ORIGINAL ARTICLE
Effects of PP1-12, a Novel Protein Phosphatase-1 Inhibitor, on Ventricular Function and Remodeling After Myocardial Infarction in Rats Yun-Yun He, MD,*† Chun-Lei Liu, MS,* Xin Li, BS,* Wu Zhong, PhD,‡ Song Li, PhD,‡ Kun-Lun He, MD, PhD,* and Li-Li Wang, PhD‡
Abstract: PP1-12, a new protein phosphatase-1 inhibitor, is designed and synthesized to modulate the endoplasmic reticulum (ER) stress apoptotic pathway, which is involved in various cardiovascular diseases. In this study, we examined the effect of PP1-12 on ventricular remodeling and heart function after myocardial infarction. Rats that survived within 24 hours after coronary ligation were randomly divided into 6 groups and treated with normal saline, vehicle, PP1-12 at 1, 3, and 10 mg$kg21$d21 and perindopril at 2 mg$kg21$d21 for 4 weeks, respectively. At the end of the follow-up point, we evaluated echocardiographic and hemodynamic parameters, myocardial pathomorphology, apoptosis, and interstitial fibrosis, as well as the expression levels of important proteins involved in ER stress and apoptosis. Left ventricular geometry and function were ameliorated by PP1-12. PP1-12 inhibited interstitial fibrosis and reduced apoptosis of cardiomyocytes in a dosedependent manner. PP1-12 decreased GRP78 and caspase-12 expression and increased p-eIF2a and Bcl-2/Bax expression. These results suggest that PP1-12 efficiently inhibits left ventricular remodeling and improves heart function. The mechanism involved may be associated with the ability of PP1-12 to depress myocardial apoptosis induced by ER stress. Key Words: protein phosphatase-1 inhibitor, ventricular remodeling, heart function, apoptosis, endoplasmic reticulum stress (J Cardiovasc Pharmacol 2014;64:360–367)
INTRODUCTION Myocardial apoptosis plays an important role in cardiac remodeling and heart failure after myocardial infarction. Cardiomyocytes are terminally differentiated cells, which have no proliferative activity. Therefore, maintenance of this cell population through prevention of apoptosis is of critical importance. The endoplasmic reticulum (ER) is primarily recognized as Received for publication March 25, 2014; accepted April 29, 2014. From the *Chinese PLA General Hospital, Beijing, China; †School of Medicine, Nankai University, Tianjin, China; and ‡Beijing Institute of Pharmacology and Toxicology, Beijing, China. Supported by the Ministry Science Foudation of the Chinese People’s Liberation Army during the 12th Five-Year Plan Perid (BWS12J048) and Major International Science and Technology Cooperation Projects (No.2013DFA31170). The authors report no conflicts of interest. Reprints: Li-Li Wang, PhD, Beijing Institute of Pharmacology and Toxicology, Taiping Road 27, Beijing 100850, China (e-mail: wangll63@126. com); Kun-Lun He, MD, PhD, Chinese PLA General Hospital, Fuxing Road 28, Beijing 100853, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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the site for protein synthesis, folding, and trafficking and serves as an intracellular Ca2+ storage organelle. A variety of stresses, such as nutrient deprivation, ischemia, and calcium depletion cause accumulation of misfolded or unfolded proteins, initiating a group of responses in the cell termed ER stress. Extensive studies have shown that ER stress is implicated in the pathogenesis of cardiovascular diseases, including atherosclerosis, cardiomyopathy, ischemia/reperfusion injury, and heart failure.1–4 Therefore, adaption to stressors is a reproducible means of improving cardiac tolerance and blocking the pathological process.5,6 Phosphorylation of eukaryotic initiation factor-2 subunit a (eIF-2a) on ser51 serves as a central regulator of protein synthesis that globally monitors the balance between nascent cargo load and folding stress in the cell. It attenuates translation initiation and reduces protein synthesis to allow cells to clear the unfolded proteins and recover from ER stress.7 However, p-eIF2a is dephosphorylated by a complex containing the serine/threonine protein phosphatase 1 (PP1) and its nonenzymatic cofactor growth arrest and DNA damage-inducible protein (GADD34).8,9 Salubrinal has been developed as a protective agent against ER stress-mediated apoptosis. It inhibits PP1/GADD34 phosphatase activity and prevents the targeting of PP1 to eIF2a, which delays its dephosphorylation.10 In our previous studies, we modified the quinoline ring terminus and thiourea unit of salubrinal and synthesized new inhibitors of PP1/GADD34. In screening 95 chemicals for compounds (we have only reported 27 of them), which protect neonatal cardiomyocytes from ER stress-induced apoptosis, we identified the small molecule PP1-12 [(E)-3-(Thiophen-2-yl)-N-(2,2,2trichloro-1-(3-(2-methoxy phenyl) thioureido) ethyl) acrylamide, Fig. 1] with markedly enhanced cardioprotective activity and low toxicity.11 PP1-12 revealed more noticeably protective effect against cell apoptosis induced by tunicamycin, a specific ER stressor in vitro. Furthermore, we showed that it ameliorates myocardial injury induced by ischemia/reperfusion.12 The purpose of this study was to investigate the effect of PP1-12 on heart function and ventricular remodeling in rats with myocardial infarction. Our preliminary results show that PP1-12 exerts its protective effect through inhibition of ER stress.
METHODS Animal Preparation All procedures used were approved to comply with recommendations of the Institutional Animal Care and Use of J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
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FIGURE 1. Chemical structure of PP1-12.
Laboratory Animals of Chinese PLA General Hospital. Adult male Sprague–Dawley rats weighing 200 to 250 g were obtained from the company of SiBeiFu Experimental Animal Science and Technology (Beijing, China). Rats were kept at an ambient temperature of 20 to 248C with a 12–12 hours light–dark cycle.
Myocardial Infarction and Grouping Acute myocardial infarction (AMI) was produced by ligation of the left anterior descending coronary artery (LAD) as previously described.13 In brief, the heart was exposed through left thoracotomy. The LAD was ligated 2 to 3 mm from its origin between the pulmonary artery conus and the left atrium using a 7-0 silk suture. Rats that survived within 24 hours after coronary ligation were randomly divided into 6 groups and treated as follows: (1) normal saline (NS), (2) vehicle, (3) PP1-12 at 1 mg$kg21$d21 (PP1-12(1)), (4) PP1-12 at 3 mg$kg21$d21 (PP1-12(3)), (5) PP1-12 at 10 mg$kg21$d21 (PP1-12(10)), and (6) Perindopril at 2 mg$kg21$d21 (Perindopril(2)). We also included sham-operation rats as controls, which had the same surgical procedures performed, except that the suture around LAD was not tied. At least 8 rats were included in each group. PP1-12 was dissolved in DMSO and administered by intraperitoneal injection. Perindopril (Servier Laboratories, France) was administered in NS by gastric gavage once a day for 4 weeks after AMI.
