Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca²⁺ via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca²⁺-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca²⁺ regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca²⁺]. Among the processes studied on myofibrils are: (1) the Ca²⁺-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.     

Passive force enhancement in single myofibrils

The purpose of this study was to gain further insight into passive force enhancement by testing whether passive force enhancement occurs in single myofibrils. Myofibrils (n = 6) isolated from rabbit psoas muscle were fixed at a sarcomere length of 2.4 μm, and then stretched passively and actively to a sarcomere length of 3.4 μm. Passive force after deactivation of the myofibrils was increased after active compared to passive stretching. Therefore, passive force enhancement, previously observed in muscle and fiber preparations, also occurs in single myofibrils. Passive force enhancement in myofibrils ranged from 86 to 145% of the steady-state force observed after passive stretch. Because titin is the main source of passive force in myofibrils, we propose that titin might be responsible for passive force enhancement observed in myofibrils. We propose that this might occur through an increase in stiffness when calcium concentration increases upon activation.     

Heterogeneity of mean sarcomere length in different fibres: effects on length range of active force production in rat muscle

Isometric active length-force characteristics of rat semimembranosus lateralis muscles [rats,n = 6, body mass = 292 (SD 9) g] were recorded. Muscles were photographed during the force plateaus of the tetani to determine lengths of proximal, intermediate and distal fibres. From the mean number of sarcomeres in series in those fibres, mean sarcomere length at different muscle lengths was calculated. The heterogeneity of mean sarcomere lengths for each muscle was quantified according to the coefficient of variation of sarcomere lengths at muscle optimal length. Absolute muscle length changes were equal to fibre length changes. At all muscle lengths, mean sarcomere lengths in the distal fibres were significantly greater than those in proximal and intermediate fibres. The heterogeneity of mean sarcomere lengths augmented the length range of active force production between muscle optimal length and active slack length by about 38% to 43%. We concluded that in a muscle with a low degree of pennation, the heterogeneity of mean sarcomere lengths should be considered as a substantial contributor to the length range over which active force can be produced. In our experiment, the length ranges of active force production between optimal and slack length showed considerable differences (range 18.7–24.9 mm). The coefficient of correlation between length ranges and mean number of sarcomeres in series in various muscle regions was extremely low (r = –0.14) and not significant. The coefficient of correlation between length ranges and heterogeneity was high (r = 0.88) and significant. These data would suggest that muscles with a similar number of sarcomeres in series can exhibit quite different functional characteristics.     

Uniform sarcomere behaviour during twitch of intact single cardiac cells

Behaviour of sarcomere length was analysed in different regions of single cardiac cells (n = 249) of the ventricle, both at rest (n = 144) and during twitch contractions (n = 57). At rest, regional distribution of sarcomere length proved to be uniform. In the leaky cell (n = 48), resting sarcomere length was not affected over longer periods of time (up to 2 h), nor by lowering the ATP concentration (from 5 mM to 2.5 mM and 500 microM), nor by increasing free calcium within subactivating ranges (5, 20, 60 microM). No statistical differences could be detected between resting cell dimensions and sarcomere length between cells isolated from left and right ventricle (n = 64), nor between cells from epicardial or endocardial layers (n = 80). During twitch contraction in the intact unloaded cardiac cell (n = 32), sarcomere lengths in different regions were analysed every 20 ms and behaved synchronously, presenting arguments for uniformity during the myocardial contraction-relaxation cycle in the free-lying intact cardiac cell.     

The shift in muscle’s length-tension relation after exercise attributed to increased series compliance

Eccentric exercise can produce damage to muscle fibres. Here damage indicators are measured in the medial gastrocnemius muscle of the anaesthetised cat after eccentric contractions on the descending limb of the muscle’s length-tension relation, compared with eccentric contractions on the ascending limb and concentric contractions on the descending limb. One damage indicator is a shift of the optimum length for peak active tension, in the direction of longer muscle lengths. The shift has been attributed to an increase in muscle compliance. It is a corollary of a current theory for the mechanism of the damage. With the intention of seeking further support for the theory, in these experiments we test the idea that other damage indicators, specifically the fall in twitch:tetanus ratio and in muscle force are due, in part, to such an increase in compliance. This was tested in an undamaged muscle by insertion of a compliant spring (0.19 mm N⁻¹) in series with the muscle. This led to a fall in tetanic tension by 17%, a shift in optimum length of 1.7 mm in the direction of longer muscle lengths and a fall in twitch tetanus ratio by 15%. The fall in tension is postulated to be due to development of non-uniform sarcomere lengths within muscle fibres. It is concluded that after a series of eccentric contractions of a muscle, the fall in force is the result of a number of interdependent factors, not all of which are a direct consequence of the damage process.