| Adiponectin (APN) is a cytokine hormone originally found to be releasedexclusively by white adipose tissue. Beyond its well-defined insulin-sensitizing,anti-atherogenic, and anti-inflammatory properties, adiponectin has recently beenfound to protect cardiomyocytes from ischemia/reperfusion (I/R) injury. Despite theimportance of adiponectin in cardiac protection, relatively little is known about theunderlying molecular mechanisms. To address this issue, in the present study, an invivo myocardial I/R model and an in vitro neonatal rat cardiomyocytehypoxia/reoxygenation (H/R) model simulating I/R injury in vivo were adopted toinvestigate whether and how the cardioprotective effects of gAd are mediated by theinhibition of endoplasmic reticulum (ER) stress.PartⅠgAd Protects Rat HeartsAgainstIschemia/reperfusion Injury In VivoObjective: An in vivo rat myocardial I/R model was adopted to investigate theeffects of gAd on myocardial I/R injury.Methods: Male Sprague-Dawley (SD) rats (220-250g,7-to-8-week-old) wereanesthetized by intraperitoneal injection of pentobarbital sodium (45mg/kg). Thetrachea was intubated for artificial ventilation with ambient air. The heart wasexposed. A5-0silk suture was passed around the left anterior descending coronaryartery, and the coronary artery was occluded by pulling the suture tightly. After45minutes of ischemia, the suture was released, and then the myocardium was reperfused for3hours. In the first set of experimental animals were divided into3groups:1) sham group (sham): sham-operated rats underwent the same surgicalprocedures except that the suture around the coronary artery was not fastened;2) I/Rgroup: rats were infused with saline10minutes before the reperfusion;3) I/R+gAdgroup: rats were infused with gAd (1μg/g)10minutes before the reperfusion (n=6).At the end of the reperfusion, blood was collected from the left ventricle, and theanterior free walls of heart were immediately frozen in liquid nitrogen and stored at-80°C for later use.Results:(1) There was no marked difference in the AAR/LV between the I/Rgroup and the I/R+gAd group. Compared with the I/R group (34.7±7.9%), theIA/AAR ratio was significantly decreased in the I/R+gAd treatment group(23.3±8.7%) at the end of reperfusion (P<0.05). As compared with the sham group,the hemodynamics were deteriorated(P<0.05), gAd improved the change(P<0.05).The cardiac enzymes LDH and CK-MB, which are indicators of myocardial cellularinjury, were also significantly increased in the I/R group (both P<0.01vs. shamgroup). gAd attenuated these enzyme levels (both P<0.05vs. I/R group) incomparison with the I/R group. TUNEL and caspase-3activity were used to evaluatethe anti-apoptotic effects of gAd. The number of TUNEL-positive cells with I/R+gAdtreatment was decreased compared with the I/R group (15.2±5.3%vs.22.3±5.2%,P<0.05). Caspase-3was activated with I/R alone, but with I/R+gAd, the activity waslower than that with I/R alone (74.0±12.2nmol/h/mg vs.49.7±14.3nmol/h/mg;P<0.05).Conclusion: gAd protects rat hearts against myocardial ischemia/reperfusioninjury. PartⅡThe protective Effects and the Signaling Mechanismsof gAd on Rat Neonatal Cardiomyoctyes withHypoxia/Reoxygenation In VitroSection1The Protective Effect of gAd on Rat NeonatalCardiomyocytes Exposed to H/R In VitroObjective: An in vitro neonatal rat cardiomyocyte hypoxia/reoxygenation (H/R)model simulating I/R injury in vivo was adopted to investigate the effects of gAd onH/R.Methods: Neonatal rat cardiomyocytes were isolated from1-to-3-day-old SDrats. In brief, the excised hearts were cut into~1mm3fragments and digested withcollagenaseⅡ in calcium free Krebs-Henseleit bicarbonate (KHB) solution for1h at37°C. The dispersed cells were resuspended and then nonattached viable cells werecollected and incubated on culture dishes supplemented with the fresh culturemedium with bromodeoxyuridine to reduce the non-myocytes. After incubation for36h, we obtained myocytes population and started experiments. For simulation ofischemia, the cardiomyocyte medium was replaced with modified Esumi’s hypoxiabuffer in a humidified hypoxic chamber for3hours, then subjected to