Catestatin attenuates endoplasmic reticulum induced cell apoptosis by activation type 2 muscarinic acetylcholine receptor in cardiac ischemia/reperfusion

Catestatin (CST) is a catecholamine secretion inhibiting peptide as non-competitive inhibitor of nicotinic acetylcholine receptor. CST play a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. Cardiomyocytes endogenously produced CST and its expression was reduced after I/R. CST pretreatment decreased apoptosis especially endoplasmic reticulum (ER) stress response during I/R. The protection of CST was confirmed in H9c2 cardiomyoblasts under Anoxia/reoxygenation (A/R). In contrast, siRNA-mediated knockdown of CST exaggerated ER stress induced apoptosis. The protective effects of CST were blocked by extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor PD90895 and phosphoinositide 3-kinase (PI3 K) inhibitor wortmannin. CST also increased ERK1/2 and protein kinase B (Akt) phosphorylation and which was blocked by atropine and selective type 2 muscarinic acetylcholine (M2) receptor, but not type 1 muscarinic acetylcholine (M1) receptor antagonist. Receptor binding assay revealed that CST competitively bound to the M2 receptor with a 50% inhibitory concentration of 25.7 nM. Accordingly, CST inhibited cellular cAMP stimulated by isoproterenol or forskolin, and which was blocked by selective M2 receptor antagonist. Our findings revealed that CST binds to M2 receptor, then activates ERK1/2 and PI3 K/Akt pathway to inhibit ER stress-induced cell apoptosis resulting in attenuation cardiac I/R injury.

Scientific RepoRts | 5:16590 | DOI: 10.1038/srep16590 that CST may play an essential role in the pathogenesis of ischemic heart diseases. More recently, Penna et al. found that CST post-conditioning or post-treatment decreased infarct area and diastolic left ventricular (LV) pressure, and increased cardiomyocyte viability during cardiac ischemic-reperfusion (I/R) 16 . PI3 K and protein kinase Cε inhibitors, mitochondrial K ATP channel blocker and reactive oxygen species (ROS) scavenger abolished the protective effects of CST in cardiac I/R 17 . Clearly, to investigate the protective mechanism of endogenous CST in cardiac diseases will be very useful and necessary for its potential clinical use.
It had been reported that activation of nAch (α 7nAchR) 18 or type 2 muscarinic Ach receptor (M2 receptor) 19 reduced cardiac I/R injury. Because CST is a noncompetitive inhibitor of nAch receptor 1 , it is likely that nAch receptor is not involved in protective effect of CST. Interestingly, Gi/o protein participated in the cardiac negative inotropism of CST 10,20 . Gi/o protein is also an important intracellular target of M2 and M4 receptors 21 . Based on these previous findings, it is reasonably to speculate that M receptor may play essential roles in CST's protective effects during cardiac I/R injury.
In the present study, we presented novel evidence that CST directly bound to and activated M2 receptor to attenuate ER-stress linked apoptosis via activation of ERK1/2 and Akt pathways in cardiac I/R.

CST pretreatment improved left ventricular (LV) function and decreased I/R injury. Ischemia
reperfusion procedure and CST pretreatment (CP) (CST supplementation for 15 min before I/R) were performed (Fig. 1a). After 30 min of ischemia and 1 h of reperfusion, cardiac endogenous ChgA mRNA (see supplementary Fig. S1 online) and protein expression were no changes, but CST fragments (21 kD and 7.5 kD) reduced comparison to control (Fig. 1b). Different dose of CST perfusion for 1 h did not induce lactate dehydrogenase (LDH) leakage (supplementary Fig. S2 online). CST (25 nM, 50 nM and 100 nM) pretreatment decreased LDH leakage and cardiac troponin I (cTNI) level in perfusate at different reperfusion time point (supplementary Fig. S3 online), which indicated that CST pretreatment lowered the ischemia reperfusion (I/R) injury, especially CST at 100 nM exhibited well protection. So we selected 100 nM CST in following experiments. As Fig. 1c showed, I/R caused global cardiac infarction, and CP significantly reduced infarct size as compared with I/R alone (9.8 ± 4.2% vs 27.5 ± 8.2%, P = 0.0001).
