Activation of skeletal muscle is controlled by a dual-filament mechano-sensing mechanism

Contraction of skeletal muscle is triggered by a transient rise in intracellular calcium concentration leading to a structural change in the actin-containing thin filaments that allows binding of myosin motors from the thick filaments. Most myosin motors are unavailable for actin binding in resting muscle because they are folded back against the thick filament backbone. Release of the folded motors is triggered by thick filament stress, implying a positive feedback loop in the thick filaments. However, it was unclear how thin and thick filament activation mechanisms are coordinated, partly because most previous studies of the thin filament regulation were conducted at low temperatures where the thick filament mechanisms are inhibited. Here, we use probes on both troponin in the thin filaments and myosin in the thick filaments to monitor the activation states of both filaments in near-physiological conditions. We characterize those activation states both in the steady state, using conventional titrations with calcium buffers, and during activation on the physiological timescale, using calcium jumps produced by photolysis of caged calcium. The results reveal three activation states of the thin filament in the intact filament lattice of a muscle cell that are analogous to those proposed previously from studies on isolated proteins. We characterize the rates of the transitions between these states in relation to thick filament mechano-sensing and show how thin- and thick-filament-based mechanisms are coupled by two positive feedback loops that switch on both filaments to achieve rapid cooperative activation of skeletal muscle

Minimum number of myosin motors accounting for shortening velocity under zero load in skeletal muscle

KEY POINTS: Myosin filament mechanosensing determines the efficiency of the contraction by adapting the number of switched ON motors to the load. Accordingly, the unloaded shortening velocity (V (0)) is already set at the end of latency relaxation (LR), ∼10 ms after the start of stimulation, when the myosin filament is still in the OFF state. Here the number of actin‐attached motors per half‐myosin filament (n) during V (0) shortening imposed either at the end of LR or at the plateau of the isometric contraction is estimated from the relation between half‐sarcomere compliance and force during the force redevelopment after shortening. The value of n decreases progressively with shortening and, during V (0) shortening starting at the end of LR, is 1–4. Reduction of n is accounted for by a constant duty ratio of 0.05 and a parallel switching OFF of motors, explaining the very low rate of ATP utilization found during unloaded shortening. ABSTRACT: The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V (0)) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca(2+)‐induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin‐attached motors per half‐thick filament during V (0) shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half‐sarcomere (hs) compliance and force during the force development following V (0) shortening. When V (0) shortening is superimposed on the maximum isometric force T (0), n decreases progressively with the increase of shortening (range 30–80 nm per hs) and, when V (0) shortening is imposed at the end of LR, n can be as low as 1–4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V (0) shortening

Intensive physiotherapic respiratory care in critically ill patients with tracheostomy after cardiac surgery

Background. Patients following major cardiac surgery are increasingly elderly and present many comorbidities. For these reasons their post-operative phase is often burdened by several complications requiring a long stay in Critical Care and prolonged mechanical ventilation. Most of these patients, when transferred to our Intensive Cardiac Rehabilitation Unit, still have a percutaneous tracheostomy due to respiratory mechanical dysfunction. The aim of our work is to present new rehabilitative care strategies in such compromised patients. Methods and materials. We studied 27 elderly critically ill tracheostomized patients who were split into 2 Groups (A = 11 and B = 16). The Groups were homogeneous for age and for left ventricular ejection fraction. Group A received a standard treatment including cautious mobilisation and respiratory unspecific physiotherapy. Group B received an earlier and more aggressive treatment with a specific respiratory physiotherapy including Positive Expiration Pressure (PEP) directly connected to the tracheostomy cannula. A protocol for tracheostomy decannulation by assessment of the Peak Expiratory Flow during cough (PCEF≥ 180 L/min.) has been defined in order to verify the patients ability to develop a mechanically effective cough to obtain weaning from tracheostomy. Besides, in the patients of Group B, we carried out a screening of the swallowing dysfunction. Results. Four patients of Group A deceased while in Group B there were no deaths. Furthermore patients of Group B showed a statistically significant improvement of mobility and respiratory indexes. In Group B only one patient was discharged with tracheostomy cannula in site because he did not reach standard criteria for decannulation and his PCEF value was not satisfactory. This patient underwent percutaneous gastrostomy. Conclusions. A precocious and intensive rehabilitation, based on specific respiratory physiotherapy, significantly improves mobility and respiratory indexes of patients with tracheostomy. The PCEF and the swallowing deficit evaluation allows an earlier tracheostomy decannulation with lower risk of complications

Does low skilled immigration increase the education of natives? Evidence from Italian provinces

While there is a vast literature considering the labour market effects of immigration, less has been done to investigate how immigration affects the educational choices of young natives. Using Italian provincial data and an instrumental variables strategy, we show that the recent rise of low skilled immigrants has increased both the probability that young native high school graduates (males in the whole country and females in the industrialized North) enrol in or attain higher education and the probability that young natives (males and females) with less than high school education stay out of further education or training. We show that our results can be explained by a standard model of educational choices if some conditions are satisfied

Activation of the myosin motors in fast-twitch muscle of the mouse is controlled by mechano-sensing in the myosin filaments

ABSTRACT: Myosin motors in resting muscle are inactivated by folding against the backbone of the myosin filament in an ordered helical array and must be released from that conformation to engage in force generation. Time‐resolved X‐ray diffraction from single fibres of amphibian muscle showed that myosin filament activation could be inhibited by imposing unloaded shortening at the start of stimulation, suggesting that filaments were activated by mechanical stress. Here we improved the signal‐to‐noise ratio of that approach using whole extensor digitorum longus muscles of the mouse contracting tetanically at 28°C. Changes in X‐ray signals associated with myosin filament activation, including the decrease in the first‐order myosin layer line associated with the helical motor array, increase in the spacing of a myosin‐based reflection associated with packing of myosin tails in the filament backbone, and increase in the ratio of the 1,1 and 1,0 equatorial reflections associated with movement of motors away from the backbone, were delayed by imposing 10‐ms unloaded shortening at the start of stimulation. These results show that myosin filaments are predominantly activated by filament stress, as in amphibian muscle. However, a small component of filament activation at zero load was detected, implying an independent mechanism of partial filament activation. X‐ray interference measurements indicated a switch‐like change in myosin motor conformation at the start of force development, accompanied by transient disordering of motors in the regions of the myosin filament near its midpoint, suggesting that filament zonal dynamics also play a role in its activation. [Image: see text] KEY POINTS: Activation of myosin filaments in extensor digitorum longus muscles of the mouse is delayed by imposing rapid shortening from the start of stimulation. Stress is the major mechanism of myosin filament activation in these muscles, but there is a small component of filament activation during electrical stimulation at zero stress. Myosin motors switch rapidly from the folded inhibited conformation to the actin‐attached force‐generating conformation early in force development