed in the supine position on a temperature-controlled platform. The chest hair was removed with a depilatory to reduce ultrasound attenuation. The images were recorded in parasternal long-axis and short-axis views. The LV wall thicknesses and dimensions were measured in parasternal short axis M-mode images. At the same time, the ejection fraction (EF), fractional shortening (FS), and LV mass were calculated using Vevo770 software. The data were averaged over 10 cardiac cycles.

Western blotting
The cardiac tissues were homogenized in lysis buffer and proteins were collected by centrifuging at 12,000 × g at 4 °C (37). Western blots were performed according to our previous studies (37). Brie y, the proteins were separated on 10% SDS-PAGE gels, and then were transferred to a nitrocellulose membrane. Quantitative densitometry was performed on the identi ed bands using a computer-based measurement system, as employed in previous studies (37).

Statistical analysis
Data were collected from the repeated experiments at least three times for in vitro studies and six animals at least for in vivo study and were presented as mean ± SD. One-way analysis of variance (ANOVA) was used to determine whether differences exist and if so, a post hoc Tukey's test was used for analysis of the difference between groups, with Origin 7.5 laboratory data analysis and graphing software. Statistical signi cance was considered as p < 0.05.

SDF-1β inhibited myocardial brosis via its receptor CXCR7 activation and by p38β MAPK-mediated KLF15 up-regulation in H9C2 cells
For the in vitro study, embryonic rat heart derived H9C2 cells were exposed to palmitate at 62.5 µM as our previous study (21). Exposure to palmitate induced signi cant KLF15 down-regulation and signi cant CTGF up-regulation in H9C2 cells (Fig. 1A). The effect of SDF-1β at 100 nM (21) on palmitate-induced brosis was also examined in H9C2 cells (Fig. 1A). SDF-1β signi cantly prevented palmitate induced KLF15 down-regulation as well as CTGF up-regulation in H9C2 cells (Fig. 1A). Furthermore, to explore the direct role of KLF15 in mediating SDF-1β's protective role on palmitate-induced H9C2 cells brosis, H9C2 cells were transfected with control siRNA or KLF15 siRNA followed by co-treatment with SDF-1β at 100 nM and palmitate at 62.5 µM for 15 h. Cell lysates were immunoblotted with anti-KLF15, anti-CTGF and anti-β-actin antibodies. Figure 1B illustrated successful down-regulation of KLF15 in KLF15 siRNA transfected cells but not in control siRNA transfected cells. Palmitate induced CTGF expression was inhibited in the presence of SDF-1β in control siRNA treated H9C2 cells but not in KLF15 siRNA transfected H9C2 cells (Fig. 1B). To directly examine the role of KLF15 in modulating palmitate-induced CTGF expression, H9C2 cells were treated with KLF15 recombinant adenovirus (rAV) or control rAV. KLF15 rAV over-expression in H9C2 cells was documented by Western-blot, KLF15 expression was markedly increased in KLF15 rAV treated cells but not in control rAV treated cells (Fig. 1C). KLF15 rAV treatment signi cantly inhibited palmitate-induced CTGF up-regulation (Fig. 1C).
In our previous study, we demonstrated that SDF-1β protected against palmitate-induced cardiac apoptosis via CXCR7-mediated p38β MAPK activation (21). Therefore, CXCR7 siRNA and p38β MAPK siRNA were used respectively to de ne the speci c role of CXCR7 and p38β MAPK on SDF-1β's protective effect against palmitate-induced cardiac brosis (Fig. 2). CXCR7siRNA or control siRNA were transfected into H9C2 cells for 48 h followed by treatment with palmitate and/or SDF-1β followed by immunoblotting with appropriate antibodies. Figure 2A demonstrated e cient silencing of CXCR7 expression in H9C2 cells in CXCR7 siRNA transfected but not in control siRNA transfected H9C2 cells. Silencing CXCR7 expression, but not control siRNA, abolished SDF-1β protection from palmitate-induced myocardial brosis, as measured by the expression of KLF15 and CTGF ( Fig. 2A). Moreover, silencing p38β MAPK expression (Fig. 2B), but not control siRNA, completely abolished the protective effect of SDF-1β on palmitate-induced myocardial brosis, as measured by the expression of KLF15 and CTGF (Fig. 2B). Similar studies were carried out using CXCR4 siRNA and p38α MAPK siRNA, however, neither CXCR4 siRNA nor p38α MAPK siRNA affected SDF-1β protection from palmitate-induced myocardial brosis measured by the expression of KLF15 and CTGF (Fig. 3A-B).