We also assessed LV diastolic pressure (LVEDP), developed LV pressure (LV systolic pressure minus LVEDP, dLVP) and maximal rate of LV pressure development (LV ± dp/dtmax) for LV function. The I/R rat heart showed greatly increased LVEDP and impaired dilation; CP lowered the LVEDP during each time point of reperfusion (All P < 0.05, Fig. 1d). CP also improved the dLVP recovery (All P < 0.05, Fig. 1e) and LV ± dp/dtmax (All P < 0.05 Fig. 1f). Thus, CST pretreating improved cardiac function during I/R.
I/R induced ER-stress response by assessing phosphorylated PERK and chaperone protein Grp78 expression. ER stress-initiated apoptosis signals evaluated by caspase-12 cleavage, Chop protein expression and phosphorylated JNK. CP also lowered the protein expression of ER stress markers and ER stress-initiated apoptosis signals in cardiac I/R (Fig. 2d,e).
To confirm the CST regulatory role in ER stress-induced apoptosis, we used siRNA knockdown of the CST precursor ChgA in H9c2 cardiomyoblasts. Firstly, we measured the cell viability by CCK8 kit and found that different CST treatment or knockdown did not affect H9c2 cell viability (see supplementary Fig. S4a,S4b online). siRNA treatment for 48 h lowered CST precursor-ChgA protein expression in H9c2 cells (Fig. 3a). In H9c2 cell with A/R, siRNA mediated knockdown of ChgA increased the number of apoptotic cells as compared with scramble siRNA (Fig. 3b). ChgA siRNA also upregulated ER stress response markers (phosphorylated PERK and JNK, Grp78 and Chop expression, and cleaved caspase-12) and an apoptosis marker (cleaved caspase-3) (Fig. 3c). Calcium overload is an important injury mechanism in I/R and an inducer of ER stress. Thapsigargin (calcium pump inhibitor induce intracellular calcium overload) is a typical ER stress inducer. Knockdown of CST also increased thapsigargin-induced cell apoptosis (Fig. 3d) and especially levels of the ER stress markers and cell apoptosis marker ( Fig. 3e and supplementary Fig. S5 online). Above results suggest that endogenous CST may reduce ER stress in part with I/R injury. ERK and PI3 K pathway is involved in the protective effect of CST. To investigate the possible signals of CST protection, we treated H9c2 cells with specific inhibitors for 30 min to assess the blocking effects. Intriguingly, PD98059 and wortmannin reversed the CST protection on cell apoptosis (Fig. 4a,b) and ER-stress response (Fig. 4c,d). Knockdown CST also lowered the phosphorylation of ERK1/2 and Akt during A/R (see supplementary Fig. S6 online). These results indicate that ERK1/2 and PI3 K pathway involved in the protection of CST. In isolated heart, we also found CP significantly increased ERK1/2, Scientific RepoRts | 5:16590 | DOI: 10.1038/srep16590 Akt and eNOS phosphorylation (Fig. 4e,f) compared with I/R, which confirmed ERK1/2 and PI3 K/Akt were partly activated by CST to reduce the ER-stress and increase cell survival.
To confirm these pathways involved in ER stress-induced cellular apoptosis by CST, we measured the effect of these inhibitors on DTT-, tunicamycin-and thapsigargin-induced cellular apoptosis. In situ staining of apoptotic cells with cleaved caspase-3 antibody revealed that CST reduced the number of apoptotic cells induced by DTT, tunicamycin and thapsigargin, and which also were blocked by PD98059 and wortmannin (see supplementary Fig. S7-S9 online). Consistently, the major apoptosis pathway of ER stress-Chop expression, caspase-12 cleavage and JNK phosphorylation, ER stress response markers including phosphorylated PERK, Grp78 protein expression-were also lowered by CP while inducing by DTT (Fig. 5a,   The ChgA protein and its cleavage fragments expression were measured by western blot. (c) Heart tissue was stained with triphenyltetrazolium chloride and infarct size (white color) was measured by volume (n = 6 for each group). Evaluation of cardiac function including LV end diastolic pressure (LVEDP)(b), LV developed pressure (dLVP, LV systolic pressure-diastolic pressure) (d), and LV ±dp/dt max (e) during I/R and CP (total 36 rats were recruited). Data are mean ± SD. *P < 0.05, **P < 0.01 vs I/R group. # P < 0.05 vs parameters before ischemia.
