Key points
Fast sarcomere‐level mechanics in contracting intact fibres from frog skeletal muscle reveal an I‐band spring with an undamped stiffness 100 times larger than the known static stiffness.
...This undamped stiffness remains constant in the range of sarcomere length 2.7–3.1 µm, showing the ability of the I‐band spring to adapt its length to the width of the I‐band.
The stiffness and tunability of the I‐band spring implicate titin as a force contributor that, during contraction, allows weaker half‐sarcomeres to equilibrate with in‐series stronger half‐sarcomeres, preventing the development of sarcomere length inhomogeneity.
This work opens new possibilities for the detailed in situ description of the structural–functional basis of muscle dysfunctions related to mutations or site‐directed mutagenesis in titin that alter the I‐band stiffness.
Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in‐series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I‐band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force–SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4 kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I‐band spring that at SL > 2.7 µm attains a maximum constant value of ∼6 pN nm−1 per half‐thick filament, two orders of magnitude larger than expected from titin‐related passive force. We conclude that a titin‐like dynamic spring in the I‐band, made by an undamped elastic element in‐series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I‐band spring plays a fundamental role in preventing the development of SL inhomogeneity.
Key points
Fast sarcomere‐level mechanics in contracting intact fibres from frog skeletal muscle reveal an I‐band spring with an undamped stiffness 100 times larger than the known static stiffness.
This undamped stiffness remains constant in the range of sarcomere length 2.7–3.1 µm, showing the ability of the I‐band spring to adapt its length to the width of the I‐band.
The stiffness and tunability of the I‐band spring implicate titin as a force contributor that, during contraction, allows weaker half‐sarcomeres to equilibrate with in‐series stronger half‐sarcomeres, preventing the development of sarcomere length inhomogeneity.
This work opens new possibilities for the detailed in situ description of the structural–functional basis of muscle dysfunctions related to mutations or site‐directed mutagenesis in titin that alter the I‐band stiffness.
The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper ...theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A. F. Huxley and his collaborators in 1981, which provides that the half-sarcomere (hs) compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length (SL) the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of hs stiffness at increasing SL, which is important either in relation to the mechanism of stabilisation of SL against the consequence of sarcomere inhomogeneity in active force generation, or for investigations on the role of titin as mechano-sensor in thick filament regulation. Moreover the model opens the possibility for understanding the functional differences related to the titin isoform of various muscle types and the mechanism by which mutations in titin gene lead to myopathies.
Key points
The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them.
In this study fast sarcomere‐level ...mechanics has been applied to Ca2+‐activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano‐kinetics of force generation by slow and fast myosin isoforms in situ.
The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform‐dependent functional diversity between skeletal muscles.
These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle.
The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere‐level mechanical methods in permeabilised fibres (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T‐500) from slow (soleus, containing the MHC‐1 isoform) and fast (psoas, containing the MHC‐2X isoform) skeletal muscle of the rabbit. The stiffness of the half‐sarcomere was determined at the plateau of Ca2+‐activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56 ± 0.04 and 1.70 ± 0.37 pN nm−1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0) was similar (∼3.3 nm) in both isoforms. Consequently the force per motor (F0 = εs0) was three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 versus 5.35 ± 1.51 pN). The fraction of actin‐attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5) was 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.
Key points
The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them.
In this study fast sarcomere‐level mechanics has been applied to Ca2+‐activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano‐kinetics of force generation by slow and fast myosin isoforms in situ.
The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform‐dependent functional diversity between skeletal muscles.
These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle.
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