Cardioprotective effect of erythropoietin on sepsis-induced myocardial injury in rats

doi:10.5847/wjem.j.issn.1920-8642.2013.03.011

Abstract

Abstract:

BACKGROUND: Sepsis-induced myocardial injury is one of the major predictors of morbidity and mortality of sepsis. The cytoprotective function of erythropoietin (EPO) has been discovered and extensively studied. However, the cardioprotective effects of EPO on sepsis-induced myocardial injury in the rat sepsis model has not been reported.
METHODS: The rat models of sepsis were produced by cecal ligation and perforation (CLP) surgery. Rats were randomly (random number) assigned to one of three groups (n=8 for each group): sham group, CLP group and EPO group (1000 IU/kg erythropoietin). Arterial blood was withdrawn at 3, 6, 12, and 24 hours after CLP. cTnI, BNP, CK-MB, LDH, AST, TNF-α, IL-6, IL-10, and CRP were tested by the ELISA assay. Changes of hemodynamic parameters were recorded at 3, 6, 12, 24 hours after the surgery. Histological diagnosis was made by hematoxylin and eosin. Flow cytometry was performed to examine cell apoptosis, myocardium mitochondrial inner membrane potential, and NF-κB (p65). Survival rate at 7 days after CLP was recorded.
RESULTS: In the CLP group, myocardial enzyme index and inflammatory index increased at 3, 6, 12 and 24 hours after CLP compared with the sham group, and EPO significantly blocked the increase. Compared with the CLP group, EPO significantly improved LVSP, LV +dp/dt_max, LV -dp/dt_min, and decreased LVEDP at different time. EPO blocked the reduction of mitochondrial transmembrane potential, suppressed the cardiomyocyte apoptosis, inhibited the activation of NF-κB, and reduced the production of proinflmmatory cytokines. No difference in the survival rate at 7 days was observed between the CLP group and the EPO group.
CONCLUSION: Exogenous EPO has cardioprotective effects on sepsis-induced myocardial injury.

Key words: Sepsis, Sepsis-induced myocardial injury, Apoptosis, Chondriosome membrane potential, Nuclear factor κB p65, Erythropoietin, Inflammatory cytokines, Rat

Yan-jun Qin, Xin-liang Zhang, Yue-qing Yu, Xiao-hua Bian, Shi-min Dong. Cardioprotective effect of erythropoietin on sepsis-induced myocardial injury in rats[J]. World Journal of Emergency Medicine, 2013, 4(3): 215-223.

Figures/Tables 10

Table 1

Table 2

Table 3

Table 4

Table 5

Figure 1.

Table 6

Figure 2.

Figure 3.

Figure 4.

