The Effects And Mechanisms Of Mst1 On Ang Ⅱ-related Cardiac Remodeling In Mice

Background and Aims:Cardiac remodeling is a highly lethal disease with a staggering economic burden worldwide.The developments of cardiac remodeling include cardiac hypertrophy,cardiac fibrosis,inflammation,mitochondria injury,cardiomyocyte autophagy and apoptosis,and finally these factors leads to heart failure.Angiotension Ⅱ（Ang Ⅱ）plays a central role in the pathogenesis of renin-angiotensin-aldosterone system（RAAS）-induced heart remodeling.Possible mechanisms for the Ang Ⅱ-induced cardiac remodeling are dependent on the increase of blood pressure,mechanically stretching,the release and elevation of inflammatory cytokines,expression of transforming growth factor-β1（TGF-β1）and the deposition of extracellular matrix（ECM）.The high morbidity and mortality of Ang Ⅱ-induced cardiac remodeling prompts the development of potential strategies of preventing cardiomyocyte dysfunction.In recent years,despite the efficacy of angiotensin converting enzyme（ACE）inhibitors and angiotensin receptor blockers（ARBs）on Ang Ⅱ-induced cardiac remodeling,their effects are limited,and the prevalence of heart remodeling is still increasing.The annoying phenomenon of "aldosterone breakthrough（aldosterone escape）" also limited the clinical gains via only blocking Ang Ⅱ receptors.Therefore,new molecular pathways directed at modulating Ang Ⅱ-related cardiac remodeling are attractive treatment strategies for patients with heart failure.Mammalian sterile 20-like kinase 1（Mst1）,a serine-threonine kinase,is a mammalian homologue of Drosophila Hippo and forms the core of Hippo signaling pathway that regulates cardiomyocyte growth and death.Our previous studies also indicated that Mst1 may play a potential role of in regulating cardiomyocyte autophagy and apoptosis.Nevertheless,the mechanisms and effects of cardiomyocyte-specific Mst1 knockout in Ang Ⅱ-induced heart remodeling are still unclear.Thus,the purposes of this paper were 1)to clarify whether cardiomyocyte-specific Mst1 knockout attenuates Ang Ⅱ-induced cardiac dysfunction and whether Mst1 specific knockout counteracts Ang Ⅱ-related mitochondria injury;2)to examine whether cardiomyocyte-specific Mst1 knockout plays a key role in Ang Ⅱ-induced cardiac hypertrophy,cardiac fibrosis and hypertension;3)to determine whether Mst1 specific knockout alleviates Ang Ⅱ-induced cardiac remodeling via adjusting cardiomyocyte autophagic flux and to further verify whether cardiomyocyte specific Mst1 knockout exerts the function independent of AT1 R and AT2 R as well as to clarify the mechanism;4)to clarify whether cardiomyocyte-specific Mst1 knockout can alleviate Ang Ⅱ-induced cardiomyocyte apoptosis and probe into the underlying mechanisms,including the role of Mst1 specific knockout counteracting ROS-mediated activation of the JNK signaling pathway and reversing the Ang Ⅱ-triggered the dissociation of Trx from ASK1.Part1: Generation of mice with cardiomyocyte specific Mst1 knockoutObjective: Construct Mst1flox/flox mice and αMHC-Mer Cre Mer Mice,and finally generate cardiomyocyte specific Mst1 knockout mice: Mst1^Δ/Δ（αMHC-Mer Cre Mer: Mst1flox/flox mice with the induction of tamoxifen）.Methods: Cardiomyocyte-specific Mst1 knockout mice were generated using standard Cre-Lox P-based gene targeting strategies.The final targeting vector（Lox P sites are A12242 to T12275 and A6695 to T6728;Exons are G13681 to A13724,T13561 to T13620,C7270 to G7320 and G4350 to G4478）for Mst1 