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Serine/Threonine‐Protein Kinase 3 Facilitates Myocardial Repair After Cardiac Injury Possibly Through the Glycogen Synthase Kinase‐3β/β‐Catenin Pathway

Background The neonatal heart maintains its entire regeneration capacity within days after birth. Using quantitative phosphoproteomics technology, we identified that SGK3 (serine/threonine‐protein kinase 3) in the neonatal heart is highly expressed and activated after myocardial infarction. This study aimed to uncover the function and related mechanisms of SGK3 on cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. Methods and Results The effect of SGK3 on proliferation and oxygen glucose deprivation/reoxygenation– induced apoptosis in isolated cardiomyocytes was evaluated using cardiomyocyte‐specific SGK3 overexpression or knockdown adenovirus5 vector. In vivo, gain‐ and loss‐of‐function experiments using cardiomyocyte‐specific adeno‐associated virus 9 were performed to determine the effect of SGK3 in cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. In vitro, overexpression of SGK3 enhanced, whereas knockdown of SGK3 decreased, the cardiomyocyte proliferation ratio. In vivo, inhibiting the expression of SGK3 shortened the time window of cardiac regeneration after apical resection in neonatal mice, and overexpression of SGK3 significantly promoted myocardial repair and cardiac function recovery after ischemia/reperfusion injury in adult mice. Mechanistically, SGK3 promoted cardiomyocyte regeneration and myocardial repair after cardiac injury by inhibiting GSK‐3β (glycogen synthase kinase‐3β) activity and upregulating β‐catenin expression. SGK3 also upregulated the expression of cell cycle promoting genes G1/S‐specific cyclin‐D1, c‐myc (cellular‐myelocytomatosis viral oncogene), and cdc20 (cell division cycle 20), but downregulated the expression of cell cycle negative regulators cyclin kinase inhibitor P 21 and cyclin kinase inhibitor P 27. Conclusions Our study reveals a key role of SGK3 on cardiac repair after apical resection or ischemia/reperfusion injury, which may reopen a novel therapeutic option for myocardial infarction.

cells. 2 Countless efforts have been made to prevent the loss of cardiomyocytes caused by ischemic heart disease. 3 Because of the low survival rate and low transformation efficiency of exogenous stem cells in the treatment of ischemic heart disease, exploring the molecular pathways capable of regulating the endogenous cardiomyocyte proliferation is expected to become the main approach for myocardial regeneration in the setting of ischemic heart disease in the future. 4 Reactivating key genes associated with the cardiac development to induce adult cardiomyocytes to reenter the cell cycle has recently become an attractive avenue. [5][6][7] Cell cycle regulatory genes, such as cell cycle dependent kinases and noncoding RNAs, which are temporarily overexpressed in the neonatal heart, can promote the adult cardiomyocyte proliferation if they are re-overexpressed in the adult heart. 7,8 Previously, we performed quantitative phosphoproteomic analysis to reveal the protein kinase network in the process of cardiac regeneration and elucidate the role of CHK1 (checkpoint kinase 1) in promoting cardiac regeneration. 6 Intriguingly, several Hippo/Yap pathway kinases (eg, large tumor suppressor kinase 1 and 2) and RAC-alpha serine/threonine-protein kinase (AKT) pathway kinases (eg, RAC-alpha serine/threonine-protein kinase 1 [AKT1] and RAC-alpha serine/threonine-protein kinase 3 [AKT3] kinases) were marginally enriched (0.05<false discovery rate<0.1) in neonatal myocardial tissue at 6 days after infarction. Evidence has been reported that both Hippo/Yap and AKT pathways exert key regulatory roles in cardiac regeneration. [9][10][11] It remains unknown if these borderline enriched kinases after cardiac injury might also play key roles in the cardiac regeneration process. Through a series of screening and validation, SGK3 (serine/threonine-protein kinase 3), which is abundantly expressed in neonatal heart and borderline activated after MI in neonatal mice, attracted our attention.
SGK3 kinase, the key protein kinase screened from the marginal enriched kinases, belongs to the AGCkinase (protein kinase A, G, and C) family and is highly conserved among species. 12 SGK3 and AKT kinases are homologous isomers, which are both downstream kinases of the phosphatidylinositol 3-kinase pathway and have the ability to recognize a host of identical downstream substrates. 13 AKT, as a key kinase in the regulation of cardiac injury repair, is known to be involved in the regulation of cardiac regeneration CLINICAL PERSPECTIVE What Is New?
• SGK3 (serine/threonine-protein kinase 3) expression was significantly downregulated on the seventh postpartum day, consistent with loss of the cardiac regeneration potential in neonatal mice, and can promote cardiomyocyte proliferation and attenuate oxygen glucose deprivation/reoxygenationinduced cardiomyocyte apoptosis in vitro and ischemia/reperfusion injury-induced cardiomyocyte apoptosis in the infarct border zone in vivo. • Overexpression of SGK3 in cardiomyocytes can promote cardiac regeneration and alleviate ischemia/reperfusion injury in mice. • SGK3 regulates cardiac repair after apical resection and ischemia/reperfusion injury by regulating the GSK-3β (glycogen synthase kinase-3β)/β-catenin pathway.
What Are the Clinical Implications?
• We demonstrated that the SGK3 gene could regulate the process of cardiac regeneration after myocardial insult, providing a new therapeutic target for improving the cardiac function after myocardial insult in clinical practice. • Cardiomyocyte-specific SGK3 overexpressionadenovirus serotype 5 (Ad5-cTNT-SGK3) and cardiomyocyte-specific SGK3 overexpressionadeno-associated virus serotype 9 (AAV9-cTNT-SGK3), 2 kinds of myocardium-specific overexpressed SGK3 vectors, have the potential to be used as gene therapy of myocardial insult. • Local injection of SGK3 protein kinase via microcatheter in the area of myocardial infarction during interventional therapy may be a new therapeutic strategy to promote cardiac repair after myocardial infarction.

