The excitation-contraction coupling mechanism in skeletal muscle

Abstract

First coined by Alexander Sandow in 1952, the term excitation-contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca²⁺ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca²⁺ from the SR and transient increase of Ca²⁺ concentration in the myoplasm, (6) activation of the myoplasmic Ca²⁺ buffering system and the contractile apparatus, followed by (7) Ca²⁺ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca²⁺ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na⁺/Ca²⁺ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca²⁺ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca²⁺ sparks.

Keywords: Ca2+ transients; Excitation–contraction coupling; Fibre types; Mitochondria; Skeletal muscle.

Fig. 1

Comparison of single Ca²⁺ transients’ kinetics recorded in muscle fibres obtained by enzymatic dissociation of flexor digitorum brevis muscles from adult mice. Different cells were loaded with each of the Ca²⁺ dyes indicated in the figure and electrically stimulated. Ca²⁺ transients were recorded in an inverted fluorescence microscope using the appropriate set of filters, a photomultiplier and a Nikon amplifier. In (a), clear kinetic differences can be recognized, mostly derived from the different dissociation constants of the dyes used, being the fastest signal that obtained with Mag-Fluo-4 (black trace) and the slowest one that obtained with Fura-2 (green trace). In (b), the records are shown in an expanded time scale to better illustrate differences in the rising part of the signal

Fig. 2

Time course of single Ca²⁺ transients of different fibre types obtained by enzymatic dissociation of extensor digitorum longus and soleus muscles from adult mice and typed by polyacrylamide gel electrophoresis. The cells were loaded with Mag-Fluo-4. A pattern can be recognized, with the pair I and IIA being the slowest and the pair IIX/D and IIB being the fastest both during decay (a) and rise (b)

Fig. 3

Subsarcolemmal (left) and inner (right) differential mitochondrial distribution in flexor digitorum brevis muscle fibres stained with Mitotracker Green. The images were acquired with a Nikon C1 confocal microscope. A pattern of paired columns of mitochondria, parallel to the short axis of the cell, can be identified in the inner or intermyofibrillar location, while single, longer rows of mitochondria, parallel to the long axis of the cell, can be identified in the subsarcolemmal region of the cell

Fig. 4

Time course of fluorescence decay after FCCP poisoning (right), in one flexor digitorum brevis fibre stained with TMRE to visualize mitochondrial potential (Ψm). On the left, confocal images of the cell indicating regions of interest (ROI, numbered squares) and time of acquisition after starting the experiment are shown. On the right, the black squares represent the mean±SEM of the measurements carried out at level of the ROI as a function of time. A rapid decrease in fluorescence, suggesting a dissipation of Ψm, is seen after the application of FCCP

Fig. 5

Increase in intramitochondrial Ca²⁺ during a sarcoplasmic reticulum (SR) depletion protocol in a flexor digitorum brevis fibre imaged in a confocal microscope. a A pseudocolor image of mitochondria loaded with Rhod-2. b Summarizes the time course of the mitochondrial mean Rhod-2 fluorescence variation in the regions of interest (white squares) marked in (a). The black circles represent the mean fluorescence in the small white squares in (a), and the black squares the mean fluorescence in the big white square in (a). The mitochondrial fluorescence increases during the protocol in which the cell is in absence of external Ca²⁺ and the SR Ca²⁺ ATPase is blocked by cyclopiazonic acid (CPA). When 5 mM Ca²⁺ external solution reaches the cell the mitochondria uptake part of the Ca²⁺ entering the fibre via the store operated Ca²⁺ entry mechanism. K indicates the moments in which an external solution with high K⁺ concentration was applied to elicit SR Ca²⁺ release in order to deplete this Ca²⁺ store

Fig. 6

Activation of store-operated Ca²⁺ entry (SOCE) in a flexor digitorum brevis fibre loaded with Fura-2 after sarcoplasmic reticulum (SR) depletion by high K⁺ exposures in the absence of external Ca²⁺ and in the presence of thapsigargin (TG) 5-10 μM. a Once depleted as shown by the absence of response to high K⁺, Ca²⁺ reintroduction in the external medium activates SOCE. The Ca²⁺ entrance can be reversibly blocked by 80 μM 2-APB. b When a similar protocol is applied to another fibre, but exposed to FCCP, SOCE cannot be activated again. It can be partially recovered after a long washout of the drug. This suggests that the mitochondrial depolarization affects the Ca²⁺ entry induced by SR depletion