Myocardial autophagy after severe burn in rats

Abstract

Background: Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn.

Methods: Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function.

Results: Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function.

Conclusion: Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction.

Figure 1. Myocardial LC3 and Beclin 1 expression following severe burns in rats.

Immunoblotting was performed on proteins extracted from myocardial tissue of rats exposed to 3^rd degree burns over 30% of the total body surface area or sham controls. A. As an indicator of autophagy, LC3 II/LC3 I ratio rose from 1 h to 12 h post-burn. The total level of LC3 protein also increased, beginning at 3 h and continuing to rise through 6 and 12 h post-burn. The sample size was n = 6 per group for sham and each of the time points, * p<0.05 vs sham. B. Beclin 1 expression was robustly expressed at 1 h post-burn and continued to follow an upward tendency. The sample size was n = 5 for each group, * p<0.05 vs sham.

Figure 2. Post-burn cardiac function in vivo and ex vivo in a Langendorff preparation.

Rats subjected to 3rd degree burns affecting 30% of total body surface area were established as a burn model. A. These cardiac mechanical parameters were obtained with an intubation method from the carotid artery of the rats under anesthesia. The left ventricular systolic pressure (LVSP) and mean arterial pressure (MAP) decreased from 1 h to 3 h post-burn and continued to decline in the following time points. The left ventricular end-diastolic pressure (LVEDP) rose from 6 h to 12 h post-burn. The sample size was n = 8 for each group, * p<0.05 vs sham. B. Reflecting cardiac contraction and relaxation, the maximal rates of left ventricular systolic pressure increase and diastolic pressure decrease (i.e., +dp/dt max and -dp/dt max, respectively) fell from 1 h to 12 h post-burn. The samples size was n = 8 for each group, * p<0.05 vs sham. C and D. In isolated hearts perfused with a Langendorff apparatus, the recorded cardiac mechanical parameters LVSP and ± dp/dt max decreased from 1 h to 12 h post-burn, similar to the changes seen in vivo. The sample size was n = 5 for each group, * p<0.05 vs sham.

Figure 3. Myocardial immunofluorescence of autophagic, apoptotic, and oncotic cell death. A.

Representative immunofluorescence images of myocardium with different staining targets in rats with 3rd degree burns over 30% of total body suface area. As indicated by the white arrows, cardiomyocytes with ubiquitin accumulation were mostly loss of nuclei and occupied partially by the positive staining. The TUNEL staining co-localized with DAPI staining in nuclei. The complement membrane attack complex C5b9 staining frequently appeared in the myocyte periphery and showed progression toward the cell center. B. Quantification of autophagic (ubiquitin positive), apoptotic (TUNEL positive), and oncotic (C5b9 positive) cardiomycyte death showed that autophagic cell death could be found at 3 h post-burn, while the other two types of cell death occurred later. All three types of cell death increased further at 6 and 12 h post-burn. The sample size was n = 8 for each group, * p<0.05 vs the 1 h group.

Figure 4. Effects of autophagic regulation on cardiac function.

The hearts were isolated from rats with 3^rd degree burns over 30% of total body surface area at 6 h post-burn. Continuous K-H buffer perfusion without medication was used as a negative control. A and B. The left ventricular systolic pressure (LVSP) and the maximal rate of the rise/drop of left ventricular pressure (± dp/dt max) decreased in the hearts perfused with the autophagy activator rapamycin. While the ± dp/dt max increased with treatment of the autophagy inhibitor, 3-methyladenine (3-MA). The sample size was n = 5 for each group, * p<0.05 vs control. C. Representative immunofluorescence images of myocardial autophagic cell death (ubiquitin positive, red) in isolated hearts perfused with rapamycin, 3-MA, or non-medication (control). D. Quantitation of autophagic cell death showed that autophagy increased in the rapamycin treated group and decreased in the 3-MA treated group. The sample size was n = 5 for each group, * p<0.05 vs control. E. Immunoblotting results confirmed the activation or inhibition effects on autophagy by rapamycin and 3-MA, respectively. The sample size was n = 5 for each group, * p<0.05 vs control.

Figure 5. Effect of enalaprilat, losartan, or DPI on autophagy and cardiac function.

The hearts were isolated from rats subjected to 3^rd degree burns over 30% of total body surface area at 6 h post-burn. Continuous K-H buffer perfusion without medication was used as a negative control. A and B. The left ventricular systolic pressure (LVSP) and the maximal rate of the rise/drop of left ventricular pressure (± dp/dt max) increased in isolated hearts perfused with enalaprilat, losartan, or DPI. The sample size was n = 5 for each group, * p<0.05 vs control. C. Representative immunofluorescence images of myocardial autophagic cell death (ubiquitin positive, red) in isolated hearts perfused with enalaprilat, losartan, DPI or non-medication (control). D. Quantitation of autophagic cell death showed that it decreased in the hearts medicated with enalaprilat, losartan, or DPI. The sample size was n = 5 for each group, * p<0.05 vs control. E. Immunoblotting results confirmed the inhibitory effects on autophagy by enalaprilat, losartan, or DPI treatment. The sample size was n = 5 for each group, * p<0.05 vs control.