conditional knockout was constructed and subsequently delivered to ES cells（C57BL/6）via electroporation,followed by drug selection,PCR screening,and Southern Blot confirmation.Then,certain clones were selected for blastocyst microinjection,followed by chimera production.Founders were confirmed as germline-transmitted via crossbreeding with wild-type（Cyagen Biosciences Inc,Chinese subsidiaries of Suzhou）.Then,Neo delete F1 heterozygous mutant mice（Mst1flox/+,3 male and 4 female）were confirmed.F2 mice were generated from F1 mice and Mst1flox/flox mice were identified from F2 mice by PCR screening.Next,we crossed Mst1flox/flox mice with αMHC-Mer Cre Mer mice（Jackson Laboratories,USA）（C57BL/6 background）as F3 mice（αMHC-Mer Cre Mer:Mst1flox/+）.By repeatedly crossing F3 mice（αMHC-Mer Cre Mer:Mst1flox/+）with Mst1flox/flox mice,F4 mice were finally generated.Then,we distinguished Mst1flox/flox mice from F4 mice and indentified the αMHC-Mer Cre Mer:Mst1flox/flox mice from Mst1flox/flox mice of F4 mice by PCR screening,the product of which is 413 bp only.Tamoxifen（Sigma Aldrich,40 mg/kg）in corn oil was administered via the IP route in 6-week-old αMHC-Mer Cre Mer:Mst1flox/flox male mice for 5 days,consecutively.After the last tamoxifen injection,all mice were allowed 7 weeks to recover before any cardiac assessments,because Cre expression in the heart can induce a transient cardiomyopathy that dissipates 5 weeks after tamoxifen-induced Cre expression.In the end,we confirmed the effectiveness of Cre recombinase in adult mice cardiomyocytes by PCR screening and Western blotting before establishing any animal mode.Result and conclusion: Cardiomyocyte specific Mst1 knockout mice were constructed successfully.Part2: Effects of cardiomyocyte specific Mst1 knockout on Ang Ⅱ-induced cardiac dysfunction and mitochondrial damageObjective: To investigate the effects of Mst1 specific knockout on Ang Ⅱ-induced cardiac dysfunction and mitochondrial damage in miceMethods: Mini-osmotic-pumps were implanted subcutaneously between the scapulae.Pumps were filled with Ang Ⅱ in sterile saline and were set to deliver Ang Ⅱ（1000ng/kg per/min）for 46 days.Control mice received an infusion of saline of comparable volume.Mst1^fl/fl and Mst1^Δ/Δ mice were randomly assigned to the control group or the Ang Ⅱ-treated group.All groups had similar blood pressure,morphometric parameters and hemodynamic indexes at baseline.The mice were divided into the following groups:（1）Mst1^fl/fl;（2）Mst1^Δ/Δ;（3）Ang Ⅱ+Mst1^fl/fl;（4）Ang Ⅱ+Mst1^Δ/Δ（n=9 mice per group）.About even weeks later,these animals were sacrificed.Before these mice were sacrificed,echocardiography was employed.On the short-axis view,LVESD and LVEDD were measured and then LVEF and LVFS were calculated by computer algorithms.Hemodynamic measurements were carried out in vivo.Furthermore,LVSP,LVEDP,positive d P/dtmax and negative d P/dtmax were assessed by invasive hemodynamic measurement before execution.JC-1 was employed to observe the change of cardiomyocyte mitochondrial membrane potential（ΔΨm）.TEM and FCM were used to determine the amplitude of mitochondrial swelling.To assess the mitochondrial biological function,enhanced ATP bioluminescent assay kit was used to detect the level of myocardial mitochondria ATP.Results: 1.Cardiomyocyte-specific Mst1 knockout attenuated Ang Ⅱ-induced left ventricular dysfunction,as evidenced by improved LVEF and LVFS,as well as decreased LVESD in mice subjected to chronic Ang Ⅱ infusion.2.LVSP and positive dp/dtmax was higher