Heng Ma

Impaired Cardiac SIRT1 Activity by Carbonyl Stress Contributes to Aging-Related Ischemic Intolerance

Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1+/− knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1+/− hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.

Aldehyde dehydrogenase 2 activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1

Cardiac aging is characterized by accumulation of damaged proteins and decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated inactivation in aged heart, we determined the benefits of activation of aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the ALDH2 KO mice progressively developed age-related heart dysfunction and showed reduction in the life span, which strongly suggests that ALDH2 ablation leads to cardiac aging. Whats more, aged hearts displayed a significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-protein adducts and protein carbonyls, impairment in the autophagy flux, and, consequently, deteriorated cardiac function after starvation. Sustained Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2 activity and abrogated these effects. Using SIRT1 deficient heterozygous (Sirt1+/−) mice, we found that SIRT1 was necessary for ALDH2 activation-induced autophagy. We further demonstrated that ALDH2 activation attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially, ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1 increased Rab7 expression, which were both necessary and sufficient for restoring autophagy flux. These results highlight that both accumulation of proteotoxic carbonyl stress linkage with autophagy decline contribute to heart senescence. ALDH2 activation is adequate to improve the autophagy flux by reducing the carbonyl modification on SIRT1, which in turn plays an important role in maintaining cardiac health during aging.

Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart

Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 mon) and aged (20 mon) Sprague Dawley (SD) rats were subjected to MI/R in vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a promising therapeutic interventional target for elderly ischemic heart disease patients.

Nuclear AMPK regulated CARM1 stabilization impacts autophagy in aged heart

Senescence-associated autophagy downregulation leads to cardiomyocyte dysfunction. Coactivator-associated arginine methyltransferase 1 (CARM1) participates in many cellular processes, including autophagy in mammals. However, the effect of CARM1 in aging-related cardiac autophagy decline remains undefined. Moreover, AMP-activated protein kinase (AMPK) is a key regulator in metabolism and autophagy, however, the role of nuclear AMPK in autophagy outcome in aged hearts still unclear. Hers we identify the correlation between nuclear AMPK and CARM1 in aging heart. We found that fasting could promote autophagy in young hearts but not in aged hearts. The CARM1 stabilization is markedly decrease in aged hearts, which impaired nucleus TFEB-CARM1 complex and autophagy flux. Further, S-phase kinase-associated protein 2(SKP2), responsible for CARM1 degradation, was increased in aged hearts. We further validated that AMPK dependent FoxO3 phosphorylation was markedly reduced in nucleus, the decreased nuclear AMPK-FoxO3 activity fails to suppress SKP2-E3 ubiquitin ligase. This loss of repression leads to The CARM1 level and autophagy in aged hearts could be restored through AMPK activation. Taken together, AMPK deficiency results in nuclear CARM1 decrease mediated in part by SKP2, contributing to autophagy dysfunction in aged hearts. Our results identified nuclear AMPK controlled CARM1 stabilization as a new actor that regulates cardiac autophagy.

Targeting ALDH2 for Therapeutic Interventions in Chronic Pain-Related Myocardial Ischemic Susceptibility

