Identification and characterization of the heat shock response and an inducible Hsp70 gene in bovine skeletal muscle.

Abstract

Experiments were conducted to quantify Hsp70 proteins and mRNAs in non-stressed bovine tissues, skeletal muscle undergoing heat shock and during cardiac muscle anoxia. Antibodies that show specificity against the inducible Hsp70 were developed and used in immunofluorescence and quantitative ELISA experiments. Tissue surveys using the ELISA assay demonstrated Hsp70 to be present in all tissues, with skeletal muscle about three fold higher than the other tissues at about 9 ng/μg. Immunofluorescence results in non-stressed ovine skeletal muscle showed Hsp70 to be mostly located in the sarcoplasm and a small but specific staining pattern suggestive of sarcoplasmic reticulum (SR) staining was observed. Skeletal muscle fractionation studies demonstrated about 1% of Hsp70 to be localized in the SR. Skeletal muscle heat shock and cardiac muscle anoxia determined that Hsp70 could be elevated by either of these insults, with heat shock increasing Hsp70 levels about two fold. Temporary cardiac anoxia experiments demonstrated that Hsp70 could be partially increased by initial handling of the heart, but after 3 hours following the release of a complete aortic clamp, Hsp70 levels had increased about four fold over basal levels. A cDNA for bovine skeletal muscle Hsp70 was isolated and used for RNAse protection assays and expression experiments. Results from RNAse protection assays demonstrated the presence of the complete cDNA in all tissues examined with a pattern of expression between tissues similar to Hsp70 protein profiles. Skeletal muscle contained the highest level of Hsp70 mRNA, about three fold higher than brain. Skeletal muscle heat shock experiments demonstrated about a four fold increase in the level of Hsp70 mRNA from 0.4 pg/μg total RNA to about 1.65 pg/μg total RNA. Bacterial expression and purification of the cDNA product demonstrated the recombinant protein to have identical mobility in polyacrylamide gels containing SDS and crossreactivity to Hsp70 antibodies as the inducible Hsp70. Two dimensional gel electrophoresis demonstrated that the recombinant protein encodes a minor isoform in skeletal muscle, and carbamylation of the recombinant protein showed the carbamylated protein to form a train similar to bovine skeletal muscle heat shock protein.