Peiyong Zhai

Sirt1 Regulates Aging and Resistance to Oxidative Stress in the Heart

Silent information regulator (Sir)2, a class III histone deacetylase, mediates lifespan extension in model organisms and prevents apoptosis in mammalian cells. However, beneficial functions of Sir2 remain to be shown in mammals in vivo at the organ level, such as in the heart. We addressed this issue by using transgenic mice with heart-specific overexpression of Sirt1, a mammalian homolog of Sir2. Sirt1 was significantly upregulated (4- to 8-fold) in response to pressure overload and oxidative stress in nontransgenic adult mouse hearts. Low (2.5-fold) to moderate (7.5-fold) overexpression of Sirt1 in transgenic mouse hearts attenuated age-dependent increases in cardiac hypertrophy, apoptosis/fibrosis, cardiac dysfunction, and expression of senescence markers. In contrast, a high level (12.5-fold) of Sirt1 increased apoptosis and hypertrophy and decreased cardiac function, thereby stimulating the development of cardiomyopathy. Moderate overexpression of Sirt1 protected the heart from oxidative stress induced by paraquat, with increased expression of antioxidants, such as catalase, through forkhead box O (FoxO)-dependent mechanisms, whereas high levels of Sirt1 increased oxidative stress in the heart at baseline. Thus, mild to moderate expression of Sirt1 retards aging of the heart, whereas a high dose of Sirt1 induces cardiomyopathy. Furthermore, although high levels of Sirt1 increase oxidative stress, moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.

NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart

NAD(P)H oxidases (Noxs) produce O2− and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4−/− (c-Nox4−/−) mice. Nox4 expression was inhibited in c-Nox4−/− mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O2− in the heart, indicating that Nox4 is a significant source of O2− in cardiac myocytes. The baseline cardiac phenotype was normal in young c-Nox4−/− mice. In response to pressure overload (PO), however, increases in Nox4 expression and O2− production in mitochondria were abolished in c-Nox4−/− mice, and c-Nox4−/− mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c-Nox4−/− mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

Silent Information Regulator 1 Protects the Heart From Ischemia/Reperfusion

Background Sirt1, a class III histone deacetylase, retards aging and protects the heart from oxidative stress. We here examined whether Sirt1 is protective against myocardial ischemia/reperfusion (I/R).Background— Silent information regulator 1 (Sirt1), a class III histone deacetylase, retards aging and protects the heart from oxidative stress. We here examined whether Sirt1 is protective against myocardial ischemia/reperfusion (I/R). Methods and Results— Protein and mRNA expression of Sirt1 is significantly reduced by I/R. Cardiac-specific Sirt1−/− mice exhibited a significant increase (44±5% versus 15±5%; P=0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15±4% versus 36±8%; P=0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4±3% versus 10±1%; P<0.003) were significantly reduced compared with nontransgenic mice. In Langendorff-perfused hearts, the functional recovery during reperfusion was significantly greater in transgenic mice with cardiac-specific overexpression of Sirt1 than in nontransgenic mice. Sirt1 positively regulates expression of prosurvival molecules, including manganese superoxide dismutase, thioredoxin-1, and Bcl-xL, whereas it negatively regulates the proapoptotic molecules Bax and cleaved caspase-3. The level of oxidative stress after I/R, as evaluated by anti-8-hydroxydeoxyguanosine staining, was negatively regulated by Sirt1. Sirt1 stimulates the transcriptional activity of FoxO1, which in turn plays an essential role in mediating Sirt1-induced upregulation of manganese superoxide dismutase and suppression of oxidative stress in cardiac myocytes. Sirt1 plays an important role in mediating I/R-induced increases in the nuclear localization of FoxO1 in vivo. Conclusions— These results suggest that Sirt1 protects the heart from I/R injury through upregulation of antioxidants and downregulation of proapoptotic molecules through activation of FoxO and decreases in oxidative stress.

A redox-dependent pathway for regulating class II HDACs and cardiac hypertrophy.