Nonstandard Abbreviations and Acronyms
AAV9 adeno-associated virus serotype 9 AKT RAC-alpha serine/threonine-protein kinase AKT1 RAC-alpha serine/threonine-protein kinase 1 AKT3 RAC-alpha serine/threonine-protein kinase 3 AR apical resection BIO 6-bromoindirubin-3'-oxime CON control cTNT cardiac troponin T CST cell signaling technology cyclin D1 G1/S-specific cyclin-D1 GSK-3β glycogen synthase kinase-3β I/R ischemia/reperfusion Figure 1. Biological characteristics of SGK3 (serine/threonine-protein kinase 3) in the mouse heart. A, The kinase-substrate network was constructed according to the proteins with upregulated phosphorylation levels (FDR ≤0.1). Blue dots mean substrates with upregulated phosphorylation levels identified in phosphoproteomics; yellow dots (including SGK3) indicate kinases with enriched upregulated substrate phosphorylation sites by iGPS; and the gray arrow links the kinase and its substrates. B, The mRNA expression of MAST3, MAST4, MASTL, NDR1, NDR2, SGK3, RSKL1, STLK3, and YANK2 in ventricular myocardium of P1 and P28 mice was detected by quantitative real-time polymerase chain reaction analysis (n=3). C, SGK3, phosphorylated (p) SGK3 Thr320 in mice ventricular myocardium from embryo to P56 were determined by Western blot. D, qRT-PCR analysis was used to detect the SGK3 expression in ventricular myocardium at P7 after myocardial infarction (MI) (n=3). E, SGK3, phosphorylated proteins of the Thr320 site in neonatal mice ventricular myocardium after MI were examined by Western blot. as a downstream target kinase of multiple genes. 11,14 Similarly, SGK3 kinase is involved in many biological processes, such as cell proliferation and survival, ion and protein transport, tumor malignant transformation, calcium and phosphorus absorption, and hair growth. [15][16][17][18] Here, we found that SGK3 expression was significantly downregulated at 7 days postpartum, which was consistent with the loss of cardiac regeneration capacity, and could be activated after MI injury in neonatal mice. In addition, many core genes capable of promoting cardiac regeneration, such as GSK-3β (glycogen synthase kinase-3β)/β-catenin, G1/ S-specific cyclin-D1 (cyclin D1), and mechanistic target of rapamycin kinase (mTOR), have been reported to be regulated by SGK3 in previous studies. 15,19,20 However, the functional role of SGK3 in cardiac regeneration and whether SGK3 can promote cardiac regeneration by regulating regenerative core genes remain elusive now.
In this study, we identified that SGK3 was significantly downregulated in myocardium beginning from postnatal day 7 to adulthood. SGK3 overexpression in the infarct border myocardial tissues resulted in markedly augmented myocardial repair and improved cardiac function after I/R injury, suggesting a regenerative and cardioprotective role of SGK3 after I/R injury.

