The Research Of Mechanisms Underlying Chronic Neuropathic Pain Increases Susceptibility To Myocardial Ischemia/reperfusion Injury

Background There are 5.5 million people suffering from pain every day all over the world,and more than 100 million of patients suffering from pain in China.Chronic pain is not an isolated pathophysiological process,but can lead to various diseases and drug abuse.How to effectively control chronic pain and pain related health problems is of great clinical and practical significance.However,the effects of chronic pain on remote organs(such as heart)and the underlying mechanisms are still poorly understood.In addition,the current used analgesic drugs also exhibit cardiovascular side effects,which limit its clinical treatment effects.For example,non-steroid anti-inflammatory drugs are used to treat acute pain,but in the meantime,these drugs also increase the incidence of cardiovascular diseases.Therefore,it is an urgent problem that how to find the effective prevention and treatment for pain related cardiovascular diseases on the basis of molecular mechanism.The purpose of this study was to investigate whether there are some comorbid mechanisms leading to abnormal cardiac function and vulnerability during chronic pain.The study of this problem will provide a broader understanding of the mechanisms of chronic pain,and provide novel insights for the development of analgesic drugs.Our previous study suggested that reactive carbonyl species produced by lipid peroxidation(represented by 4-HNE,4-hydroxy-2-nonenal)can attack the protein side chain,resulting in carbonyl formation and leading to the inactivation and denaturation of protein,which was called carbonyl stress.Moreover,recent study has indicated that 4-HNE is strongly associated with pain.On one hand,4-HNE is an important pain trigger in response to tissue injury,which can directly activated by transient receptor potential(TRP)channels.On the other hand,4-HNE level increased in both acute and inflammatory pain models of rodent.4-HNE can be produced by pain and 4-HNE can leads to the aggravation of pain.Due to the high diffusion properties,4-HNE can reach the whole body through the lipid membrane.Therefore,peripheral pain may be associated with cardiac carbonyl stress,and 4-HNE may play an important role in this process.Mitochondrial aldehyde dehydrogenase(ALDH2)is the most important aldehyde metabolic enzyme and is the key mechanism of the detoxification of endogenous aldehydes.The activation of ALDH2 can convert cytotoxic aldehyde to acid.Our previous study and other studies have demonstrated that the activation of ALDH2 can effectively inhibit myocardial injury induced by 4-HNE,and reduce the aldehyde load and carbonyl stress,contributing its cardioprotection.The new problem is that,as an important endogenous cardioprotective factor,whether ALDH2 can enhance the protection against pain related myocardial ischemia injury? If so,what is the key signaling mechanism? SIRT1(Silent information regulator 1)is an NAD+-dependent histone deacetylase.Previous studies have investigated the biological function of SIRT1 in cell proliferation,apoptosis,differentiation,and senescence.Other studies have shown that activation of SIRT1 can inhibit myocardial ischemia/reperfusion(MI/R)injury.On the other hand,SIRT1 deletion or deficiency can significantly damage the ability against myocardial ischemia,resulting in increased MI/R damage and vulnerability to myocardial ischemia.However,how does SIRT 1 protect the myocardium against MI/R by deacetylation? The specific signaling still needs to be further investigated.More importantly,our previous studies have confirmed that SIRT1 is an important target of carbonyl stress,and SIRT1 carbonylation can significantly inhibit its activity.So,is SIRT1 carbonylation inactivation involved in the changes of cardiac function in chronic pain? That is a question worth exploring.In summary,based on the preliminary work and the latest discovery,we propose that chronic pain may induce the carbonyl stress;SIRT1 is an important target of carbonyl stress;SIRT1 carbonylation inactivation may lead to myocardial ischemia vulnerability;ALDH2 may be an effective target to protect the myocardium in chronic pain conditions via inhibition of carbonyl stress.The current study aims to provide a new strategy for the prevention and treatment of pain related cardiovascular events by using in vitro and in vivo animal experiments,combined with the ALDH2 gene knockout mice(ALDH2 KO)and gene intervention.Aims 1.To determine the effect of chronic neuropathic pain on the myocardial ability against