Echocardiographic and Hemodynamic Analysis Echocardiography was measured at the end of the follow-up point. In brief, transthoracic echocardiographic assessment was performed at 4 weeks in anesthetized rats (3% pentobarbital sodium) using an echocardiographic system equipped with a 14.4-MHz transducer (SEQUOI A 512; Siemens, Germany). The parameters of heart rate, left ventricular end-diastolic diameter (LVEDD), LV endsystolic diameter (LVESD), ejection fraction (EF), and fractional shortening (FS) were recorded from M-mode images using averaged measurements from 3 to 5 consecutive cardiac cycles. Hemodynamic parameters were measured in anesthetized rats at the end of the echocardiogram. A polyethylene tubing catheter (0.8-mm internal diameter, PE-50) was inserted into the right carotid artery and advanced into the LV to measure LV systolic pressure and LV enddiastolic pressure (LVEDP) as the mean of measurements of 5 consecutive pressure cycles. The maximal rate of LV pressure increase (+dP/dt) and decrease (2dP/dt) were also recorded.
Morphometric Analysis Rat cardiac tissues were harvested with the atrial and right ventricular tissues discarded. The LV (including septum) was 2014 Lippincott Williams & Wilkins
PP1-12 Improved Heart Function
isolated and weighed, and ratio of LV to body weight (LVW/ BW) was calculated. Hearts were fixed in 4% paraformaldehyde and then embedded in paraffin. The cross-sectional area of myocytes was processed with hematoxylin–eosin staining using a standard protocol. Masson’s trichrome staining was performed to assess myocardial fibrosis. The index of collagen volume fraction was calculated as the percentage of fibrosis. Slices of tissue were observed under light microscopy.
TUNEL Assay The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s protocol using a commercially available kit (In Situ Cell Death Detection Kit, POD; Roche Company, Germany). The labeled cardiomyocytes were analyzed under light microscopy. Apoptotic index, which means the numbers of apoptotic cells (AI) per 100 cells, was calculated. Cells in 5 different random fields in noninfarcted tissue of the heart were counted.
Western Blot Analysis Total proteins were extracted from rat heart tissues using lysis buffer [62.5 mmol/L Tris-HCl (pH 6.8 at 258C), 2% wt/vol SDS (sodium dodecyl sulfate), 10% glycerol, and 50 mmol/L DTT (dithiothreitol)]. The homogenates were heated and centrifuged to obtain protein whose concentrations were determined using the bicinchoninic acid assay. Proteins were subjected to SDS/PAGE followed by western blotting. The primary antibodies used were p-eIF2a, eIF2a, caspase12, Bcl-2, Bax (1:1000; Cell Signaling Technology), and GRP78 (1:100; Abcam). GAPDH (1:1000; Zhongshan Golden Bridge Biotechnology Company, China) was used as internal calibration to account for differences in protein. Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology Company) at a 1:5000 dilution. Protein bands were detected with the Alpha Imager 5500 system (Alpha Innotech).
Statistical Analysis
All values are shown as mean 6 SEM. Statistical analyses were performed by one-way analysis of variance followed by the least significant difference test for multiple comparisons. All tests were carried out using SPSS version 15.0 statistical software. Differences were considered statistically significant at P , 0.05.
RESULTS Effect of PP1-12 on LV Geometry and Heart Function Echocardiographic assessments of LV geometry and function at 4 weeks in all of the groups are shown in Table 1. After 4 weeks of AMI, LVEDD and LVESD were significantly higher, whereas EF and FS were lower in the NS and vehicle groups compared with the sham-operated group. There were no significant differences in LVEDD, LVESD, EF, and FS between the NS and vehicle groups. LV geometry www.jcvp.org |
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TABLE 1. The Echocardiographic Assessments of Left Ventricular Geometry and Function MI Group n HR, bpm LVEDD, mm LVESD, mm LVEF, % FS, %
Sham 10 380 6 6.3 6 3.9 6 76.1 6 38.2 6
11 0.3 0.2 1.1 0.9
399 7.6 5.7 53.4 25.2
NS
Vehicle
PP1-12(1)
8 6 6 6 6 6
8 6 6 6 6 6
8 6 6 6 6 6
10 0.4* 0.3† 3.8† 2.2†
371 8.0 6.1 53.3 24.3
13 0.3† 0.3† 2.7† 1.5†
404 7.3 5.4 56.5 26.1
PP1-12(3)
12 0.5* 0.5† 3.2† 2.0*
381 6.7 4.7 63.7 30.6
PP1-12(10)
8 6 13 6 0.2‡ 6 0.2*§ 6 3.4*§ 6 2.3*§
9 370 6 8 7.0 6 0.3‡ 4.7 6 0.2*‡ 66. 9 6 2.1†§ 32.3 6 1.5†§
Perindopril(2) 392 6.5 4.3 69.0 34.1
10 6 14 6 0.2§ 6 0.2§ 6 1.5*§ 6 1.1*§
Values are mean 6 SEM. *P , 0.05. †P , 0.01 versus sham. ‡P , 0.05. §P , 0.01 versus vehicle. HR, heart rate; LVEF, left ventricular ejection fraction; bpm, beats per minute.
and function were ameliorated by drug treatment except for PP1-12(1) compared with vehicle group rats. Furthermore, the effect of PP1-12(10) and perindopril were significantly greater (P , 0.01). Hemodynamic parameters and LV weights at 4 weeks are shown in Table 2. At 4 weeks post-MI, the NS and vehicle groups showed significant increase in LVW/BW and LVEDP and decrease in +dp/dtmax and 2dp/dtmax compared with the sham-operated group. The PP1-12(10) and perindopril groups had a lower LVW/BW compared with the vehicle-treated group. In addition, PP1-12 and perindopril significantly ameliorated the increase in LVEDP and decrease in +dp/dtmax and 2dp/dtmax post-AMI, suggesting an improvement in heart function.
