Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat

Larkins NT, Murphy RM, Lamb GD. Absolute amounts and diffusibility of HSP72, HSP25, and alpha B-crystallin in fast-and slow-twitch skeletal muscle fibers of rat. Am J Physiol Cell Physiol 302: C228-C239, 2012. First published October 5, 2011; doi: 10.1152/ajpcell.00266.2011.-Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and alpha B-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only similar to 1.1 and 4.6 mu mol/kg wet wt, respectively, and HSP25 content approximately twofold greater (similar to 3.4 and similar to 8.9 mu mol/kg, respectively). alpha B-crystallin content of EDL muscle was similar to 4.9 mu mol/kg but in SOL muscle was similar to 30-fold higher (similar to 140 mu mol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and alpha B-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas similar to 95% remained tightly bound in fibers from SOL muscles heated to 45 degrees C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo) plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or alpha B-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of alpha B-crystallin, suggesting its importance in normal cellular function without upregulation.