ischemia.2.To explore the role of carbonyl stress in myocardial ischemia vulnerability induced by chronic neuropathic pain.3.To clarify the role of SIRT1 carbonylation inactivation in the above pathophysiological processes,and to analyze the related signaling mechanisms.To elucidate the signaling mechanism by which myocardial ischemia vulnerability is induced by chronic neuropathic pain.4.To determine whether ALDH2 activation can inhibit carbonyl stress and protect the SIRT1 signaling,resulting in the effect of pain relieving and cardioprotection.Methods 1.Male wild-type(WT)mice and ALDH2 KO mice(all C57BL/6 background)were used.ALDH2 KO mice were backcrossed into the C57BL/6 background(generation N6).2.Chronic compression of the dorsal root ganglion(CCD)operations was performed as previously described,known as CCD group.The sham-operated group was non-CCD group(Control).3.Nociception was assessed using von Frey filaments,which was applied to the plantar surface of the hind paw.4.After two weeks of CCD,the in vivo regional myocardial ischemia-reperfusion injury model(ischemia: 30 min;reperfusion: 4 h)was established in mice.5.Cardiac function was determined using echocardiography.TTC staining was used to assess the infarction size.Cardiac caspase-3 activity was assessed.6.Isolated heart perfusion was used to assess cardiac function.7.Assessment of circulating 4-HNE level.8.SIRT1 activity assay.9.Cardiac ALDH2 activity assay.10.Assessment of cardiac protein carbonylation.11.Detection of SIRT1 carbonylation,LKB1 acetylation,and LKB1-AMPK interaction using co-IP.12.Detection of ALDH2,SIRT1,LKB1,AMPK and p-AMPK using Western blot.13.Mice were subcutaneous injected with Alda-1(3 mg/kg BW/per injection).14.Recombinant adeno-associated virus(serotype 9)vectors carrying mouse ALDH2(accession number BC005476)with a c-TNT promoter(AAV9-c TNT-ALDH2).AAV9-c TNT-GFP used as a negative control.AAV9-c TNT-ALDH2,or AAV9-c TNT-GFP was injected into the left ventricular cavity through a 27 G catheter while the aorta and pulmonary artery were cross-clamped for 50 s.Results 1.Chronic pain sensitizes heart to MI/R injury The PWMT decreased dramatically in WT mice 1 day after CCD surgery and remained stable for more than 3 weeks.The successful establishment of CCD was also confirmed using Von Frey filament test.In comparison with WT mice,the reduced PWMT was more significant in ALDH2 KO mice.Similar allodynia was observed when the nociceptive score was estimated using Von Frey filament test(P < 0.05,n=5).The data above indicated that ALDH2 was strongly associated with pain,and ALDH2 deficiency led to hyperalgesia.Two weeks after CCD,WT and ALDH2 KO mice were subjected to 30 min of ischemia followed by 4 h of reperfusion.CCD-WT mice hearts demonstrated an increase in myocardial infarct size compared with no-CCD WT control hearts.Caspase-3 activity,ejection fraction,and fractional shortening were worsened in CCD-WT mice.MI/R induced mortality was increased in CCD-WT mice than in no-CCD WT control mice.These results suggested that CCD could aggravate MI/R injury.More importantly,CCD-ALDH2 KO mice showed more severe myocardial injury and higher MI/R induced mortality(P < 0.05,n=5).These data indicated that more exacerbated MI/R injury was induced in ALDH2 deficiency heart.2.Chronic pain induces carbonyl stress In WT mice,the circulating 4-HNE levels were increased as early as 1 day post-CCD,becoming statistically significant 3 days post-CCD,peaking 2 weeks post-CCD(increased by ~10-flod),and remaining elevated for more that 3 weeks,which is consistent with the time of increased susceptibility to ischemia.Moreover,cardiac protein carbonylation in CCD mice increased significantly compared with non-CCD mice(P < 0.05,n=5).These results suggest that chronic neuropathic pain produced reactive aldehydes and sufficient to mediate cardiac carbonyl stress.ALDH2 deficiency enhanced aldehydic overload.3.Chronic pain induces SIRT1 carbonylation inactivation The results of co-IP suggested that chronic pain(2 week post-CCD)increased SIRT1 carbonylation and caused a ~40% decrease in SIRT1 activity in WT mice heart compared with non-CCD WT heart(P < 0.05,n=5).Similarly,the SIRT1 carbonylation and inactivation were further deteriorated after CCD surgery in ALDH2 KO hearts.4.LKB1-AMPK interaction was impaired in ischemic heart in CCD mice Activated AMPK has important cardioprotection during ischemia,and LKB1 is the upstream kinase of AMPK.LKB1 deacetylation is regulated by SIRT1 and that this in turn activates AMPK.Our results suggested that LKB1 acetylation was significantly higher in CCD-WT hearts(vs.non-CCD