Bogdan Iorga

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.

Kinetics of cardiac sarcomeric processes and rate-limiting steps in contraction and relaxation

The sarcomere is the core structure responsible for active mechanical heart function. It is formed primarily by myosin, actin, and titin filaments. Cyclic interactions occur between the cross-bridges of the myosin filaments and the actin filaments. The forces generated by these cyclic interactions provide the molecular basis for cardiac pressure, while the motion produced by these interactions provides the basis for ejection. The cross-bridge cycle is controlled by upstream mechanisms located in the membrane and by downstream mechanisms inside the sarcomere itself. These downstream mechanisms involve the Ca(2+)-controlled conformational change of the regulatory proteins troponin and tropomyosin and strong cooperative interactions between neighboring troponin-tropomyosin units along the actin filament. The kinetics of upstream and downstream processes have been measured in intact and demembranated myocardial preparations. This review outlines a conceptual model of the timing of these processes during the individual mechanical heart phases. Particular focus is given to kinetic data from studies on contraction-relaxation cycles under mechanical loads. Evidence is discussed that the dynamics of cardiac contraction and relaxation are determined mainly by sarcomeric downstream mechanisms, in particular by the kinetics of the cross-bridge cycle. The rate and extent of ventricular pressure development is essentially subjected to the mechanistic principles of cross-bridge action and its upstream and downstream regulation. Sarcomere relengthening during myocardial relaxation plays a key role in the rapid decay of ventricular pressure and in early diastolic filling.

The slow skeletal muscle isoform of myosin shows kinetic features common to smooth and non-muscle myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the β-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to ∼1000 s–1, a very similar rate constant to MHC-2, but 10–15% of the complex must go through a slow isomerization (∼20 s–1) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin·ADP and provide evidence of three closely related actin-myosin·ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (∼20 s–1), and a third complex that releases ADP too slowly (∼6 s–1) to be on the main ATPase pathway. The role of these actin-myosin·ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Why choose myofibrils to study muscle myosin ATPase

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction. In the absence of calcium, myofibrillar ATPase activity is basal, no shortening occurs and no active force is developed. In the presence of calcium, myofibrillar ATPase is activated and myofibrils either shorten with no external load (native myofibrils) or contract isometrically (cross-linked myofibrils). With this organised system, both chemical and mechanical studies can be carried out. For a decade, our laboratory has been using rabbit psoas myofibrils for exploring myosin ATPase activity. The first challenge was to successfully apply rapid kinetic approaches, such as rapid-flow-quench, to this organised system. Another challenge was to work with myofibrils in cryoenzymic conditions, i.e. in the presence of organic solvents and at sub-zero temperatures. In this overview, we highlight differences between the myosin ATPase in organised systems (myofibrils or fibres) and that of contractile proteins in solution (S1 or actoS1) that we observed using these approaches. We discuss the importance of these differences in terms of mechanochemical coupling. It is concluded that great care should be taken when extrapolating mechanochemical properties of the contractile proteins in solution to the whole muscle.

Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling

Sudden Ca2+ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152–2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (∼10xa0ms) after Ca2+ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (∼24xa0ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca2+ removal until troponin C is switched off. However, this switch off occurs ∼5xa0times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.

At Physiological Temperatures the ATPase Rates of Shortening Soleus and Psoas Myofibrils Are Similar

We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4-35 degrees C) with energies of activation for soleus myofibrils 155 kJ mol(-1) (activated) and 78 kJ mol(-1) (relaxed). With psoas myofibrils, the energies of activation were 71 kJ mol(-1) (activated) and 60 kJ mol(-1) (relaxed). When extrapolated to 42 degrees C the ATPase rates of the two types of myofibril were identical: 50 s(-1) (activated) and 0.23 s(-1) (relaxed). Whereas with psoas myofibrils the K(m) for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3 microM at 4 degrees C to 66.5 microM at 35 degrees C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties.

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (kACT) after Ca2+ activation and of force decay (τREL−1) during relaxation upon Ca2+ removal, isometric force at maximal (Fmax) or partial Ca2+ activations, and force response to an external stretch applied to the relaxed myofibril (Fpass). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that kACT, Fmax, Ca2+ sensitivity of the force, and Fpass were comparable and τREL−1 was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease–related mutations in sarcomeric proteins in the zebrafish model.

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M. ADP.Pi states predominate on the cross-bridge cycle.

The ATPases (±Ca2+) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different Ea: −Ca2+, 78 and 60 kJ/mol and +Ca2+, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) BiophysJ85: 3132–3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the Ea data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4°C. We used two methods: rapid flow quench (`cold ATP chase: titration of active sites, ATP binding kinetics, kcat; `Pi burst: ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free Pi; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M·ADP·Pi. We propose a reaction pathway that includes several (A)M·ADP·Pi sub-states that are either `weak or `strong, depending on the mechanical condition.

Paradoxical effects of endurance training and chronic hypoxia on myofibrillar ATPase activity.

This study aimed to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and myosin heavy chain (MHC) isoform expression after endurance training and/or chronic hypoxic exposure. Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N; n = 14), normoxic endurance trained (NT; n = 14), hypoxic sedentary (H; n = 10), and hypoxic endurance trained (HT; n = 14). Rats lived and trained in normoxia at 760 mmHg (N and NT) or hypobaric hypoxia at 550 mmHg (approximately 2,800 m) (H and HT). m-ATPase activity was measured by rapid flow quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. Endurance training significantly increased m-ATPase (P < 0.01), although an increase in MHC-I content occurred (P < 0.01). In spite of slow-to-fast transitions in MHC isoform distribution in chronic hypoxia (P < 0.05) no increase in m-ATPase was observed. The rate constants of m-ATPase were 0.0350 +/- 0.0023 s(-1) and 0.047 +/- 0.0050 s(-1) for N and NT and 0.033 +/- 0.0021 s(-1) and 0.038 +/- 0.0032 s(-1) for H and HT. Thus, dissociation between variations in m-ATPase and changes in MHC isoform expression was observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation, could not explain the variations in m-ATPase. Myosin posttranslational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.

ATP binding and cross‐bridge detachment steps during full Ca2+ activation: comparison of myofibril and muscle fibre mechanics by sinusoidal analysis

•u2002 Sinusoidal length change was employed to study the single myofibril mechanics during full Ca2+ activation for the first time, and the tension time course results were compared with those of single muscle fibres. •u2002 With myofibrils, the rate constants of exponential processes (tension transients) B and C were very close to each other at 8 mm phosphate, indicating that they could not be analysed independently. Thus, the sum and the product of the two rate constants were fitted to a cross‐bridge model. •u2002 The results demonstrate that the association constant of MgATP to cross‐bridge is K1= 2.91 mm−1, and the rate constants of the cross‐bridge detachment step are k2= 288 s−1 and k−2= 10 s−1. These values compare to those for fibres: K1= 2.35 mm−1, k2= 243 s−1, and k−2= 6 s−1, which are respectively not significantly different from myofibril values. •u2002 With inspection of video records, we did not observe any local shortening, wave propagation, or sarcomere inhomogeneity along myofibrils during isometric contraction or while applying sinusoidal length oscillations, indicating the integrity of the analysis method and the data acquired.