Physiological consequences of thin filament cooperativity for vertebrate striated muscle contraction: a theoretical study

Bindings of both myosin and Ca²⁺ to the thin filament of vertebrate striated muscle are known to be strongly cooperative. Here the relation between these two sources of cooperativity and their consequences for physiological properties are assessed by comparing two models, with and without Monod-type myosin-binding cooperativity. In both models a thin filament regulatory unit (RU) is in either ‘off’ or ‘on’ state, and the equilibrium between them (K _on) is [Ca²⁺]-dependent. The calculations predict the following: (1) In both models, myosin binding stabilizes the RU in the ‘on’ state, causing troponin to trap Ca²⁺. This stabilization in turn increases the Ca²⁺-binding cooperativity, ensuring efficient regulation to occur in a narrow [Ca²⁺] range. (2) In the cooperative model, the RU is stabilized with a relatively low myosin affinity for actin (K∼ ∼1), while the non-cooperative model requires a much higher affinity (K∼ ∼ 10) to produce the same effect. (3) The cooperative model reproduces the known effects of [Ca²⁺] on the rate of force development and shortening velocity with a low K, but again the non-cooperative model requires a higher value. (4) Because of the finite value of K _on, the thin filaments can never be fully activated by increasing [Ca²⁺], indicating that contracting muscles are under strong influence of thin-filament cooperativity even at saturating [Ca²⁺]. Interpretation of data on muscle mechanics without considering these cooperative effects could therefore lead to a substantial (10-fold) overestimate of cross-bridge binding properties.     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

Residual sarcoplasmic reticulum Ca<Superscript>2+</Superscript> concentration after Ca<Superscript>2+</Superscript> release in skeletal myofibers from young adult and old mice

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca²⁺ or significant residual Ca²⁺ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca²⁺ indicators targeted to the SR that exhibit low-Ca²⁺ affinity, a fast biosensor: Ca²⁺ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca²⁺ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca²⁺ with aging. We found CatchER a proper tool to investigate SR Ca²⁺ depletion in young adult and older adult mice, consistently tracking SR luminal Ca²⁺ release in response to brief and repetitive stimulation. We evoked SR Ca²⁺ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca²⁺ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca²⁺ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca²⁺ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.     

A reappraisal of the Ca<Superscript>2+</Superscript> dependence of fast inactivation of Ca<Superscript>2+</Superscript> release in frog skeletal muscle

Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca²⁺ Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca²⁺ release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca²⁺-free conditions. 2-APB applied extracellularly at 100 μM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between −45 and −35 mV from 7.99 ± 0.73 nC/μF (N = 17) before drug application to 6.27 ± 0.68 nC/μF in the presence of 2-APB. This effect was mostly on the delayed Q_γ component. In fibers treated with the SERCA ATPase inhibitor CPA the Q_γ component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca²⁺ release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 μM. 2-APB also inhibited the L-type Ca²⁺ current (20 ± 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 μM, a typical concentration used for inhibiting SOCE, to fibers held at −80 mV inhibited the charge moved in response to pulses ranging −45 to −30 mV from 7.95 ± 2.59 nC/μF to 3.48 ± 0.9 nC/μF (N = 12). A parallel reduction of Ca²⁺ release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to −100 mV. SKF-96365 also inhibited the L-type Ca²⁺ current (41 ± 8%, N = 4) and increased its rate of inactivation.     

Voltage-gated Ca<Superscript>2+</Superscript> channel mRNAs and T-type Ca<Superscript>2+</Superscript> currents in rat gonadotropin-releasing hormone neurons

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the neuroendocrine regulation of reproduction. We have previously reported that rat GnRH neurons exhibit voltage-gated Ca²⁺ currents. In this study, oligo-cell RT-PCR was carried out to identify subtypes of the α₁ subunit of voltage-gated Ca²⁺ channels in adult rat GnRH neurons. GnRH neurons expressed mRNAs for all five types of voltage-gated Ca²⁺ channels. For T-type Ca²⁺ channels, α_1H was dominantly expressed in GnRH neurons. Electrophysiological analysis in acute slice preparations revealed that GnRH neurons from adult rats exhibited T-type Ca²⁺ currents with fast inactivation kinetics (~20 ms at −30 mV) and a time constant of recovery from inactivation of ~0.6 s. These results indicate that rat GnRH neurons express subtypes of the α₁ subunit for all five types of voltage-gated Ca²⁺ channel, and that α_1H was the dominant subtype in T-type Ca²⁺ channels.     

