Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach

摘要

One hundred and eighty six enzymatically dissociated murine muscle fibres were loaded with Mag-Fluo-4 AM, and adhered to laminin, to evaluate the effect of modulating cytosolic Ca2+ buffers and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), mitochondria, and Na+/Ca2+ exchanger (NCX) on the differential tetanic Ca2+ transient kinetics found in different fibre types. Tetanic Ca2+ transients were classified as morphology type I (MT-I) or type II (MT-II) according to their shape. The first peak of the MT-I (n = 25) and MT-II (n = 23) tetanic Ca2+ transients had an amplitude (a dagger F/F) of 0.41 +/- A 0.03 and 0.83 +/- A 0.05 and a rise time (ms) of 1.35 and 0.98, respectively. MT-I signals had a time constant of decay (tau(1), ms) of 75.9 +/- A 4.2 while MT-II transients showed a double exponential decay with time constants of decay (tau(1) and tau(2), ms) of 18.3 +/- A 1.4 and 742.2 +/- A 130.3. Sarcoendoplasmic reticulum Ca2+ ATPase inhibition demonstrated that the decay phase of the tetanic transients mostly rely on SERCA function. Adding Ca2+ chelators in the AM form to MT-I fibres changed the morphology of the initial five peaks to a MT-II one, modifying the decay phase of the signal in a dose-dependent manner. Mitochondria and NCX function have a minor role in explaining differences in tetanic Ca2+ transients among fibre types but still help in removing Ca2+ from the cytosol in both MT-I and MT-II fibres. Cytoplasmic Ca2+ buffering capacity and SERCA function explain most of the different kinetics found in tetanic Ca2+ transients from different fibre types, but mitochondria and NCX have a measurable role in shaping tetanic Ca2+ responses in both slow and fast-twitch muscle fibre types. We provided experimental evidence on the mechanisms that help understand the kinetics of tetanic Ca2+ transients themselves and explain kinetic differences found among fibre types.