RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation

RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation RGFP966通过抑制YAP通路活化减轻长时程低温保存诱导的心功能损伤 1 first-author 1 2 3 1 4 1 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:bchenyy@zju.edu.cnshenyueliang@zju.edu.cn †E-mail:bchenyy@zju.edu.cnshenyueliang@zju.edu.cn bchenyy@zju.edu.cn 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:bchenyy@zju.edu.cnshenyueliang@zju.edu.cn †E-mail:bchenyy@zju.edu.cnshenyueliang@zju.edu.cn bchenyy@zju.edu.cn Department of Basic Medicine Sciences, Zhejiang University School of Medicine, Hangzhou 310058, China Department of Basic Medicine Sciences, Zhejiang University School of Medicine, Hangzhou 310058, China Department of Orthopaedics, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China Department of Orthopaedics, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China Department of Pharmacology, Hangzhou Medical College, Hangzhou 310053, China Department of Pharmacology, Hangzhou Medical College, Hangzhou 310053, China Department of Obstetrics, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China Department of Obstetrics, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4–6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP–transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway. 目的 2 观察组蛋白脱乙酰化酶3(HDAC3)抑制剂RGFP966是否能够促进低温保存心脏复灌期心功能的恢复,并探讨Hippo-YAP信号通路是否参与了RGFP966的心肌保护作用. 创新点 2 首次证实了在Celsior保存液中添加RGFP966可以减轻长时程低温保存引起的心脏功能障碍.该研究结果为临床实践中的心脏移植提供有价值且可行的策略,为患者获得远距供体心脏提供了可能. 方法 2 将大鼠离体心脏置于含有或不含有RGFP966的Celsior保存液中低温保存12 h后,于Langendorff灌流系统中复灌60 min,测定复灌期各项心功能指标.用Western blotting法分析Mst1和YAP蛋白表达和磷酸化水平.用TUNEL法测定心肌细胞凋亡情况. 结论 2 RGFP966可以促进低温保存心脏复灌期心功能的恢复,其作用机制可能是通过抑制Hippo-YAP信号传导通路的激活,增强心肌抗氧化能力,抑制心肌细胞凋亡而实现的. Hypothermic preservation RGFP966 Yes-associated protein (YAP) Oxidative stress Apoptosis 低温保存 RGFP966 YAP 氧化应激 凋亡 Introduction 1 Heart transplantation is a major strategy to improve the quality of life and prolong survival of patients with end-stage heart failure (Mehra et al., 2016 2016 ). Donor hearts are typically hypothermically preserved in preparation for transplantation. Oxidative stress and apoptosis are the key factors that lead to cardiomyocyte injury in hypothermically preserved hearts (Korkmaz-Icöz et al., 2019 2019 ), which restricts their preservation time to within 4–6 h. Therefore, to prolong their preservation time and enhance the source of donor hearts, alleviation of prolonged hypothermic preservation-induced oxidative stress and inhibition of apoptosis would be appropriate goals. Histone deacetylase (HDAC) inhibitors have been proven to protect against cardiac injury during different cardiovascular conditions. Pan-HDAC inhibitors can improve cardiac function in heart failure animal models. The improvement is associated with attenuation of oxidative stress and inflammation, and improvement of cardiac metabolism (McKinsey, 2012 2012 ; Lkhagva et al., 2015 2015 ). Inhibition of class I HDAC restored left ventricular contractile function in isolated ischemia-reperfusion rat hearts by increasing mitochondrial superoxide dismutase (SOD) and catalase (CAT) expression (Aune et al., 2014 2014 ). HDAC3 is a key regulator of cell proliferation during cardiac development (Poleshko et al., 2017 2017 ), and plays an important role in maintaining myocardial energy metabolism, cardiac electrophysiological property, and contractile function (Ferguson and McKinsey, 2015 2015 ; Brundel et al., 2020 2020 ). RGFP966 is a selective inhibitor of HDAC3 and is a more efficient inhibitor than other class I HDAC inhibitors (Malvaez et al., 2013 2013 ). Janczura et al. ( 2018 2018 ) reported that RGFP966 decreased Tau phosphorylation and Aβ-42 protein expression