Assessment of the mechanical activity of cardiac isomyosins V1 and V3 by the in vitro motility assay with regulated thin filament

The dependences of thin filament sliding velocity on the calcium concentration in solution (pCa 5 to 8) for rabbit cardiac myosin isoforms V1 and V3 were determined in a set of experiments using an in vitro motility assay with a reconstructed thin filament. The constructed pCa-versus-velocity curves had a sigmoid shape. It was demonstrated that the sliding velocity of regulated thin filament at the saturating calcium concentration (pCa 5) did not differ from the actin sliding velocity for each isoform. The determined values of Hill’s cooperativity coefficient for isomyosins V1 and V3 were 1.04 and 0.75, respectively. It was demonstrated that isomyosin V3 was more sensitive to calcium as compared with isomyosin V1. Using the same assay, the dependence of thin filament sliding velocity on the concentration of the actin-binding protein α-actinin (analog of a force-velocity dependence) was determined at the saturating calcium concentration for each myosin isoform (V1 and V3). The results suggest that the calcium regulation of V1 and V3 contractile activity follows different mechanisms.     

Signal transduction and protein phosphorylation in smooth muscle contraction

Smooth muscles are important constituents of vertebrate organisms that provide for contractile activity of internal organs and blood vessels. Basic molecular mechanism of both smooth and striated muscle contractility is the force-producing ATP-dependent interaction of the major contractile proteins, actin and myosin II molecular motor, activated upon elevation of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, whereas striated muscles display a proportionality of generated force to the [Ca²⁺]_i level, smooth muscles feature molecular mechanisms that modulate sensitivity of contractile machinery to [Ca²⁺]_i. Phosphorylation of proteins that regulate functional activity of actomyosin plays an essential role in these modulatory mechanisms. This provides an ability for smooth muscle to contract and maintain tension within a broad range of [Ca²⁺]_i and with a low energy cost, unavailable to a striated muscle. Detailed exploration of these mechanisms is required to understand the molecular organization and functioning of vertebrate contractile systems and for development of novel advances for treating cardiovascular and many other disorders. This review summarizes the currently known and hypothetical mechanisms involved in regulation of smooth muscle Ca²⁺-sensitivity with a special reference to phosphorylation of regulatory proteins of the contractile machinery as a means to modulate their activity.     

Overexpression of SERCA2 ATPaase in vascular smooth muscle cells treated with oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca²⁺ _i homeostasis and altered two Ca²⁺ release mechanisms in these cells (IP₃ and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca²⁺ pump. One of the primary Ca²⁺ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP₃ and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca²⁺ homeostasis in VSMC chronically exposed to atherogenic oxLDL.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Study of the interaction between rabbit cardiac contractile and regulatory proteins. An <Emphasis Type="Italic">in vitro</Emphasis> motility assay

A series of experiments was performed in an in vitro motility assay with reconstructed thin filaments to obtain pCa-force relationships for cardiac isomyosins V1 and V3. Two concentrations of each isomyosin (200 and 300 microg/ml) on the surface of a flow cell were tested. Isometric force was estimated as the amount of actin-binding protein, alpha-actinin, stopping thin filament movement. It was found that the amount of alpha-actinin stopping the movement at saturating calcium concentration for V3 was twice higher than for V1 at both concentrations of isoforms. Hill coefficients of cooperativity (h) were determined for pCa-force relationships. The value of h did not differ significantly for isoforms at 300 microg/ml of protein (h was 1.56 for V1 and 1.54 for V3). However, the Hill coefficient was higher for V3 isoform at 200 microg/ml (h = 2.00 and 1.76 for V3 and V1, respectively). Importantly, the Hill coefficient increased for both isoenzymes when their concentrations were decreased. The connection between Hill coefficient and cooperative interactions between cardiac contractile and regulatory proteins is analyzed in detail.     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca²⁺ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca²⁺ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca²⁺ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Calcium current kinetics in young and aged human cultured myotubes

