Elevation in heat shock protein 72 mRNA following contractions in isolated single skeletal muscle fibers.
The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n=26) or not stimulated to contract (REST; n=14). The relative mean developed tension of EX fibers decreased to 29+/-7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 (n=9), 2 (n=8), or 4 h (n=9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac alpha-actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly (P<0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5+/-7.5-fold) or cardiac alpha-actin (14.7+/-4.3-fold), although not at the 1- or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac alpha-actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources.
Division of Physiology, Department of Medicine, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0623, USA.
Creed M
CM
Stary
Brandon J
BJ
Walsh
Amy E
AE
Knapp
David
D
Brafman
Michael C
MC
Hogan
D000199:Actins / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000378:metabolism; D004558:Electric Stimulation; D005260:Female; D050884:HSP72 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D009119:Muscle Contraction; D018763:Muscle Fatigue; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D000199:Actins; D018834:Chaperonin 60; D050884:HSP72 Heat-Shock Proteins; D012333:RNA, Messenger; D029867:Xenopus Proteins