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This symposium celebrates the seminal discovery of troponin by Professor Ebashi. In the 1960s we knew quite a bit about how muscle functions. It was established that contraction was the result of the interaction of ATP with the complex formed from actin and myosin. The filamentous structure and the constancy of the A-band of striated muscle led to the sliding filament theory., A detailed model relating muscle mechanics with the cross bridge cycle was produced. A change in orientation of the cross bridge between rest and rigor in insect muscle was also demonstrated. However, we were ignorant regarding how muscles stay relaxed.

Plasmodia of Physarum polycephalum shows vigorous cytoplasmic streaming by changing direction every few minutes. This oscillatory streaming is regulated by Ca and is thought to be driven by a conventional myosin, i.e., by a myosin II isoform., While working as an assistant professor in Professor Ebashi’s laboratory at the University of Tokyo, one of the present authors (K.K.) induced the superprecipitation of actomyosin preparation or myosin B from the plasmodia to examine the effect of Ca. It superprecipitated without requiring Ca. When Ca at μM level was present, the superprecipitation was inhibited. This calcium inhibition was quite the opposite of the superprecipitation of actomyosin from vertebrate muscles, and we expected that the inhibitory mode could be involved in the plant cytoplasmic streaming. With the finding of the diverse classes of unconventional myosin such as myosin I and V in vertebrate muscles, the inhibitory mode was shown to play a role in cell motility in both animal and plant kingdoms. In this case the myosins have calmodulin (CaM) as the light chains and are regulated by interaction of Ca with CaM, which exerts an inhibitory effect on activity.

In early 1960s clear evidence was presented by Professor S. Ebashi for the fact that contraction-relaxation cycle of living muscle is regulated by calcium ion (Ca) (cf. ). He then inquired into the mechanism of the action of Ca and disclosed that the regulation of contractile reaction by Ca requires the presence of a protein component other than myosin and actin (). A few years later, he showed that the protein component is a complex of a known protein, tropomyosin, and a new protein, troponin ().

Ca released through the Ca release channel triggers muscle contraction. The Ca release channel in the sarcoplasmic reticulum (SR) of the striated muscles is referred to as the ryanodine receptor (RyR), and is so named because of its binding ability of the open state with a high affinity to ryanodine.– Three genetically distinct isoforms (RyR1-3) are identified in mammals: RyR1 is the primary isoform in the skeletal muscle, RyR2 in the cardiac muscle, and RyR3 is ubiquitously expressed, although in a minuscule amount. In non-mammalian vertebrate skeletal muscles, e.g., chicken, frog, and fish, two isoforms referred to as α- and β-RyR are expressed in almost equal amounts. Further studies show that α- and β-RyR are homologs of RyR1 and RyR3, respectively, and that RyR3 is much degenerated and almost disappears in adult mammalian skeletal muscles except diaphragm and soleus.,,

Ca-ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) is an integral membrane protein of 110K and the best characterised member of the P-type (or E1/E2-type) ion translocating ATPases. It was first identified by Ebashi in the “relaxing factor” of muscle contraction and gave rise to the calcium theory that Ca is a fundamental and ubiquitous factor in the regulation of intracellular processes. There are several types of Ca-ATPases in different tissues; all transfer Ca from the cytoplasm to the opposite side of the membrane and countertransport H. Stoichiometry of Ca: ATP may be variable but it is well established that SERCA1a can transfer two Ca per ATP hydrolysed. In the sarcoplasmic reticulum (SR) membrane, Ca-ATPase pumps Ca, released into muscle cells during muscle contraction, back into SR, thereby relaxes muscle cells. This pump runs as long as ATP and Ca are present, and establishes more than 10-fold concentration gradient across membranes. According to the classical E1/E2 theory, transmembrane Ca-binding sites have high affinity and face the cytoplasm in E1; in E2, the binding sites have low affinity and face the lumen of SR (extracellular side)., Actual transfer of bound Ca is thought to take place between two phosphorylated intermediates, E1P and E2P, in exchange of H. Because 2 Ca are transferred in the forward direction and 2 to 3 protons in the opposite direction, active transport of Ca is an electrogenic process. Although no H gradient is built up across the SR membrane because it is leaky to H, this Ca/H exchange may cause pathological pH effects with plasma membrane Ca-ATPase.