References 29

1	Fernandes CJ Jr, Akamine N, Knobel E. Cardiac troponin: a new serum marker of myocardial injury in sepsis. Intensive Care Med 1999; 25:1165-1168.
2	Blanco J, Muriel-Bombin A, Sagredo V, Taboada F, Gandia F, Tamayo L, et al. Incidence, organ dysfunction and mortality in severe sepsis: a Spanish multicentre study. Crit Care 2008; 12:R158. pmid: 19091069
3	Ducrocq N, Kimmoun A, Furmaniuk A, Hekalo Z, Maskali F, Poussier S, et al. Comparison of equipressor doses of norepinephrine, epinephrine, and phenylephrine on septic myocardial dysfunction. Anesthesiology 2012; 116:1083-1091.
4	Fineschi V, Michalodimitrakis M, D'Errico S, Neri M, Pomara C, Riezzo I, et al. Insight into stress-induced cardiomyopathy and sudden cardiac death due to stress. A forensic cardio-pathologist point of view. Forensic Sci Int 2010; 194:1-8.
5	Burniston JG, Ellison GM, Clark WA, Goldspink DF, Tan LB. Relative toxicity of cardiotonic agents: some induce more cardiac and skeletal myocyte apoptosis and necrosis in vivo than others. Cardiovasc Toxicol 2005; 5:355-364. doi: 10.1385/CT:5:4
6	Chen L, Tang H, Liang YB, Chen ZB, Li ZY, Huang ZT, et al. Expressions of SOCS-1 and SOCS-3 in the myocardium of patients with sudden cardiac death. World J Emerg Med 2010; 1:99-103.
7	Fisher JW. Erythropoietin: physiology and pharmacology update. Exp Biol Med (Maywood) 2003; 228:1-14.
8	Rathod DB, Salahudeen AK. Nonerythropoietic properties of erythropoietin: implication for tissue protection. J Investig Med 2011; 59:1083-1085. doi: 10.2310/JIM.0b013e31822cf86e pmid: 22011619
9	Lipsic E, Schoemaker RG, van der Meer P, Voors AA, van Veldhuisen DJ, van Gilst WH. Protective effects of erythropoietin in cardiac ischemia: from bench to bedside. J Am Coll Cardiol 2006; 48:2161-2167.
10	Aoshiba K, Onizawa S, Tsuji T, Nagai A. Therapeutic effects of erythropoietin in murine models of endotoxin shock. Crit Care Med 2009; 37:889-898. pmid: 19237893
11	Singleton KD, Wischmeyer PE. Distance of cecum ligated influences mortality, tumor necrosis factor-alpha and interleukin-6 expression following cecal ligation and puncture in the rat. Eur Surg Res 2003; 35:486-491.
12	Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 2009; 4:31-36. doi: 10.1038/nprot.2008.214 pmid: 19131954
13	Singh NP. A rapid method for the preparation of single-cell suspensions from solid tissues. Cytometry 1998; 31:229-232.
14	Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184:39-51. pmid: 7622868
15	Zydowo MM, Swierczynski J, Nagel G, Wrzolkowa T. The respiration and calcium content of heart mitochondria from rats with vitamin D-induced cardionecrosis. Biochem J 1985; 226:155-161. doi: 10.1042/bj2260155 pmid: 2983684
16	Fernandes CJ, Jr. Akamine N, Knobel E. Myocardial depression in sepsis. Shock 2008; 30 Suppl 1: 14-17.
17	Xing XZ, Wang HJ, Huang CL, Yang QH, Qu SN, Zhang H, et al. Prognosis of patients with shock receiving vasopressors. World J Emerg Med 2013; 4:59-62. pmid: 25215094
18	Silva E, Passos Rda H, Ferri MB, de Figueiredo LF. Sepsis: from bench to bedside. Clinics (Sao Paulo) 2008; 63:109-120.
19	Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell 1996; 87:13-20.
20	Schmitz ML, Baeuerle PA. Multi-step activation of NF-kappa B/Rel transcription factors. Immunobiology 1995; 193:116-127. pmid: 8530134
21	Simmonds RE, Foxwell BM. Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation. Rheumatology (Oxford) 2008; 47:584-590.
22	Cinel I, Dellinger RP. Advances in pathogenesis and management of sepsis. Curr Opin Infect Dis 2007; 20:345-352. pmid: 17609592
23	Su Q, Li L, Liu YC, Zhou Y, Wen WM. Effect of metoprolol on myocardial apoptosis after coronary microembolization in rats. World J Emerg Med 2013; 4:138-143. doi: 10.5847/wjem.j.issn.1920-8642.2013.02.010 pmid: 25215108
24	Ge XH, Zhu GJ, Geng DQ, Zhang ZJ, Liu CF. Erythropoietin attenuates 6-hydroxydopamine-induced apoptosis via glycogen synthase kinase 3b-mediated mitochondrial translocation of Bax in PC12 cells. Neurol Sci 2012; 33:1249-1256.
25	Caetano AM, Vianna Filho PT, Castiglia YM, Golim MA, de Souza AV, de Carvalho LR, et al. Erythropoietin attenuates apoptosis after ischemia-reperfusion-induced renal injury in transiently hyperglycemic Wister rats. Transplant Proc 2011; 43:3618-3621.
26	Brealey D, Singer M. Mitochondrial dysfunction in sepsis. Curr Infect Dis Rep 2003; 5:365-371. doi: 10.1007/s11908-003-0015-9 pmid: 13678565
27	Fink MP. Bench-to-bedside review: Cytopathic hypoxia. Crit Care 2002; 6:491-499. pmid: 12493070
28	Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 2001; 153:1265-1276. pmid: 11402069
29	Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-1312. pmid: 9721092

