Waveless mammalian muscle

Ca²⁺-induced Ca²⁺ release (CICR) refers to the mechanism through which a Ca²⁺ channel sitting in the membrane of an intracellular Ca²⁺ storage organelle opens as the consequence of a rise in Ca²⁺ at its cytoplasmic face. The resulting self-amplified rise in cytosolic Ca²⁺ is a powerful biological signal, as classically exemplified in cardiac cells where action-potential-induced Ca²⁺ entry triggers opening of type 2 ryanodine receptor (RyR2) release channels in the sarcoplasmic reticulum (SR) to build the rise in Ca²⁺ that elicits contraction.

CICR was discovered in skeletal muscle ∼40 years ago. Still, despite keeping a number of lab benches busy since then, the question of its physiological relevance in muscle has remained controversial and is a still-debated issue. The purist view of CICR restrictively includes Ca²⁺-dependent activation of the channels, independent of any other necessary stimuli (Endo, 2009). This obviously does not fit with physiological activation of SR Ca²⁺ release in skeletal muscle cells which is under the strict control of the plasma membrane voltage and requires a molecular interaction between voltage-sensing Cav1.1 proteins in the transverse tubule membrane and type 1 ryanodine receptor channels (RyR1) in the adjacent SR terminal cisternae membrane. Now, if we temporarily forget the purist view, the question of whether a Ca²⁺-gated component secondarily joins in, is standing and is of severe importance for the comprehension of excitation–contraction (E–C) coupling. Progress along the years has highlighted a possible CICR contribution from one out of every two RyR1 channels that is lacking Cav1.1 partners in the T-tubule membrane (see for instance Stern et al. 1997) but no definite consensus has so far been reached. More recent years have seen the emergence of coherent sets of findings pin-pointing the potentially critical need for the number 3 isoform of RyR for CICR to operate; indeed, there is now consistent evidence that differentiated adult frog muscle that benefits from a much larger ratio of (its own homologous forms of) RyR3 to RyR1 than mammalian muscle, is in a much better condition to physiologically produce and use CICR signals for normal E–C coupling, probably under the form of Ca²⁺ sparks. Along the same trend, forced expression of RyR3 in mouse muscle fibres was found to be functionally associated with the presence of either Ca²⁺ sparks (spontaneous and voltage-activated) and/or of voltage-independent Ca²⁺ release phenomena yielding variable properties in terms of amplitude, frequency, spatial spread and propagation (see Pouvreau et al. 2007; Legrand et al. 2008).

The paper by Figueroa et al. in a recent issue of The Journal of Physiology further tackles the problem by exploring the archetypal issue of whether a pure rise in Ca²⁺ can trigger physiologically significant Ca²⁺ release in adult muscle fibres.

The authors used 2-photon excitation of an optimized Ca²⁺-cage molecule together with simultaneous fast confocal Ca²⁺ imaging within permeabilized muscle fibres from either frog or mouse. This way, they were able to reliably compare in the two preparations, the consequences of a synthetic Ca²⁺ transient featuring properties consistent with those of the ones physiologically triggered by membrane depolarization (we are talking of a fast, large increase in Ca²⁺). A specifically neat feature of their experiments was that synthetic Ca²⁺ transients were elicited outside the fibres, allowing clear separation of the trigger and of the response, while also limiting potential artifacts and complications due to 2-photon excitation within the cells.

Well, the difference between frog and mouse is striking: frog fibres react to the trigger Ca²⁺ by a propagating Ca²⁺ wave whereas mouse fibres do not, unless RyR1 sensitizing pharmacological tools are used. The waves in frog fibres mobilize a very substantial Ca²⁺ release flux and appear to fulfill certain classical CICR criteria. Also quite remarkably, the free Ca²⁺ level necessary to trigger the waves is less than the one faced by the channels during physiological E–C coupling, which definitely supports the possibility that CICR can operate under physiological conditions. Data convincingly establish that CICR underlies these travelling Ca²⁺ signals and it is more than tempting to make a parallel between the difference reported here between frog and mouse and the fact that frog fibres produce voltage-dependent Ca²⁺ sparks whereas mouse fibres do not.

As clearly acknowledged by the authors, observations were made in conditions which were far from physiological so that unequivocal demonstration that CICR operates in intact frog muscle fibres during E–C coupling will need further efforts. For the same reason, one could still suspect that the experimental conditions may have hindered the potency of mouse fibres to produce CICR because of, for instance, a lost physiological RyR1-sensitizing component necessary in mouse but not in frog. Further speculation could go as far as prospecting that, thus far, collected data tending to deny CICR function in mammalian fibres have missed fulfilling an anonymous crucial criterion for CICR to operate. At the moment, however, as suggested by Figueroa et al. the most likely explanation for the difference is that RyR3 is the required component.

Independent of this issue, data from Figueroa et al. will definitely establish or at least further strengthen our confidence in the capability of frog muscle fibres to use CICR during normal activity; this is an important step towards a comprehensive view of the physiological role of this mechanism in muscle.