METHODS
The data that support the findings of this study are available from the corresponding author on request. The raw data related to phosphorylation proteomics are uploaded to the ProteomeXchange Consortium. 6 Additional methods can be found in Data S1.

Availability of Data and Material
All data generated or analyzed during this study are included in this article.

Ethical Statement
All animal studies were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85-23, revised 1996).

Experimental Animals
All animal experiments were performed in accordance with the Guide for the Use and Care of Laboratory Animals and approved by Animal Care and Use Committee of Nanjing Medical University. Wild-type Institute of Cancer Research mice were obtained from the animal center of Nanjing Medical University and reared in a specific pathogen-free environment.

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction Analysis
Total RNA from the myocardial tissue used in our study was extracted with TRIzol reagent (Thermo Fisher Scientific) according the manufacturer's instructions. The PrimeScript RT Master Mix kit (Takara Bio, Kusatsu, Japan) was used to synthesize complementary DNA. Relative RNA levels analysis, detected by quantitative real-time polymerase chain reaction analysis, was performed on a Roche Lightcycler 96 by using the SYBR Green (Vazyme Biotech, Nanjing, China; Q131-02). 18S ribosomal DNA was used as an internal control, and determined genes sequences for quantitative realtime polymerase chain reaction analysis primers in this study are shown in Table S1.

Immunofluorescence Staining
Hearts were harvested and fixed overnight in 4% paraformaldehyde and then embedded in paraffin. Paraffinembedded hearts were cut into 5-μm tissue sections and boiled in antigen retrieval buffer (BD Pharmingen) for 10 minutes. Then, the sections were immersed in PBS containing 0.2% Triton X-100 for 15 minutes and blocked with 5% bovine serum albumin for 2 hours. To identify cell-cycle activities and cytokinesis, the Click-iT EdU Imaging Kits (Thermo Fisher), anti-Ki67 antibody (Abcam; ab16667), anti-Aurora B antibody (Abcam; ab2254), and anti-phosphorylated-histone 3 (pH3) antibodies (CST; 9701) were used to culture sections and formaldehyde-fixed cardiomyocytes. Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) staining was performed to determine the cardiomyocyte apoptosis in vivo. WGA (wheat germ agglutinin) staining (Thermo Fisher; w32466) was performed to stain the cell membrane, anti-cardiac troponin T (cTNT) (Abcam; ab8295) was used to label cardiomyocytes, and N-acetyl-diaminopimelate deacetylase was used to mark nuclei. Quantitative data were obtained by measuring confocal microscope (Zeiss, Oberkochen, Germany).
In vitro, neonatal mouse cardiomyocytes were cultured in 24-well plates. After being treated with different adenoviruses or reagents, the nonadherent cells were washed out with PBS, and the adherent cardiomyocytes were then fixed with 4% paraformaldehyde for 20 minutes. After being sealed with 10% goat serum for 1 hour, 5-ethynyl-2′-deoxyuridine (EDU), Ki67, pH3, WGA, and TUNEL staining were performed. Finally, a Zeiss microscope (Carl Zeiss Microscopy, Jena, Germany) was used to take pictures and count the randomly selected visual field. The statistical data of all immunofluorescence staining in this study are shown in Table S2.

Statistical Analysis
All the statistical data were analyzed using SPSS 22.0 (IBM, Armonk, USA) and were presented as mean±SEM using GraphPad (La Jolla, CA) Prism 8.0 software. For the comparison of mean values between 2 groups, an unpaired Student t test was performed when the data were shown to be normally distributed. For the comparisons among multiple groups, 1-way ANOVA (with the Tukey multiple comparisons test) was performed to determine the statistical differences. A log-rank (Mantel-Cox) test was performed to analyze the survival rate. Differences were considered statistically significant when P≤0.05.