reoxygenationfor6hours with standard incubator.To study the effect of gAd, cardiomyocytes werethen divided into7groups:1) control group;2) gAd group: cells were incubated with2μg/ml gAd under normoxic conditions;3) H/R group: cardiomyocytes were treatedwith H/R;4) H/R+gAd group: cells were incubated with gAd at the point ofreoxygenation;5) H/R+4-PBAgroup: cells were incubated with1mM4-PBA (Sigma)at the point of reoxygenation, a classic ER stress inhibitor;6) thapsigargin (TG)group: cells were incubated for24hours with3μM TG (Sigma), a classic ER stress inducer; and7) TG+gAd group: cells were pretreated with gAd and then incubatedwith TG.Results: At the end of reoxygenation, LDH activity in the H/R group wasmarkedly increased over the control (P<0.01). H/R+gAd treatment significantlydecreased LDH compared with the H/R group (862±161.0U/L vs.1445.0±202.8U/L,P<0.05). TUNEL-positive cells made up fewer than5%of cells in the control group.After3hours of hypoxia followed by6hours of reoxygenation, more than20%of thecells were TUNEL positive. Cells treated with H/R showed a high rate of apoptosis,which was significantly improved by gAd treatment (25.3±3.5%vs.13.7±5.5%,P<0.05). To confirm the results obtained from the TUNEL assay, caspase-3activitywas performed. Caspase-3activity was increased with H/R treatment but was lowerwith H/R+gAd treatment than with H/R alone (54.0±11.8nmol/h/mg vs.83.0±12.5nmol/h/mg, P<0.05).Conclusion: gAd reduced H/R-induced cardiomyocyte injury in adose-dependent manner.Section2gAd Attenuates Endoplasmic Reticulum Stress in NeonatalRat Cardiomyocytes Exposed to H/RObjective: An in vitro neonatal rat cardiomyocyte hypoxia/reoxygenation (H/R)model simulating I/R injury in vivo was adopted to investigate the effects of gAd onendoplasmic reticulum stress (ERS).Methods: To further investigate the mechanisms responsible for the protectiveeffect of gAd, we assessed the ER stress-responsive markers GRP78, CHOP andcaspase-12by quantitative real-time PCR and western blot. TG, a typical inducer ofER stress, was used to study the potential mechanisms of H/R injury. Meanwhile, 4-PBA, an ER stress inhibitor, was used as a positive control.Results: TG treatment, caused significant upregulation of mRNA and proteinexpression levels of GRP78, CHOP and activated caspase-12compared with thecontrol (P<0.05or P<0.01). Similar to TG, H/R led to several-fold increases in boththe mRNA and protein levels of GRP78, CHOP and caspase-12compared with thecontrol (P<0.05or P<0.01). These increases were normalized by4-PBA. Collectively,these results confirm the involvement of ER stress in H/R injury. Most importantly,similar to4-PBA, I/R+gAd treatment significantly attenuated the upregulation ofGRP78, CHOP and caspase-12compared with H/R alone (P<0.05, Figs.3A,3B).Interestingly, gAd also ameliorated the overexpression of ER stress-responsive geneinduced by TG (P<0.05, Figs.3A,3B), which indicated the direct inhibitory effect ofgAd on ER stress.Conclusions: gAd inhibits H/R-induced ER stress in neonatal ratcardiomyocytes.Section3gAd Restores SERCA Activity and Maintains Ca2+Homeostasis by Upregulating p-PLN Expression in Neonatal RatCardiomyocytes Exposed to H/RObjective: An in vitro neonatal rat cardiomyocyte hypoxia/reoxygenation (H/R)model simulating I/R injury in vivo was adopted to investigate the effects of gAd onsarcoplasmic reticulum stress Ca2+-ATPase (SERCA).Methods: To further investigate the mechanisms responsible for the protectiveeffect of gAd, we assessed the SERCA activity, and SERCA regulatorphospholamban (PLN) and SERCA protein expression by western blot.Results: Our results demonstrated that H/R or TG treatment significantly decreased the SERCA activity in neonatal rat cardiomyocytes (P<0.01). gAdenhanced the activity of SERCAcompared with H/R alone or TG alone (P<0.01). Theprotective action of gAd on ER stress seemed to correlated with the restoration ofSERCAactivity.p-PLN were increased in the H/R group and the TG group compared with thecontrol group (P<0.05), and gAd induced a further increase (P<0.05), suggesting thatgAd improved SERCA activity through increase in PLN