Assessment of O2 uptake dynamics in isolated single skeletal myocytes

Abstract

The purpose of this research was to develop a technique for rapid measurement of O2 uptake (V˙o 2) kinetics in single isolated skeletal muscle cells. Previous attempts to measure single cellV˙o 2 have utilized polarographic-style electrodes, thereby mandating large fluid volumes and relatively poor sensitivity. Thus our laboratory has developed an ∼100-μl, well-stirred chamber for the measurement ofV˙o 2 in isolated Xenopus laevismyocytes using a phosphorescence quenching technique [Ringer solution with 0.05 mM Pd- meso-tetra(4-carboxyphenyl)porphine] to monitor the fall in extracellular Po 2 (which is proportional to cellular V˙o 2 within the sealed chamber). V˙o 2 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 V˙o 2 to steady state (SS) was 16.7 ± 1.3 ml · 100 g−1 · min−1 (range 13.0–21.9 ml · 100 g−1 · min−1; n = 6). The rise in V˙o 2at contractions onset ( n = 6) was fit with a time delay (2.1 ± 1.2 s, range 0.0–7.7 s) plus monoexponential rise to SS (time constant = 9.4 ± 1.5 s, range 5.2–14.9 s). Furthermore, in two additional myocytes,V˙o 2 increased progressively as contraction frequency increased (ramp protocol). This technique for measuring V˙o 2 in isolated, single skeletal myocytes represents a novel and powerful investigative tool for gaining mechanistic insight into mitochondrial function andV˙o 2 dynamics without potential complications of the circulation and other myocytes.