An original method to induce heat stress was used to clarify the time course of changes in heat shock proteins (HSPs) in rat skeletal muscles during recovery after a single bout of heat stress. One hindlimb was inserted into a stainless steel can and directly heated by raising the air temperature inside the can via a flexible heater twisted around the steel can. Muscle temperature was increased gradually and maintained at 42 degrees C for 60 min. Core rectal and contralateral muscle temperatures were increased <1.5 degrees C during the heat stress. HSP60, HSP72, and heat shock cognate (HSC) 73 content in the slow soleus and fast plantaris in both limbs were determined immediately (0 h) and 2, 4, 8, 12, 24, 36, 48, or 60 h after heat stress. Within 0-4 h, all HSPs were approximately 1.5- to 2.2-fold higher in heat-stressed than contralateral soleus. Compared with the contralateral plantaris, the heat-stressed plantaris had a higher (1.5-fold) HSP60 content immediately and 2 h after heat stress and a higher (2.5- to 6.8-fold) HSP72 content between 24 and 48 h after heat stress. Plantaris HSC73 content was not affected by heat stress. This unique heat-stress method provides advantages over existing systems; muscle temperature can be controlled precisely during heating and the HSP response can be compared between muscles in heat-stressed and contralateral limbs of individual rats. Results show a differential response of HSPs in the soleus and plantaris during recovery after heat stress; soleus demonstrated a more rapid and broader HSP response to heat stress than plantaris.

Changes in the expression of heat shock protein 72 (HSP72) in response to atrophic-inducing perturbations of muscle involving chronic mechanical unloading and denervation were determined. Adult male Wistar rats were assigned randomly to a sedentary cage control (CON), hind limb unloading (HU, via tail suspension), HU plus tenotomy (HU + TEN), HU plus denervation (HU + DEN), or HU + TEN + DEN group. Tenotomy and DEN involved cutting the Achilles tendon and removing a segment of the sciatic nerve, respectively. After 5 days, HSP72 levels in the soleus of the HU + DEN and HU + TEN + DEN groups were 42 (P < 0.05) and 53% (P < 0.01) less than CON, respectively. Soleus weight decreased in both groups. Heat shock protein 72 levels in the plantaris of the HU + TEN, HU + DEN, and HU + TEN + DEN groups were 31, 25, and 30% lower than CON, respectively (P < 0.05). Plantaris weight decreased in the HU + DEN and HU + TEN + DEN, but not in the HU + TEN group. Hind limb unloading alone had little effect on the HSP72 level in either muscle. Reduced levels of HSP72 were associated with a decreased soleus (r=0.62, P < 0.01) and plantaris (r=0.78, P < 0.001) weight. These results indicate that the levels of HSP72 in both a slow and a fast rat plantarflexor are responsive to a chronic decrease in the levels of loading and/or activation and suggest that the neuromuscular activity level and the presence of innervation of a muscle are important factors that induce HSP72 expression.

Clenbuterol, a beta2-agonist, administration results in hypertrophy of fast fibres and an increase in the fast myosin heavy chain (MHC) composition of both fast and slow muscles. The present study was designed to determine the phenotypic response at the single fibre level. Clenbuterol was added to the drinking water (30 mg L(-1)) of adult male Wistar rats for 4 weeks. Single fibres from the soleus muscle of control (10 rats; 555 fibres) and clenbuterol-treated (10 rats; 577 fibres) were dissected and their MHC isoform composition was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. Body, heart, and soleus weights were 9, 24, and 27% higher in clenbuterol-treated than control rats. The mean cross-sectional areas of fast and slow/fast hybrid fibres were approximately 64 and approximately 74% larger in the clenbuterol-treated than control rats, whereas the size of the slow fibres were similar in the two groups. Fibres from control soleus showed three MHC patterns: pure type I (84%), pure type IIa (4%), and type I + IIa (12%) MHC. Some fibres from clenbuterol-treated soleus showed a de novo expression of type IIx MHC resulting in the following fibre type proportions: pure type I (62%), pure type IIa (2%), type I + IIa (26%), type I + IIa + IIx (6%), and type IIa + IIx (1%). In those fibres containing multiple MHCs, there was a shift towards the faster MHC isoforms after clenbuterol treatment. These data indicate that clenbuterol results in muscle fibre hypertrophy, stimulates a de novo expression of type IIx MHC and increases the percentage of fibres containing multiple MHC isoforms in the rat soleus muscle. These phenotypic changes at the single fibre level are consistent with a clenbuterol-related shift in the functional properties of the soleus towards those observed in a faster muscle.

