Mechanical Stretch-Induced Protection against Myocardial Ischemia-Reperfusion Injury Involves AMP-Activated Protein Kinase

Jia Hao; Hun-Sik Kim; Woong Choi; Tae Sun Ha; Hee-Yul Ahn; Chan-Hyung Kim

doi:10.4196/kjpp.2010.14.1.1

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Hao, Kim, Choi, Ha, Ahn, and Kim: Mechanical Stretch-Induced Protection against Myocardial Ischemia-Reperfusion Injury Involves AMP-Activated Protein Kinase

Original Article

Korean J Physiol Pharmacol 2010;14(1):1-9.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.1.1

Mechanical Stretch-Induced Protection against Myocardial Ischemia-Reperfusion Injury Involves AMP-Activated Protein Kinase

Jia Hao¹, Hun-Sik Kim², Woong Choi², Tae Sun Ha³, Hee-Yul Ahn², Chan-Hyung Kim^2,

¹Institute of Biomedical and Pharmaceutical Technology (IBPT), Fuzhou University, Fuzhou, 350002, PR China

²Department of Pharmacology, College of Medicine, BK21 Chungbuk Biomedical Science Center, School of Medicine, Chungbuk National University, Cheongju 361-763, Korea

³Department of Pediatrics, College of Medicine, BK21 Chungbuk Biomedical Science Center, School of Medicine, Chungbuk National University, Cheongju 361-763, Korea

Corresponding to: Chan-Hyung Kim, Department of Pharmacology, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea. (Tel) 82-43-261-2868, (Fax) 82-43-272-1603, (E-mail) kch@chungbuk.ac.kr

Received 1 December 2009 Revised 31 December 2009 Accepted 18 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

AMP-activated protein kinase (AMPK) protects various tissues and cells from ischemic insults and is activated by many stimuli including mechanical stretch. Therefore, this study investigated if the activation of AMPK is involved in stretch-induced cardioprotection (SIC). Intraventricular balloon and aorto-caval shunt (ACS) were used to stretch rat hearts ex vivo and in vivo, respectively. Stretch preconditioning reduced myocardial infarct induced by ischemia-reperfusion (I/R) and improved post-ischemic functional recovery. Phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase (ACC) were increased by mechanical stretch and ACC phosphorylation was completely blocked by the AMPK inhibitor, Compound C. AMPK activator (AICAR) mimicked SIC. Gadolinium, a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of AMPK and ACC, whereas diltiazem, a specific L-type calcium channel blocker, did not affect AMPK activation. Furthermore, SIC was abrogated by Compound C and gadolinium. The in vivo stretch induced by ACS increased AMPK activation and reduced myocardial infarct. These findings indicate that stretch preconditioning can induce the cardioprotection against I/R injury, and activation of AMPK plays an important role in SIC, which might be mediated by SACs.

Keywords: AMP-activated protein kinase, Stretch, Cardioprotection, Ischemia-reperfusion

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Fig. 1.

Protocols for each experimental group, showing the components and the time course of various treatments. All the hearts underwent 30 minutes sustained ischemia followed by 1 hour reperfusion. Prior to the sustained ischemia, the hearts were assigned to each experimental group with specific treatment; no treatment, three cycles of a 5 minutes ischemia, 5 minutes stretch, 10 minutes AICAR treatment, CC with 5 minutes stretch, and Gd³⁺ with 5 minutes stretch. I/R Con, ischemia-reperfusion control; SPC, stretch preconditioning; IPC, ischemia preconditioning; AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; CC, compound C; Gd³⁺, gadolinium.

Fig. 2.