in the brain, and improved spatial learning and memory in an Alzheimer’s disease mouse model. RGFP966 can also prevent diabetes-induced liver damage and cardiac injury, and ameliorate cerebral ischemia reperfusion injury in diabetic animal models (Xu et al., 2017 2017 ; Zhang J et al., 2018 2018 ; Zhao et al., 2019 2019 ). In an experimental model of atrial fibrillation, RGFP966 delayed progression of atrial fibrillation and preserved contractile function (Zhang DL et al., 2018 2018 ). Antioxidant and anti-apoptosis properties of RGFP966 have also been reported in animal models of optic nerve injury (Schmitt et al., 2018 2018 ) and acute lung injury (Joshi et al., 2015 2015 ). So we hypothesized that RGFP966 might have potential for the alleviation of cardiac dysfunction induced by hypothermic preservation. Recent studies have demonstrated that HDAC inhibitors can alleviate oxidative stress (Yu et al., 2018 2018 ), regulate inflammation response (Rivera-Reyes et al., 2018 2018 ), manipulate cell cycle (Ren et al., 2019 2019 ), and inhibit apoptosis (Jung et al., 2017 2017 ) by targeting the Hippo pathway. The Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are the key downstream effectors of the Hippo pathway (Li et al., 2018 2018 ; Fu et al., 2019 2019 ). When the Hippo pathway is inactive, YAP/TAZ is dephosphorylated and imported into the nucleus. In the nucleus, YAP/TAZ binds to transcriptional enhanced associate domains (TEADs), and then promotes gene transcription. HDAC8 has been reported to enhance the migration of breast cancer cells by activating Hippo-YAP signals (An et al., 2019 2019 ). Overexpression of HDAC3 caused a significant increase in TEAD4 expression and resulted in negative regulation of Schwann cell myelination (Rivera-Reyes et al., 2018 2018 ). In the present study, we aimed to investigate whether RGFP966 could protect against prolonged hypothermic preservation-induced cardiac injury, and explored the role of Hippo-YAP signaling in hypothermically preserved rat hearts. Materials and methods 1 Animals 2 Male Sprague-Dawley rats (each weighing 250–300 g) were purchased from the Experimental Animal Center of Zhejiang University (Hangzhou, China). All experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA) and approved by the Ethics Committee on Animal Experimentation of Zhejiang University. Animal grouping 2 Rats were randomly divided into following four groups ( n =8): (1) control group, rat hearts were not preserved in Celsior solution; (2) Celsior group, rat hearts were preserved in Celsior solution for 12 h at 4 °C; (3) RGFP966 group, rat hearts were preserved in Celsior solution containing RGFP966 (at 0.25, 0.50, or 0.75 μmol/L; Selleck Chemicals, Houston, TX, USA) for 12 h at 4 °C; (4) RGFP966+verteporfin (VP) group, rat hearts were preserved in Celsior solution containing 0.75 μmol/L RGFP966 and 10 μg/mL VP (Selleck Chemicals, Houston, TX, USA) for 12 h at 4 °C. After that, all hearts in the above groups were perfused with Krebs-Henseleit solution for 60 min. Components of the Celsior solution (pH 7.4) included: NaOH 100 mmol/L, KCl 15 mmol/L, MgCl 2 13 mmol/L, CaCl 2 0.25 mmol/L, mannitol 60 mmol/L, lactobionate 80 mmol/L, histidine 30 mmol/L, and glutamate 20 mmol/L. Components of the Krebs-Henseleit solution (pH 7.4) included: NaCl 118 mmol/L, NaHCO 3 3.25 mmol/L, KH 2 PO 4 1.2 mmol/L, MgSO 4 1.2 mmol/L, KCl 4.7 mmol/L, and glucose 10 mmol/L. The Krebs-Henseleit solution was saturated with 95% O 2 and 5% CO 2 . Perfusion of isolated rat hearts 2 Isolated rat hearts were hypothermically preserved and reperfused as previously reported (Chen et al., 2016 2016 ). Briefly, after the rats were anesthetized intraperitoneally with 