Increased Generation of Intracellular Nitric Oxide During Contraction of Isolated Skeletal Muscle Fibres

Resumen

keletal muscle continually produces nitric oxide (NO) which is involved in excitation-contraction coupling, mitochondrial respiration and gene expression. The aim of this study was to quantify the real-time generation of intracellular NO in resting and electrically stimulated isolated mature mouse muscle fibres.Single muscle fibres were isolated from the Flexor Digitorum Brevis muscle of mice and cultured on collagen coated plates. Muscle fibres were loaded with the nitric oxide specific probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) and measurements were taken in gray scale by fluorescence microscopy. The addition of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) resulted in a large rise in intracellular fluorescence. The introduction of a 30 minute post-incubation period following DAF-FM loading resulted in a decreased response to SNAP indicating DAF-FM leaks from muscle fibres following loading. Baseline and electrical stimulation experiments were therefore performed over 15 minutes to minimise the loss of DAF-FM. Electrical stimulation of fibres resulted in a 44% increase in NO generation compared to control resting fibres. This increase was totally abolished by pre-incubation of fibres in the nitric oxide synthase inhibitor NG-methyl –L-arginine.These data describe a novel technique that will provide a valuable tool for measuring the real-time generation of intracellular NO in isolated muscle fibres and will improve knowledge of the roles of NO in muscle function.