in Ang Ⅱ+Mst1^fl/fl group versus Mst1^fl/fl group,suggesting an enhanced cardiac systolic function to help counteract Ang Ⅱ-induced pressure overload.There were no significant differences in LVSP and positive dp/dtmax between Ang Ⅱ+Mst1^fl/fl group and Ang Ⅱ+Mst1^Δ/Δ group.3.LVEDP and negative dp/dtmax deteriorated in the Ang Ⅱ+Mst1^fl/fl group relative to Mst1^fl/fl group.Mst1^Δ/Δ improved cardiac diastolic function to counteract Ang Ⅱ-induced volume overload as evidence of decreased LVEDP and improved negative dp/dtmax in the Ang Ⅱ+Mst1^Δ/Δ group relative to Ang Ⅱ+Mst1^fl/fl group.4.Primary cardiomyocytes transduced with AD-sh-Mst1 exhibited an increased ratio of JC-1 red to green in the presence of Ang Ⅱ.5.TEM revealed that Ang Ⅱ induced mitochondria ultrastructure injury and there was improved mitochondrial ultrastructure in the Ang Ⅱ+Mst1^Δ/Δ group compared with the Ang Ⅱ+Mst1^fl/fl group.6.The relative mitochondria FSC/SSC indicated that Mst1^Δ/Δ could attenuate Ang Ⅱ-induced mitochondria swelling.7.Mst1^Δ/Δ could increase the mitochondrial ATP content in Ang Ⅱ-treated groups.Conclusions:1.Cardiomyocyte specific Mst1 knockout effectively alleviates Ang Ⅱ-induced cardiac dysfunction.2.The Ang Ⅱ-induced mitochondria damage was also improved in Mst1 specific knockout mice.Part3: Effects of Mst1 specific knockout on Ang Ⅱ-induced cardiac hypertrophy,cardiac fibrosis and hypertension.Objective: To explore the effects of cardiomyocyte specific Mst1 knockout on Ang Ⅱ-induced cardiac compensatory concentric hypertrophy,cardiac maladaptive pattern of eccentric hypertrophy,cardiac fibrosis and hypertension.Methods: The hearts were swiftly removed to calculate for the HW/BW and HW/TL after execution.Then these sections of left ventricles were stained with H&E staining.LVWT and IVST were also measured on H&E-stained sections by using the software of 3DHISTECH.The cardiomyocyte major size,cardiomyocyte minor size and cardiomyocyte cross-sectional areas were measured on slides with H&E staining and WGA staining by using the software of Image-Pro Plus 6.0.Considering that the left ventricle can also subject to maladaptive eccentric hypertrophy,which produces cardiomyocyte elongation and undergoes apoptosis according to the Laplace law.Therefore,the myocardium specimens from each group were also serially cut into 6 μm sections and three sections per specimens were selected randomly for the TUNEL assay.The relative ANF and β-MHC m RNA expression and protein expression were detected by RT-PCR and Western blotting.The sections of left ventricles were then stained with Masson’s trichrome reagent and Picro Sirius Red Stain.Slides were viewed with a scanner and measured using the software of the pannoramic viewer.The noninvasive blood pressure acquisition system for mice was used for all tail cuff measurements.By this way,blood pressure and heart rate measurements were carried out every two days.Results: 1.Despite the fact that the LVWT,IVST,HW/BW and HW/TL increased in mice subjected to chronic Ang Ⅱ infusion,there were no significant differences between the Ang Ⅱ+Mst1^fl/fl group and the Ang Ⅱ+Mst1^Δ/Δ group.The cardiomyocyte major diameter,minor diameter and cross-sectional area,determined by WGA staining and H&E staining,also showed the same trend.2.In vivo and in vitro,the m RNA and protein levels of ANF and β-MHC also revealed that cardiomyocyte specific Mst1 knockout failed to alleviate Ang Ⅱ-induced myocardial compensatory concentric hypertrophy.3.Masson’s trichrome and