1% (0.01 g/mL) sodium pentobarbital (50 mg/kg), their hearts were quickly removed and fixed on a Langendorff perfusion device. Then they were perfused with Krebs-Henseleit solution at 37 °C and a constant pressure of 76 mmHg. After 30 min of balanced perfusion, the hearts were transferred into Celsior solution (4 °C) to stop heart palpitations. Then the hearts were preserved in Celsior solution for 12 h with or without RGFP966/VP, followed by 60 min of reperfusion. Measurement of cardiac function 2 A balloon sensor was inserted into left ventricle through the left atrial appendage of each perfused isolated heart. Cardiac functions were measured using a MedLab Biological Signal Acquisition and Processing System (Nanjing, China). The parameters included left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), maximal systolic and diastolic velocity of left ventricular pressure (±d P /d t max ), heart rate (HR), and coronary flow. Western blotting 2 Total cellular protein was extracted by lysing left ventricle apical tissue using radioimmunoprecipitation assay (RIPA) buffer containing phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China). Nuclear protein was obtained using a Cytoplasmic and Nuclear Extraction kit (Invent Biotechnologies, Plymouth, MN, USA). The equivalent protein (20 μg) was loaded on a 10% (0.1 g/mL) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After electrophoresis (Bio-Rad, Hercules, CA, USA), proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% (0.05 g/mL) skimmed milk for 1 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies (Cell Signaling Technology, Danvers, MA, USA) were as follows: anti-HDAC3 (1:1000 diluted; Cat. No. 85057), anti-phosphorylated mammalian STE20-like kinase-1 (p-Mst1) (1:1000; Cat. No. 49332), anti-Mst1 (1:1000; Cat. No. 14946), anti-p-YAP (Ser127) (1:1000; Cat. No. 13008), anti-YAP (1:1000; Cat. No. 14074), anti-cleaved caspase-3 (1:1000; Cat. No. 9661), anti-B-cell lymphoma protein 2 (Bcl-2) (1:1000; Cat. No. 3498), anti-Bcl-2 associated X (Bax) (1:1000; Cat. No. 14796), anti-Histone H3 (1:1000; Cat. No. 4499), and anti-β-actin (1:1000; Cat. No. 3700). The membrane was then incubated with near-infrared-labeled secondary antibodies (1:5000, Li-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. Strips were detected using an Odyssey CLx near infrared dual-color fluorescence imaging system (Li-COR Biosciences, Lincoln, NE, USA) and quantified using Image Studio Ver. 5.2 software. Real-time quantitative polymerase chain reaction 2 Left ventricular myocardium tissue was homogenized in RNAiso Plus buffer (TaKaRa Bio, Inc., Mountain View, CA, USA) containing chloroform. Total RNA was precipitated and purified with isopropanol and ethanol, and then reverse-transcribed into complementary DNA (cDNA) using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Bio, Inc.). Polymerase chain reaction (PCR) reaction solution including 10 μL 2×TB Green Premix Ex Taq II, 0.8 μL PCR forward primer (10 μmol/L), 0.8 μL PCR reverse primer (10 μmol/L), 0.4 μL 50×ROX Reference Dye, 2 μL cDNA, and 6 μL diethyl pyrocarbonate (DEPC) water was prepared in an ice bath. PCR amplification was performed using an ABI 7500 Fast Real-Time PCR system (Life Technologies, Austin, TX, USA). Primer sequences were as follows (forward and reverse, respectively): SOD , 5'-GGCC AAGGGAGATGTTACAA-3' and 5'-GAACCTTGGA CTCCCACAGA-3'; GPx , 5'-CGGTTTCCCGTGCAA TCAGT-3' and 5'-ACACCGGGGACCAAATGATG-3'; CAT , 5'-CCTGACATGGTCTGGGACTT-3' and 5'-CA AGTTTTTGATGCCCTGGT-3'; Bcl-2 , 5'-ATGTGTG TGGAGAGCGTCAACC-3' and 5'-CCAGGAGAAA TCAAACAGAGGC-3'; GAPDH , 5'-GACATCAAG AAGGTGGTG-3' and 5'-CAGCATCAAAGGTGGA AG-3'. The relative expression of genes was calculated by