Clinical observations have demonstrated a link between chronic pain and increased ischemic heart disease mortality, but the mechanisms remain elusive. Reactive aldehydes have recently been confirmed as a new player in pain pathologies, while our previous study demonstrated that reactive aldehydes (4-HNE) induced carbonyl stress contributing to myocardial ischemic intolerance. The aim of this study was to explore whether chronic pain increases susceptibility to myocardial ischemia/reperfusion (MI/R) injury and to investigate the underlying mechanisms focusing on toxic aldehyde and carbonyl stress. Methods: Chronic pain was induced by chronic compression of the dorsal root ganglion (CCD). After 2 weeks CCD, aldehyde dehydrogenase (ALDH2) KO or wild-type (WT) littermate mice were then subjected to in vivo MI/R. Results: In CCD-WT mice, heightened nociception paralleled circulating aldehyde (4-HNE) accumulation and cardiac protein carbonylation. Mechanistically, CCD-induced 4-HNE overload provoked cardiac Sirtuin 1 (SIRT1) carbonylative inactivation and inhibited Liver kinase B1 (LKB1) - AMP-activated protein kinase (LKB1-AMPK) interaction, which resulted in exacerbated MI/R injury and higher mortality compared with non-CCD WT mice. ALDH2 deficiency further aggravated CCD-induced susceptibility to MI/R injury. Exogenous 4-HNE exposure in peripheral tissue mimicked chronic pain-induced aldehyde overload, elicited sustained allodynia and increased MI/R injury. However, cardiac-specific ALDH2 upregulation by AAV9-cTNT-mediated gene delivery significantly ameliorated chronic pain-induced SIRT1 carbonylative inactivation and decreased MI/R injury (minor infarct size, less apoptosis, and improved cardiac function). Conclusion: Collectively, chronic pain-enhanced carbonyl stress promotes myocardial ischemic intolerance by SIRT1 carbonylative inactivation and impairment of LKB1-AMPK interaction. ALDH2 activation and prevention of protein carbonylation may be a potential therapeutic target for myocardial ischemic vulnerability in chronic pain patients. Our results newly provided overlapping cellular mechanisms of chronic pain and myocardial dysfunction interplay.

EXERCISE TRAINING IMPROVES ISCHAEMIC TOLERANCE OF THE SENESCENT HEART BY AMPK-AUTOPHAGY CASCADE

Objectives Exercise promotes myocardial resistance to ischaemic injury. But the mechanisms underlying exercise induced anti-aged cardioprotection are incompletely understood. Here, we report that swimming training (ST) modulation of AMPK dependent autophagy prevents ischaemic injury in aged heart. Methods and Results— young (4 months) and aged (22 months) mice maintained 10-week free-loading exercise training (swimming 1 h/day, 5 days/week). Results Exercise significantly promoted basal autophagy in aged heart, which was accompanied by AMPK activation and mammalian target of rapamycin (mTOR) inhibition. As thus, exercise training improve the tolerance of aged hearts to ischaemia/reperfusion (I/R) injury as evidenced by reduced infarct size and cardiomyocytes damage, ameliorated the recovery of LV function after ischaemia as well as improved cardiomyocyte contractile function under hypoxic condition. Exercise training also restores the energy in response to I/R and cardiomyocyte mitochondrial membrane potential (MMP). Additionally, exercise induced cardioprotctive effect and cardiac autophagy upregulation were impaired in AMPK KD mice. Furthermore, hypoxia stress–induced cardiomyocytes death in aged heart was promoted by autophagy depression. Conclusions Excitation AMPK-autophagic flux by exercise training in senescence may be attributed to enhance intrinsic myocardial resistance to myocardial ischaemic injury in aged individuals.

Melatonin attenuates chronic pain related myocardial ischemic susceptibility through inhibiting RIP3-MLKL/CaMKII dependent necroptosis

Chronic pain aggravates cardiovascular injury via incompletely understood mechanisms. While melatonin may participate in the pathophysiological process of chronic pain, its cardiovascular effects under chronic pain states remains unknown. In this study, chronic pain was induced by spared nerve injury model (SNI) for 4 weeks. We showed decreased the ipsilateral hind paw withdrawal mechanical threshold (PWMT) in SNI mice. High dose melatonin treatment (60 mg/kg, i.p.) could reversed nociceptive threshold in SNI mice. To verify the effect of chronic pain on the cardiac tolerance to ischemic stress, mice were subjected to myocardial ischemia-reperfusion (MI/R) in vivo. SNI mice showed exaggerated MI/R-induced detrimental effects and myocardial necroptosis compared with control group (P < .05). Mechanically, an increased level of tumor necrosis factor-α (TNF-α) was found in SNI group following by a robust interaction of RIP1/RIP3. RIP3-induced phosphor-MLKL and CaMKII more significantly in SNI mice (P < .05). We found that RIP3 deficiency provided a comparable protection against MI/R-induced necroptosis under chronic pain conditions. More importantly, low dose melatonin (20 mg/kg, i.p.) treatment 10 min before reperfusion decreased the level of TNF-α following with a negatively regulating the RIP3 induced phosphor-MLKL/CaMKII signaling, thus significantly reduced ROS production and cardiomyocyte necroptosis and ameliorated cardiac function. In summarize, our results demonstrated that chronic pain sensitizes heart to MI/R injury and myocardial necrosis plays an important role in this pathophysiological process. We also define melatonin acted as triple cardioprotective effects: ameliorating TNF-α level, suppressing RIP3-MLKL/CaMKII signaling induced necroptosis and exerting analgesia effect.