Effects of membrane depolarization and changes in extracellular [K<Superscript>+</Superscript>] on the Ca<Superscript>2+</Superscript> transients of fast skeletal muscle fibers. Implications for muscle fatigue

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K⁺ gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K⁺ ( \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } ) concentration ( [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} ) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca²⁺ transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} and membrane potentials. Depolarization resulting from current injection or raised [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} produced an increase in the resting [Ca²⁺]. Ca²⁺ transients elicited by single stimulation were potentiated by depolarization from −80 to −60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca²⁺ transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca²⁺ transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca²⁺ transients along the trains were associated with impaired or abortive APs. Raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 10 mM potentiated Ca²⁺ transients elicited by single and tetanic stimulation, while raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 15 mM markedly depressed both responses. The effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on Ca²⁺ transients, but not those of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } might be mediated by depolarization dependent changes in resting [Ca²⁺] and Ca²⁺ release, and that additional mechanisms might be involved in the effects of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on force generation.     

Altered intracellular Ca<Superscript>2+</Superscript> regulation in chronic rat heart failure

Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF have not been clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake is required for decreased contractility, we investigated the cytosolic Ca²⁺ transients and SR Ca²⁺ content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca²⁺ transients were markedly reduced in amplitude in HF myocytes (ΔF/F ₀ = 12.3 ± 0.8) compared with control myocytes (ΔF/F ₀ = 17.7 ± 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca²⁺ content (HF: ΔF/F ₀ = 12.4 ± 1.1, control: ΔF/F ₀ = 32.4 ± 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.     

cGMP reduces the sarcoplasmic reticulum Ca<Superscript>2+</Superscript> loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca<Superscript>2+</Superscript> by cGMP

Ca²⁺ and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca²⁺ and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca²⁺ storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca²⁺ has not been completely understood. To evaluate this role, intracellular Ca²⁺ concentration ([Ca²⁺]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca²⁺ from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca²⁺]i undershoot. Caffeine-induced Ca²⁺ transient peak and [Ca²⁺]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca²⁺]i undershoot and reduced the subsequent caffeine peak (SR-Ca²⁺ loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca²⁺ ATPase with cyclopiazonic acid inhibited the [Ca²⁺]i undershoot as well as the SR-Ca²⁺ loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca²⁺ loading. Cyclic GMP enhanced [Ca²⁺]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca²⁺ content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca²⁺ loading and the simultaneous release of SR-Ca²⁺. The reduction of SR-Ca²⁺ content induced by cGMP might be a putative mechanism limiting releasable Ca²⁺ in response to a particular stimulus.     

Lower active force generation and improved fatigue resistance in skeletal muscle from desmin deficient mice

The mechanical effects of the intermediate filament protein desmin was examined in desmin deficient mice (Des−/−) and their wild type control (Des+/+). Active force generation was determined in intact soleus muscles and in skinned single fibres from soleus and psoas. A decreased force generation of skinned muscle fibres from Des−/− mice and a tendency towards decreased active force in intact soleus muscle were detected. Concentrations of the contractile protein actin and myosin were not altered in Des−/− muscles. Ca²⁺-sensitivity of skinned single fibres in Des−/− muscles was unchanged compared to Des+/+. Using a protocol with repeated short tetani an increased fatigue resistance was found in the intact soleus muscles from Des−/− mice. In conclusion, desmin intermediate filaments are required for optimal generation or transmission of active force in skeletal muscle. Although other studies have shown that the desmin intermediate filaments appear to influence Ca²⁺-handling, the Ca²⁺-sensitivity of the contractile filaments is not altered in skeletal muscle of Des−/− mice. Previous studies have reported a switch towards slower myosin isoforms in slow skeletal muscle of Des−/− mice. The increased fatigue resistance show that this change is reflected in the physiological function of the muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Late sodium current contributes to diastolic cell Ca<Superscript>2+</Superscript> accumulation in chronic heart failure

We elucidate the role of late Na⁺ current (I_NaL) for diastolic intracellular Ca²⁺ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca²⁺ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). I_NaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca²⁺ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca²⁺ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective I_NaL blocker ranolazine (10 μM) or the prototypical Na⁺ channel blocker tetrodotoxin (2 μM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of I_NaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that I_NaL contributes to diastolic Ca²⁺ accumulation and spontaneous Ca²⁺ release in HF.     