the 2 −ΔΔ C T method (Lerman et al., 2018 2018 ). Determination of LDH activity 2 Lactate dehydrogenase (LDH) activity of coronary flow was determined using an LDH determination kit according to the manufacturer’s protocol (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Optical density was measured at 440 nm by a microplate reader (Thermo scientific, Waltham, MA, USA). Detection of SOD activity, CAT activity, MDA content, and GPx activity 2 Left ventricular myocardium tissue was homogenized in lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The supernatant was collected after centrifugation at 12 000 r/min, at 4 °C for 10 min. The SOD activity, CAT activity, malondialdehyde (MDA) content, and glutathione peroxidase (GPx) activity were determined using SOD activity test kit (Beyotime Institute of Biotechnology), CAT determination kit (Nanjing Jiancheng Institute of Bioengineering), MDA test kit (Beyotime Institute of Biotechnology), and GPx detection kit (Beyotime Institute of Biotechnology), respectively. Optical densities were measured at 450, 405, 532, and 340 nm, respectively, by a microplate reader (Thermo scientific). Immunohistochemistry 2 Left ventricular tissue was fixed in 10% neutral formalin solution, and then embedded in paraffin. After being dewaxed and dehydrated, 4-μm thick tissue slices were dyed with hematoxylin and eosin. The slices were observed under an optical microscope (Olympus, Japan). Detection of apoptosis using TUNEL method 2 Apoptotic cells were detected using an In Situ Cell Death Detection kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, after being dewaxed and hydrated, slices of left ventricular tissue were soaked in permeabilisation solution (containing 0.1% Triton X-100 and 0.1% sodium citrate solution) for 10 min. Then slices were incubated with terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) reaction solution at 37 °C for 1 h in darkness. After being thoroughly washed, specimens were incubated with converter-peroxidase (POD) at 37 °C for 30 min in darkness and dyed with diaminobenzidine (DAB) and hematoxylin. The number of apoptotic cells (brown) was calculated from observations under an optical microscope (Olympus, Japan) (Chen et al., 2018 2018 ). Statistical analysis 2 Data are expressed as mean±standard error of mean (SEM). Differences between groups were analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test using Prism 8.0.1 (GraphPad Software, Inc., La Jolla, CA, USA). Results 1 Inhibition of HDAC3 protein expression by RGFP966 in rat hearts subjected to hypothermic preservation 2 Hypothermic preservation significantly increased HDAC3 protein expression in rat hearts. The HDAC3 inhibitor RGFP966 inhibited the increase of hypothermic preservation-induced HDAC3 protein expression in a concentration-dependent manner ( P < 0.01; Fig. 1 1 ). (a) Representative immunoblot obtained with histone deacetylase 3 (HDAC3) and β-actin antibodies. (b) Densitometric analysis showing the expression of HDAC3. β-Actin served as an internal control. Data are shown as mean±standard error of mean (SEM) of four independent experiments, expressed as a fold increase relative to the value of the control group. ** P < 0.01 vs. control group; ## P < 0.01 vs. Celsior group Recovery of cardiac function promoted by RGFP966 in hypothermically preserved rat hearts 2 At the end of balanced perfusion, cardiac functions among the different groups were similar ( P > 0.05, data not shown). At the end of 60 min of reperfusion after hypothermic preservation, LVEDP was higher than that of the control group, while LVDP, ±d P /d t max , HR, and coronary flow were lower than those of the control group ( P < 0.01; Fig. 2 2 ). These