(Fig. 2A). Masson’s trichrome staining also showed that PP1-12 attenuated collagen deposition in the remote-infarct area because the collagen volume fraction was significantly reduced at the dose of 10 mg$kg21$d21 (Fig. 2B). We observed that TUNEL-positive cardiomyocytes were mainly located in the border zones adjacent to infarct scars. Scattered apoptotic cells were also observed in the remote myocardium. There were few apoptotic cells in the sham-operated heart. Treatment with PP1-12 significantly suppressed cellular apoptosis, and this effect increased with increasing PP1-12 concentrations. The antiapoptotic effect of PP1-12(10) was also more significant than that of perindopril (0.07 vs. 0.16, P , 0.01) (Fig. 3).
Effect of PP1-12 on Myocardial Injury and Apoptosis
Effect of PP1-12 on ER Stress-associated Proteins and Apoptotic Markers
The histomorphology of tissue and AI at 4 weeks is shown in Figures 2 and 3, respectively. Compared with vehicle group rats, HE staining showed that PP1-12 inhibited hypertrophy of cardiomyocytes, hyperemia, myocardial fiber fracture, and myocardial pericardial inflammatory infiltration
To confirm the antiapoptotic effect and mechanism of PP1-12, we evaluated the expression levels of important proteins involved in ER stress, including GRP78, p-eIF2a, and the apoptotic markers caspase-12 and Bcl-2/Bax by western blotting (Fig. 4). Compared with the sham group,
TABLE 2. The Hemodynamic Parameters and Left Ventricular Weights MI Group BW, g LVW/BW, mg/g HR, bpm MAP, mm Hg LVSP, mm Hg LVEDP, mm Hg +dp/dtmax, mm Hg/s 2dp/dtmax, mm Hg/s
Sham 374.5 2.11 404 105.5 127.6 2.8 6384 25262
6 6 6 6 6 6 6 6
NS 8.8 0.04 12 3.9 3.4 0.9 212 301
368.3 2.44 422 112.9 131.5 13.3 4072 23377
6 6 6 6 6 6 6 6
Vehicle 9.9 0.07† 7 5.2 6.7 3.0† 131 146†
364.6 2.35 403 105.9 125.9 14.5 3950 23334
6 6 6 6 6 6 6 6
5.4 0.06† 9 4.4 5.6 2.0† 235 290†
PP1-12(1) 370.8 2.29 414 115.5 114.8 7.0 4682 24185
6 6 6 6 6 6 6 6
7.1 0.08* 8 8.0 10.8 3.1‡ 457 492*‡
PP1-12(3) 364.7 2.27 395 108.8 123.5 5.8 4449 23561
6 6 6 6 6 6 6 6
6.1 0.06* 18 7.1 7.3 2.0§ 214 215†
PP1-12(10) 372.3 2.21 406 113.6 134.3 3.2 4745 24305
6 6 6 6 6 6 6 6
8.2 0.03‡ 9 7.4 13.4 1.2§ 338 125*‡
Perindopril(2) 366.1 2.11 422 104.5 135.3 7.2 4966 24120
6 6 6 6 6 6 6 6
6.1 0.02§ 14 3.2 6.6 2.3§ 319†§ 183†‡
Values are mean 6 SEM. *P , 0.05. †P , 0.01 versus sham. ‡P , 0.05. §P , 0.01 versus vehicle. HR, heart rate; bpm, beats per minute; MAP, mean arterial pressure; LVSP, left ventricular systolic pressure; 6dp/dtmax, the maximal rate of LV pressure rise and decrease.
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FIGURE 2. The effect of PP1-12 on myocardial morphology and interstitial fibrosis. A and B, Results of hematoxylin–eosin staining (·20) and Masson trichrome staining (·40), respectively. a, Sham-operated group; (b) vehicle group; (c) 1 mg$kg21$d21 of PP1-12; (d) 3 mg$kg21$d21 of PP1-12; (e) 10 mg$kg21$d21 of PP1-12; (f) 2 mg$kg21$d21 of perindopril. The fibrotic area is stained blue and the viable area is stained red in Masson staining. The lower panel exhibits the mean data of collagen volume fraction from 3 heart tissues of each sample in 5 different fields. Results are mean 6 SEM. The size of the bar is 100 mm. **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle. 2014 Lippincott Williams & Wilkins
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FIGURE 3. The effect of PP1-12 on myocardial apoptosis (TUNEL assay, ·40). A, Sham-operated group; (B) vehicle group; (C) 1 mg$kg21$d21 of PP1-12; (D) 3 mg$kg21$d21 of PP1-12; (E) 10 mg$kg21$d21 of PP1-12; (F) 2 mg$kg21$d21 of perindopril. The apoptotic cells are stained by brown in TUNEL assay. The lower panel exhibits the mean data of AI from 3 heart tissues of each sample in 5 different fields. Results are mean 6 SEM. The size of the bar is 100 mm. *P , 0.05, **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle.
enhanced level of GRP78 and p-eIF2a in the vehicle group indicated the activation of the ER stress. Compared with the vehicle group, treatment with PP1-12 for 4 weeks significantly decreased GRP78 expression, whereas PP1-12(10)
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further increased eIF2a phosphorylation (Fig. 4A and B). It also revealed that PP1-12 administration significantly increased the ratio of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (Fig. 4D). PP1-12 significantly 2014 Lippincott Williams & Wilkins
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FIGURE 4. The effect of PP1-12 on ER stress-associated proteins and apoptotic markers. The protein levels of p-eIF2a and total eIF-2a (A), GRP78 (B), caspase-12 (C), as well as Bcl-2 and Bax (D) from LV were analyzed by western blotting. Similar results were obtained in 3 independent experiments. The lower panels exhibit the mean data and results are mean 6 SEM. *P , 0.05, **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle.
inhibited the activation of caspase-12, which suggested that the antiapoptotic effect of PP1-12 was linked to suppression of ER stress (Fig. 4C).
