Michihiko Maeiwa

Mechanism of postmortem autolysis of skeletal muscle

Male Wistar rats were treated with the carboxyl, thiol, and serine protease inhibitors, pepstatin, Ep-475[L-trans-epoxysuccinyl-leucylamide(3-methyl) butane; E-64-c], and chymostatin. Then the femoral muscles of these rats and control animals were used for preparation of myofibril proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the degradation of these myofibril proteins with time (day) after death. The protease activities of the muscle were also measured. Tropomyosin was degraded most rapidly, followed by the heavy chain of myosin, alpha-actinin, and light chains of myosin (L1 and L2). Actin and troponin-T were degraded slowly, still remaining unchanged 2 weeks after death. The degradation of protein was not inhibited by pepstatin but was inhibited strongly by Ep-475 and very strongly by chymostatin. Chymostatin inhibited degradation of all components except alpha-actinin more strongly than Ep-475. Data on enzyme activities were consistent with these findings. These results suggest that after death the components of myofibrils are degraded with various proteases at various rates depending on their properties or their structure and that the proteases involved in the degradation show some specificity.

Comparison of postmortem autolysis in cardiac and skeletal muscle.

To understand the mechanism in postmortem autolysis better, processes in the postmortem degradation of myofibril proteins in the presence of protease inhibitors were studied. Male Wistar rats were given injections of the carboxyl-, thiol-, and serine-protease inhibitors, pepstatin, Ep-475[L-transepoxysuccinyl-leucylamide(3-methyl) butane; E-64-C], and chymostatin, via the femoral vein. Control rats were similarly treated with saline. Then, myofibril proteins were isolated from their cardiac and femoral muscles and from those of control animals at various times after death, and degradation of these myofibril proteins with time was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In cardiac muscle, alpha-actinin was degraded rapidly, followed by the heavy chain of myosin and light chain of myosin (L2). Actin and the light chain of myosin (L1) were degraded slowly. the degradations of the heavy chain of myosin, alpha-actinin, tropomyosin and L2 after 14 days were not inhibited by pepstatin, but were inhibited by Ep-475 and chymostatin. In skeletal muscle, L1 and L2 were degraded rapidly, followed by the heavy chain of myosin and alpha-actinin. Actin was degraded slowly and was still unchanged 2 weeks after death. The degradations of protein components were inhibited by pepstatin, Ep-475 and chymostatin. These results indicated that after death the components of myofibrils are degraded by various proteases at various rates depending on their properties or structures. This degradation is fundamentally the same in cardiac and skeletal muscles, but inhibitors have somewhat different effects on the postmortem degradation processes after death in the two types of muscle.

Experimental studies on death by fire in automobiles and exhaust gas poisoning

Studies were made on the acid-base balance, blood gases carbon monoxide (CO), cyanide sulfur dioxide concentrations in the blood of albino rabbits that died from automobile exhaust gas poisoning (group I) or fires in cares (complete combustion, group II; incomplete combustion, group III). In group I, the temperature and CO concentration increased gradually to 35°C and 5.2% in 70 min. The animals died after 9 min, when the values were 20°C and 5.2%, respectively. In group II the animals died after 9 min, when the values were 55°C and 1.95%, respectively. In group III, the temperature was very high (870°C), but the CO concentration was not (0.6–1.3%) after 4 min. The animals died after 5 min.In all experimental groups, marked acidosis and hypoxemia were seen, but the CO2 tension (PCO2) was high, in contrast to previous studies on pure CO poisoning. In group I, the level of carboxyhemoglobin (CO-Hb) was significantly higher (91.2 ± 3.4% in arterial blood, 87.5 ± 8.1% in venous blood; p < 0.01) than in groups II and III. Although the O2 tensions of venous and arterial blood (PvO2, PaO2) were very low, that of arterial blood was higher, suggesting that O2 was still being utilized in the tissues at the time of death. In group II, CO-Hb was high (57.7 ± 16.0% in arterial blood, 61.2 ± 20.6% in venous blood) and the acid-base balance indicated marked acidosis. In group III, the CO-Hb, Pco2 and cyanide levels in the blood were very high. CO and CO2 might be produced by either type of combustion cyanide by a pyrolytic reaction of nitrogenous material. The high Pco2 value suggested respiratory acidosis induced by inhibition of the central respiratory center by CO and/or cyanide. PaO2 and PvO2 were similar, suggesting that intracellular respiration was blocked by cyanide, the level of which was significantly higher than in group II (p < 0.01). Sulfur dioxide was not detected in any of the groups.It is concluded that in group II and more especially group I, CO may be the main lethal factor and that marked acidosis was induced by asphyxiation owing to CO and deficient O2. In group III, the primary cause of death may have been CO poisoning, with consequent inhibition of the cytochrome system by cyanide and O2 deficiency.