Hypothermia and postconditioning after cardiopulmonary resuscitation reduce cardiac dysfunction by modulating inflammation, apoptosis and remodeling

Abstract

Background: Mild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia.

Methodology/principal findings: Thirty pigs (28-34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38 degrees C, hypothermia at 33 degrees C or hypothermia at 33 degrees C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34+/-7 versus 57+/-12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1beta and IL-6 mRNA up-regulation, and (iv) IL-1beta protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached.

Conclusions/significance: Hypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression.

Figure 1. Experimental time line.

Thirty pigs were subjected to cardiac arrest following left anterior descending (LAD) coronary artery ischemia. After 7 minutes of ventricular fibrillation (VF), pigs were resuscitated (CPR). After successful return of spontaneous circulation (ROSC; n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized either to normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. Five animals were sham operated. cTnT indicates cardiac troponin T; TEE, transesophageal echocardiography.

Figure 2. Myocardial damage.

A) Evans blue (EB) staining to determine the area at risk (AAR; three images in the top row, corresponding to pigs subjected to normothermia (NT), hypothermia (HT), or hypothermia and sevoflurane (HT+SEV), respectively). AAR was subjected to triphenyltetrazolium chloride (TTC) staining to determine infarct size (corresponding images in the lower row). B) AAR expressed as percentage of left ventricle (LV). Infarct size expressed as percentage of the AAR. Data are expressed as dot plots and mean. C) Serum cardiac troponin T (cTnT) release at baseline (BL), after return of spontaneous circulation (ROSC) and 1, 3, 5, 7, 13 and 24 hours after ROSC. Data are expressed as mean ± SD. §p<0.05, #p<0.01 vs. NT.

Figure 3. Transesophageal echocardiography.

A) Left ventricular ejection fraction, B) fractional shortening, C) E/A ratio and D) myocardial performance index at baseline (BL), after left anterior descending (LAD) coronary artery occlusion, and 24 hours after return of spontaneous circulation in the normothermia (NT), hypothermia (HT) and hypothermia and sevoflurane (HT+SEV) group. *p<0.05, †p<0.01, ‡p<0.001 vs. baseline; §p<0.05, #p<0.01 vs. NT.

Figure 4. Myocardial infiltration of immune cells and apoptosis.

A) Representative hematoxylin and eosin staining of the ischemic myocardium (400×magnification) corresponding to pigs subjected to normothermia (NT), hypothermia (HT), hypothermia and sevoflurane (HT+SEV) or sham control are shown. Myocardial samples from all animals used in the study were processed for histopathological analysis. B) Quantification of myeloperoxidase (MPO) activity in myocardial tissue for area at risk (AAR) and area at no risk (AANR). C, D) Myocardial protein expression of procaspase-3 and β-actin were determined by western blotting from all animals used in the study. Representative blots of pigs subjected to NT, HT, HT+SEV, and sham control are shown. E) Representative anti-caspase 3 immunfluorescence staining of the AAR (400×magnification). Data are expressed as dot plots and mean. *p<0.05, †p<0.01 vs. Sham. §p<0.05 vs. NT.

Figure 5. Myocardial cytokine expression.

Transcript levels of the cardiac cytokines interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α and intercellular adhesion molecule (ICAM)-1 were determined by quantitative RT-PCR (A, B), and protein concentrations of IL-1β were determined by a swine specific enzyme-linked-immunosorbent assay (C). AAR indicates area at risk; AANR, area at no risk; NT, normothermia; HT, hypothermia; HT+SEV, hypothermia combined with sevoflurane. Data are expressed as mean±SD (A, B) and dot plots/mean (C), respectively. *p<0.05, †p<0.01 vs. Sham. §p<0.05 vs. NT.

Figure 6. Effects of hypothermia and pharmacological postconditioning on MMP-9 and MMP-2 activity.

Activity of matrix metalloproteinase (MMP)-9 and MMP-2 was evaluated by gelatine zymography employing ischemic (AAR) and non-ischemic (AANR) myocardial tissue of the same animal. A) One representative gel out of three is shown. White bands represent areas of enzymatic activity of MMP-9 and MMP-2, respectively. Mean intensity of the zymography bands was normalized to sham control (dashed line) separately for MMP-9 (B) and MMP-2 (C). AAR indicates area at risk; AANR, area at no risk; NT, normothermia; HT, hypothermia; HT+SEV, hypothermia combined with sevoflurane. Data are expressed as mean±SD. All experiments were repeated three times. *p<0.001 vs. Sham, §p<0.05, #p<0.001 vs. NT.