RESULTS
SGK3 Is Upregulated After MI in the Neonatal Heart pH3-positive cell staining confirmed that the cardiomyocyte proliferation ability was significantly decreased in P7 and P28 hearts compared with the P1 heart, which was consistent with the lost regeneration capacity of mammalians at P7 ( Figure S1). Quantitative proteomic analysis of protein and phosphorylation level in infarct margin myocardial tissue of P1 MI mice at 6 days after infarction was performed in our previous study. Besides 11 activated kinases (false discovery rate≤0.05), 6 several borderline enrichment kinases, which belong to Hippo/Yap pathway kinases (eg, large tumor suppressor kinase 1 and 2) and AKT signaling kinases (eg, AKT1 and AKT3) were defined at 6 days after infarction of P1 MI mice (0.05<false discovery rate<0.1) ( Figure 1A and Table S3).
9 out of the 35 marginally enriched protein kinases that could mediate cell proliferation but were not reported in cardiovascular field, and detected their mRNA levels using quantitative real-time polymerase chain reaction analysis in neonatal (P1) and adult (P28) myocardial tissue. Results indicated that mRNA expression of SGK3 (one of the borderline enrichment kinases; P=0.0039, false discovery rate=0.0632), was significantly downregulated in P28 myocardial tissues than that in P1 mice ( Figure 1B). Total protein and phosphorylation levels of SGK3 were also decreased remarkably in the heart of P7 mice as compared with that in P1 mice ( Figure 1C). Additionally, the mRNA expression of SGK3 did not change significantly ( Figure 1D), but the phosphorylated-SGK3/SGK3 ratio was elevated significantly after MI in neonatal mice (P=0.0031; Figure 1E). Nuclear cytoplasmic separation experiments and Western blot analysis revealed that SGK3 mainly existed in the cytoplasm ( Figure 1F). Finally, conservative analysis showed high homology of SGK3 gene between human and mouse ( Figure S2).
P<0.0001) showed that SGK3 did not affect the apoptosis rate of neonatal cardiomyocytes under physiological conditions, but could significantly reduce the apoptosis rate of cardiomyocytes induced by oxygen glucose deprivation/reoxygenation ( Figure 2L).

SGK3 Overexpression Enhances Cardiomyocyte Proliferation in Neonatal Mice
To identify the effects of SGK3 on the proliferation, apoptosis, heart weight/body weight ratio, and cardiomyocyte size in neonatal mice without heart injury, adeno-associated virus serotype 9 (AAV9):cTNT-SGK3 (1.1×10 12 vg/mL, total stock solution volume=8 μL/ mouse) or AAV9:cTNT-CON (1.1×10 12 vg/mL, total stock solution volume=8 μL/mouse) were intraperitoneally injected into P1 mice, and EDU solution (5 μg/ μL, total volume=70 μL) was then injected at different time points (details shown in Figure 3A). The transfection efficiency of AAV9:cTNT-SGK3 was confirmed by an elevated expression of SGK3 in ventricular myocardium of neonatal mice by Western blot analysis ( Figure S4). Immunofluorescence staining results indicated that overexpression of SGK3 promoted the ratio of cell proliferation, as represented by the increase of EDU + (
To further explore the mechanism of SGK3 in regulating cardiomyocyte proliferation, the cardiomyocytes were transfected with Ad5:cTNT-SGK3 or Ad5:cTNT-CON for 48 hours, and mRNA expressions of cell cycle regulatory genes were determined. Quantitative realtime polymerase chain reaction analysis revealed that SGK3 overexpression in cardiomyocytes significantly upregulated the cell cycle promoting genes of cyclin D1, c-myc, and cdc20 (cell division cycle 20) while downregulating the cell cycle inhibiting genes of cardiomyocytes from P21 (cyclin kinase inhibitor P21) and P27 (cyclin kinase inhibitor P27) mice as compared with Ad-CON cardiomyocytes ( Figure 8A). Cycline D1 and c-myc are important downstream target genes of β-catenin, and we have shown that SGK3 can upregulate the expression of β-catenin. SGK3 alone or with β-catenin short hairpin RNA were transfected in cardiomyocytes for 48 hours, and the transfection efficiency was evidenced by Western blot analysis ( Figure S7B). Immunofluorescence staining showed that the proportion of cardiomyocytes reentering the cell cycle (Ki67 + ) promoted by Ad5:cTNT-SGK3 was obviously blocked by β-catenin short hairpin RNA (P<0.0001) ( Figure S7C). In vivo, specific AAV9:cTNT-SGK3 or/and AAV9:cTNT-β-catenin RNA interference intramyocardial injection was made by a microinjector in P56 adult mice underwent I/R injury. At 14 days after reperfusion, Western blot analysis verified the transfection efficiency of AAV9:cTNT-SGK3 and AAV9:cTNT-βcatenin RNAi (Figure 8B). At 28 days after reperfusion, I/R injury-impaired cardiac function (EF: 43.3±2.8%, FS: 21.5±1.5%) in Ad:cTNT-CON group mice was significantly improved after SGK3 overexpression (EF: 52.5±2.2%, FS: 27.1±1.3%), while β-catenin knockdown resulted in further reduced EF and FS values (EF: 27.6±3.1%, FS: 13.3±1.6%). In addition, EF and FS values in SGK3 overexpression combined with β-catenin knockout group mice were lower (EF: 42.6±3.9%, P=0.0112; FS: 21.6±2.3%, P=0.014) than in the SGK3 overexpression group ( Figure 8C). Meanwhile, SGK3 overexpression-induced reduction in scar size after I/R injury (20.0±2.3%) was blocked in AAV9:cTNT-SGK3 and AAV9:cTNT-β-catenin RNA interference cotransfected hearts (26.0±1.3%, P=0.0001) (Figure 8D and 8E). Overall, in view of the significant downregulation of SGK3 expression after P7, we speculate that endogenous SGK3 in cardiomyocytes could hardly affect cardiac repair after ischemic injury in adult hearts. Here, we show that exogenous overexpression of SGK3, especially in cardiomyocytes, could promote myocardial regeneration and inhibit apoptosis, and thus play a protective role in I/R-induced cardiac injury. The role and potential mechanism of SGK3 on neonatal cardiomyocyte regeneration and the therapeutic effect on cardiac repair after I/R injury in adult mice are illustrated in Figure 8F.