Picro Sirius Red staining showed that there were no significant differences between the Ang Ⅱ+Mst1^fl/fl and Ang Ⅱ+Mst1^Δ/Δ groups.4.There were no significant differences in heart rate,systolic blood pressure and diastolic blood pressure between all groups at the beginning of the experiment.Forty-six days later,the Ang Ⅱ groups had higher HR,SBP and DBP compared with their corresponding control groups.However,there were no significant differences in the HR,SBP and DBP between the Ang Ⅱ+Mst1^Δ/Δ group and the Ang Ⅱ+Mst1^fl/fl group.Conclusions:1.Cardiomyocyte-specific Mst1 knockout not only reverses the maladaptive pattern of eccentric hypertrophy by alleviating Ang Ⅱ-induced cardiac apoptosis and improving cardiac diastolic function to counteract Ang Ⅱ-induced volume overload but also does not weaken myocardial concentric hypertrophy,which can maintain cardiac function to counteract the Ang Ⅱ-induced pressure overload to some extent.2.The effects of cardiomyocyte-specific Mst1 knockout on cardiac fibrosis and hypertension were marginal.Part4: The effects and mechanism of cardiomyocyte specific Mst1 knockout on cardiomyocyte autophagic flux in Ang Ⅱ-treated miceObjective: To determine whether cardiomyocyte specific Mst1 knockout alleviates Ang Ⅱ-induced cardiac remodeling by improving cardiomyocyte autophagic flux and whether these functions depend on Ang Ⅱ type1 receptor and Ang Ⅱ type2 receptor.Methods: The expression level of LC3-Ⅱ/LC3-I,p62,Beclin1,JNK,p-JNK,Erk1/2,p-Erk1/2,Cleaved caspase-3/caspase-3 and Bax were determined by Western blotting both in vivo and in vitro.In vivo,we also analyzed the level of autophagy by IHC and IF assays of LC3.For in vitro experiments,primary ventricular cardiomyocytes were prepared from 1-to 3-day-old wild type C57BL/6 neonatal mice.Cells were cultured in DMEM supplemented with 10% Fetal bovine serum and 1% penicillin/streptomycin and maintained at 37 °C with 5% CO2.The adenoviruses harbouring Mst1 sh RNA（Ad-sh-Mst1）and control vectors for Mst1 sh RNA（Ad-Lac Z）were transduced 24 hours after transduction of the adenoviruses harbouring green/red fluorescent protein-LC3（GFP-m RFP-LC3）.After 36 hours,the primary cardiomyocytes were treated with either Ang Ⅱ（10μmol/l）or equal volume of PBS for 24 hours.The primary cardiomyocytes were randomly allocated into the following groups:（i）Control;（ii）Control+Ad-Lac Z;（iii）Control+Ad-sh-Mst1（iv）Ang Ⅱ;（v）Ang Ⅱ + Ad-Lac Z;（vi）Ang Ⅱ+ Ad-sh-Mst1.Besides,AO red puncta was observed by fluorescence microscopic as a rough marker of autolysosomes and fluorescence microscopic detection of P62 was also conducted.Furthermore,to test whether the increased autophagic flux triggered by cardiomyocyte specific Mst1 knockout played a beneficial role in the heart under chronic Ang Ⅱ infusion and whether cardiomyocyte specific Mst1 knockout increased autophagic flux independent of Ang Ⅱ receptors,the new groupings were implemented by treatment with 3-MA or losartan.Mice were injected intraperitoneally with autophagy inhibitor 3-MA（10 mg/kg per day）for 14 days.All groups had similar blood pressure,morphometric parameters and hemodynamic indexes at the beginning of the experiment.The mice were divided into the following groups:（1）Ang Ⅱ+Mst1^fl/fl;（2）Ang Ⅱ+Mst1^fl/fl+3-MA;（3）Ang Ⅱ+Mst1^Δ/Δ;（4）Ang Ⅱ+Mst1^Δ/Δ+3-MA（n=5 mice per group）.Seven weeks later,echocardiography was carried out to calculate the LVEF,LVFS,LVESD and LVEDD of these mice.Furthermore,other mice were administered with the angiotensin 1 receptor