phosphorylation.Compared with the control group, the intensity of [Ca2+]ifluorescence detectedby confocal laser scanning microscopy were significantly increased in the H/R groupand the TG group (P<0.01). The increased [Ca2+]iin cardiomyocytes weresignificantly reduced by gAd treatment (P<0.05). These results indicate that therecovered SERCA activity by gAd may explain its effect on inhibiting [Ca2+]ioverloading during H/R.Conclusion: The effect of gAd on ERS is related with SERCA activityupregulation.Section4gAd Modulates SERCA Activity and ER Stress through theProtective PI3K/Akt Signaling Pathway In Neonatal RatCardiomyocytes Exposed to H/R In VitroObjective: To further explore the potential mechanisms through which gAdregulates SERCA activity and ER stress, we examined the involvement of thePI3K/Akt pathway in gAd’s effects.Methods: To further investigate the mechanisms responsible for the protectiveeffect of gAd, we assessed the activation of PI3K/Akt signaling pathway usingwestern blot. Results: Compared with the control group, H/R treatment increased the ratio ofp-Akt/Akt by130%(P<0.05). Compared with the H/R group, gAd markedlyincreased the ratio of p-Akt/Akt by139%(P<0.05), but this effect was reversed bythe PI3K inhibitor LY294002(P<0.05). Thus, we confirm that gAd can activatePI3K/Akt signaling pathway. To further explore whether activation of PI3K/Akt wasnecessary for gAd mediated regulation of SERCA activity and ER stress, wepretreated the neonatal rat cardiomyocytes with LY294002before gAd treatment andexamined the expression of ER stress-responsive genes and SERCA activity afterreoxygenation. LY294002effectively abolished the effect of gAd on ER stress andSERCA activity as evidenced by an decrease in SERCA activity and downregulationof expression of GRP78and CHOP (P<0.01or P<0.05, respectively).Conclusion: These data indicate that blocking the PI3K/Akt signaling pathwayby LY294002abolished the beneficial effect of gAd.PartⅢgAd Improves Myocardial SERCA Activity and ERStress and these Effects are Abolished by PI3K Inhibitor inRats with Myocardial I/R In VivoObjective: An in vivo rat myocardial I/R model was adopted to investigatewhether gAd could improve ER stress and SERCA activity in rat myocardium andwhether these changes are mediated through the PI3K/Akt pathway.Methods: we pretreated rats with gAd in the presence or absence of LY294002and then examined the expression levels of p-Akt, Akt, GRP78, caspase-12, CHOPand SERCA activity of myocardium after I/R.Results: Western blots indicated that I/R treatment induced significant ER stresscompared with the sham group (P<0.01). gAd markedly attenuated the I/R-induced activation of ER stress. The expression of GRP78, caspase-12and CHOP in theI/R+gAd treatment group were all decreased compared with rats that underwent I/Rtreatment alone (P<0.05). We also found that SERCA activity was significantlysuppressed in the I/R group compared with the control group (P<0.01), and thissuppression of SERCA activity was recovered by gAd administration (P<0.01).To investigate the role of Akt in the protective effect of gAd, LY294002wasused to suppress Akt activity. Compared with I/R treatment alone, I/R+gAd treatmentincreased the p-Akt/Akt ratio in rat myocardium by1.67-fold (P<0.05), and additionof LY294002attenuated gAd’s effects by94%. These results provide direct evidencethat gAd also activates the PI3K/Akt pathway in rats with I/R in vivo. Furthermore,we explored whether the PI3K/Akt pathway was involved in the effect of gAd onmodulating SERCA activity. The result showed that LY294002treatment decreasedAkt phosphorylation, which abolished gAd-elicited protection of SERCA activityduring I/R (P<0.01). Finally, LY294002also abolished the protection provided bygAd against ER stress (P<0.05or P<0.01).Conclusion: Our findings showed that PI3K/Akt inhibitor LY294002, not onlyinhibited the activity of PI3K/Akt signaling, but also blocked gAd’s improvement ofmyocardial SERCA activity and ER stress in rats with I/R in vivo. These effects werein line with the results obtained from our in vitro experiment. |
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