DISCUSSION
In the present study, we elaborate that SGK3, which functions as the upstream of the GSK-3β/β-catenin pathway and some kinds of cell cycle regulatory genes, is capable of promoting cardiomyocytes reentering cell cycle through in vitro and in vivo experiments. Along with cardiomyocyte proliferation, SGK3 overexpression robustly attenuated oxygen glucose deprivation/ reoxygenation-induced cardiomyocyte apoptosis in vitro and I/R injury-induced cardiomyocyte apoptosis in the infarct border zone in vivo, which is also of great significance for the improvement of and myocardial repair and cardiac function after I/R injury. SGK3 overexpression brings beneficial effects in terms of reducing the ratio of infarction area/area at risk in the acute phase and the long-term fibrous scar size after I/R injury, indicating a strong effect of alleviating postinfarction cardiac remodeling with this strategy. Mechanistically, the beneficial effects are mediated through the GSK-3β/βcatenin pathway. To the best our knowledge, this is the first report describing the effects of SGK3 and related mechanism after cardiac insult.
Based on gain-and loss-of-function study in vitro, we indicated that SGK3 overexpression obviously increased, whereas SGK3 knockdown significantly inhibited the proportion of cardiomyocytes in DNA synthesis and mitotic state. In line with our results, Scortegagna et al also reported that inhibition of SGK3 can significantly reduce the expression of cyclin D1 and the phosphorylation of P70S6K, resulting in the arrest of melanoma cell cycle in the Gap 1 phase. 22 In contrast, overexpression of SGK3 could increase the phosphorylation level of GSK-3β on Ser9, thus suppressing β-catenin degradation and promoting cell cycle gene CCND1 expression in hepatocellular carcinoma. 19,23 These results suggest that SGK3 plays an important role in cell cycle regulation. Furthermore, it was also demonstrated that SGK3 overexpression could attenuate oxygen glucose deprivation/reoxygenation and I/R-induced cardiomyocyte apoptosis in vitro and in vivo. The above results indicate that SGK3 can simultaneously exert a dual role in promoting proliferation and inhibiting apoptosis of cardiomyocytes.
Our results are consistent with previous findings, which showed that modulating functional genes could protect against heart injury through promoting proliferation and suppressing apoptosis of cardiomyocytes. For instance, Hauck et al and Magadum et al revealed that Pkm2 (pyruvate kinase muscle isozyme 2) played a crucial role in promoting myocardial proliferation, inhibiting oxidative stress and apoptosis after MI injury. 24,25 In the neonatal heart, cardiomyocyte-specific SGK3 overexpression adenovirus was used to transfect SGK3 to isolated cardiomyocytes, and the expression of reported downstream proteins of SGK3 related to cardiomyocyte regeneration and cardiac repair was explored. 19,23,26 We found that SGK3 significantly promoted the phosphorylation level of GSK-3β on Ser9 and β-catenin expression, but did not affect the expression of phosphorylated-mTOR and mTOR, suggesting that SGK3 played a role in cardiomyocyte proliferation through mTOR-independent pathways. We also demonstrated that SGK3 can regulate a series of cell cycle genes including β-catenin targets. In cardiomyocytes, GSK-3β is activated by SGK3, which could lead to ubiquitination and degradation of β-catenin in the cytoplasm, and promote the accumulation of β-catenin in the nucleus. Subsequently, as a transcription coactivator of T-cell factor/lymphoid enhancer factor transcription factors, β-catenin could translocate to the nucleus and initiate the transcription events of target genes. 27,28 These mechanistic data collectively demonstrate that SGK3 might be a critical regulator of cardiomyocyte regeneration and cardiac repair through regulating GSK-3β/β-catenin and cell cycle regulator genes.
The GSK-3β/β-catenin signaling pathway is generally considered as the downstream common pathway to drive the proliferation of cardiomyocytes. For instance, Hippo/Yap, insulin-like growth factor, peroxisome promoter activated receptor delta, and neuregulin/erb-b2 receptor tyrosine kinase 2 signaling pathways have all been reported to be involved in the enhancement of downstream β-catenin signaling to stimulate the cardiomyocyte proliferation. 11,[29][30][31] However, Quaife-Ryan et al recently showed that when the β-catenin signal was directly activated, it could just promote cell proliferation in immature cardiomyocytes, while the activation of the β-catenin signaling pathway can induce cardiomyocyte hypertrophy instead of cardiomyocyte proliferation in mature cardiomyocytes. 32 The possible reason for this phenomenon might be that the transcriptional responses of immature and mature cardiomyocytes to active β-catenin signal differ significantly. β-catenin signal could drive the transcriptional regulatory network of cell cycle-related target genes in immature cardiomyocytes,whereas in mature cardiomyocytes, it is converted to the regulation of the transcriptional program of inflammation, apoptosis, and metabolism-related target genes. [32][33][34] Although the activation of β-catenin cannot induce mature cardiomyocyte proliferation, it can give rise to cardioprotection response and decrease in scar size after MI in the adult heart. 