DISCUSSION We demonstrated that PP1-12, a novel small molecule, reduced myocardial apoptosis and ameliorated ventricular function and remodeling in a rat model of AMI. PP1-12 enhanced eIF2a phosphorylation and reduced GRP78 and caspase-12, indicating that the protective effect of PP1-12 may be mediated by modulating ER stress. PP1 is a holoenzyme consisting of a catalytic subunit associated with an ancillary protein.14 A previous study showed that that at the level of cardiac sarcoplasmic reticulum, inhibition of PP1 is protective against the progression of cardiac hypertrophy and heart failure but it focused on catalytic activity.15 Regulatory proteins target PP1 to appropriate subcellular compartments, and this could dictate substrate specificity and modulate its catalytic activity. Therefore, a second approach to inhibit the association of PP1 to PP1-interacting proteins is direct targeting of the regulatory subunit.16,17 In 2014 Lippincott Williams & Wilkins
2005, Boyce et al10 found that salubrinal, a selective inhibitor of PP1/GADD34, protects PC-12 cell from apoptosis. Salubrinal has a protective effect on ischemia/reperfusion-induced brain injury and b-adrenergic receptor-stimulated cardiomyocyte apoptosis.18,19 In our previous study, salubrinal was also assayed for its ability to inhibit cardiomyocyte apoptosis from tunicamycin and hypoxia exposure.20 Recently, Liu et al21 found that therapy with salubrinal reduces ER stress, alters the natural history of heart failure after myocardial infarction, and improves cardiac function. PP1-12, a novel PP1/GADD34 inhibitor, was designed and synthesized on the basis of salubrinal. Our previous reports have shown that, in vitro, PP1-12 protects the viability of cardiomyocyte treated with tunicamycin, a specific ER stressor through inhibition of protein glycosylation. Multiparametric analysis of apoptosis has shown that PP1-12 reduces nuclear condensation, increases mitochondrial membrane potential, and decreases the reorganization of actin cytoskeleton. PP1-12 considerably increases p-eIF2a/eIF2a levels and inhibits expression of CHOP protein, which is specific to ER stress-induced apoptosis. Additionally, in a rat model of myocardial ischemia/reperfusion injury, infarct www.jcvp.org |
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size is decreased with increasing doses of PP1-12.12 In this study, we examined the effect of PP1-12 on LV remodeling and heart function after AMI in a rat model of coronary ligation. We observed remarkable cytoprotective activity in heart ischemia. We found that PP1-12 treatment for 4 weeks inhibited myocardial hypertrophy, decreased apoptosis of cardiomyocytes, attenuated unfavorable myocardial remodeling, inhibited interstitial fibrosis, and preserved heart function. GRP78 is a critical regulator of ER function. Consistent with previous study,22 our observation revealed that GRP78 and p-eIF2a protein levels were significantly induced 4 weeks after AMI in rats, which suggested that ER stress was induced in our model. Salubrinal intraperitoneally treated over 28 days abrogates the increase of GRP78 and reduces cyclosporine nephrotoxicity in rat kidneys.23 Augmentation of eIF2a phosphorylation with systemically administered salubrinal minimizes motoneuronal injury induced by long-term intermittent hypoxia exposure.24 In our study, PP1-12 ameliorated the elevated expression of GRP78 post-AMI, which suggested that the ER stress was inhibited. PP1-12 enhanced eIF2a phosphorylation, which could attenuate translation initiation and reduce protein synthesis to allow cells to clear unfolded proteins and recover from ER stress. By eIF2a phosphorylation, cells attempt to adapt to stressors, and this improves cardiac tolerance.25 ER stress is a “double-edged sword,” and the fate of cells, whether it is survival or death, is determined by the degree of the stress. If stress is prolonged and excessive, the protective mechanisms are not sufficient to restore normal ER function, and damaged cells undergo ER stress-induced apoptosis. Caspase-12, which is localized on the ER membrane, can only be activated in ER stress-signaling pathway.26 In our study, activated caspase-12 and increased apoptotic cardiomyocytes in the vehicle group showed that the ER stress apoptotic pathway was involved in the heart after AMI. PP1-12 significantly inhibited the ischemia-induced cleavage of caspase-12 and reduced apoptosis of cardiomyocytes. The Bcl-2 family is one of the most important proteins involved in the regulation of apoptosis. Proteins of this family are initiated by the mitochondrial-dependent or intrinsic pathway to induce cell apoptosis. Recent studies have shown that Bcl-2 family proteins are located at the ER and regulate apoptotic pathways in response to many cellar stresses.27,28 There are some limitations to our study. In vitro, we previously found that the antiapoptotic effect of PP1-12 is more effective than that of salubrinal.11 However, in vivo, although PP1-12 at 1 mg$kg21$d21 could inhibit apoptosis of cardiomyocytes and partly improve heart function, this effect is not as significant as salubrinal at the same concentration. This discrepancy may be due to the different characters of drugs in vivo and in vitro. Additional studies are required to clarify this issue. Although phosphorylation of eIF2a can restore cellular homeostasis, the long-term effect of sustained activation of eIF2a phosphorylation is still unknown. Finally, we speculate that PP1-12 is not only specially fit for ischemic heart diseases, but may also be applicable to other human disorders involved in protein misfolding or unfolding. More research needs to be performed to examine whether other mechanisms are involved in the protective effect of PP1-12.
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CONCLUSIONS In summary, this study preliminarily shows that PP1-12 effectively improves cardiac function and reduces ER stress. The mechanisms responsible for this protective effect could be the result of improving cardiac tolerance, restoring cell homeostasis, and blocking the pathological process. Our findings suggest that PP1-12 and PP1 inhibitor-like small molecules may represent a potential treatment. However, regulation of p-eIF2a in response to ER stress is a complex process, and the effect of these compounds needs to be further verified. REFERENCES
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PP1-12 Improved Heart Function 23. Pallet N, Bouvier N, Bendjallabah A, et al. Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death. Am J Transplant. 2008;8:2283–2296. 24. Zhu Y, Fenik P, Zhan G, et al. Eif-2a protects brainstem motoneurons in a murine model of sleep apnea. J Neurosci. 2008;28:2168–2178. 25. Fullwood MJ, Zhou W, Shenolikar S. Targeting phosphorylation of eukaryotic initiation factor-2alpha to treat human disease. Prog Mol Biol Transl Sci. 2012;106:75–106. 26. Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates ERspecific apoptosis and cytotoxicity by amyloid- beta. Nature. 2000; 403:98–103. 27. Hetz CA. ER stress signaling and the Bcl-2 family of proteins: from adaptation to irreversible cellular damage. Antioxid Redox Signal. 2007;9:2345–2355. 28. Szegezdi E, Macdonald DC, Ní Chonghaile T, et al. Bcl-2 family on guard at the ER. Am J Physiol Cell Physiol. 2009;296:C941–C953.
