Mechanisms Of ER Stress Mediated-cardiomyocytes Apoptosis And Heart Failure

The endoplasmic reticulum (ER) is a central organelle of each eukaryotic cell as theplace of calcium storage,lipid synthesis,protein folding and protein maturation.Disturbances in any of these functions such as accumulation of unfolded protein (unfoldedprotein response,UPR) or excessive protein traffic can lead to so-called ER stress.ERstress is the initial cell response to stress and the common pathway leading to manyintracellular stress responses,such as oxidative stress in mitochondria.ER stress is apro-survival response to reduce the accumulation of unfolded proteins and restore normalER function.However,if stress prolongs,signaling switches from pro-survival topro-apoptotic.It has been reported that ER stress is involved in many heart diseases thatcontribute to heart failure at last,including artherosclerosis,myocardial ischemia,dilatedcardiomyopathy.However,the exact mechanisms of ER stress interfering with heart failurestill remain unclear.This study investigated the inducement and signaling pathway of ERstress in failing heart.We found that heart failure really induced ER stress and theER-initiated apoptosis,and confirm the inducement,pathway and interaction of ER stressin heart failure.We went first to confirm the induction of ER stress in human failing heart from hearttransplant recipients by Western Blots analysis,evaluated by GRP78 increase,PERK-eIF2αpathway and JNKs pathway activation.Then we use two kinds of rattus models with cardiac hypertrophy and heart failure,overload pressure (abdominal aortic constriction,AAC) and sustainedβ₁-adrenergic stimulation (isoproterenol injection,Iso),to investigate the exact pathway of ER stress inheart failure.And we treated the experiment animals with metoprolol,a selectiveβ₁ blocker.By examination of morphology,pathology,and hemodynamics,we found cardiachypertrophy and left ventricular function impairment induced by overload pressure orchronicβ₁-adrenergic stimulation.And compared with control groups,ER stress wasincreased in the failing hearts of both AAC and Iso rattus,evaluated by increase of GRP78,activation of IRE1-XBP1 and induction of CHOP,by analysis of Western Blots.And wefound KDEL positive cells increased in AAC rattus hearts by histochemical analysis.TUNEL examination showed more apoptosis cells in Iso rattus hearts.Treatment ofmetoprolol attenuated ER stress and apoptosis as well as improving hypertrophy and leftventricular function.The data indicates that overload pressure or sustainedβ₁-adrenergicstimulation induce ER stress leading to cardiomyocytes apoptosis,cardiac hypertrophy andheart failure.To investigate whetherβ₁-adrenergic stimulation induces ER stress,we treatedH9c2(2-1) cells with isoproterenol (Iso) for 24 hours,and found GRP78 expressionincreased.We pretreated H9c2(2-1) cells with PKI,a specific inhibitor of PKA,KN93,aspecific inhibitor of CaMKⅡδ,metoprolol or propranolol for 1 hour before exposing themto Iso.After 24 hours,we found that there was no significant change of GRP78 proteinlevel in PKI,KN93 and metoprolol,but pretreatment of propranolol reduced GRP78expression induced by Iso.It revealed that ER stress induced byβ₁-adrenergic stimulationmay not go through the typical PKA pathway or CaMKⅡδpathway.It may be some wayindependent.Nonselectiveβ-blocker propranolol down-regulation the ER chaperonsinduced by Iso.We pretreated H9c2(2-1) cells with metoprolol or propranolol before exposing them toER stressor TG or TM.By Western Blots analyses,Hoechst staining and Anexin V/PIapoptosis tests,we found pretreatment of metoprolol partially reduced GRP78 andattenuated apoptosis.And propranolol nearly completely cut off the GRP78 and inhibited the activation of pro-caspase-12.We next examined the effect of the propranolol isomerD-propranolol,which compared to L-isomer is approximately 50-fold less potent as aβ-adrenoceptor antagonist,but also plays as a membrane-stabilizer,as well as mexiletine,which is a membrane stabilizer,but not a beta-blocker.We found they also attenuated ERstress and inhibited apoptosis pathway.These data supports that the effect ofpropranolol toattenuate ER stress is dependent of itsβ-blockage.It may be associated with its effects asmembrane stabilizer.To investigate the association of membrane stability and ER stress,we administrateAAC rattus with propranolol or mexiletine.We found that treatment with propranolol andmexiletine both reduced ER stress,assessed by GRP78 decrease,PERK-eIF2αpathwayinhibition,and CHOP reduction.They both improve the left ventricular function of AACrattus,and propranolol attenuated cardiac hypertrophy.We presumed thatβblockade andinhibition of increase Na+ influx in heart failure may attenuate ER stress and decreaseapoptosis.As noted above,membrane-stabilizers unexpectedly improved the heart function andprevented ER stress both in vivo and in cultured cells.We know that the membranestability depends on the sodium homeostasis in fact.To investigate whether ER stress isassociated with sodium channel action,which determins the membrane stability,wepretreated H9c2(2-1) cells with veratridine,a sodium channel activator.GRP78 wasincrease at 30 min,and phosphorylated PERK and CHOP significantly increase after 24hours,suggesting that activation of sodium channel induced ER stress.We then pretreatedcells with TTX (tetrodotoxin),a specific sodium channel blocker,before adding TG or TM.It showed that GRP78 was remarkably decrease and expression of phosphorylated PERKand CHOP was blocked completely.The data yielded evidence that inhibition ofoveractivation of sodium channel blocked ER stress effectually.Taken together,the datasupposed that sodium channel action played an important role in ER stress.In summary,we have systemically investigated the mechanisms and pathway of ER stress in heart failure.We propose that ER stress is associated with sustainedβ₁-adrenergicstimulation and intracellular Na⁺ increase in heart failure.ER stress induces cardiomyocytesapoptosis by activation of ER chaperons such as GRP78 and CHOP,PERK-eIF2αpathway,IRE1-XBP1 pathway,caspase-12 and JNKs pathway.Blockade of the key molecular of ERstress signaling pathway can inhibit heart failure,suggesting that it is a potential target fordiagnosis and treatment of heart failure.