Scientific RepoRts | 5:16590 | DOI: 10.1038/srep16590 CST acts as an M2 receptor agonist. CST might activate G i/o protein 20 . In 5 subtypes (M1 to M5) muscarinic acetylcholine receptors, only M 2 and M 4 receptors bind with G i/o protein 21 . Thus, muscarinic acetylcholine receptor signaling might be involved in the cardioprotective effect of CST. To verify this hypothesis, the nonspecific M receptor antagonist atropine, selective M 1 receptor antagonist pirenzepine, and selective M 2 receptor antagonist AF-DX116 were used. We found that carbamoylcholine lowered Grp78 and Chop protein expression and increased phosphorylated ERK1/2 and Akt levels effect which was similar to the CST (Fig. 6a). Atropine and AF-DX116 but not pirenzepine blocked the CST-altered level of these proteins (Fig. 6a). Therefore, M 2 but not M 1 receptor may mediate the CST action. To confirm this finding, we used another selective M 2 receptor antagonist-methoctramine, and the selective M 2 /M 4 receptor antagonist himbacine, which also blocked the effect of CST on the ER stress response and ERK1/2 and Akt phosphorylation during A/R (Fig. 6b). In isolated neonatal rat cardiomyocytes, CP also reduced cell apoptosis (PARP and caspase-3 cleavage), ER-stress response (Grp78 and Chop protein expression) by I/R, and increased phosphorylated ERK1/2 and Akt. The protection were also blocked with selective M2 receptor inhibitors (Fig. 6c,d). In isolated heart, AF-DX116 also blocked the protective effects of CP on infarct size, LDH leakage and cTNI level (supplementary Fig. S13a online). These data suggested that M 2 receptor may be a target protein interacting with CST.
To investigate the interaction of CST with M 2 receptor and as an endogenous ligand, we used radioactive isotope [ 125 -I]-labeled CST (0.4 μ M) as a ligand, and N-methylscopolamine (NMS:0.045 nM to 0.3 μ M) as a "cold" ligand were co-incubated with membrane protein (200 μ g) in H9c2 cardiomyblasts. Radioactivity of binding CST was assessed and the competitive inhibition curve is in Fig. 7a. The half-maximal inhibition (IC 50 ) of binding occurred with 25.7 nM NMS (Fig. 7a). Scatchard analysis revealed a single binding site with KD 18.6 nM (Fig. 7b). To confirm the specific binding of CST to the M 2 receptor, we added AF-DX116 (final concentration: 50 nM, IC 50 50 nM for M 2 receptors), and the IC 50 of binding was significantly increased to 36.0 nM (Fig. 7a); scatchard analysis revealed a KD of 45.8 nM (Fig. 7b). Therefore, CST specifically binds to the M2 receptor.
Activation M2 receptor can decrease intracellular cAMP level under catecholamine stimulation. To identify the biological function of CST binding to the M2 receptor, we measured cAMP level with carbamoylcholine as a positive control. CST and carbamoylcholine lowered cAMP level induced by isoproterenol in H9c2 cells (Fig. 7c). AF-DX116 and himbacine did not affect increased cAMP level with isoproterenol, but blocked the effect of CST. To distinguish the β -receptor inhibition, we used the adenylyl-cyclase activator forskolin to directly increase intracellular cAMP level. CST also lowered cAMP  level, which was blocked by AF-DX116 and himbacine but not pirenzepin (Fig. 7d). Thus, CST functions as an agonist of the M2 receptor.