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Effects of PP1-12, a Novel Protein Phosphatase-1 Inhibitor, on Ventricular Function and Remodeling After Myocardial Infarction in Rats Yun-Yun He, MD,*† Chun-Lei Liu, MS,* Xin Li, BS,* Wu Zhong, PhD,‡ Song Li, PhD,‡ Kun-Lun He, MD, PhD,* and Li-Li Wang, PhD‡
Abstract: PP1-12, a new protein phosphatase-1 inhibitor, is designed and synthesized to modulate the endoplasmic reticulum (ER) stress apoptotic pathway, which is involved in various cardiovascular diseases. In this study, we examined the effect of PP1-12 on ventricular remodeling and heart function after myocardial infarction. Rats that survived within 24 hours after coronary ligation were randomly divided into 6 groups and treated with normal saline, vehicle, PP1-12 at 1, 3, and 10 mg$kg21$d21 and perindopril at 2 mg$kg21$d21 for 4 weeks, respectively. At the end of the follow-up point, we evaluated echocardiographic and hemodynamic parameters, myocardial pathomorphology, apoptosis, and interstitial fibrosis, as well as the expression levels of important proteins involved in ER stress and apoptosis. Left ventricular geometry and function were ameliorated by PP1-12. PP1-12 inhibited interstitial fibrosis and reduced apoptosis of cardiomyocytes in a dosedependent manner. PP1-12 decreased GRP78 and caspase-12 expression and increased p-eIF2a and Bcl-2/Bax expression. These results suggest that PP1-12 efficiently inhibits left ventricular remodeling and improves heart function. The mechanism involved may be associated with the ability of PP1-12 to depress myocardial apoptosis induced by ER stress. Key Words: protein phosphatase-1 inhibitor, ventricular remodeling, heart function, apoptosis, endoplasmic reticulum stress (J Cardiovasc Pharmacol 2014;64:360–367)
INTRODUCTION Myocardial apoptosis plays an important role in cardiac remodeling and heart failure after myocardial infarction. Cardiomyocytes are terminally differentiated cells, which have no proliferative activity. Therefore, maintenance of this cell population through prevention of apoptosis is of critical importance. The endoplasmic reticulum (ER) is primarily recognized as Received for publication March 25, 2014; accepted April 29, 2014. From the *Chinese PLA General Hospital, Beijing, China; †School of Medicine, Nankai University, Tianjin, China; and ‡Beijing Institute of Pharmacology and Toxicology, Beijing, China. Supported by the Ministry Science Foudation of the Chinese People’s Liberation Army during the 12th Five-Year Plan Perid (BWS12J048) and Major International Science and Technology Cooperation Projects (No.2013DFA31170). The authors report no conflicts of interest. Reprints: Li-Li Wang, PhD, Beijing Institute of Pharmacology and Toxicology, Taiping Road 27, Beijing 100850, China (e-mail: wangll63@126. com); Kun-Lun He, MD, PhD, Chinese PLA General Hospital, Fuxing Road 28, Beijing 100853, China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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the site for protein synthesis, folding, and trafficking and serves as an intracellular Ca2+ storage organelle. A variety of stresses, such as nutrient deprivation, ischemia, and calcium depletion cause accumulation of misfolded or unfolded proteins, initiating a group of responses in the cell termed ER stress. Extensive studies have shown that ER stress is implicated in the pathogenesis of cardiovascular diseases, including atherosclerosis, cardiomyopathy, ischemia/reperfusion injury, and heart failure.1–4 Therefore, adaption to stressors is a reproducible means of improving cardiac tolerance and blocking the pathological process.5,6 Phosphorylation of eukaryotic initiation factor-2 subunit a (eIF-2a) on ser51 serves as a central regulator of protein synthesis that globally monitors the balance between nascent cargo load and folding stress in the cell. It attenuates translation initiation and reduces protein synthesis to allow cells to clear the unfolded proteins and recover from ER stress.7 However, p-eIF2a is dephosphorylated by a complex containing the serine/threonine protein phosphatase 1 (PP1) and its nonenzymatic cofactor growth arrest and DNA damage-inducible protein (GADD34).8,9 Salubrinal has been developed as a protective agent against ER stress-mediated apoptosis. It inhibits PP1/GADD34 phosphatase activity and prevents the targeting of PP1 to eIF2a, which delays its dephosphorylation.10 In our previous studies, we modified the quinoline ring terminus and thiourea unit of salubrinal and synthesized new inhibitors of PP1/GADD34. In screening 95 chemicals for compounds (we have only reported 27 of them), which protect neonatal cardiomyocytes from ER stress-induced apoptosis, we identified the small molecule PP1-12 [(E)-3-(Thiophen-2-yl)-N-(2,2,2trichloro-1-(3-(2-methoxy phenyl) thioureido) ethyl) acrylamide, Fig. 1] with markedly enhanced cardioprotective activity and low toxicity.11 PP1-12 revealed more noticeably protective effect against cell apoptosis induced by tunicamycin, a specific ER stressor in vitro. Furthermore, we showed that it ameliorates myocardial injury induced by ischemia/reperfusion.12 The purpose of this study was to investigate the effect of PP1-12 on heart function and ventricular remodeling in rats with myocardial infarction. Our preliminary results show that PP1-12 exerts its protective effect through inhibition of ER stress.
METHODS Animal Preparation All procedures used were approved to comply with recommendations of the Institutional Animal Care and Use of J Cardiovasc Pharmacol Volume 64, Number 4, October 2014
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FIGURE 1. Chemical structure of PP1-12.
Laboratory Animals of Chinese PLA General Hospital. Adult male Sprague–Dawley rats weighing 200 to 250 g were obtained from the company of SiBeiFu Experimental Animal Science and Technology (Beijing, China). Rats were kept at an ambient temperature of 20 to 248C with a 12–12 hours light–dark cycle.
Myocardial Infarction and Grouping Acute myocardial infarction (AMI) was produced by ligation of the left anterior descending coronary artery (LAD) as previously described.13 In brief, the heart was exposed through left thoracotomy. The LAD was ligated 2 to 3 mm from its origin between the pulmonary artery conus and the left atrium using a 7-0 silk suture. Rats that survived within 24 hours after coronary ligation were randomly divided into 6 groups and treated as follows: (1) normal saline (NS), (2) vehicle, (3) PP1-12 at 1 mg$kg21$d21 (PP1-12(1)), (4) PP1-12 at 3 mg$kg21$d21 (PP1-12(3)), (5) PP1-12 at 10 mg$kg21$d21 (PP1-12(10)), and (6) Perindopril at 2 mg$kg21$d21 (Perindopril(2)). We also included sham-operation rats as controls, which had the same surgical procedures performed, except that the suture around LAD was not tied. At least 8 rats were included in each group. PP1-12 was dissolved in DMSO and administered by intraperitoneal injection. Perindopril (Servier Laboratories, France) was administered in NS by gastric gavage once a day for 4 weeks after AMI.