WT mice,P < 0.05,n=5),which was consistent with SIRT1 carbonylation inactivation.In the CCD-WT hearts,the ischemic LKB1-AMPK interaction and AMPK phosphorylation were mitigated.Theses results indicated that impaired LKB1-AMPK interaction might be the mechanism of increased susceptibility to ischemia after SIRT1 carbonylation inactivation.Moreover,ALDH2 deficiency further blocked LKB1-AMPK interaction and AMPK phosphorylation during ischemia in the CCD-ALDH2 KO hearts(vs.CCD WT heart,P < 0.05,n=5).5.4-HNE elicits sustained allodynia and increased MI/R injury Our results shown that reactive aldehydes 4-HNE are produced by chronic pain.We next asked whether circulating 4-HNE can elicit pain-related cardiac intolerance to MI/R injury.Injection of 4-HNE(150 nmol/50?l per paw)into hind paws of mice induced a robust and sustained allodynia lasting for >120 min,as evidenced by decreased the nociceptive threshold.In addition,plasma 4-HNE levels were significantly increased by ~10-flod,which approached to the peaking level at 2 week post-CCD.4-HNE treatment induced cardiac protein carbonyls and SIRT1 carbonylation inactivation,and decreased p-AMPK level in ischemic heart.Isolated hearts from WT mice with or without 4-HNE treatment were subjected to Langendorff heart perfusion,and we found that exposure of 4-HNE increased cardiac vulnerability to ischemic injury(vs.vehicle,P < 0.05,n=5).These data suggested that 4-HNE induced carbonyl stress is a critical mechanism to induce pain and increased cardiac vulnerability to ischemic injury.6.ALDH2 activation mediates the cardioprotection against MI/R injury in chronic pain mice As ALDH2 metabolizes acetaldehyde,we investigated that activating ALDH2 would protect the hearts from MI/R injury in individuals suffering from chronic pain.We found that administration of Alda-1(3 mg/kg BW/per injection,3 doses per day,2 weeks)increased PWMT and reversed nociceptive threshold in CCD WT mice,which is opioid-receptor signaling independent.We found that CCD mice received Alda-1 treatment inhibited cardiac protein carbonyl level,effectively suppressed the chronic pain induced SIRT1 carbonylation and LKB1 hyperacetylation,enhanced LKB1-AMPK interaction,and increased p-AMPK level.MI/R-induced infarct size was reduced,caspase-3 activity was decreased and cardiac function was improved in Alda-1 treated CCD mice.Finally,treatment with Alda-1 also improved the survival rate of post-MI/R CCD mice.These data suggested that ALDH2 activation might represent a mechanism-based analgesic and cardioprotective therapeutic strategy for chronic pain related vulnerability to MI/R injury.7.Cardiac ALDH2 upregulation ameliorates MI/R injury in chronic pain mice The above results suggested that carbonyl stress is linked to key factor of chronic pain related susceptibility to MI/R injury,and SIRT1-LKB1-AMPK signaling is the target of carbonyl stress.The concomitant problem is how to protect the heart in the presence of chronic pain.We used an AAV of serotype 9 expressing ALDH2 under the control of the cardiac-specific c TNT promoter(AAV9-c TNT-ALDH2).In vivo gene transfer of AAV9-c TNT-ALDH2 or GFP(used as a negative control)were performed via intra-ventricular cavity injections while the aorta and pulmonary artery were cross-clamped for 50 s.Using immunoblot and immunohistochemical analysis,ALDH2 protein level was overexpressed by 3-fold in the heart compared with AAV9-c TNT-GFP-injected animals.Our results demonstrated that cardiac-specific ALDH2 upregulation in CCD mice markedly decreased cardiac protein carbonylation,protected SIRT1-LKB1-AMPK signaling,and enhanced protection against MI/R injury in individual with chronic pain.Conclusions 1.Chronic pain sensitizes heart to MI/R injury.2.Carbonyl stress plays an important role in the pathophysiology of increased vulnerability to myocardial ischemia induced by chronic neuropathic pain,in which SIRT1 carbonylation inactivation is a critical player.3.Regulation of LKB1 acetylation and LKB1-AMPK interaction is one of the mechanisms of cardioprotection conferred by SIRT1.4.ALDH2 activation exhibits both analgesic and cardioprotective effects.5.Cardiac specific ALDH2 upregulation significantly reduces pain related hypersensitivity to myocardial I/R injury.The current study indicates that carbonyl stress is the important comorbid mechanism between chronic pain and myocardial dysfunction.And prevention of protein carbonylation might be a potential therapeutic target than symptom reduction analgesics.Our findings thus elucidate the potential novel mechanisms of therapeutic targeting between chronic pain and myocardial injury.