Conditional knockout of cardiac speci c Klf15 gene aggravated the cardiac dysfunction and
weakened protection of SDF-1β in T2D mice Echocardiography (Table 1) revealed that T2D induced cardiac dysfunction and Klf15-cKO could also lead to cardiac dysfunction. Klf15-cKO aggravated T2D-induced cardiac dysfunction. The cardiac dysfunction was con rmed by decreased EF and FS (Table 1). SDF-1β treatment signi cantly improved EF and FS of the WT group, and it also signi cantly improved EF in Klf15-cKO group (Table 1), thus demonstrating that the protective effect of SDF-1β on DCM was not always dependent on the KLF15 pathway.
3.3 Conditional knockout of cardiac speci c Klf15 gene aggravated the cardiac brosis and abolished protection of SDF-1β in T2D mice To validate the in vitro ndings we examined effects of in vivo loss of KLF15 expression with or without T2D on cardiac brosis. To this end, Klf15-cKO and WT mice were fed with HFD for 3 months to induce insulin resistance, and then these mice were injected with a single dose of STZ at 100 mg/kg body weight to induce T2D mouse model. After the onset of hyperglycemia, all T2D and age-matched control mice were treated with or without SDF-1β at 5 mg/kg body-weight twice a week for 3 months. At the end of the treatment, although blood glucose levels, total cholesterol and triglyceride levels were signi cantly increased in the T2D group both in WT group and Klf15-cKO group, SDF-1β treatment did not modulate these levels in both control and T2D groups (Fig. 4A-C). However, T2D as well as SDF-1β treatment did not affect the heart weight/tibia length ratio (Fig. 4D). Fibrotic response was examined by Sirius-red staining of collagen (38) (Fig. 4E). T2D was associated with a signi cant increase in cardiac brosis and Klf15-cKO signi cantly aggravated the damage of diabetes to myocardium, as shown by Sirius-red staining of collagen deposition (Fig. 4E). SDF-1β treatment signi cantly, but incompletely prevented the diabetes-induced cardiac brosis in WT group. However, in Klf15-cKO group, cardiac brosis was not alleviated by SDF-1β treatment (Fig. 4E). These results suggest that T2D-induced down-regulation of KLF15 was critical to cardiac brosis and the protective effect of SDF-1β on cardiac brosis was mediated by upregulating KLF15 expression in the heart, thus making KLF15 a key regulator of the cardioprotective effect of SDF-1β.
Next, the expressions of KLF15 and CTGF in the cardiac tissues of the Klf15-cKO and WT mice were measured by Western blotting. In WT group, T2D signi cantly decreased KLF15 protein level and signi cantly increased CTGF protein level (Fig. 5A), and SDF-1β signi cantly prevented the downregulation of KLF15 expression and up-regulation of CTGF caused by T2D (Fig. 5A). Klf15-cKO signi cantly increased CTGF expression compared to the WT group, while the expression of CTGF was not different among groups with and without SDF-1β in Klf15-cKO/T2D mice group (Fig. 5A).
In both WT group and Klf15-cKO groups, T2D signi cantly increased collagen I, bronectin and TGF-β1 protein levels in the cardiac tissues, and in WT group, SDF-1β signi cantly inhibited T2D-induced collagen I, bronectin and TGF-β1 up-regulation. In Klf15-cKO/T2D mice, a more signi cant increase in collagen I, bronectin and TGF-β1 protein levels were detected as compared to Klf15-cKO group, and the expression of collagen I, bronectin and TGF-β1 were unaffected by SDF-1β treatment in Klf15-cKO/T2D mice (Fig. 5B).