Echocardiographic and Hemodynamic Analysis Echocardiography was measured at the end of the follow-up point. In brief, transthoracic echocardiographic assessment was performed at 4 weeks in anesthetized rats (3% pentobarbital sodium) using an echocardiographic system equipped with a 14.4-MHz transducer (SEQUOI A 512; Siemens, Germany). The parameters of heart rate, left ventricular end-diastolic diameter (LVEDD), LV endsystolic diameter (LVESD), ejection fraction (EF), and fractional shortening (FS) were recorded from M-mode images using averaged measurements from 3 to 5 consecutive cardiac cycles. Hemodynamic parameters were measured in anesthetized rats at the end of the echocardiogram. A polyethylene tubing catheter (0.8-mm internal diameter, PE-50) was inserted into the right carotid artery and advanced into the LV to measure LV systolic pressure and LV enddiastolic pressure (LVEDP) as the mean of measurements of 5 consecutive pressure cycles. The maximal rate of LV pressure increase (+dP/dt) and decrease (2dP/dt) were also recorded.
Morphometric Analysis Rat cardiac tissues were harvested with the atrial and right ventricular tissues discarded. The LV (including septum) was 2014 Lippincott Williams & Wilkins
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isolated and weighed, and ratio of LV to body weight (LVW/ BW) was calculated. Hearts were fixed in 4% paraformaldehyde and then embedded in paraffin. The cross-sectional area of myocytes was processed with hematoxylin–eosin staining using a standard protocol. Masson’s trichrome staining was performed to assess myocardial fibrosis. The index of collagen volume fraction was calculated as the percentage of fibrosis. Slices of tissue were observed under light microscopy.
TUNEL Assay The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s protocol using a commercially available kit (In Situ Cell Death Detection Kit, POD; Roche Company, Germany). The labeled cardiomyocytes were analyzed under light microscopy. Apoptotic index, which means the numbers of apoptotic cells (AI) per 100 cells, was calculated. Cells in 5 different random fields in noninfarcted tissue of the heart were counted.
Western Blot Analysis Total proteins were extracted from rat heart tissues using lysis buffer [62.5 mmol/L Tris-HCl (pH 6.8 at 258C), 2% wt/vol SDS (sodium dodecyl sulfate), 10% glycerol, and 50 mmol/L DTT (dithiothreitol)]. The homogenates were heated and centrifuged to obtain protein whose concentrations were determined using the bicinchoninic acid assay. Proteins were subjected to SDS/PAGE followed by western blotting. The primary antibodies used were p-eIF2a, eIF2a, caspase12, Bcl-2, Bax (1:1000; Cell Signaling Technology), and GRP78 (1:100; Abcam). GAPDH (1:1000; Zhongshan Golden Bridge Biotechnology Company, China) was used as internal calibration to account for differences in protein. Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology Company) at a 1:5000 dilution. Protein bands were detected with the Alpha Imager 5500 system (Alpha Innotech).
Statistical Analysis
All values are shown as mean 6 SEM. Statistical analyses were performed by one-way analysis of variance followed by the least significant difference test for multiple comparisons. All tests were carried out using SPSS version 15.0 statistical software. Differences were considered statistically significant at P , 0.05.
RESULTS Effect of PP1-12 on LV Geometry and Heart Function Echocardiographic assessments of LV geometry and function at 4 weeks in all of the groups are shown in Table 1. After 4 weeks of AMI, LVEDD and LVESD were significantly higher, whereas EF and FS were lower in the NS and vehicle groups compared with the sham-operated group. There were no significant differences in LVEDD, LVESD, EF, and FS between the NS and vehicle groups. LV geometry www.jcvp.org |
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TABLE 1. The Echocardiographic Assessments of Left Ventricular Geometry and Function MI Group n HR, bpm LVEDD, mm LVESD, mm LVEF, % FS, %
Sham 10 380 6 6.3 6 3.9 6 76.1 6 38.2 6
11 0.3 0.2 1.1 0.9
399 7.6 5.7 53.4 25.2
NS
Vehicle
PP1-12(1)
8 6 6 6 6 6
8 6 6 6 6 6
8 6 6 6 6 6
10 0.4* 0.3† 3.8† 2.2†
371 8.0 6.1 53.3 24.3
13 0.3† 0.3† 2.7† 1.5†
404 7.3 5.4 56.5 26.1
PP1-12(3)
12 0.5* 0.5† 3.2† 2.0*
381 6.7 4.7 63.7 30.6
PP1-12(10)
8 6 13 6 0.2‡ 6 0.2*§ 6 3.4*§ 6 2.3*§
9 370 6 8 7.0 6 0.3‡ 4.7 6 0.2*‡ 66. 9 6 2.1†§ 32.3 6 1.5†§
Perindopril(2) 392 6.5 4.3 69.0 34.1
10 6 14 6 0.2§ 6 0.2§ 6 1.5*§ 6 1.1*§
Values are mean 6 SEM. *P , 0.05. †P , 0.01 versus sham. ‡P , 0.05. §P , 0.01 versus vehicle. HR, heart rate; LVEF, left ventricular ejection fraction; bpm, beats per minute.
and function were ameliorated by drug treatment except for PP1-12(1) compared with vehicle group rats. Furthermore, the effect of PP1-12(10) and perindopril were significantly greater (P , 0.01). Hemodynamic parameters and LV weights at 4 weeks are shown in Table 2. At 4 weeks post-MI, the NS and vehicle groups showed significant increase in LVW/BW and LVEDP and decrease in +dp/dtmax and 2dp/dtmax compared with the sham-operated group. The PP1-12(10) and perindopril groups had a lower LVW/BW compared with the vehicle-treated group. In addition, PP1-12 and perindopril significantly ameliorated the increase in LVEDP and decrease in +dp/dtmax and 2dp/dtmax post-AMI, suggesting an improvement in heart function.