hypothermic preservation-induced injuries were significantly alleviated by supplementation with RGFP966 ( P < 0.05; Fig. 2 2 ). RGFP966 (0.75 μmol/L) was chosen to further explore the mechanism of RGFP966-induced cardioprotection. (a) Left ventricular developed pressure (LVDP); (b) Left ventricular end-diastolic pressure (LVEDP); (c) Maximal systolic velocity of left ventricular pressure (+d P /d t max ); (d) Maximal diastolic velocity of left ventricular pressure (−d P /d t max ); (e) Heart rate; (f) Coronary flow. Data are shown as mean±standard error of mean (SEM) ( n =8). ** P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. Celsior group; && P < 0.01 vs. RGFP966 (0.75 μmol/L) group. VP: verteporfin Inhibition of the hypothermic preservation-induced activation of Hippo-YAP signaling pathway by RGFP966 2 After hypothermic preservation, total Mst1 protein expression did not change, but the p-Mst1/Mst1 ratio increased significantly ( P < 0.01; Figs. 3a and 3b 3a and 3b ). Hypothermic preservation also decreased total YAP protein expression and increased the p-YAP/YAP ratio ( P < 0.01; Figs. 3a, 3d 3a, 3d , and 3e 3e ). Compared with the Celsior group, RGFP966 (0.75 μmol/L) significantly inhibited the increase of the p-Mst1/Mst1 and p-YAP/YAP ratios induced by hypothermic preservation, and prevented a reduction in total YAP protein expression ( P < 0.01; Fig. 3 3 ). (a) Representative immunoblot obtained with phosphorylated mammalian STE20-like kinase-1 (p-Mst1), Mst1, p-Yes-associated protein (YAP), YAP, and β-actin antibodies. (b–e) Densitometric analysis showing the expression of p-Mst1/Mst1 (b), total Mst1 (c), p-YAP/YAP (d), and total YAP (e). β-Actin served as an internal control. Data are shown as mean±standard error mean (SEM) of three independent experiments, expressed as a fold increase relative to the value of the control group. ** P < 0.01 vs. control group; ## P < 0.01 vs. Celsior group Abolishment of RGFP966-induced improvement of cardiac function by VP 2 Hypothermic preservation reduced the nuclear YAP protein level; however, this was prevented by RGFP966 ( P < 0.01; Figs. 4a and 4b 4a and 4b ). VP, a small molecular inhibitor of YAP–TEAD interaction, partially abolished the protective effect of RGFP966 on cardiac function ( P < 0.01; Fig. 2 2 ). RGFP966 also prevented hypothermic preservation-induced increases of LDH activity and MDA content. This was also reversed by VP ( P < 0.01; Figs. 4c and 4d 4c and 4d ). (a) Representative immunoblot obtained with Yes-associated protein (YAP) and histone H3 antibodies. (b) Densitometric analysis showing the expression of nuclear YAP. Histone H3 served as a nuclear internal control. Data are mean±standard error of mean (SEM) of four independent experiments, expressed as a fold increase relative to the value of the control group. (c, d) Lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. Data are shown as mean±SEM ( n =8). ** P < 0.01 vs. control group; ## P < 0.01 vs. Celsior group; && P < 0.01 vs. RGFP966 group. VP: verteporfin Inhibition of hypothermic preservation-induced oxidative stress by RGFP966 2 Hypothermic preservation reduced the expression of antioxidant genes ( SOD , GPx , and CAT ) and inhibited SOD, GPx, and CAT activity. Compared with the Celsior group, RGFP966 significantly increased SOD , GPx , and CAT mRNA expression and enhanced their activity. The beneficial effects of RGFP966 were cancelled by addition of VP ( P < 0.05; Figs. 5a−5c 5a−5c , Fig. 6 6 ). (a) Expression of superoxide dismutase ( SOD ); (b) Expression of glutathione peroxidase ( GPx ); (c) Expression of catalase ( CAT ); (d) Expression of B-cell lymphoma protein 2 ( Bcl-2 ). Data are shown as mean±standard error of mean (SEM) ( n =3). ** P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. Celsior group; & P < 0.05, && P < 0.01 vs. RGFP966 group. VP: verteporfin (a) Activity of superoxide dismutase (SOD); (b) Activity of glutathione peroxidase (GPx); (c) Activity of catalase (CAT). Data are shown as mean±standard error of mean (SEM) ( n =8). ** P < 0.01 vs. control group; ## P < 0.01 vs. Celsior group; && P < 0.01 vs. RGFP966 group. VP: verteporfin 3.6 Inhibition of hypothermic preservation-induced apoptosis by RGFP966 2 After hypothermic preservation, the arrangement of myocardial fibers was disordered and the gaps between them widened (Fig. 7a 7a ). The number of apoptotic cells also increased ( P < 0.01; Figs. 7b and 7c 7b and 7c ). RGFP966 improved myocardial morphology and reduced the number of apoptotic cells. The above effect was prevented by supplementation with VP (Fig. 7 7 ). (a) Cardiac tissue morphology in hypothermically preserved rat hearts. (b) Cell apoptosis was detected using a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. The nuclei of apoptotic cells were stained brown, and nuclei of living cells were stained blue. The arrows indicate apoptotic cells. (c) Statistical analysis showed the percentage of TUNEL-positive cells. Data are shown as mean±standard error of mean (SEM) ( n =8). ** P < 0.01 vs. control group; ## P < 0.01 vs. Celsior group; && P < 0.01 vs. RGFP966 group. VP: verteporfin RGFP966 inhibited hypothermic preservation-induced overexpression of Bax and cleaved caspase-3, and enhanced Bcl-2 mRNA and protein expression ( P < 0.01; Figs. 8 8 and 5d 5d ). Compared with the RGFP966 group, Bax and cleaved caspase-3 protein were higher, and Bcl-2 mRNA and protein were lower in the RGFP966+VP group ( P < 0.01; Figs. 8 8 and 5d 5d ). (a) Representative immunoblot obtained with B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X (Bax), cleaved caspase-3, and β-actin antibodies. (b–d) Densitometric analysis showing the expression of Bcl-2 (b), Bax (c), and cleaved caspase-3 (d). β-Actin served as an internal control. Data are shown as mean±standard error of mean (SEM) of four independent experiments, expressed as a fold increase relative to the value of the control group. ** P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. Celsior group; & P < 0.05 vs. RGFP966 group. VP: verteporfin Discussion 1 Both in vitro and in vivo studies indicate that overexpression of HDAC3 is involved in cardiac ischemia-reperfusion injury, development of hypertension, heart failure, and atrial fibrillation, while inhibition of HDAC3 alleviates cardiac injury and cardiac remodeling (Sharifi-Sanjani et al., 2014 2014 ; Chen et al., 2017 2017 ; Zhang DL et al., 2018 2018 ; Ryu et al., 2019 2019 ). Moreover, it has been proved that RGFP966 can alleviate cerebral ischemia-reperfusion injury by reducing HDAC3 protein expression in diabetic mice (Zhao et al., 2019 2019 ). In the present study, we confirmed that the HDAC3 protein was overexpressed in rat hearts subjected to 12 h of hypothermic preservation, while downregulation of HDAC3 by RGFP966 could prevent hypothermic preservation-induced cardiac dysfunction and promote cardiac function recovery during reperfusion. LVDP and +d P /d t max were used to evaluate the cardiac systolic function, while LVEDP and −d P /d t max were used to indicate the cardiac diastolic function. It has been reported that d P /d t max is determined not only by ventricular contractility, but also by preload and afterload (Hamlin and del Rio, 2012 2012 ). In the Langendorff perfusion system, an isolated rat heart is perfused at a fixed perfusion pressure (afterload). The balloon containing a known volume of fluid inserted into the left ventricle also