| Abstract: | Adriamycin (ADR, Doxorubicin) effects on actin and other proteins in cultured neonatal rat cardiac myocytes were investigated. Heart cells were exposed to ADR in doses of 10(-8) M to 10(-5) M for 24 hours. Cells were harvested in 2 mM of Tris buffer containing Triton X-100, homogenized and centrifuged in a microfuge. Parallel dishes of cultured cardiac myocytes were washed in buffered saline and were fixed at 4 degrees C in Karnovsky's fixative. The supernatant solutions were dialyzed and then incubated with pancreatic DNAase I to quantify actin by enzyme inhibition. In parallel studies, both cell supernatant solutions and pellets were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the stained polypeptide bands were quantified by densitometry. Results showed that heart cells exposed to 10(-6) M of ADR for 24 hours had unpolymerized actin levels reduced to 7.7 micrograms/10(6) cells (as measured by DNAase I inhibition or by sodium dodecyl sulfate polyacrylamide gel electrophoresis along with densitometry) compared to 11.0 micrograms/10(6) cells in untreated culture heart cells. When ADR concentration was 10(-7) or 10(-8) M, unpolymerized actin levels were similar to the levels of untreated heart cells. Protein content of extract solutions of untreated and ADR-treated myocytes were 1.2 mg/ml and 0.8 mg/ml, respectively. Gel densitometry of electrophoretograms showed actin to account for 12 to 16% of total density of bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Comparative densitometry of ADR-treated cells treated with 10(-6) M of ADR show depolymerized actin to account for 77% of total actin. Ultrastructural results show a large clear cytoplasmic zone of disorganized 12 to 14-nm filaments in cultured myocytes exposed to 10(-6) M ADR. Little change in myocyte ultrastructure was seen at 10(-7) M or 10(-8) M ADR exposure. Data support ADR as a cellular disruptor with toxic effects on cardiac cytoplasmic and contractile proteins and filaments. This ADR effect on heart cells in culture is dose-related. |