(Fig. 2A). Masson’s trichrome staining also showed that PP1-12 attenuated collagen deposition in the remote-infarct area because the collagen volume fraction was significantly reduced at the dose of 10 mg$kg21$d21 (Fig. 2B). We observed that TUNEL-positive cardiomyocytes were mainly located in the border zones adjacent to infarct scars. Scattered apoptotic cells were also observed in the remote myocardium. There were few apoptotic cells in the sham-operated heart. Treatment with PP1-12 significantly suppressed cellular apoptosis, and this effect increased with increasing PP1-12 concentrations. The antiapoptotic effect of PP1-12(10) was also more significant than that of perindopril (0.07 vs. 0.16, P , 0.01) (Fig. 3).
Effect of PP1-12 on Myocardial Injury and Apoptosis
Effect of PP1-12 on ER Stress-associated Proteins and Apoptotic Markers
The histomorphology of tissue and AI at 4 weeks is shown in Figures 2 and 3, respectively. Compared with vehicle group rats, HE staining showed that PP1-12 inhibited hypertrophy of cardiomyocytes, hyperemia, myocardial fiber fracture, and myocardial pericardial inflammatory infiltration
To confirm the antiapoptotic effect and mechanism of PP1-12, we evaluated the expression levels of important proteins involved in ER stress, including GRP78, p-eIF2a, and the apoptotic markers caspase-12 and Bcl-2/Bax by western blotting (Fig. 4). Compared with the sham group,
TABLE 2. The Hemodynamic Parameters and Left Ventricular Weights MI Group BW, g LVW/BW, mg/g HR, bpm MAP, mm Hg LVSP, mm Hg LVEDP, mm Hg +dp/dtmax, mm Hg/s 2dp/dtmax, mm Hg/s
Sham 374.5 2.11 404 105.5 127.6 2.8 6384 25262
6 6 6 6 6 6 6 6
NS 8.8 0.04 12 3.9 3.4 0.9 212 301
368.3 2.44 422 112.9 131.5 13.3 4072 23377
6 6 6 6 6 6 6 6
Vehicle 9.9 0.07† 7 5.2 6.7 3.0† 131 146†
364.6 2.35 403 105.9 125.9 14.5 3950 23334
6 6 6 6 6 6 6 6
5.4 0.06† 9 4.4 5.6 2.0† 235 290†
PP1-12(1) 370.8 2.29 414 115.5 114.8 7.0 4682 24185
6 6 6 6 6 6 6 6
7.1 0.08* 8 8.0 10.8 3.1‡ 457 492*‡
PP1-12(3) 364.7 2.27 395 108.8 123.5 5.8 4449 23561
6 6 6 6 6 6 6 6
6.1 0.06* 18 7.1 7.3 2.0§ 214 215†
PP1-12(10) 372.3 2.21 406 113.6 134.3 3.2 4745 24305
6 6 6 6 6 6 6 6
8.2 0.03‡ 9 7.4 13.4 1.2§ 338 125*‡
Perindopril(2) 366.1 2.11 422 104.5 135.3 7.2 4966 24120
6 6 6 6 6 6 6 6
6.1 0.02§ 14 3.2 6.6 2.3§ 319†§ 183†‡
Values are mean 6 SEM. *P , 0.05. †P , 0.01 versus sham. ‡P , 0.05. §P , 0.01 versus vehicle. HR, heart rate; bpm, beats per minute; MAP, mean arterial pressure; LVSP, left ventricular systolic pressure; 6dp/dtmax, the maximal rate of LV pressure rise and decrease.
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FIGURE 2. The effect of PP1-12 on myocardial morphology and interstitial fibrosis. A and B, Results of hematoxylin–eosin staining (·20) and Masson trichrome staining (·40), respectively. a, Sham-operated group; (b) vehicle group; (c) 1 mg$kg21$d21 of PP1-12; (d) 3 mg$kg21$d21 of PP1-12; (e) 10 mg$kg21$d21 of PP1-12; (f) 2 mg$kg21$d21 of perindopril. The fibrotic area is stained blue and the viable area is stained red in Masson staining. The lower panel exhibits the mean data of collagen volume fraction from 3 heart tissues of each sample in 5 different fields. Results are mean 6 SEM. The size of the bar is 100 mm. **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle. 2014 Lippincott Williams & Wilkins
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FIGURE 3. The effect of PP1-12 on myocardial apoptosis (TUNEL assay, ·40). A, Sham-operated group; (B) vehicle group; (C) 1 mg$kg21$d21 of PP1-12; (D) 3 mg$kg21$d21 of PP1-12; (E) 10 mg$kg21$d21 of PP1-12; (F) 2 mg$kg21$d21 of perindopril. The apoptotic cells are stained by brown in TUNEL assay. The lower panel exhibits the mean data of AI from 3 heart tissues of each sample in 5 different fields. Results are mean 6 SEM. The size of the bar is 100 mm. *P , 0.05, **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle.
enhanced level of GRP78 and p-eIF2a in the vehicle group indicated the activation of the ER stress. Compared with the vehicle group, treatment with PP1-12 for 4 weeks significantly decreased GRP78 expression, whereas PP1-12(10)
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further increased eIF2a phosphorylation (Fig. 4A and B). It also revealed that PP1-12 administration significantly increased the ratio of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (Fig. 4D). PP1-12 significantly 2014 Lippincott Williams & Wilkins
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FIGURE 4. The effect of PP1-12 on ER stress-associated proteins and apoptotic markers. The protein levels of p-eIF2a and total eIF-2a (A), GRP78 (B), caspase-12 (C), as well as Bcl-2 and Bax (D) from LV were analyzed by western blotting. Similar results were obtained in 3 independent experiments. The lower panels exhibit the mean data and results are mean 6 SEM. *P , 0.05, **P , 0.01 versus sham; #P , 0.05, ##P , 0.01 versus vehicle.
inhibited the activation of caspase-12, which suggested that the antiapoptotic effect of PP1-12 was linked to suppression of ER stress (Fig. 4C).