blocker（losartan）in drinking water（0.8 g/L）.The mice were divided into the following groups:（1）Mst1^fl/fl;（2）Ang Ⅱ+Mst1^fl/fl;（3）Ang Ⅱ+Mst1 Δ / Δ;（4）Ang Ⅱ+Mst1^fl/fl+losartan;（5）Ang Ⅱ+Mst1^Δ/Δ+losartan（n=5 mice per group）.All groups also had similar blood pressure,morphometric parameters and hemodynamic indexes at base level.In vitro study,RNA interference plasmids（p AT1a）targeting the AT1 a receptor gene（Agtr1a）and AT2 R blocker PD123319 were employed to knockdown AT1 R and block AT2 R.Results: 1.In vitro and in vivo,mildly up-regulated LC3-Ⅱ/LC3-I and Beclin1 expression as well as mildly down-regulated P62 expression were observed in the Ang Ⅱ groups compared to their corresponding control groups.IF and IHC assays of LC3 also showed that Ang Ⅱ administration triggered a mild enhancement in the expression of LC3.In vitro,a mild increase in AO red puncta and a mild decrease in P62 puncta,were observed in the Ang Ⅱ groups compared with their corresponding control groups.In primary cardiomyocytes transduced with Ad-GFP-m RFP-LC3,Ang Ⅱ treatment mildly increased the number of autophagosomes and autolysosomes compared with their corresponding control groups.3-MA,an autophagy inhibitor,aggravated Ang Ⅱ-induced cardiac remodeling by the evidence that further depressed LVEF and LVFS expression as well as further increased cleaved caspase-3/caspase-3 and Bax expression in Ang Ⅱ-related mice following the treatment with 3-MA were observed.2.In vivo and in vitro,there were no significant differences in LC3-Ⅱ/LC3-I,P62 and Beclin1 between Mst1^Δ/Δ group and Ang Ⅱ+Mst1^Δ/Δ group.Besides,in vivo,the IHC and IF assays of LC3 revealed there were no significant differences in the expression of LC3 between Mst1^Δ/Δ group and Ang Ⅱ+Mst1^Δ/Δ group.Furthermore,in neonatal mouse primary cardiomyocytes transduced with AD-GFP-m RFP-LC3,there were no significant differences in autophagosomes and autolysosomes between Ad-sh-Mst1 group and Ang Ⅱ+Ad-sh-Mst1 group.3.Following Ang Ⅱ treatment,Mst1 specific knockout increased LC3-Ⅱ and Beclin1 expression as well as decreased P62,cleaved caspase-3/caspase-3 and Bax expression compared with their corresponding control mice(Mst1^fl/fl mice).More typical autophagosomes accompanied by less damaged mitochondria were also observed by electron microscopy in Ang Ⅱ-treated Mst1^Δ/Δ mice.4.In vitro,the downregulation of Mst1 promoted cardiomyocyte autophagic flux,as demonstrated by more GFP-m RFP-LC3 puncta per cell,more pronounced increase in the number of AO red puncta per cell and more pronounced decrease in the accumulation of P62 puncta per cell in the presence of Ang Ⅱ.Mst1 inhibition could still increase the number of autophagosomes and autolysosomes,despite the knockdown of AT1 R.Increased LC3-Ⅱ and decreased P62 expression both in the presence and absence of chloroquine were observed in Ang Ⅱ-treated Mst1-inhibited neonatal primary cardiomyocytes.5.The benefits of improving cardiac function and suppressing Ang Ⅱ-induced cardiomyocyte apoptosis by cardiomyocyte specific Mst1 knockout disappeared after stimulation with 3-MA,an autophagy inhibitor.6.IF assays of LC3 showed losartan inhibited the number of LC3 puncta in cardiomyocyte,and Mst1^Δ/Δ could still elevate the number of LC3 puncta even when AT1 R was blocked by losartan.TUNEL assay showed that the Ang Ⅱ-induced cardiomyocytes apoptosis was partly negated by losartan,and there was a further mitigated apoptosis rate in the Ang Ⅱ+losartan+Mst1^Δ/Δ group compared with the Ang Ⅱ+losartan+Mst1^fl/fl