There is evidence that the complex process of sarcopenia in human aged skeletal muscle is linked to the modification of mechanisms controlling Ca²⁺ homeostasis. To further clarify this issue, we assessed the changes in the kinetics of activation and inactivation of T- and L-type Ca²⁺ currents in in vitro differentiated human myotubes, derived from satellite cells of healthy donors aged 2, 12, 76 and 86 years. The results showed an age-related decrease in the occurrence of T- and L-type currents. Moreover, significant age-dependent alterations were found in L-(but not T) type current density, and activation and inactivation kinetics, although an interesting alteration in the kinetics of T-current inactivation was observed. The T- and L-type Ca²⁺ currents play a crucial role in regulating Ca²⁺ entry during satellite cells differentiation and fusion into myotubes. Also, the L-type Ca²⁺ channels underlie the skeletal muscle excitation–contraction coupling mechanism. Thus, our results support the hypothesis that the aging process could negatively affect the Ca²⁺ homeostasis of these cells, by altering Ca²⁺ entry through T- and L-type Ca²⁺ channels, thereby putting a strain on the ability of human satellite cells to regenerate skeletal muscle in elderly people.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Molecular structure of troponin C from chicken skeletal muscle at 3Å resolution

Troponin C is the Ca²⁺-binding subunit of the troponin complex and is involved in the calcium control of muscle contraction. The X-ray structure of chicken TnC has been determined at 3Å resolution using a single heavy atom derivative and application of a novel phase improvement and phase extension procedure. The protein has an unusual dumbbell-shape with a length of about 70A. The N- and C-domains are connected by a single long α-helix of about 9 turns. Two metal binding sites (the Ca²⁺-Mg²⁺ sites) in the C-domain are occupied by metal ions in the crystals and the helix-loop-helix Ca²⁺ -binding folds are very similar to those in other known Ca²⁺ -binding proteins. In contrast, the Ca²⁺ -specific sites in the N-domain appear unoccupied and the two putative Ca²⁺ -binding folds have a vastly different structural arrangement. The conformational rearrangements in the N-domain upon Ca²⁺ binding are believed to be the trigger for a cascade of protein-protein interaction alterations which lead to muscle contraction.     

Oxygen free radicals and calcium homeostasis in the heart

Many experiments have been done to clarify the effects of oxygen free radicals on Ca²⁺ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca²⁺ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca²⁺ binding is increased, Ca²⁺ influx through Ca²⁺ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca²⁺ can enter the myocytes is Na⁺?Ca²⁺ exchanger. Although, the activities of Na⁺?K⁺ ATPase and Na⁺?H⁺ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na⁺ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na⁺?Ca²⁺ exchange in Ca²⁺ influx process from extracellular space. Another question is ‘which way does Na⁺?Ca²⁺ exchange work under oxidative stress? Net influx or efflux of Ca^2+?’ Membrane permeability for Ca²⁺ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca²⁺-pump ATPase activity is depressed by oxygen free radicals, Ca²⁺ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca²⁺ release from sarcoplasmic reticulum and inhibit Ca²⁺ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca²⁺ handling systems could cause the Ca²⁺ overload due to oxygen free radicals.     

Sarcoplasmic calcium-binding proteins in protochordate and cyclostome muscle

Summary A sarcoplasmic calcium-binding protein (SCP) has been purified from the muscle of the protochordate Amphioxus and shown to be more similar to invertebrate SCP's than to their counterpart found in vertebrates, i.e. parvalbumins. The Amphioxus protein has a pI of 4.9, is rich in tyrosine and tryptophan, has a molecular weight of 22,000 and binds strongly 2Ca²⁺ with a pK of 7.88. Magnesium competes with calcium for only one of the two metal-binding sites and induces positive cooperativity in Ca²⁺ binding.In cyclostome muscle (lamprey and hagfish), no protein with high affinity for Ca²⁺ or Mg²⁺ could be found, irrespective of molecular weight. Instead, a protein with moderate affinity for Ca²⁺ (10⁵ m ^–1) was detected: it has a molecular weight of 60,000 and might be quite ubiquitous, as the presence of a similar protein has been reported both in red and white muscle of vertebrates such as chicken and rabbit.     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.