Discussion
Cardiac brosis is a hallmark feature of pathologic remodeling of the heart in response to hemodynamic or neurohormonal stress. In DCM, the pathogenesis is multifactorial and myocardial brosis is the frequently proposed mechanism to explain cardiac changes (42). The anti-brosis action of KLF15 is an important part of the cardioprotective mechanisms (24,26,27). Previous studies have shown that SDF-1 has a protective effect on the survival of hypoxic cardiomyocytes after myocardial infarction and ischemia/reperfusion injury (18)(19)(20)43). In the current study we demonstrated that SDF-1β signi cantly inhibited myocardial brosis and signi cantly prevented the down-regulation of myocardial KLF15 expression caused by T2D (Fig. 5). Our ndings shed new light on the mechanisms by which SDF-1β attenuates cardiac brosis through KLF15 dependent pathway in T2D mice. Here, we presented evidences that support the regulatory effect of KLF15 by SDF-1β in T2D mice.
KLF15 is highly expressed in the mouse heart tissue (44). Fisch et.al have identi ed KLF15 as a novel inhibitor of the heart's response to pressure overload through inhibition of the MEF2 and GATA4 transcriptional pathway (27). Wang et.al found that adenoviral overexpression of KLF15 also signi cantly inhibited the basal expression of CTGF of neonatal rat ventricular broblasts and the expression of CTGF induced by TGF-β1. After ligating the aorta of Klf15 knockout mice, the expression of CTGF in the heart increased and the degree of cardiac brosis increased (26). The above studies have fully demonstrated that KLF15 is a key factor that negatively regulates myocardial brotic signaling pathway. In our study, we demonstrated that EF signi cantly declined (Table 1), with signi cant decrease in KLF15 expression and concurrent increase in CTGF expression in T2D group compared to WT group (Fig. 5). Moreover, in Klf15-cKO mice, EF signi cantly declined and the expression of CTGF signi cantly increased in the Klf15-cKO/T2D group compared with the Klf15-cKO group, con rming that KLF15 is a key negative regulator in modulating cardiac dysfunction and myocardial brosis induced by T2D (Table 1 and Fig. 5). After cotreatment with SDF-1β, EF signi cantly improved, and the expression of KLF15 in the heart of T2D mice was signi cantly increased (Table 1 and Fig. 5). We also demonstrated that the protective effect of SDF-1β on myocardial brosis in Klf15-cKO/T2D mice disappeared, proving that SDF-1β exerts anti-brosis effects through KLF15. However, in Klf15-cKO group, SDF-1β still signi cantly improved EF (Table 1). Previous studies have proved that in addition to myocardial brosis, there are a variety of molecular mechanisms that act synergistically to impair cardiac function and promote cardiomyocyte injury in diabetes, such as metabolic disturbances (45), small vessel disease (46), cardiac autonomic neuropathy (47), and insulin resistance (48), thus the protective effect of SDF-1β on DCM may not have merely been through inhibiting myocardial brosis.
SDF-1 regulates many essential biological processes, which has been considered predominantly through binding chemokine receptor, CXCR4 (7,14). However, recent studies from us and others have implicated that SDF-1 can also bind chemokine receptor CXCR7 and can illicit different functions compared with CXCR4, while playing an important role in mediating cell survival or the anti-apoptotic effect of SDF-1 (14,21). Ding et al have con rmed the different effects of CXCR4 and CXCR7 on chronic liver injury, and found that during chronic liver injury, selective CXCR7 activation in liver sinusoidal endothelial cells promoted anti-brotic signaling, while loss of CXCR7 expression and the upregulation of CXCR4 expression promoted liver brosis (49). In our previous study, we have demonstrated that both CXCR4 and CXCR7 are expressed in cardiac H9C2 cells (21). In vitro studies, we also demonstrated that CXCR7 siRNA transfection completely blocked the myocardial protection of SDF-1β in palmitate treated H9C2 cells ( Fig. 2A). However, CXCR4 siRNA transfection had no effect on the protective effect of SDF-1β on brosis of H9C2 cells (Fig. 3A). These ndings suggested that the protective effect of SDF-1β on palmitate-induced cardiomyocyte brosis was mediated via binding to CXCR7 receptor.
Studies have shown that CXCR7 can activate MAPK signaling pathways (50). The MAPK signaling pathway allows cells to process a wide range of external signals and respond appropriately, generating a plethora of different biological effects. The diversity and speci city of cellular outcomes is achieved by functionally distinct p38 MAPK isoforms (51). The cardioprotective effect of p38β MAPK has been con rmed in different animal models. Venkatakrishnan et al. have shown that activation of the p38β MAPK attenuated doxorubicin-induced cardiotoxicity (52). In our previous study, we have also demonstrated that p38β MAPK activation was required for SDF-1β's cardiac protection from palmitateinduced cardiac cell death (21). In this study, we demonstrated that silencing p38β MAPK expression prevented upregulation of KLF15 expression in the presence of palmitate and SDF-1β, suggesting the direct requirement of p38β MAPK for the cardiac protection by SDF-1β (Fig. 2B).

Conclusions
In summary, the current study utilized Klf15-cKO mice for the rst time and demonstrated that in T2D mice, SDF-1β attenuates cardiac brosis through KLF15 dependent pathway. The cardiac protective effect of SDF-1β was mediated by binding to CXCR7 receptor and by p38β MAPK-mediated KLF15 upregulation. These ndings demonstrate that KLF15 is a key negative regulator in the process of T2Dinduced myocardial brosis, which may nd serving as a new molecular target for the prevention and/or treatment of T2D-induced myocardial brosis, and may lead to the development of new drugs that promote KLF15 expression and treat DCM. Furthermore, these ndings also provide a novel insight into the mechanism by which SDF-1β induced cardiac protection in T2D and SDF-1β may serve as a therapy in the prevention and treatment of DCM. All animal protocols were approved by the Animal Ethics Committee of Jilin University.

Consent for publication
Not applicable.
Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by grants from the National Science Foundation of China (81670221, 81974227 to YGZ).
Authors' contributions YYT, ZYW, YGZ analyzed data, and wrote the manuscript. LC and MR interpreted experimental data and prepared and reviewed the manuscript. XYZ, SHC, WJS, ZJ discussed project progression, researched data, and reviewed the manuscript. YGZ contributed to the initial formulation of the project, discussed project progression, and wrote, reviewed, and edited the manuscript. YGZ is the guarantor of this work, and, as such, had full access to all the data in the study and takes full responsibility for the integrity of data and the accuracy of data analysis.