group.The results of Western Blotting were consistent with the TUNEL and IF assays.When AT1 R was blocked with losartan,Mst1^Δ/Δ not only continued to elevate the expression of Beclin1 and LC3-Ⅱ/LC3-I,and reduce the expression of P62 but also continued to decrease the Ang Ⅱ-induced activation of caspase-3 and Bax.7.In vitro,Mst1 inhibition could reverse the Ang Ⅱ-induced phosphorylation of JNK,and this protective effect remained after the knockdown of AT1 R.However,Mst1 inhibition could not decrease the Ang Ⅱ-induced activation of p-ERK1/2,a central signaling molecule downstream of the AT1 R pathway.Conclusions:1.The compensatory effects of Ang Ⅱ on upregulated autophagy were associated with Mst1 inhibition.2.Cardiomyocyte-specific Mst1 knockout alleviates Ang Ⅱ-induced cardiac remodeling by enhancing cardiomyocyte autophagic flux and these effects were independent of activating Ang Ⅱ receptors.3.Despite the efficacy of angiotensin receptor blockers（ARBs）on cardiac remodeling,the knockdown or antagonization of AT1 R also inhibited cardiomyocyte autophagic flux,which may represent a threat to cardiac function and aggravate cardiac remodeling.It reveals the limitations of ARBs.4.Cardiomyocyte-specific Mst1 knockout enhances cardiomyocyte autophagic flux to reverse Ang Ⅱ-induced heart remodeling via coordinately increasing the express of Beclin1 and decreasing the expression of p-JNK.5.Cardiomyocyte specific Mst1 knockout could still reverse Ang Ⅱ-induced phosphorylation of JNK and elevate the expression of Beclin1 after the knockdown of AT1 R.Part5: The mechanisms of cardiomyocyte specific Mst1 knockout on alleviating cardiomyocyte apoptosis in Ang Ⅱ-treated miceObjective: To further reveal the underlying mechanisms of cardiac-specific Mst1 knockout alleviating Ang Ⅱ-induced cardiomyocyte apoptosis.Methods: The Ang Ⅱ-treated animal model refers to above.Furthermore,for elimination of ROS in vivo,mice were injected intraperitoneally with ROS scavenger NAC（50mg/kg per day）.Mst1^fl/fl mice and Mst1^Δ/Δ mice were randomly assigned to Ang Ⅱ groups or Ang Ⅱ+NAC groups.All groups had similar blood pressure,morphometric parameters and hemodynamic indexes before carry out trials.Mice were divided into four groups:（1）Ang Ⅱ+Mst1^fl/fl;（2）Ang Ⅱ+Mst1^fl/fl +NAC;（3）Ang Ⅱ+Mst1^Δ/Δ;（4）Ang Ⅱ+Mst1^Δ/Δ+NAC（n=6 mice per group）.46 days later,Echocardiography was carried out to calculate the LVEF,LVFS,LVESD and LVEDD.Then,all mice were executed for further H&E,IHC assay,TUNEL assay and IF assay.IHC of p-JNK was performed in paraffin sections using a mircowaved-based antigen retrieval method.For measurement of tissue superoxide,part of left ventricle myocardium was immediately frozen in OCT embedding agent,and these tissues were cut into 8 μm frozen sections by freezing microtome.Three sections per left ventricle were selected randomly.Sections were incubated with fluorescent probe DHE（5mmol/l）for 30 min at 37 °C away from light.The fluorescence intensity of DHE probe was evaluated by fluorescence microscope.In vivo experiments,TUNEL assays were performed again.The two chamber’s cardiac sections were captured with a scanner and viewed with the software of Pannormamic viewer.Sequential method of immunofluorescence double stainings was employed to detect the co-localization of Trx and ASK1.COIP was performed to detect the Trx/ASK1 interaction in myocardium.In vitro,primary cultures of cardiomyocyte were harvested from the ventricle of neonatal C57BL/6 mice.Adenoviruses harbouring a short