Since the initial discovery of the regulatory role of intracellular Ca signals in skeletal muscle contraction (; ), the list of cellular functions that are regulated by Ca signals has expanded. Now, it is recognized that intracellular Ca signals are involved in the regulation of various cell functions including fertilization, secretion, transcription, immunity, learning and memory (). One of the striking features of Ca signals is that they display complex spatiotemporal distributions such as Ca waves and oscillations. An oscillatory change in Ca concentration was first observed in skinned fiber experiments by Endo and collaborators in 1970 (). When skinned skeletal muscle fibers were immersed in a solution mimicking intracellular conditions and caffeine was added to the solution at millimolar concentrations, the skinned fibers underwent periodic contractions because of the periodic release of Ca from the sarcoplasmic reticulum, in the absence of membrane potential changes. With the advent of methods of measuring intracellular Ca concentration, Ca oscillation has been observed in many types of intact cell. In 1986, Cobbold and collaborators () observed Ca oscillation in agonist-stimulated hepatocytes using aequorin, a Ca-sensitive luminescent protein. The introduction of fluorescent Ca indicators further facilitated the observation of intracellular Ca transients (). Initially, the physiological significance of Ca oscillation was not fully appreciated, because they were observed only in cell lines or in isolated cells.

It has been a great honor and a particular pleasure to participate in this meeting to celebrate the fortieth anniversary of the discovery of troponin by Professor Ebashi, whom I have been privileged to know for many years of my scientific life. I thought therefore it would be appropriate to described briefly what was happening in studies of another aspect of muscle contraction over somewhat the same time period.

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

A molecular motor in striated muscle, myosin II, is a non-processive motor that is unable to perform physiological functions as a single molecule and acts as an assembly of molecules. It is widely accepted that a myosin II motor is an independent force generator; the force generated at a steady state is usually considered to be a simple sum of those generated by each motor. This is the case at full activation (pCa <5 in the presence of MgATP); however, we found that the myosin II motors show cooperative functions, i.e., non-linear auto-oscillation, named SPOC (SPontaneous Oscillatory Contraction), when the activation level is intermediate between those of contraction and relaxation (that is, at the intermediate level of pCa, 5∼6, for cardiac muscle, or at the coexistence of MgATP, MgADP and inorganic phosphate (Pi) at higher pCa (>7) for both skeletal and cardiac muscles). Here, we summarize the characteristics of SPOC phenomena, especially focusing on the physiological significance of SPOC in cardiac muscle. We propose a new concept that the auto-oscillatory property, which is inherent to the contractile system of cardiac muscle, underlies the molecular mechanism of heartbeat. Additionally, we briefly describe the dynamic properties of the thin filaments, i.e., the Ca-dependent flexibility change of the thin filaments, which may be the basis for the SPOC phenomena. We also describe a newly developed experimental system named “bio-nanomuscle,” in which tension is asserted on a single reconstituted thin filament by interacting with crossbridges in the A-band composed of the thick filament lattice. This newly devised hybrid system is expected to fill the gap between the single-molecule level and the muscle system.

Muscle contraction is caused by relative sliding movement between interdigitating actin and myosin filaments. It has been thought that myosin heads protruding from the myosin filament rotate between two orientations, while they repeat detachment from and attachment to actin filament coupled to the ATP hydrolysis cycle and the rotation of the head may cause the sliding. Recently atomic structure obtained from X-ray crystallography supports the rotation of the myosin head relative to the actin filament. A small conformational change in the ATP binding domain is transmitted to a neck domain that connects a motor domain (head) and tail domain, depending on the chemical state of nucleotide bound. Thus the neck domain acts as a lever-arm that can cause a displacement of 5–10 nm for the muscle myosin. This lever-arm swinging model has been a paradigm not only for the muscle myosin but also for unconventional myosins. Large stepsize of unconventional processive myosin V motor can be explained by its large lever arm within the frame of the lever-arm swinging model.