32 In our study, we observed that the cardiomyocyte-specific overexpression of SGK3 kinase activated downstream β-catenin signal in neonatal and adult cardiomyocytes. The protective effect of SGK3 on the cardiac function recovery and the Figure 8. The cardiac function improvement of SGK3 (serine/threonine-protein kinase 3) on ischemia/reperfusion (I/R)induced cardiac injury was partially reversed by β-catenin knockdown. A, Quantitative real-time polymerase chain reaction analysis was performed to detect the messenger RNA (mRNA) expression of SGK3, cyclin D1, c-myc (cellular-myelocytomatosis viral oncogene), cdc20 (cell division cycle 20), P21 (cyclin kinase inhibitor P21), and P27 (cyclin kinase inhibitor P27). B, The AAV9:cTNT-SGK3 was transfected into myocardium with or without AAV9:cTNT-β-catenin RNAi after ischemia, and reperfusion was given after 45 minutes of ischemia. Western blot analysis was then performed to determine the expression of SGK3 and β-catenin after 14 days of I/R. C, Cardiac function of ejection fraction and fractional shortening was tested in each group of mice (I/R+AAV9:cTNT-CON group, n=8; I/R+AAV9:cTNT-β-catenin RNAi group, n=14; I/R+AAV9:cTNT-SGK3 group, n=9; I/R+AAV9:cTNT-SGK3+AAV9:cTNT-β-catenin RNAi group, n=12). D and E, Masson staining of mouse ventricular cross-sections was performed to determine the scar size in each group mice (I/R+AAV9:cTNT-CON group, n=7; I/R+AAV9:cTNT-β-catenin RNAi group, n=7; I/R+AAV9:cTNT-SGK3 group, n=8; I/R+AAV9:cTNT-SGK3i+AAV9:cTNT-β-catenin RNAi group, n=8). F, A model illustration of the role and mechanism of SGK3 in promoting cardiomyocyte regeneration and cardiac function recovery in neonatal and adult heart. Transient high expression of SGK3 in the neonatal heart maintains the regeneration ability within 7 days after birth. In the adult heart, the expression and activity of SGK3 is significantly downregulated. Exogenous administration of SGK3 significantly promotes the cardiomyocyte proliferation and cardiac repair after I/R injury. Data are presented as mean±SEM. *P≤0.05; **P≤0.01; ***P≤0.001. 18S indicates 18S ribosomal DNA; AAV, adeno-associated virus serotype; AAV9:cTNT-CON, control adeno-associated virus serotype 9; AAV9:cTNT-SGK3, cardiomyocyte-specific SGK3 overexpression adeno-associated virus serotype 9; CON, control; cyclin D1, G1/ S-specific cyclin-D1; dpi, days post infarction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK-3β, glycogen synthase kinase-3β; MI, myocardial infarction; NS, no significance; P21, postnatal day 21; P27, postnatal day 27; and RNAi, RNA interference. fibrous scar size reduction after I/R injury in adult mice was significantly reversed by cardiomyocyte-specific AAV9:cTNT-β-catenin RNA interference, indicating that the β-catenin pathway mediated the protective effect of SGK3 on adult I/R injury.
It is known that AKT and its downstream pathways, including the AKT/GSK-3β pathway, play an important role in cardiac repair after MI injury. 35,36 After cardiac injury, inhibition of GSK-3β activity downstream of AKT improved cardiac function and promoted myocardial repair by enhancing cardiomyocyte proliferation and reducing apoptosis. 37,38 In this study, SGK3 overexpression promoted cardiomyocyte proliferation and ameliorated adult I/R injury, and increased phosphorylation level of GSK-3β at ser9, which is linked to GSK-3β activity inhibition. In line with these results, inhibition of GSK-3β activity significantly blocked the inhibitory effect of SGK3 knockdown on cardiomyocyte proliferation and cardiac function recovery after AR in neonatal mice. Although SGK3 is highly homologous with AKT in structure, and multiple substrates of SGK3 are also regulated by AKT, including GSK-3β/β-catenin signaling, 23 some downstream AKT signaling is not mediated by AKT in some types of cells, but is dependent on other signaling, such as SGK3. 39,40 Previously, Liu et al also reported the role of SGK3 in the progression of hepatocellular carcinoma via AKT independent pathways. 23 In view of the complexity of the PI3K pathway, future studies are warranted to explore if other downstream signaling of SGK3 pathways also mediated the effects of exogenous introduction of SGK3 and promoted cardiomyocyte regeneration and cardiac repair in adult mice post MI, in addition to the defined GSK-3β/βcatenin pathway.
Although our current results provide convincing evidence to support the effects of SGK3 in cardiac repair after I/R, several issues should be considered in interpretation of present results. One of the concerns is the specificity of SGK3 action. As a potential carcinogenic kinase, it is necessary to achieve transientspecific overexpression in myocardial tissue after MI, so as to improve cardiac repair and reduce adverse effects on other organ systems. Another limitation of this study is that no commercial isoform-specific SGK3 inhibitors are available currently, which hindered further investigating on the molecular mechanism of SGK3 in promoting cardiomyocyte proliferation.
In conclusion, we explored the potential cardioprotective effect of SGK3 kinase, and proved the cardiac protective role of SGK3 through GSK3β and β-catenin. The present findings extend our knowledge on the kinase regulatory network of myocardial regeneration and indicate that targeting the SGK3 and GSK3β/βcatenin pathway might have therapeutic implication on cardiac repair after heart injury.