Discussion
In the present study, we identify the CST is an endogenous ligand of the M2 receptor. CST pretreatment activates M2 receptor, thereby activates PI3 K and ERK1/2 pathway results in reducing ER stress-induced cell apoptosis and increasing cell survival (Fig. 8), thus lowers infarct size and recovers cardiac pump function under I/R.
Firstly we identified the endogenous CST reducing after I/R, but its precursor ChgA expression were no changes. That means increased enzymes (which catalyzed ChgA to generate CST) activity during I/R, although the enzymes were not identified until now. CST pretreatment compensated to endogenous CST decrease, thus lowered the infarct size and improved cardiac function in rats under I/R, which agreed with studies of CST post-conditioning 16,17 . However, human wild type CST (100 nM) treatment for 2 h during reperfusion phase increased, but Gly 364 Ser variant peptide lowered infarct size; because of lower inhibitory activity on nicotinic cholinergic receptor 22 . In our present study or CST post-conditioning, short-times supplementation reduced the inhibitory effects on nicotinic cholinergic receptor, which might partly explain the paradoxical effects of CST on cardiac I/R. Apoptosis occurs in cardiomyocytes during I/R. Genetic or pharmacological inhibition of cell apoptosis reduced infarct size and improved cardiac function 23 . CST pretreatment similar with CST post-conditioning 24 , decreased apoptosis during I/R. In contrast, knockdown CST increased apoptosis. ER stress induces apoptosis involved in regulation during I/R 25 . CST pretreatment lowered ER stress induced apoptosis during I/R, even directly stimulated by ER-stress inducers; oppositely, knockdown CST aggravated ER-stress induced apoptosis, suggesting endogenous CST stabilized ER-stress response to anti-apoptosis. Next, we assessed the possible signal involved in the CST regulation. A PI3 K inhibitor blocked the Ca 2+ transient inhibition of CST after β -adrenergic stimulation 11 and cardiac function recovery with   CST post-conditioning 17 . We found that wortmannin blocked the effect of CST on ER stress-linked apoptosis; and knockdown CST lowered phosphorylated Akt. Therefore, the PI3 K-Akt pathway is an important signal cascade of CST in cardiac diseases. The canonical mitogen-activated protein kinases (MAPKs) JNK, ERK1/2 and p38 are activated in response to ER stress, each with different roles. JNK is considered a downstream target of IRE1-dependent ER stress 26 . I/R-induced ROS leads to sustained calmodulin-dependent protein kinase II activation, which promotes JNK signaling 27 , thereby promoting apoptosis 26 . Here we found that CST inhibited JNK phosphorylation, and siRNA knockdown of CST activated JNK. Thus, the JNK signal is involved in inhibiting ER stress-induced apoptosis with CST. In general, ERK1/2 activation is considered to promote cell survival. ER stress-promoted activation of ERK1/2 is associated with increased cell proliferation and decreased apoptosis 28 . Pharmacological or genetic inhibition of ERK1/2 promoted ER stress-induced apoptosis 29 . We found that PD98059-inhibited MAPK kinase reduced ERK1/2 activation and reversed the protection of CST on ER stress, so CST increase ERK1/2 phosphorylation to promote cell survival. These findings indicate that PI3 K and ERK1/2 pathway involved in the protection of endogenous CST during I/R.
We asked how CST activates PI3 K and ERK1/2 signal and which the receptor of CST is. Here we showed CST was similar to carbamoylcholine, reduced ER-stress response and activated ERK1/2 and PI3 K signals. This effect was blocked by a selective M2 but not M1 receptor antagonist. Receptor binding assay showed that CST is a ligand of the M2 receptor. CST bound to M2 receptor activation directly activating G i protein, thus reducing cAMP production while stimulated by isoproterenol 30 or forskolin; and the effect was also blocked by M2 selective antagonism, suggesting CST functions as an M2 receptor agonist. M2 receptor activation increases the NOS/NO cGMP pathway 31 , which also explains in part the CST anti-adrenergic effect by NOS/NO cGMP 11 . All these findings demonstrate that endogenous CST as an agonist of M2 receptor to participate in pathogenesis of the cardiac I/R.