Thioredoxin 1 (Trx1) facilitates the reduction of signaling molecules and transcription factors by cysteine thiol-disulfide exchange, thereby regulating cell growth and death. Here we studied the molecular mechanism by which Trx1 attenuates cardiac hypertrophy. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II histone deacetylases (HDACs), master negative regulators of cardiac hypertrophy. Both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 are oxidized and form intramolecular disulfide bonds in response to reactive oxygen species (ROS)-generating hypertrophic stimuli, such as phenylephrine, whereas they are reduced by Trx1. Whereas reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export, independently of its phosphorylation status. Our study reveals a novel regulatory mechanism of cardiac hypertrophy through which the nucleocytoplasmic shuttling of class II HDACs is modulated by their redox modification in a Trx1-sensitive manner.

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2

Yasuhiro Maejima1,4, Shiori Kyoi1, Peiyong Zhai1, Tong Liu2, Hong Li2, Andreas Ivessa1, Sebastiano Sciarretta1, Dominic P. Del Re1, Daniela K. Zablocki1, Chiao-Po Hsu3, Dae-Sik Lim5, Mitsuaki Isobe4, and Junichi Sadoshima1,6 1Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, Rutgers, The State University of New Jersey, New Jersey Medical School, Newark, NJ 07103Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

Oxidative Stress Stimulates Autophagic Flux During Ischemia/Reperfusion

Autophagy is a bulk degradation process in which cytosolic proteins and organelles are degraded through lysosomes. To evaluate autophagic flux in cardiac myocytes, we generated adenovirus and cardiac-specific transgenic mice harboring tandem fluorescent mRFP-GFP-LC3. Starvation significantly increased the number of mRFP-GFP-LC3 dots representing both autophagosomes and autolysosomes per cell, suggesting that autophagic flux is increased in cardiac myocytes. H(2)O(2) significantly increased autophagic flux, which was attenuated in the presence of N-2-mercaptopropionyl glycine (MPG), an antioxidant, suggesting that oxidative stress stimulates autophagy in cardiac myocytes. Myocardial ischemia/reperfusion (I/R) increased both autophagosomes and autolysosomes, thereby increasing autophagic flux. Treatment with MPG attenuated I/R-induced increases in oxidative stress, autophagic flux, and Beclin-1 expression, accompanied by a decrease in the size of myocardial infarction (MI)/area at risk (AAR), suggesting that oxidative stress plays an important role in mediating autophagy and myocardial injury during I/R. MI/AAR after I/R was significantly reduced in beclin1(+/-) mice, whereas beclin1(+/-) mice treated with MPG exhibited no additional reduction in the size of MI/AAR after I/R. These results suggest that oxidative stress plays an important role in mediating autophagy during I/R, and that activation of autophagy through oxidative stress mediates myocardial injury in response to I/R in the mouse heart.

Cardiac-specific overexpression of AT1 receptor mutant lacking Gαq/Gαi coupling causes hypertrophy and bradycardia in transgenic mice

Ang II type 1 (AT1) receptors activate both conventional heterotrimeric G protein-dependent and unconventional G protein-independent mechanisms. We investigated how these different mechanisms activated by AT1 receptors affect growth and death of cardiac myocytes in vivo. Transgenic mice with cardiac-specific overexpression of WT AT1 receptor (AT1-WT; Tg-WT mice) or an AT1 receptor second intracellular loop mutant (AT1-i2m; Tg-i2m mice) selectively activating G(alpha)q/G(alpha)i-independent mechanisms were studied. Tg-i2m mice developed more severe cardiac hypertrophy and bradycardia coupled with lower cardiac function than Tg-WT mice. In contrast, Tg-WT mice exhibited more severe fibrosis and apoptosis than Tg-i2m mice. Chronic Ang II infusion induced greater cardiac hypertrophy in Tg-i2m compared with Tg-WT mice whereas acute Ang II administration caused an increase in heart rate in Tg-WT but not in Tg-i2m mice. Membrane translocation of PKCepsilon, cytoplasmic translocation of G(alpha)q, and nuclear localization of phospho-ERKs were observed only in Tg-WT mice while activation of Src and cytoplasmic accumulation of phospho-ERKs were greater in Tg-i2m mice, consistent with the notion that G(alpha)q/G(alpha)i-independent mechanisms are activated in Tg-i2m mice. Cultured myocytes expressing AT1-i2m exhibited a left and upward shift of the Ang II dose-response curve of hypertrophy compared with those expressing AT1-WT. Thus, the AT1 receptor mediates downstream signaling mechanisms through G(alpha)q/G(alpha)i-dependent and -independent mechanisms, which induce hypertrophy with a distinct phenotype.

Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress

Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen-inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiac-specific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

Rheb is a Critical Regulator of Autophagy During Myocardial Ischemia Pathophysiological Implications in Obesity and Metabolic Syndrome

Background— Rheb is a GTP-binding protein that promotes cell survival and mediates the cellular response to energy deprivation (ED). The role of Rheb in the regulation of cell survival during ED has not been investigated in the heart. Methods and Results— Rheb is inactivated during cardiomyocyte (CM) glucose deprivation (GD) in vitro, and during acute myocardial ischemia in vivo. Rheb inhibition causes mTORC1 inhibition, because forced activation of Rheb, through Rheb overexpression in vitro and through inducible cardiac-specific Rheb overexpression in vivo, restored mTORC1 activity. Restoration of mTORC1 activity reduced CM survival during GD and increased infarct size after ischemia, both of which were accompanied by inhibition of autophagy, whereas Rheb knockdown increased autophagy and CM survival. Rheb inhibits autophagy mostly through Atg7 depletion. Restoration of autophagy, through Atg7 reexpression and inhibition of mTORC1, increased cellular ATP content and reduced endoplasmic reticulum stress, thereby reducing CM death induced by Rheb activation. Mice with high-fat diet–induced obesity and metabolic syndrome (HFD mice) exhibited deregulated cardiac activation of Rheb and mTORC1, particularly during ischemia. HFD mice presented inhibition of cardiac autophagy and displayed increased ischemic injury. Pharmacological and genetic inhibition of mTORC1 restored autophagy and abrogated the increase in infarct size observed in HFD mice, but they failed to protect HFD mice in the presence of genetic disruption of autophagy. Conclusions— Inactivation of Rheb protects CMs during ED through activation of autophagy. Rheb and mTORC1 may represent therapeutic targets to reduce myocardial damage during ischemia, particularly in obese patients.

Thioredoxin 1 Negatively Regulates Angiotensin II–Induced Cardiac Hypertrophy Through Upregulation of miR-98/let-7

Rationale: Thioredoxin (Trx)1 inhibits pathological cardiac hypertrophy. MicroRNAs (miRNAs) are small noncoding RNAs that downregulate posttranscriptional expression of target molecules. Objectives: We investigated the role of miRNAs in mediating the antihypertrophic effect of Trx1 on angiotensin II (Ang II)–induced cardiac hypertrophy. Methods and Results: Microarray analyses of mature rodent microRNAs and quantitative RT-PCR/Northern blot analyses showed that Trx1 upregulates members of the let-7 family, including miR-98, in the heart and the cardiomyocytes therein. Adenovirus-mediated expression of miR-98 in cardiomyocytes reduced cell size both at baseline and in response to Ang II. Knockdown of miR-98, and of other members of the let-7 family, augmented Ang II–induced cardiac hypertrophy, and attenuated Trx1-mediated inhibition of Ang II–induced cardiac hypertrophy, suggesting that endogenous miR-98/let-7 mediates the antihypertrophic effect of Trx1. Cyclin D2 is one of the predicted targets of miR-98. Ang II significantly upregulated cyclin D2, which in turn plays an essential role in mediating Ang II–induced cardiac hypertrophy, whereas overexpression of Trx1 inhibited Ang II–induced upregulation of cyclin D2. miR-98 decreased both expression of cyclin D2 and the activity of a cyclin D2 3′UTR luciferase reporter, suggesting that both Trx1 and miR-98 negatively regulate cyclin D2. Overexpression of cyclin D2 attenuated the suppression of Ang II–induced cardiac hypertrophy by miR-98, suggesting that the antihypertrophic actions of miR-98 are mediated in part by downregulation of cyclin D2. Conclusions: These results suggest that Trx1 upregulates expression of the let-7 family, including miR-98, which in turn inhibits cardiac hypertrophy, in part through downregulation of cyclin D2.