Supplemental Material
Data S1.

Animal model construction and intervention
Apical resection (AR) on neonatal (P1) mice (n=139, and 117 survived the operation.) was carried out as previously described. 6 Briefly, P1 mice were anesthetized by cooling on an ice bed for 3 min. Lateral thoracotomy at the fourth intercostal space was performed by blunt dissection of the intercostal muscles following skin incision.
Approximately one square millimeter of the apical myocardium was removed perpendicular to the long axis of the heart. After AR, the thoracic wall and skin incision was sutured with 6.0 non-absorbable silk sutures. All P1 mice were then placed under a heat lamp until recovery and returned to their mother. The injection of adeno-associated virus injection (I/R+AAV9: cTNT-SGK3, n=76, I/R+AAV9:cTNT-CON, n=83) was performed in P56 mice following the ligation of LAD and 125 mice survived. A microsyringe with a 36G needle was used for adenovirus injection around the apex of the P56 heart (The total amount of virus injected was 1.5*10 9 v.g.). The needle was inserted into the left ventricle from apex, and injection was made on three locations around the apex (the front, middle and back: 3µl for each point) respectively from the endocardium. Cardiac function was assessed by echocardiography at 1dpi and 28dpi (n=10 mice each group). The infarct area was assessed by Masson staining.
The adeno-associated virus (I/R+ Ad5:cTNT-SGK3, n=25, I/R+Ad5:cTNT-CON, n=27) was injected in P56 mice following the ligation of LAD, of which 41 mice survived. The injection procedure is same as I/R+AAV9 groups listed above (The total amount of Ad5 injected was 7.5*10 7 PFU). Cardiac function was assessed by echocardiography at 1 hour and 2 days post I/R operation. TTC and TUNEL staining were performed 2 days post operation.