DISCUSSION We demonstrated that PP1-12, a novel small molecule, reduced myocardial apoptosis and ameliorated ventricular function and remodeling in a rat model of AMI. PP1-12 enhanced eIF2a phosphorylation and reduced GRP78 and caspase-12, indicating that the protective effect of PP1-12 may be mediated by modulating ER stress. PP1 is a holoenzyme consisting of a catalytic subunit associated with an ancillary protein.14 A previous study showed that that at the level of cardiac sarcoplasmic reticulum, inhibition of PP1 is protective against the progression of cardiac hypertrophy and heart failure but it focused on catalytic activity.15 Regulatory proteins target PP1 to appropriate subcellular compartments, and this could dictate substrate specificity and modulate its catalytic activity. Therefore, a second approach to inhibit the association of PP1 to PP1-interacting proteins is direct targeting of the regulatory subunit.16,17 In 2014 Lippincott Williams & Wilkins
2005, Boyce et al10 found that salubrinal, a selective inhibitor of PP1/GADD34, protects PC-12 cell from apoptosis. Salubrinal has a protective effect on ischemia/reperfusion-induced brain injury and b-adrenergic receptor-stimulated cardiomyocyte apoptosis.18,19 In our previous study, salubrinal was also assayed for its ability to inhibit cardiomyocyte apoptosis from tunicamycin and hypoxia exposure.20 Recently, Liu et al21 found that therapy with salubrinal reduces ER stress, alters the natural history of heart failure after myocardial infarction, and improves cardiac function. PP1-12, a novel PP1/GADD34 inhibitor, was designed and synthesized on the basis of salubrinal. Our previous reports have shown that, in vitro, PP1-12 protects the viability of cardiomyocyte treated with tunicamycin, a specific ER stressor through inhibition of protein glycosylation. Multiparametric analysis of apoptosis has shown that PP1-12 reduces nuclear condensation, increases mitochondrial membrane potential, and decreases the reorganization of actin cytoskeleton. PP1-12 considerably increases p-eIF2a/eIF2a levels and inhibits expression of CHOP protein, which is specific to ER stress-induced apoptosis. Additionally, in a rat model of myocardial ischemia/reperfusion injury, infarct www.jcvp.org |
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size is decreased with increasing doses of PP1-12.12 In this study, we examined the effect of PP1-12 on LV remodeling and heart function after AMI in a rat model of coronary ligation. We observed remarkable cytoprotective activity in heart ischemia. We found that PP1-12 treatment for 4 weeks inhibited myocardial hypertrophy, decreased apoptosis of cardiomyocytes, attenuated unfavorable myocardial remodeling, inhibited interstitial fibrosis, and preserved heart function. GRP78 is a critical regulator of ER function. Consistent with previous study,22 our observation revealed that GRP78 and p-eIF2a protein levels were significantly induced 4 weeks after AMI in rats, which suggested that ER stress was induced in our model. Salubrinal intraperitoneally treated over 28 days abrogates the increase of GRP78 and reduces cyclosporine nephrotoxicity in rat kidneys.23 Augmentation of eIF2a phosphorylation with systemically administered salubrinal minimizes motoneuronal injury induced by long-term intermittent hypoxia exposure.24 In our study, PP1-12 ameliorated the elevated expression of GRP78 post-AMI, which suggested that the ER stress was inhibited. PP1-12 enhanced eIF2a phosphorylation, which could attenuate translation initiation and reduce protein synthesis to allow cells to clear unfolded proteins and recover from ER stress. By eIF2a phosphorylation, cells attempt to adapt to stressors, and this improves cardiac tolerance.25 ER stress is a “double-edged sword,” and the fate of cells, whether it is survival or death, is determined by the degree of the stress. If stress is prolonged and excessive, the protective mechanisms are not sufficient to restore normal ER function, and damaged cells undergo ER stress-induced apoptosis. Caspase-12, which is localized on the ER membrane, can only be activated in ER stress-signaling pathway.26 In our study, activated caspase-12 and increased apoptotic cardiomyocytes in the vehicle group showed that the ER stress apoptotic pathway was involved in the heart after AMI. PP1-12 significantly inhibited the ischemia-induced cleavage of caspase-12 and reduced apoptosis of cardiomyocytes. The Bcl-2 family is one of the most important proteins involved in the regulation of apoptosis. Proteins of this family are initiated by the mitochondrial-dependent or intrinsic pathway to induce cell apoptosis. Recent studies have shown that Bcl-2 family proteins are located at the ER and regulate apoptotic pathways in response to many cellar stresses.27,28 There are some limitations to our study. In vitro, we previously found that the antiapoptotic effect of PP1-12 is more effective than that of salubrinal.11 However, in vivo, although PP1-12 at 1 mg$kg21$d21 could inhibit apoptosis of cardiomyocytes and partly improve heart function, this effect is not as significant as salubrinal at the same concentration. This discrepancy may be due to the different characters of drugs in vivo and in vitro. Additional studies are required to clarify this issue. Although phosphorylation of eIF2a can restore cellular homeostasis, the long-term effect of sustained activation of eIF2a phosphorylation is still unknown. Finally, we speculate that PP1-12 is not only specially fit for ischemic heart diseases, but may also be applicable to other human disorders involved in protein misfolding or unfolding. More research needs to be performed to examine whether other mechanisms are involved in the protective effect of PP1-12.
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CONCLUSIONS In summary, this study preliminarily shows that PP1-12 effectively improves cardiac function and reduces ER stress. The mechanisms responsible for this protective effect could be the result of improving cardiac tolerance, restoring cell homeostasis, and blocking the pathological process. Our findings suggest that PP1-12 and PP1 inhibitor-like small molecules may represent a potential treatment. However, regulation of p-eIF2a in response to ER stress is a complex process, and the effect of these compounds needs to be further verified. REFERENCES
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PP1-12 Improved Heart Function 23. Pallet N, Bouvier N, Bendjallabah A, et al. Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death. Am J Transplant. 2008;8:2283–2296. 24. Zhu Y, Fenik P, Zhan G, et al. Eif-2a protects brainstem motoneurons in a murine model of sleep apnea. J Neurosci. 2008;28:2168–2178. 25. Fullwood MJ, Zhou W, Shenolikar S. Targeting phosphorylation of eukaryotic initiation factor-2alpha to treat human disease. Prog Mol Biol Transl Sci. 2012;106:75–106. 26. Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates ERspecific apoptosis and cytotoxicity by amyloid- beta. Nature. 2000; 403:98–103. 27. Hetz CA. ER stress signaling and the Bcl-2 family of proteins: from adaptation to irreversible cellular damage. Antioxid Redox Signal. 2007;9:2345–2355. 28. Szegezdi E, Macdonald DC, Ní Chonghaile T, et al. Bcl-2 family on guard at the ER. Am J Physiol Cell Physiol. 2009;296:C941–C953.
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