hairpin sh RNA2 directed against Mst1（Ad-sh-Mst1）and harboring control vectors for Ad-sh-Mst1（Ad-Lac Z）were constructed and selected successfully.The titers of adenoviruses were 1.26*1010 PFU/ml.The multiplicity of infection used was 100:1.The sh RNA sequence targeting mouse Mst1 was CCCGTTTGTTAAGAGTGCCAAAGGA.The primary cardiomyocytes were treated with either Ang Ⅱ（10 μmol/l,Merck 1656）or equal volume of PBS for 24 hours after transduction of adenoviruses harbouring Mst1 sh RNA（Ad-sh-Mst1）and control vectors for Mst1 sh RNA（Ad-Lac Z）for 36 hours.Then primary cardiomyocyte were treated with or without the antioxidant NAC（1mmol/L）at the same time.Furthermore,primary cardiomyocytes cultured on confocal dishes were labeled with DCFH-DA fluorescent probe to measure the intracellular ROS in vitro.JNK nuclear translocation,which reflects the activation of JNK,was examined under fluorescence microscope in vitro.IF of AO/EB was also used to count the number of live apoptotic cells and the necrotic cells in vitro.Intracellular MDA,SOD,T-AOC and mitochondrial ATP level were measured using commercial assay kits.Results: 1.TUNEL assay revealed that cardiac-specific Mst1 knockout decreased Ang Ⅱ-induced myocardial apoptosis relative to Ang Ⅱ+Mst1^fl/fl mice.Western blotting also showed that the ratios of cleaved caspase-3/caspase-3 and Bax were higher in the Ang Ⅱ+Mst1^fl/fl group than in control animals and that Mst1 specific knockout in the presence of Ang Ⅱ suppressed cardiomyocyte apoptosis.2.Ang Ⅱ stimulated the expression of p47 phox.Mst1 specific knockout attenuated the protein level of p47 phox in mice subjected to chronic Ang Ⅱ infusion.3.Ang Ⅱ treatment increased DHE fluorescence intensity in myocardial tissues of Ang Ⅱ+Mst1^fl/fl mice,an effect that was abrogated by cardiomyocyte specific loss of Mst1.Furthermore,Ang Ⅱ induced JNK phosphorylation in Mst^fl/fl mice,while Mst1 specific deficiency abrogated Ang Ⅱ-triggered JNK activation.Western blotting analysis also revealed that Ang Ⅱ increased the phosphorylation of both JNK and ERK1/2.Cardiomyocyte specific Mst1 knockout suppressed JNK/SAPK activation（Thr183/Tyr185）in the presence of Ang Ⅱ,although ERK1/2 phosphorylation was unaffected.4.In Mst1^fl/fl mice with Ang Ⅱ infusion,treatment with NAC abrogated the Ang Ⅱ-induced production of superoxide radical and suppressed Ang Ⅱ-triggered ROS production,as evidenced by the decreases in DHE fluorescence intensity and MDA level as well as the upregulation of SOD and T-AOC.In NAC-treated mice with chronic Ang Ⅱ infusion,cardiomyocyte specific Mst1 knockout failed to further inhibit the production of active oxygen radicals.Besides,in the presence of Ang Ⅱ,NAC treatment reduced JNK phosphorylation in Mst1^fl/fl mice as compared with their control mice.In Ang Ⅱ infused mice with NAC treatment,cardiomyocyte specific Mst1 knockout failed to further reverse this decrease in JNK phosphorylation level.In NAC-treated Mst1^fl/fl mice with Ang Ⅱ infusion,NAC alleviated Ang Ⅱ-induced pathological changes and apoptosis by the evidence of decreased apoptotic index in the TUNEL assays.However,there were no significant differences in the apoptotic index between Mst1^fl/fl and Mst1^Δ/Δ mice treated with Ang Ⅱ and NAC.5.In vitro,Mst1 knockdown alleviated Ang Ⅱ-induced primary cardiomyocyte apoptosis,as evidenced by the downregulation of cleaved caspase-3 and Bax in Ang Ⅱ-treated primary cardiomyocytes.Beside,the ratio of apoptotic primary cardiomyocytes+necrotic primary cardiomyocytes/total