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Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte - PubMed

Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte

Lingjia Qian et al. Cell Stress Chaperones. 2004 Autumn.

Abstract

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.

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Figures

Fig 1.
Fig 1.

Cardiomyocyte apoptosis in heat-stressed rats. (A) Electron micrographs of cardiomyocyte apoptosis induced by heat stress. The rats were exposed to heat, and the myocardium was selected and examined by electron microscopy (JEM 1200 EXIT). (a) The characteristic apoptotic condensation and surrounding distribution of nuclear chromatin in the cardiomyocyte of heat-stressed rats. (b) The alterations of the mitochondria in cardiomyocytes of heat-stressed rats including reduction of the number of cristae membranes, disruption of the surrounding membranes, and swelling of the organelles. (B). Agarose gel electrophoresis analysis of deoxyribonucleic acid fragmentation from cardiomyocytes of heat-stressed rats

Fig 2.
Fig 2.

Time course of apoptosis and necrosis of cardiomyocytes under heat stress. Cardiomyocytes were isolated from 3-day-old neonatal Wistar rats and exposed to 37°C, 39°C, 41°C, or 43°C heat for 40 minutes. The apoptosis rate was detected with flow cytometry, and necrosis rate was assessed with trypan blue staining method at different times after heat stress. Each point represents the mean ± SEM of 6 separate experiments, *P < 0.05, **P < 0.01 vs 37°C group

Fig 3.
Fig 3.

Effect of heat stress H+-ATPase synthesis activity of mitochondria in heat-stressed cardiomyocytes. Cardiomyocytes were isolated from 3-day-old neonatal Wistar rats and exposed to 37°C, 39°C, 41°C, or 43°C heat for 40 minutes. Mitochondria were isolated from the cardiomyocyte, and mitochondrial H+-ATPase synthesis activity was measured by fluorescence bioluminescence. The values are the mean ± SEM, n = 8, *P < 0.05, **P < 0.01 vs 37°C group

Fig 4.
Fig 4.

Changes of Ca2+ metabolism in cardiomyocytes of heat-stressed rats. The rats were exposed to heat of different temperatures. The cardiomyocytes and the mitochondria were isolated from myocardium immediately after heat stress. Ca2+-ATPase activity of mitochondria was determined using bioluminescent method, Ca2+ content in mitochondria was assessed using inductively coupled plasma atomic emission spectrometry (ICP) and intracellular Ca2+ concentration of cardiomyocyte was measured by Fura-2 fluorescence method. The values given are the mean ± SEM of 6 separate experiments, *P < 0.05, **P < 0.01 vs 37°C group. (A) Ca2+-ATPase activity of mitochondria in cardiomyocytes of heat-stressed rats. (B) Ca2+ content in mitochondria in cardiomyocytes of heat-stressed rats. (C) Intracellular Ca2+ concentration in cardiomyocyte of heat-stressed rats

Fig 5.
Fig 5.

Influence of heat stress on the mitochondrial membrane permeability transition (MPT) in cardiomyocyte. (A) The changes of mitochondrial MPT of cardiomyocyte under the special environmental conditioning induced by heat stress. Isolated mitochondrial MPT was measured with spectrophotometer after exposure to Ca2+ concentration up to 200 μmol/L, 100 μmol/L H2O2, or to acidifying mitochondrial surroundings (pH 6.5) and given ruthenium red, SOD, or correcting acid-base imbalance in reactive system (n = 3). (B) The changes of A540 nm of mitochondria in cardiomyocytes from heat-stressed rats. The swelling of mitochondrial suspension after permeability transition pore opening was assayed by measuring the change of turbidity absorbance at 540 nm before and after 150 nm Ca2+. The values are the mean ± SEM, n = 6, *P < 0.05, **P < 0.01 vs 37°C group. (C) The influence of heat stress on opening of mitochondrial MPT pore in cardiomyocytes of heat-stressed rats. The analysis was started by the addition of 1 mg mitochondria with 0.2 μM Rhodamine 123. After incubation at 25°C for 5 minutes, the fluorescence intensity of the reaction medium was determined immediately using fluorescence spectrophotometer. CaCl2 (150 μM) was added after 1 minute, and the change of fluorescence intensity in 15 minutes was recorded. The change of membrane potential after Ca2+ addition reflected the MPT original state. The values are the mean ± SEM, n = 6, *P < 0.05, **P < 0.01 vs 37°C group

Fig 6.
Fig 6.

The effect of heat stress on the cytochrome c (cyto c) release from mitochondria and caspase-3 activity in cardiomyocytes. (A) The changes of cyto c distribution in cardiomyocytes of heat-stressed rats. Proteins in the supernatant (15 μL) of mitochondria preparation and from the S-100 fraction of cardiomyocytes were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10–15%). Immunoblots for the determination of cyto c release were performed using a specific cyto c monoclonal antibody, n = 4. (B) The effect of heat stress on the caspase-3 activity in cardiomyocytes. The 10 mg lysate protein of cardiomyocytes was incubated with 5 mM Ac-DEVD-MCA at 37°C for 5 minutes. The release of 7-amino-4-metrylcouumarin was measured by a spectrofluorometer (Hitachi F-2000) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The values are the mean ± SEM, n = 8, *P < 0.05, **P < 0.01 vs 37°C group

Fig 7.
Fig 7.

The protective effects of bcl-2 transfection on the mitochondria of cardiomyocytes under heat stress. (A) Immunohistochemical expression of the cardiomyocytes transfected with the plasmid PCI-neo without bcl-2 (-bcl-2 transfected cardiomyocytes) and the cardiomyocytes transfected with plasmid PCI-neo containing bcl-2 complementary deoxyribonucleic acid (cDNA) (bcl-2 transfected cardiomyocytes) (200×). I. Lymphatic tissue as positive control. II. The cardiomyocytes transfected with plasmid PCI-neo containing bcl-2 cDNA. III. The cardiomyocytes transfected with the plasmid PCI-neo without bcl-2. Cells expressing Bcl-2 show a yellow immunostaining. (B) Effect of bcl-2 transfection on H+-adenosine triphosphatase synthesis activity of mitochondria in heat-stressed cardiomyocytes. The values are the mean ± SEM, n = 8. *P < 0.05, **P < 0.01 vs -bcl-2 transfected group, #P < 0.05, ##P < 0.01 vs 37°C group. (C) Effect of bcl-2 transfection on the cytochrome c release from mitochondria. The values are the mean ± SEM, n = 5. *P < 0.05, **P < 0.01 vs -bcl-2 transfected group #P < 0.05, ##P < 0.01 vs 37°C group. (D) The effect of Bcl-2 overexpression on caspase-3 activity of heat-stressed cardiomyocytes. The values are the mean± SEM, n = 8. *P < 0.05, **P < 0.01 vs -bcl-2 transfected group, #P < 0.05, ##P < 0.01 vs 37°C group

Fig 8.
Fig 8.

The effect of Bcl-2 overexpression on apoptosis and necrosis rate of heat-stressed cardiomyocytes. (A) The effect of Bcl-2 overexpression on apoptosis rate of heat-stressed cardiomyocytes. The values are the mean ± SEM, n = 8. *P < 0.05, **P < 0.01 vs -bcl-2 transfected group, #P < 0.05, ##P < 0.01 vs 37°C group. The effect of Bcl-2 overexpression on necrosis rate of heat-stressed cardiomyocytes. The values are the mean ± SEM, n = 8. *P < 0.05, **P < 0.01 vs -bcl-2 transfected group, #P < 0.05, ##P < 0.01 vs 37°C group

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