Remote Ischemic Perconditioning Ameliorates Myocardial Ischemia and Reperfusion-Induced Coronary Endothelial Dysfunction and Aortic Stiffness in Rats

Figure 1.. Experimental protocol. Rats were allocated to (1) Sham group, in which rats underwent sham operation without any intervention; (2) MIR group, in which rats underwent 30 minutes LAD ischemia followed by 3 days or 4 weeks reperfusion with no conditioning therapy; (3) remote ischemic perconditioning (RIPerc) group, in which 3 cycles of 5 minutes of IR of a hindlimb performed during myocardial ischemia. Cardiac and vessel segments (septal artery and aorta) were collected 3 days (short-term) or 4 weeks (long-term) after reperfusion, respectively. Cardiac function was assessed by transthoracic echocardiography 4 weeks after LAD occlusion. To measure the levels of oxidative stress, plasma MDA was measured by high performance liquid chromatography. LAD indicates left anterior descending artery; MIR, myocardial ischemia/reperfusion; MDA, malondialdehyde.

Figure 2.. Septal artery vascular function following myocardial ischemia and reperfusion. Representative pictures about the septal coronary artery preparation without (A) and with (B) myocardial ischemia and reperfusion in rat hearts. Graph showing (C) endothelium-dependent relaxation in response to ACh and (D) endothelium independent relaxation induced by sodium nitroprusside (SNP) in septal artery rings from wt and Sham, MIR and RIPerc rats. Segments were collected 3 days after the induction of myocardial ischemia. Data are expressed as mean ± SEM, n = 4-6/group, *P < 0.05 and *P < 0.01 Sham vs MIR; # P < 0.05 MIR vs RIPerc using two-way ANOVA. Ach indicates acetylcholine; SNP, sodium nitroprusside (SNP); Ao, aorta; RV, right ventricular; LV, left ventricular; MIR, myocardial ischemia/reperfusion; SA, septal artery. White circle represent Sham group, black circles represent MIR group, and gray circles represent RIPerc group.

Figure 3.. Expression of inflammatory cytokines, P-selectin and eNOS in infarcted and non-infarcted cardiac tissue. RT-qPCR data of LV infarcted and non-infarcted tissue samples from Sham, MIR, and RIPerc rats. Samples were collected 3 days after myocardial infarction. Upper panel showing the mRNA expression MCP-1 (A), TNF-α (B), P-selectin (C), IL-6 (D), and eNOS (E) in infarcted cardiac tissue samples. Lower panel showing the mRNA expression MCP-1 (F), TNF-α (G), P-selectin (H), IL-6 (I), and eNOS (J) in non-infarcted cardiac tissue samples. Data are expressed as mean ± SEM, n = 5-6/group; *P < 0.05, **P < 0.01, ***P < 0.001 using one-way ANOVA. MCP-1 indicates monocyte chemoattractant protein 1; TNF-alpha, tumor necrosis factor alpha; IL-6, Interleukin 6; eNOS, endothelial nitric oxide synthase; MIR, myocardial ischemia/reperfusion; RIPerc, remote ischemic perconditioning.

Figure 4.. Plasma levels of TNF-α 3 days after myocardial reperfusion and the effect of TNF-α on septal artery vascular function. (A) Plasma levels of TNF-α in MIR and RIPerc group 3 days after the initiation of reperfusion. Graph showing the reactivity of septal artery segments to (B) ACh in the absence or presence of rh TNF-α for (1000 pg/ml; 2 hours incubation period) and to (C) SNP. Data are expressed as mean ± SEM; n = 5-6/group; *P < 0.05, using unpaired Student’s t-test (A) and two-way ANOVA (B and C), respectively. Ach indicates acetylcholine; TNF-α, tumor necrosis factor alpha; MIR, myocardial ischemia/reperfusion; RIPerc, remote ischemic perconditioning. Dotted circle represent vehicle group (baseline); black circles represent vehicle treatment group, white circle represent rh TNF-α group (baseline); and grey represent rh TNF-α (2 hours incubation) treated group.

Figure 5.. Aortic vascular function and stiffness and levels of malondialdehyde in plasma. Vascular reactivity and aortic stiffness were assessed by using wire myograph in aorta segments were taken 4 weeks after myocardial infarction. Graph showing (A) endothelium-dependent relaxation in response to ACh and (B) endothelium-independent vasorelaxation by sodium nitroprusside (SNP) in aortic rings from wt and Sham, MIR and RIPerc rats. (C) Aortic stiffness was assessed by using wire myograph and the passive property of vessel segments was used to measure the aortic stiffness. (D) Levels of MDA in plasma in Sham, MIR and RIPerc group 4 weeks after myocardial infarction. Data are expressed as mean ± SEM, n = 4-7/group, *P < 0.05 Sham vs MIR, and ***P < 0.001 Sham vs MIR and RIPerc, using one-way ANOVA and two-way ANOVA, respectively. Ach indicates acetylcholine; MDA, malondialdehyde; MIR, myocardial ischemia/reperfusion; RIPerc, remote ischemic perconditioning. White circle represent Sham group; black circle represent MIR group and grey circle represent RIPerc group.

Figure 6.. Left ventricular function and morphology. RIPerc significantly improves LVEF, FS and LV dilation. Graphs showing (A) left ventricular ejection fraction, (B) fractional shortening, (C) left ventricular end-systolic, and (D) end-diastolic diameter following 4 weeks after myocardial infarction. Data are expressed as mean ± SEM, n = 4-6/group, *P < 0.05, **P < 0.001, and ***P < 0.0001, using one-way ANOVA. LVEF indicates left ventricular ejection fraction; FS, fractional shortening; LVESD, ventricular end-systolic diameter; LVEDD, ventricular end-diastolic diameter; MIR, myocardial ischemia/reperfusion; RIPerc, remote ischemic perconditioning.