The hind-limb suspension is an established method to unload hind-limb muscles and is largely known to induce muscle fiber atrophy, especially in an antigravity slow soleus muscle. The ankle joints of rat are hyper-extended in response to hind-limb suspension, therefore, the soleus muscles are passively shortened and tension development is inhibited even when the active electromyogram (EMG) is present [1,2]. The hind-limb unloading (HU) changes the myosin heavy chain (MHC) phenotypes and contractile properties in the rat soleus muscle toward the "fast" type [3][4][5][6]. These responses are very similar to those observed after exposure to actual microgravity [1,[7][8][9][10][11]. Thus the functional properties of the soleus muscle seem to depend on the gravitational and/or weight-bearing conditions.Heat shock proteins (HSPs) are observed in any types of tissues or organs [12]. Within several types of HSPs, a 70 kDa HSP called HSP72 is known as a heat-or stress-inducible protein in mammalian skeletal muscles [13][14][15]. It is proposed that HSPs including HSP72 have an important role to maintain the homeostasis of cells and to protect tissues and organs from heat or other types of stresses [12,13]. Although the exact functions are not clear in mammalian skeletal muscles, some studies suggest that HSP72 can protect muscle damage or atrophy [16,17]. We have previously reported that HSP72 was decreased in the slow soleus and fast plantaris muscles of rats following short-term (5 d) of HU combined with tenotomy and/or denervation, and the expression level of HSP72 was paralleled to the muscle weight [18]. Our results Japanese Journal of Physiology Vol. 53, No. 4, 2003 281Japanese Journal of Physiology, 53, 281-286, 2003 Key words: heat shock protein, hind-limb unloading, reloading, soleus, rat. Abstract:To clarify the changes of heat shock protein (HSP) 72 in the rat soleus muscle after hind-limb unloading (HU) and during reloading, 7-week-old male Wistar rats were hind-limb-suspended for 9 weeks, thereafter ambulatory recovery was permitted for 8 weeks. The body and absolute soleus weights were significantly lower in the HU than in the age-matched control group after HU and during reloading. The soleus weight relative to body weight was also significantly lower in the HU than in the age-matched control group at the end of 9 weeks of suspension, but returned to the control level after 2 weeks of reloading. The HSP72 content decreased to 38%of the control level after HU and conversely increased to 165 and 175% of the control level after 2 and 4 weeks of reloading, respectively. The HSP72 content returned to the control level after 8 weeks of reloading. Thus our results showed that the expression of HSP72 was downregulated by HU and upregulated temporally over the level of the control during the reloading period, and they suggested that these down-and up-regulations of HSP72 may be related to many factors including mechanical stress or load applied to the muscle.

In a recent study, we showed that the rat slow soleus and fast plantaris muscles exhibited different time courses for the response of specific heat shock proteins (HSPs) after 1 h of heat stress. We hypothesized that these differential responses were related, in part, to the varying fiber type composition of these muscles. To further test this hypothesis, we now have determined the responses of Hsp60, Hsp72, and Hsc73 during the 60 h following exposure to a single bout of heat stress in the deep (relatively high percentage of slow fibers) and superficial regions (only fast fibers) of the adult rat gastrocnemius muscle. The temperature of the musculature in the left hindlimb was elevated to approximately 42 degrees C for 1 h, while the right hindlimb served as a control. Two hours after the heat stress, the Hsp60 levels were increased by 1.3- and 2.0-fold in the deep and superficial regions, respectively. The Hsp72 levels were increased (1.8-fold) in the deep region at 8 h after heat stress, whereas in the superficial region these levels were increased between 4 and 48 h (peak at 36 h by 10-fold) after the heat stress. No changes were observed for Hsc73 in either region of the muscle. Combined with our previous data, the results indicate that the responses of HSPs in the rat hindlimb muscles after a single exposure to heat stress are related to fiber type composition of the muscle or muscle region or to the inherent properties of each HSP. From a clinical viewpoint, these data indicate that specific regions (most likely based on fiber type composition) within a muscle may be affected differentially by any intervention inducing HSPs.