Muscarinic receptor expression is abundant in ventricular muscle. Acetylcholine has protective role in cardiac I/R in vitro, but it is non-selective ligand and activates all cholinergic receptor subtype, which limited its application due to enormous side effects. Here we identified that CST is a selective ligand of M2 receptor with high affinity and its intracellular signal (cAMP, PI3 K and ERK1/2) is consistent with M2 receptor agonist. Berberine also has potential binding ability to M2 receptor (KD values 5.4 μ M) and has protective effects in cardiac diseases 32 . These data indicate that M2 receptor might be a new target for cardiac I/R, but it should be confirmed in cardiomyocytes specific M2 receptor knockout mice in the future. Different amino acid variant in CST affected CST inhibitory activity on nicotinic cholinergic receptor 33 . Thus, we also increasing CST binding to M2 receptor by modification replacement amino acid, if the sites and model of CST binding to M2 receptor are clear. A synthesized small-molecule chemical according to the pharmacophore model of CST lowered blood pressure in hypertensive animals 34 . According to the model, more selective and effective M2 agonist might be designed and synthesized for treating cardiac ischemic diseases. Langendorff perfusion procedure and experimental protocol. Rats fed a standard diet were heparinized (200 IU) and anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). Hearts were excised and rapidly moved to a Langendorff perfusion system as described 35 . After 30-min stabilization, hearts underwent 30-min normoxia, zero-flow global ischemia for 30 min, followed by 60-min reperfusion (I/R group). For the CP group, CST (100 nM) was infused for 15 min before ischemia (Fig. 1A). Cardiac function including left ventricular end diastolic pressure (LVEDP), LV systolic pressure (LVSP), and maximal and minimal LV pressure development (± dp/dt max ) were recorded.

Animals and materials.
At the end of reperfusion, hearts were frozen at − 20 °C, then cut into 2-mm thin slices, which were stained with 1% triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4) at 37 °C for 10 min, then fixed in 4% paraformaldehyde solution to enhance the contrast of the stain. Images in each slice were analyzed by image pro plus software. The volume of infarcted tissue (white) and the risk zone (total) was then calculated by each area plus slice thickness and summing the results. The infarct size was expressed as the percentage of the risk zone 36 . Total 12 rats were sacrificed for the infarct size assay. Other rats, while finished I/R procedure, the apex of heart (about 100 mg) was quickly removed for western blot assay. The residual heart were cut about 5 mm high (from the site of apex), embedded with OCT then the frozen slice were prepared for apoptosis staining (Methods of western blot and apoptosis assay are available in the Supplementary methods online).

Anoxia/reoxygenation (A/R) and induced ER stress in H9c2 and neonatal cardiomyo-
cytes. Ten two-day-old Sprague-Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), and then decapitated by heavy scissors. Their ventricles were excised and dissociated using trypsin digestion as described previously 37 . The isolated cells were pre-plated for 0.5 h in minimal essential medium with 10% fetal calf serum. Nonattached cells were re-plated in another plate in the same medium with 0.1 mM bromodeoxyuridine (BrDU) for 4 days and then were removed. The neonatal cardiomyocytes were used for experiments after a further 2 or 3 days.
H9c2 rat cardiomyoblasts or neonatal cardiomyocytes were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C, 5% CO 2 . Medium was changed to fresh medium (serum-free), and cells were cultured under anoxia with 95% N 2 and 5% CO 2 for 12 h at 37 °C, then incubated in a standard incubator for 4 h (A/R) 38  Cells at 40% to 50% confluence were transfected with siRNA or scramble siRNA (50 pM) by use of Lipofectamine 2000 (Invitrogen), then incubated at 37 °C in a CO 2 incubator for 48 h before A/R.
Competitive receptor binding assay. Membranes were prepared from H9c2 cells as described 39 . Data are presented as mean ± SD. Differences among groups were analyzed by one-way ANOVA, then Student-Newman-Keuls test. Differences between 2 groups were analyzed by two-tail nonparametric t test.