allows cardiac contraction independent of ventricular preload (end-diastolic volume). So, in this study, the preload and afterload were held constant, and the d P /d t max was considered mainly as a sensitive indicator of myocardial contractility. The coronary flow, which is associated with coronary artery diameter at a constant perfusion pressure, indicates the extent of coronary dilation or myocardial blood supply. Recent studies have shown that the beneficial effect of HDAC inhibitors is dependent on Hippo signaling (Jung et al., 2017 2017 ; Rivera-Reyes et al., 2018 2018 ; Yu et al., 2018 2018 ). The Hippo pathway is a kinase cascade reaction, in which the p-Mst1/2 (ortholog of Drosophila Hippo) activates long acting thyroid stimulator 1/2 (LATS1/2) kinases, and in turn phosphorylates YAP/TAZ. The p-YAP/TAZ is then sequestered in the cytoplasm and degraded via ubiquitination. When dephosphorylated, YAP/TAZ is translocated into nucleus and induces TEADs-mediated gene transcription, inhibits apoptosis, and promotes cell survival (Jung et al., 2017 2017 ). Inhibition of HDAC3 attenuated oxidative stress, decreased neuronal apoptosis in the hippocampi, and improved the spatial memory function of Alzheimer’s disease model mice by reducing p-Mst1 and p-YAP expression levels (Yu et al., 2018 2018 ). HDAC inhibitor could also inhibit mammalian target of rapamycin (mTOR) signaling, upregulate protein kinase R (PKR)-like endoplasmatic reticulum kinase (PERK) and ataxia telangiectasia mutated (ATM)-and RAD3-related kinases 6 (ATR6)-mediated unfolded protein response, and increase autophagy via downregulation of YAP protein (Rivera-Reyes et al., 2018 2018 ). Our present study showed that hypothermic preservation increased phosphorylation of Mst1 and YAP. RGFP966 suppressed phosphorylation of YAP, stabilized total YAP expression, and increased nuclear localization of YAP. VP, an inhibitor of YAP–TEAD interaction, prevented RGFP966-induced improvement of cardiac function in hypothermically preserved rat hearts, suggesting that the Hippo-YAP signaling pathway was involved in the beneficial effect of RGFP966 on rat hearts subjected to hypothermic preservation. The Hippo-YAP signaling pathway plays an important role in cardiac physiological and pathophysiological conditions (Wang et al., 2018 2018 ). Hippo-YAP signaling acts as a “signal integrator” in the regulation of oxidative stress and apoptosis. This is because YAP–TEAD interaction can lead to a higher expression of anti-apoptotic, antioxidant, and mitochondrial biogenesis genes that are critical for regulation of various cellular events (Roy et al., 2016 2016 ; Zhang et al., 2016 2016 ; Wang et al., 2018 2018 ). SOD and glutathione gene expression was upregulated by YAP in riboflavinogenic fungus Ashbya gossypii (Kavitha and Chandra, 2014 2014 ). Restoration of YAP activity upregulated the expression of the antioxidant genes CAT and SOD , and alleviated cardiac ischemia-reperfusion injury through a forkhead box O1 (FoxO1)-mediated mechanism (Shao et al., 2014 2014 ). The Hippo-YAP signaling pathway is also involved in myocardial reperfusion injury associated with the dissociation of Bcl-xL from Bax, thereby promoting apoptosis (Nakamura et al., 2016 2016 ). Overexpression of YAP reduced Bak and Bid expression, inhibited caspase-3 activation, decreased apoptosis, and delayed senescence of human periodontal ligament stem cells (Jia et al., 2018 2018 ). Our present study showed that RGFP966 attenuated oxidative stress and increased expression of the antioxidant enzymes SOD, CAT, and GPx. RGFP966 also increased Bcl-2 expression, and inhibited caspase-3 activity and apoptosis. Inhibition of YAP and TEAD interaction abolished