This has been a long time in the making but I am absolutely delighted to share our latest paper published in @PNASNews where we use time-resolved X-ray diffraction to elucidate the regulatory roles of the thick and thin filaments during muscle relaxation.

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RT @jploenneke: Interesting paper titled "Calcium binding to troponin C is required for activation of the myosin-containing thick filaments….

At high calcium (pCa 4.7), there was no change in force in E76A samples, or any structural changes indicative of ON thick filaments as determined by X-ray diffraction and fluorescent probes on the RLC of myosin. This is in direct contrast to samples retaining their native TnC.

To test this, the native TnC in permeabilised rat trabeculae was substituted with its E76A variant which is unable to bind calcium. E76A and native TnC samples were then activated by a temperature-jump protocol in differing calcium concentrations (pCa's 9, 7, 6.07, 4.7).

Three signalling pathways have been proposed for the release of myosin motors during the heartbeat: thick filament mechano-sensing, thin-to-thick filament signalling, and calcium signalling on the thick filament. Here, the third hypothesis is tested.

Excited to share the latest work from our lab, led by Michaeljohn Kalakoutis, where we showed no evidence for direct activation of the thick filaments by calcium in rat trabeculae. Calcium binding to troponin-C is required for thick filament activation.

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For a bite-sized summary of our main findings @esrfsynchrotron have kindly written a brief overview of our paper:.

To summarise, removing load is more effective at relaxing the muscle than Ca2+. When Ca2+ is removed, a conformational change in troponin may initiate the initial detachment of myosin from actin. Yielding of weak sarcomeres and tendon compliances determine muscle relaxtion.

When examining sarcomere length, SL remains constant during the first isometric phase of relaxation (blue). When force starts to decline, weak sarcomeres being to yield and stretch as identified by two populations of sarcomeres, eventually resulting in exponential force decay.

In this period called "isometric relaxation" motors begin to detach as signalled by a decrease in the equatorial intensity ratio as myosin moves away from actin (blue), meaning the strain per attached motor increases.

If both the thick and thin filaments are remaining ON given the high load and low Ca2+ after electrical stimulation, how does myosin escape their actin-attached, force-generating state?.

IAL2 increases as motors attach to actin and tropomyosin moves into the actin grooves. When load is removed and myosin detaches, IAL2 ↓ despite the high Ca2+ as tropomyosin is allowed to move out of the grooves between actin. When Ca2+ ↓, the change in IAL2 is similar to force

When load is removed (grey shade) the thick filament begins to switch off as motors detach, and occurs much faster than force due to the small number of motors required to drive shortening. When Ca2+ is removed IML1 remains low (blue shade) and slowly returns to its OFF state

X-ray diffraction patterns were collected and reflections ascribed to certain sarcomere structures. (See ALT text for description of each reflection examined). I want to draw attention to two reflections in particular - the ML1 and AL2 reflections

To test this, we visited ID02 at @esrfsynchrotron to perform time-resolved X-ray diffraction on mouse EDL muscles. To separate out the contribution of load and [Ca2+]i we imposed ramp shortening to remove load when [Ca2+]i was high, and by allowing reuptake of Ca2+ by the SR.

This paradox poses a fundamental question: how does a muscle relax following contraction given the above paradox of high load and actin-bound motors preventing the thin filament switching off.

Muscle relaxation has been long viewed as a reversal of this activation paradigm, but actin-bound myosin motors prevent the return of tropomyosin back to the "blocked" position, suggesting that motor detachment rather than Ca2+ dissociation may be a key determinant of relaxation.

When [Ca2+]i increases on electrical stimulation, troponin becomes saturated with Ca2+ triggering a movement of troponin and tropomyosin exposing binding sites. Motors then leave their helical tracks in a load-dependent manner. High load + mechanosensing = rapid activation.