Quantification of infarct size
Mice were re-anesthetized and re-intubated 2 days after reperfusion. The LAD coronary artery was re-occluded by ligating the suture in the same position. Animals were executed and then 1 ml of 1% Evans Blue dye was infused i.v. to define the area at risk (AAR, representing the myocardium lacking blood flow, i.e. negative for blue dye staining). After OCT embedding, transversal sections (n=8, 1-2mm) were cut from the apex to the ventricle. Slices were incubated in triphenyltetrazolium chloride (1% TTC, Sigma) at 37℃ for 30 min to identify the infarcted myocardium. The sections were emerged in formaldehyde overnight. A camera was used to take pictures of four sections of heart tissue in sequence. Regions negative for Evans Blue staining (AAR) and TTC (infarcted myocardium) were then re-photographed and quantified with ImageJ software. % of AAR was determined as the percentage of the area of AAR to LV, and % of infarct size was determined as the percentage of the area of infarcted myocardium to AAR averaged from the four sections.
At 28 dpi post-reperfusion, mice were sacrificed under deep anesthesia (n=7), then hearts were harvested and fixed overnight in 4% paraformaldehyde, and then embedded in paraffin. Transverse serial sections (5 μm) were obtained from apex to ventricle at an interval of more than 150 μm, then dewaxed by gradient alcohol and rehydrated for Masson staining under standard procedures and infarct area (blue regions) versus the total left ventricular area was quantified using Image J software.

Cardiomyocyte isolation, culture and intervention
Neonatal mice CMs were isolated from 1-3-day-old ICR mice (50-150 mice each time) as previously described. 42 Briefly, after 75% alcohol epidermis disinfection, neonatal mice ventricular myocardium were collected. The ventricular muscle was then shredded and washed out most of red blood cells. Heart tissues were digested with 20 ml digestive solution containing 0.06 g/100ml trypsin (Sigma, USA) and 0.04 g/100ml collagenase II (Worthington, USA) for 6-7 minutes each time until the myocardia tissues were digested into single cells. After incubation with DMEM containing 10% FBS for 45 min, the adherent fibroblasts were removed. The collected suspension was then centrifuged with Percoll liquid (3000 rpm, 30 minutes, slowly rising and falling) to separate CMs from fibroblasts. After discarding the fibroblasts in the upper layer, the CMs in the middle layer were collected and cultured in incubator with 5% CO2 at 37 ℃.
After 24 hours of culture, the cells without adherence were washed out, and the cells were incubated for another 24 hours and then transfected with plasmids or adenoviruses for different experiments.

Preparation of nuclear and cytoplasmic extracts
The myocardial tissue was grinded and lysed with cytoplasmic lysate (including 0.1% phenylmethylsulfonyl fluoride, 0.1% protease inhibitor, 1% phosphatase inhibitor, 1% cocktail) and then placed on ice for 1 hour. After centrifugation (4 ℃, 14000rpm, 20min), the supernatant is cytoplasmic protein. Then, the precipitate was washed three times with precooled PBS, and then added with nuclear cracking solution. After blowing and mixing, the precipitate was ultrasonic (energy 25%, 4 ℃, 3 seconds) for three times, and then the cracking solution was placed on ice for about 1 hour. The supernatant obtained after centrifugation is nucleoprotein. Cytoplasmic and nuclear fractionations were homogenized according to nuclear and cytoplasmic extraction reagent kit (Thermo Fisher, USA).

Histological and immunohistochemical staining
To determine the effects of SGK3 on the infarct size, we collected the hearts after MI with SGK3 specifically overexpression in myocardial tissue. The collected hearts were embedded in paraffin, and 5 μm thick cross section was prepared. Each heart was cut into 5 transversal sections (5µm) at 150-200µm interval from apex to base, and each section was stained by Masson staining according to the standard procedure. Finally, the infarct area and total left ventricular area were quantified by Image J software.

Figure S2. SGK3 homology analysis between human and mouse.
SGK3 homology analysis between human and mouse genome was conducted using BLAST.