primary cardiomyocytes was increased by Ang Ⅱ treatment.Mst1 knockdown attenuated Ang Ⅱ-induced primary cardiomyocyte damage,as evidenced by the decreased numbers of apoptotic cardiomyocytes+necrotic primary cardiomyocytes.Furthermore in vitro,the results of the TUNEL assay showed that Ang Ⅱ induced primary cardiomyocyte apoptosis while Mst1 knockdown abrogated this effect,consistent with in vivo findings.6.In primary cardiomyocyte,NAC treatment and Mst1 knockdown both reversed the Ang Ⅱ-induced increase in intracellular ROS level.However,Mst1 knockdown failed to further decrease ROS production following NAC treatment.JNK nuclear translocation was enhanced in the presence of Ang Ⅱ and this effect was reversed by NAC treatment or Mst1 knockdown.However,in Ang Ⅱ-treated primary cardiomyocytes,Mst1 knockdown failed to further suppress JNK nuclear translocation after NAC treatment.Western blotting showed that Ang Ⅱ induced the phosphorylation of JNK（Thr183/Tyr185）and ERK1/2,whereas NAC treatment abolished the activation of JNK and ERK1/2.Mst1 knockdown reversed JNK activation（Thr183/Tyr185）in the presence of Ang Ⅱ but had no effect on ERK1/2 phosphorylation status,which is in accordance with the in vivo results.Interestingly,in Ang Ⅱ-treated primary cardiomyocytes,Mst1 knockdown did not further reverse JNK phosphorylation after NAC treatment.7.In vitro,Mst1 deficiency decreased Ang Ⅱ-induced intracellular ROS production and improved Ang Ⅱ-induced mitochondria dysfunction,as seen by the increased levels of SOD and T-AOC as well as downregulation of MDA and DCFH-DA.Meanwhile,Mst1 deficiency enhanced mitochondrial metabolism.In Ang Ⅱ-treated primary cardiomyocytes,loss of Mst1 did not further improve mitochondrial metabolism following treatment with NAC.8.The results of COIP showed that cardiomyocyte specific Mst1 knockout enhanced the interaction between Trx and ASK1 in the presence of Ang Ⅱ,which suppressed ASK1 and downstream apoptotic signaling.Meanwhile,Ang Ⅱ+Mst^Δ/Δ mice showed markedly decreased JNK phosphorylation（Thr183/Tyr185）as well as ASK1 and p47 phox expression as compared to the Ang Ⅱ+Mst^fl/fl group.Immunofluorescence double-labeling experiments of Trx and ASK1 revealed greater co-localization of Trx and ASK1 in the Ang Ⅱ+Mst^Δ/Δ mice as compared to the Ang Ⅱ+Mst^fl/fl group,indicating that loss of Mst1 promoted Trx and ASK1 binding in the presence of Ang Ⅱ.Conclusions:1.Ang Ⅱ increased intracellular reactive oxygen species（ROS）production and cardiomyocyte apoptosis;these were reversed by administration of the ROS scavenger N-acetylcysteine and by Mst1 specific deficiency,which suppressed c-Jun N-terminal kinase（JNK）phosphorylation and its downstream signaling.However,cardiomyocyte specific Mst1 knockout failed to alleviate Ang Ⅱ-induced phosphorylation of extracellular signal-regulated kinase 1/2,suggesting that Mst1 specific knockout had the specificity of MAPKs signaling transduction.2.The protective effect of cardiomyocyte specific Mst1 knockout leading to the attenuation of Ang Ⅱ-induced cardiomyocyte apoptosis is dependent on the suppression of Ang Ⅱ-triggered ROS production.3.Furthermore,Mst1 specific knockdown enhances mitochondrial function by reversing Ang Ⅱ-induced oxidative stress.4.Cardiomyocyte specific Mst1 knockout inactivated apoptosis signal-regulating kinase1（ASK1）by promoting its association with thioredoxin（Trx）,which reversed the Ang Ⅱ-induced activation of the ASK1–JNK pathway and suppressed Ang Ⅱ-induced cardiomyocyte apoptosis.