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Articles in PresS, published online ahead of print October 11, 2002
J Appl Physiol, 10.1152/jap.00559.2002
Submitted on June 26, 2002
Accepted on September 28, 2002
1 Department of Medicine, Physiology Division, University of California-San Diego, La Jolla, CA, USA
* To whom correspondence should be addressed. E-mail: ckindig{at}ucsd.edu.
The purpose of this research was to develop a technique for rapid measurement of oxygen uptake (VO2) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cell VO2 have utilized polarographic-style electrodes thereby mandating large fluid volumes and relatively poor sensitivity. Thus, our laboratory has developed an ~100 µ1, well-stirred chamber for the measurement of VO2 in isolated Xenopus laevis myocytes using a phosphorescence quenching technique (Ringers solution with 0.05 mM Pd-meso-tetra [4-carboxyphenyl] porphine) to monitor the fall in extracellular PO2 (which is proportional to cellular VO2 within the sealed chamber). VO2 in single living myocytes dissected from Xenopus lumbrical muscles was measured from rest across a bout of repetitive tetanic contractions (0.33 Hz) and in response to a ramp protocol utilizing an increasing contraction frequency. In response to the square-wave contraction bout, the increase in VO2 to steady-state (SS) was 16.7±1.3 ml/100g/min (range, 13.0-21.9 ml/100g/min; n=6). The rise in VO2 at contractions onset (n=6) was fit with a time delay (TD; 2.1±1.2 s; range 0.0-7.7 s) + mono-exponential rise to SS (time constant = 9.4±1.5 s; range, 5.2-14.9 s). Furthermore, in 2 additional myocytes, VO2 increased progressively as contraction frequency increased (ramp protocol). This technique for measuring VO2 in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function and VO2 dynamics without potential complications of the circulation and other myocytes.
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