Proceedings of The Physiological Society

University of Cambridge (2004) J Physiol 555P, C35


Genomic responses to chronic systemic hypoxia in skeletal muscle of rats in vivo

Katie E. Glen, Janice M. Marshall, D. Candinas* and Deborah M. Stroka*

Department of Physiology, Medical School, University of Birmingham, Birmingham B15 2TT, UK and * VCHL, University Hospital Bern, Switzerland

Our recent studies on rats showed that in the first 1-7 days of chronic hypoxia there is vasodilatation (Walsh & Marshall, 1999) and by 3 weeks there is capillary angiogenesis (Deveci et al. 2001) in skeletal muscle. In the present study we have investigated the genomic events that may underlie these changes.

Experiments were performed on chronically hypoxic (CH) rats housed in a hypoxic chamber at 12 % O2 for 2, 6, 12 h and 1, 3, 7 and 14 days (n = 6 in each case) and on 6 normoxic (N) rats that breathed air. Under anaesthesia (Sagatal 60 mg kg-1, Tibialis Anterior (TA; mainly glycolytic), Soleus (SOL; oxidative) and Spinotrapezius (SP; mixed fibre types) muscles were removed and frozen in liquid N2-cooled 2-methylbutane. All animals were humanly killed by anaesthetic overdose. mRNA was quantified by real-time PCR using probes and primers to VEGF, iNOS and eNOS. Values are expressed as fold increases (mean ± S.D.) in CH relative to N rats. All changes described below were significant (P < 0.05) when analysed by ANOVA followed by Dunnett's post hoc test when appropriate.

In SOL there was an increase in iNOS, VEGF and eNOS mRNA expression. iNOS mRNA showed a time-dependent increase from 2hr reaching a 6.2 ± 1.7-fold increase at 1day and remained 3-fold greater than N rats at 3, 7 and 14 days. VEGF mRNA increased from 1-14days reaching a maximum 4.8 ± 1.3-fold increase at 3days, but falling to 2.3 ± 0.9-fold at 14days. eNOS mRNA was increased by 2.2 ± 1.0-fold at days 7 and 14.In TA, there was a 2-2.5-fold increase in iNOS mRNA at all time points. Similarly VEGF mRNA showed a prolonged increase reaching 2.0 ± 0.8-fold at 6hr. eNOS mRNA was similar to that seen in SOL: 2.0 ± 0.6-fold increase at day 7.In SP, iNOS mRNA showed a similar time-dependent increase to SOL: 3.7 ± 1.3-fold, at 1day. Any change in VEGF mRNA did not reach statistical significance. In contrast to SOL and TA, eNOS mRNA in SP was increased earlier: at 6hr by 2.3 ± 1.0-fold.These results suggest chronic hypoxia induces genomic responses in skeletal muscle of different time-dependencies and magnitudes in muscles of different fibre type. Increases in iNOS and VEGF mRNA were particularly pronounced and prolonged in SOL; this may be consistent with capillary angiogenesis being more substantial in oxidative SOL than in TA (Deveci et al. 2001). The increases in iNOS mRNA in all muscles and the sustained increase in eNOS mRNA in SP is consistent with tonic NO-dependent dilatation (Walsh & Marshall, 1999).

We gratefully acknowledge the support of the British Heart Foundation, UK.

Where applicable, experiments conform with Society ethical requirements