Effects of stretch preconditioning in ex vivo hearts. (A) After each experimental protocol, slices of heart were stained with TTC and (B) normalized infarct size was calculated and compared. There was no significant difference among IPC, SPC and AICAR group. All data are means±SEM of N≥6 in each experimental group. ^∗p<0.05 compared with I/R Con group, ^#p<0.05 compared with SPC group. I/R Con, ischemia-reperfusion control; SPC, stretch preconditioning; IPC, ischemia preconditioning; AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; CC, compound C; Gd³⁺, gadolinium. (C) Functional recovery of ex vivo stretched hearts. During post-ischemic period, cardiac function was evaluated in terms of 4 parameters; cardiac functional recovery, Left ventricular end-diastolic pressure (LVEDP), heart rate, and coronary blood flow. The changes of 4 parameters were traced during 60 minutes post-ischemia-reperfusion period. There was no significant difference among IPC, SPC and AICAR group. All data are means±SEM of N≥6 in each experimental group. , ischemiareperfusion control; ❍, ischemic preconditioning; ▾, stretch preconditioning; ∇, AICAR; ▪, stretch+Compound C; ☐, stretch + gadolinium.

Fig. 3.

The phosphorylation of AMPK and ACC in the ex vivo hearts All the hearts underwent 30 minutes sustained ischemia. (A) The phosphorylation of AMPK and ACC was analyzed with immunoblot and (B, C) semi-quantitative densitometry. The phosphorylation level before and after the ischemia with various pretreatment was compared. (D) Effects of gadolinium and diltiazem on AMPK and ACC phosphorylation induced by SPC. Hearts were treated with gadolinium (10 μM) or diltiazem (1, 3, and 10 μM) during SPC. Results are representative from 6 independent experiments and means±SEM. ^∗p< 0.05 compared with I/R Con group. ^#p<0.05 compared with SPC group. N, normal control; I/R Con, ischemiareperfusion control; SPC, stretch preconditioning; CC, compound C; Gd³⁺, gadolinium.

Fig. 4.

Phosphorylation of AMPK and ACC after aorto-caval shunt. (A) Left ventricular wall stress caused by aorto-caval shunt (ACS). Central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP) were monitored after ACS. ^∗p<0.05, N=6. (B) After ACS was fitted to the rats, phosphorylation of AMPK and ACC was analyzed at various time points with immunoblot and (C, D) semi-quantitative densitometry. Results are representative from 6 independent experiments. ^∗p<0.05 compared with I/R Con group. I/R C, ischemia-reperfusion control; ACS, aorto-caval shunt.

Fig. 5.

Effects of stretch preconditioning on the infarct size and the functional recovery of in vivo rat hearts. (A) Infarct size was measured using TTC staining after 30 minutes ischemia and 1 hour reperfusion and (B) normalized infarct size was calculated and compared. All data are means±SEM of N≥6 in each experimental group. ^∗p<0.05 compared with I/R Con group. I/R Con, ischemia-reperfusion control; ACS Sham, sham-operated hearts; ACS, aorto-caval shunt. (C) Functional recovery of in vivo stretched rat hearts. Cardiac function was evaluated in terms of 4 parameters; cardiac functional recovery, Left ventricular end-diastolic pressure (LVEDP), heart rate, and coronary blood flow. The changes of 4 parameters were traced during 60 minutes post-ischemic reperfusion period. All data are means±SEM of N≥6 in each experimental group. ^∗p<0.05 compared with control group. , ischemia-reperfusion control; ❍, Sham; ▾, 10 minutes ACS; ∇, 20 minutes ACS; ACS, aorto-caval shunt.

Table 1.

Lactate concentration in the langendorff perfusate of ex vivo hearts and in the tissue of in vivo hearts

Perfusate lactate (mol/min/g wet wt)			Tissue lactate (μmol/g wet wt)
Normal control		Normal control
0 minute	1.87±0.41
30 minutes	3.59±0.44		0.475±0.001
60 minutes	3.57±0.72
Stretch		ACS
0 minute	1.91±0.23	0 minute	0.450±0.001
30 minutes	2.35±0.31	30 minutes	0.534±0.003
60 minutes	3.33±0.65	60 minutes	0.444±0.002
Ischemia		Ischemia
10 minutes	11.63±0.52	10 minutes	3.674±0.232

Each value represents the means±SEM for six to eight hearts.

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