Parameters	3 h	6 h	12 h	24 h
LVSP (mmHg)
Sham	176.3±4.5	178.5±3.9	175.4±4.0	177.5±4.9
CLP	102.1±5.3^*	67.4±6.1^#	85.6±8.9^#	92.7±7.6^#
EPO	132.2±5.0^*▲	119.1±5.1^*▼	135.4±6.7^*▼	138.3±5.9^*▼
LVEDP (mmHg)
Sham	4.12±0.45	3.98±0.32	4.01±0.47	4.16±0.28
CLP	4.52±0.73	6.93±0.98^#	7.03±1.21^#	7.14±1.23^#
EPO	4.63±0.82	6.49±1.04^#	5.32±0.92^#▼	4.98±0.86^#▼
+dp/dt_max(mmHg)
Sham	6840±121.7	6884±127.5	6900±122.3	6850±121.7
CLP	4002±375.7^#	2821±372.6^#	4366±473.8^#	4496±504.1^#
EPO	5471±180.3^#▼	5277±153.2^#▼	5350±211. 6^#▼	5379±143.9^#▼
-dp/dt_max (mmHg)
Sham	-5737±184.7	-5740±178.6	-5800±180.2	-5772±170.8
CLP	-3821±187.3^#	-1351±151.3^#	-3081±416.1^#	-3321±490.1^#
EPO	-4546±112.1^#▼	-4121±149.5^#▼	-4214±109.2^#▼	-4350.3±173.1^#▼

Parameters	3 h	6 h	12 h	24 h
CRP (mg/L)
Sham	0.80±0.16	0.75±0.20	0.77±0.19	0.84±0.16
CLP	1.26±0.18	2.31±0.33^#	1.89±0.29^#	1.40±0.27
EPO	1.12±0.13	1.86±0.23^#▼	1.45±0.20^#▼	1.17±0.15
TNF-α (ng/L)
Sham	12.92±0.91	13.24±0.77	12.65±0.95	13.07±0.85
CLP	63.69±6.85^#	45.58±3.96^#	41.46±5.04^#	25.99±5.08^#
EPO	53.57±5.48^#▼	33.31±4.21^#▼	29.44±2.68^#▼	17.98±2.13^*▼
IL-6 (ng/L)
Sham	68.71±11.48	62.50±13.22	69.47±10.25	70.03±10.72
CLP	1768.9±195.1^#	958.7±141.6^#	449.9±56.28^#	198.05±58.10^#
EPO	1129.5±151.3^#▼	666.5±191.5^#▼	309.3±65.4^#▼	122.4±34.5^*▼
IL-10 (pg/mL)
Sham	49.90±10.34	48.70±11.55	47.24±10.68	50.29±9.47
CLP	198.6±42.1^#	711.3±84.9^#	528.7±108.7^#	334.2±44.2^#
EPO	297.8±43.2^#▼	1345.6±171.5^#▼	798.8±61.4^#▼	392.3±52.8^#▲

Parameters	3 h	6 h	12 h	24 h
cTnI (ng/L)
Sham	0.47±0.20	0.49±0.12	0.38±0.11	0.46±0.14
CLP	386.1±137.7^*	1184.3±385.4^*	2320.6±447.4^*	1173.5±249.6^*
EPO	344.2±111.9^*	671.4±349.8^*▲	1134.7±247.6^*▼	878.6±184.9^*▲
CK (U/L)
Sham	732±107.5	750±102.7	743±105.8	749±104.2
CLP	4632±638.4^*	2704±415.8^*	8638±1995.4^*	10604±3660.1^*
EPO	2580±941.6^*▼	1638±750.8^*▼	6060±1766.5^*▲	8728±1975.1^*
AST (U/L)
Sham	473±103.3	450±110.2	466±108.3	470±102.9
CLP	582±213.5	1233±429.1^*	1865±349.1^*	2543±598.4^*
EPO	546±127.9	743±107.2^*▼	1199±217.1^*▼	2445±774.8^*
LDH (U/L)
Sham	763±96.2	780±85.7	769±80.8	774±90.2
CLP	1282±357.7^#	1755±446.3^#	2945±744.9^*	4246±771.9^*
EPO	1035±189.5^#	1319±527.9^#	1933±546.3^#▼	4073±552.6^*

Groups	Early apoptosis rate (%)	Late stage apoptosis rate (%)
Sham	0.27±0.31	2.88±0.35
CLP	8.54±1.33^#	23.51±5.67^#
EPO	1.81±0.79^*▼	9.02±2.32^#▼

Groups	Mitochondrial membrane potential
Sham	340.42±25.90
CLP	122.26±23.69^#
EPO	236.25±24.02^*▼