Adiponectin protects rat myocardium against chronic intermittent hypoxia-induced injury via inhibition of endoplasmic reticulum stress.

Obstructive sleep apnea syndrome (OSAS) is associated with many cardiovascular disorders such as heart failure, hypertension, atherosclerosis, and arrhythmia and so on. Of the many associated factors, chronic intermittent hypoxia (CIH) in particular is the primary player in OSAS. To assess the effects of CIH on cardiac function secondary to OSAS, we established a model to study the effects of CIH on Wistar rats. Specifically, we examined the possible underlying cellular mechanisms of hypoxic tissue damage and the possible protective role of adiponectin against hypoxic insults. In the first treatment group, rats were exposed to CIH conditions (nadir O2, 5-6%) for 8 hours/day, for 5 weeks. Subsequent CIH-induced cardiac dysfunction was measured by echocardiograph. Compared with the normal control (NC) group, rats in the CIH-exposed group experienced elevated levels of left ventricular end-systolic dimension and left ventricular end-systolic volume and depressed levels of left ventricular ejection fraction and left ventricular fractional shortening (p<0.05). However, when adiponectin (Ad) was added in CIH + Ad group, we saw a rescue in the elevations of the aforementioned left ventricular function (p<0.05). To assess critical cardiac injury, we detected myocardial apoptosis by Terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling (TUNEL) analysis. It was showed that the apoptosis percentage in CIH group (2.948%) was significantly higher than that in NC group (0.4167%) and CIH + Ad group (1.219%) (p<0.05). Protein expressions of cleaved caspase-3, cleaved caspase-9, and cleaved-caspase-12 validated our TUNEL results (p<0.05). Mechanistically, our results demonstrated that the proteins expressed with endoplasmic reticulum stress and the expression of reactive oxygen species (ROS) were significantly elevated under CIH conditions, whereas Ad supplementation partially decreased them. Overall, our results suggested that Ad augmentation could improve CIH-induced left ventricular dysfunction and associated myocardial apoptosis by inhibition of ROS-dependent ER stress

Possible mechanisms of myocardium apoptosis induced by CIH.

<p>(A) and protective roles of Ad (B). A: CIH can induce the ROS and then further causes ER stress represented by generation of misfold proteins, which combine Bip released from IRE1, PERK and ATF6. Upon Bip release, IRE1, PERK and ATF6 are activated. All of the three pathways finally upregulate the expression of CHOP, which may further trigger cell apoptosis. The IRE1 pathway also activates the JNK and P38 MAPK, which can trigger apoptosis. In addition, caspase-12 is activated during ER stress, which sequentially activates caspase-9 and/or caspase-3, leading to apoptosis. B: Through inhibition of ROS, Ad supplement may further suppress ERS and therefore the myocardial apoptosis may be reduced.</p

PERK pathway activation in heart.

<p>The protein levels of p-PERK, PERK, p-eIF2α, eIF2α. Western blot bands of p-PERK and p-eIF2α were separately normalized to PERK and eIF2α. *P<0.05 versus NC group; #p<0.05 versus CIH group.</p

The protein levels of caspase-12, caspase-9, caspase-3.

<p>Western blot bands of cleaved caspase-12, cleaved caspase-9 and cleaved caspase-3 were separately normalized to caspase-12, caspase-9 and caspase-3. *P<0.05 and **P<0.01 versus NC group; #p<0.05 and ##P<0.01 versus CIH group.</p

The protein levels of JNK1/2, P38 MAPK in heart.

<p>The protein levels of p-JNK1/2, JNK1/2, p-P38 MAPK and P38 MAPK. Western blot bands of p-JNK1/2 and p-P38 MAPK were separately normalized to JNK1/2 and p-P38 MAPK. *P<0.05 versus NC group; #p<0.05 versus CIH group.</p

Serum adiponectin levels in the three groups.

<p>NC, normal control group; CIH, chronic intermittent hypoxia; CIH + Ad, chronic intermittent hypoxia and adiponectin supplement; **P<0.01 versus NC group; #P<0.05 versus CIH.</p

ATF6 pathway activation in heart.

<p>The protein levels of pro-ATF6; *P<0.05 versus NC group; #p<0.05 versus CIH group.</p

Echocardiographic data —5 weeks.

<p>Values are means ± SD; n, number of animals exposed to CIH or room air for 5 weeks; HR, heart rate; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameter; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; LVEF%, left ventricular ejection fraction; LVFS%, left ventricular percent fractional shortening. *P<0.05 versus NC group; #p<0.05 versus CIH.</p

IRE1 pathway activation in heart.

<p>The protein levels of p-IRE1, IRE1, XBP-1 (s). Western blot band of p-IRE1 was normalized to IRE1. *P<0.05 versus NC group; #p<0.05 versus CIH.</p

The UPR induction after CIH.

<p>A: Western blot analysis of GRP78 and CHOP in whole heart tissue homogenates. WB bands were normalized to β-actin. B: mRNA expressions of GRP78, CHOP in heart of three groups; PCR fluorescent signals for GRP78 and CHOP were standardized to PCR fluorescent signals obtained from an endogenous reference (β-actin). C: the densitometric evaluation of the independent western blot of GRP78; D: the densitometric evaluation of the independent western blot CHOP; *P<0.05 and **P<0.01 versus NC group; #p<0.05 and ##P<0.01 versus CIH group.</p