Aerobic Interval Training Regulated SIRT3 Attenuates High-Fat-Diet-Associated Cognitive Dysfunction

Cognitive dysfunction is an important complicated disease in obesity. Exercise ameliorates obesity and the related cognitive dysfunction. However, the underlying mechanism is still unclear. In this study, we investigated whether aerobic interval training (AIT) could attenuate high-fat-diet- (HFD-) associated cognitive dysfunction and the possible mechanism of SIRT3-MnSOD pathway. C57BL/6 wild-type (WT) mice and SIRT3 knockout (KO) mice were randomized into control (Con) or HFD group with or without AIT training for 6 weeks. The spatial learning and memory ability were impaired in HFD group compared to the control group. The levels of mitochondrial protein acetylation were increased in the hippocampus of HFD group. The acetylation level of antioxidative MnSOD was increased as well. As a result, the ROS and MDA levels were significantly increased, which leads to the neuron apoptosis in the hippocampus. SIRT3 deficiency further aggravated HFD-induced cognitive dysfunction and susceptibility to oxidative stress injury. However, AIT upregulated neuron SIRT3 expression and decreased the acetylation of MnSOD. The hippocampus neuron oxidative stress and apoptosis were both decreased compared to untrained HFD group, which finally improved cognitive function of HFD mice. Collectively, AIT attenuates HFD-associated cognitive dysfunction through SIRT3 upregulation and improvement of antioxidative MnSOD activity.

GW24-e0624 Impairted cardiac SIRT1 activity by carbonyl stress increase susceptibility of aged hearts to ischaemia reperfusion injury

Objectives Ageing increases susceptibility to myocardial ischaemia/reperfusion (I/R) injury. The present study is designed to evaluate the protective effects of ALDH2 activation on I/R tolerance in aged heart and to elucidate the underlying mechanisms with a focus on SIRT1. Methods Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to I/R (30 min/4h). Cardiac ALDH2, SIRT1 activity and aldehydes-protein adduct formation were assessed. Immunoblotting was used to evaluate the SIRT1 carbonylation. Results Cardiac ALDH2 activity is impaired and toxic aldehydes (4-HNE)-protein adduct formation is enhanced with ageing (all P < 0.05). 4-HNE increased the carbonyl modifications on SIRT1 concomitant with decreased SIRT1 activity in vitro, and thus reduced myocardial tolerance to hypoxia/oxidative (all P < 0.05). But all these effects were blocked by Alda-1 (ALDH2 activator). The endogenous 4-HNE-protein adduct formation in aged hearts was 1.7-fold larger than that in young mice during I/Rin vivo. The levels of carbonylation on SIRT1 in I/R aged hearts was higher than that seen in their young counterparts. ALDH2 activation by Alda-1 significantly reduced the carbonylation on SIRT1 in the I/R aged hearts (P < 0.05). Alda-1 treatment significantly improved the tolerance of aged hearts to I/R injury, which was evidenced by reduced plasma creatine kinase activity and infarct size. However, Alda-1 treatment failed to decreased infarct size in Sirt1 +/− knockout mice hearts. Conclusions These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischaemic intolernce.