these RGFP966-induced antioxidant and anti-apoptosis effects. The results suggest that the Hippo-YAP pathway participates in RGFP966-induced cardiac protection via regulation of oxidative stress and apoptosis. However, VP did not fully abolish the protective effect of RGFP966, suggesting that other signaling pathways beyond YAP might also be involved. Some studies demonstrated that the beneficial effect of RGFP966 could be accounted for by an epigenetic modulation mechanism (Xu et al., 2017 2017 ; Hitchcock et al., 2019 2019 ). However, other studies reported that HDAC3 regulates embryonic development and lipid metabolism in a deacetylase-independent manner (Sun et al., 2013 2013 ; Lewandowski et al., 2015 2015 ). HDAC inhibitor could also protect neurons from exogenous oxidative stress via an HDAC-independent mechanism (Olson et al., 2015 2015 ). In rat cortical and spinal astrocytes, RGFP966 has been reported to exhibit its anti-inflammatory property through both HDAC3-dependent and HDAC3-independent mechanisms (Morioka et al., 2016 2016 ). Whether RGFP966 regulates the YAP pathway in an HDAC3-dependent or HDAC3-independent manner is worthy of further investigation. Therefore, supplementation of Celsior solution with RGFP966 can attenuate cardiac dysfunction induced by prolonged hypothermic preservation. The protective effect of RGFP966 on hypothermic preservation-induced myocardial injury may provide a valuable and feasible strategy for heart transplantation in clinical practice, making it possible for patients to obtain donor hearts from more distant locations, thereby expanding possible donor heart resources in the future. There were some limitations in this study. Firstly, both YAP and TAZ are downstream signaling molecules in the Hippo pathway. It has been reported that YAP has a stronger effect on regulation of cellular function than TAZ (Plouffe et al., 2018 2018 ). So, in the present study, we explored only the role of YAP in the effect of RGFP966. The role of TAZ needs to be further investigated. Secondly, LDH, troponin, and creatine kinase-MB (CKMB) are three classic markers of myocardial injury. Only LDH was determined in the present study. Lack of evaluations of troponin and CKMB is a critical limitation. Finally, although the protective effect of RGFP966 on hypothermic preservation-induced myocardial injury has been demonstrated, further confirmation is needed before RGFP966 can be used in heart transplantation in clinical practice. Conclusions 1 The results suggest that supplementation of Celsior solution with RGFP966 attenuated cardiac dysfunction induced by prolonged hypothermic preservation in rat hearts. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway. References PP An , JX Li , LL Lu , 等 . Histone deacetylase 8 triggers the migration of triple negative breast cancer cells via regulation of YAP signals . Eur J Pharmacol , 2019 . 845 16 - 23 . 30582912 DOI: 10.1016/j.ejphar.2018.12.030 http://doi.org/10.1016/j.ejphar.2018.12.030 . 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Inhibition of HDAC3 ameliorates cerebral ischemia reperfusion injury in diabetic mice in vivo and in vitro Compliance with ethics guidelines Xiao-he ZHENG, Lin-lin WANG, Ming-zhi ZHENG, Jin-jie ZHONG, Ying-ying CHEN, and Yue-liang SHEN declare that they have no conflict of interest. The research was conducted in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All experiments were approved by the Ethics Committee on Animal Experimentation of Zhejiang University (Hangzhou, China). 10.1631/jzus.B2000026 Project supported by the National Natural Science Foundation of China (No. 81871541) 2020-01-21 2020-05-11 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 21