Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers

Abstract

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: <3 mmHg) or AMP kinase activation had no effect on HSP72 mRNA levels. These results suggest that the intermittent cytosolic Ca(2+) transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

Keywords: ATP; Ca2+; HSP70; O2; RTq-PCR; exercise; fatigue; gene.

Fig. 1.

Contraction-induced heat shock protein 72 (HSP72) transcriptional activation occurs independently of fatigue development. A: individual tracings of developed tension in single skeletal muscle fibers stimulated to contract at either low frequency (0.1 Hz, top) or high (0.33 Hz, bottom). B: high-frequency (0.33 Hz) electrical stimulation results in a significant decrease in peak developed tension, described as the development of fatigue; a.u., arbitrary units. C: both low-intensity (nonfatiguing) and high-intensity (fatiguing) contractions result in a significant increase in HSP72 mRNA levels. Data are means ± SD. *Significant (P < 0.05) difference from time-matched control.

Fig. 2.

Effect of excitation-contraction (E-C) uncoupling. A: tracings of developed tension from control (top) and N-benzyl-p-toluene sulfonamide (BTS)-treated (bottom) single skeletal muscle fibers. B: treatment with BTS resulted in significant impairment of cross-bridge cycling, resulting in reduction in the development of tension throughout the stimulation time-course. C: fura-2 fluorescence (representing changes in [Ca²⁺]_cyt) in control (top) and BTS treated (bottom) single fibers demonstrate preservation of cytosolic Ca²⁺ cycling with electrical stimulation, which persisted throughout the stimulation time course (D). E: despite uncoupling of Ca²⁺ from cross-bridge cycling with BTS, electrical stimulation resulted in an increase HSP72 mRNA levels comparable to control fibers. In the absence of electrical stimulation, treatment of single fibers with BTS, low Po₂, or the AMP-kinase activator 5-aminoimidazole-4-carboxamide-1-b-riboside-Z-riboside (AICAR) did not result in any change in HSP72 levels (E). Data are means ± SD. *Significant (P < 0.05) difference from time-matched control.

Fig. 3.

A model of contraction-induced HSP72 transcription in skeletal muscle. Depolarization induces Ca²⁺ release from the sarcoplasmic reticulum (SR), which results in cross-bridge cycling and the production of force. E-C coupling alters the phosphorylation potential through consumption of ATP and generation of ADP and inorganic phosphate (P_i), which is exacerbated by reductions in O₂ availability to maintain ATP regeneration through oxidative phosphorylation. Alterations in the phosphorylation potential may either directly or indirectly (via AMP kinase) activate transcription. Uncoupling of SR Ca²⁺ release from cross-bridge cycling with BTS results in a similar elevation in HSP72 mRNA following electrical stimulation as untreated muscle, suggesting factors resulting from cross-bridge cycling (i.e, cytomechanical stress/reductions in O₂ availability/alterations in the phosphorylation) have a more minimal effect (dashed lines) on activation of HSP72 transcription. While SR Ca²⁺ cycling may generate alterations in the phosphorylation potential, neither a reduction in O₂ availability nor direct activation of AMP kinase results in HSP72 transcriptional activation, suggesting a direct action of cytosolic Ca²⁺ on HSP72 transcriptional activation.