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca²⁺ and voltage-gated potassium (BK_Ca) channels. Here, we explored the effect of LPI on intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels using excised inside-out patches from endothelial cells. The functional expression of IK_Ca was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK_Ca channels with a slope conductance of 39 pS in symmetric K⁺ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK_Ca single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK_Ca single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.     

Ca<Superscript>2+</Superscript> regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca²⁺ chelator or inhibitors of Ca²⁺-permeable channels or Ca²⁺-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca²⁺ channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca²⁺-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the γ subunit of epithelial Na⁺ channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 μM amiloride or 10 μM phenamil. Taken together, these findings suggest that [Ca²⁺]_c regulated by endoplasmic Ca²⁺-pump and Ca²⁺-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na⁺ across the basolateral membrane in marginal cells.     

Osteoblasts modulate Ca<Superscript>2+</Superscript> signaling in bone-metastatic prostate and breast cancer cells

Metastatic prostate and breast cancers display a predilection for the skeleton. The high incidence of skeletal metastasis may be a reflection of favorable reciprocal interactions between the bone microenvironment and disseminated cancer cells. Here we show that bone-metastatic PC3-ML prostate cancer cells and MDA-231 breast cancer cells—when co-cultured with human osteoblasts—down-regulate the increase in cytosolic free calcium (Ca²⁺) induced by agonist stimulation. This osteoblast promoted alteration of Ca²⁺ signaling develops and reverts in a time-dependent manner. Most importantly, the Ca²⁺ responses of cancer cells lacking bone metastatic potential are not affected by osteoblasts. The limited increase in cytosolic Ca²⁺ observed in bone-metastatic cells does not result from depleted intracellular Ca²⁺ stores but rather a decreased entry of Ca²⁺ from the extracellular space. Interestingly, the inhibition of histone deacetylase in cancer cells replicates the changes in Ca²⁺ signaling induced by osteoblasts, suggesting the participation of epigenetic mechanisms. Finally, cancer cells harvested from skeletal metastases induced in mice showed Ca²⁺ responses identical to cells co-cultured with osteoblasts. However, Ca²⁺ signaling in cancer cells recovered from metastases to soft-tissues was not affected, emphasizing the role of the bone microenvironment in regulating the functional behavior of bone-metastatic cells. We propose that osteoblasts protect selected malignant phenotypes from cell death caused by an excessive increase in cytosolic Ca²⁺, thereby facilitating their progression into macroscopic skeletal metastases.     

Differential effects of arginine,glutamate and phosphoarginine on Ca<Superscript>2+</Superscript>-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

KB-R7943 inhibits Na<Superscript>+</Superscript>-dependent Mg<Superscript>2+</Superscript> efflux in rat ventricular myocytes

Na⁺-dependent Mg²⁺ efflux activity was studied with the fluorescent Mg²⁺ indicator furaptra in the presence of various potential antagonists known to inhibit other transporters and channels. Among the compounds tested, KB-R7943, an inhibitor of Na⁺/Ca²⁺ exchange, most potently inhibited the Na⁺/Mg²⁺ exchange with half inhibitory concentrations (IC₅₀) of 21 μM (25°C) and 16 μM (35°C). These IC₅₀ values were a factor of three to four lower than those of imipramine, a widely used inhibitor of Na⁺/Mg²⁺ exchange. Apart from the inhibitory effect on Na⁺/Mg²⁺ exchange, relatively high concentrations of KB-R7943 (100 μM at 25°C and ≥20 μM at 35°C), in combination with prolonged UV-illumination, caused cell shortening, probably because of the phototoxicity of the compound and the formation of rigor crossbridges. We conclude that KB-R7943 may be a useful tool to study cellular Mg²⁺ homeostasis if care is taken to minimize its phototoxicity.     

Titin/connectin-based modulation of the Frank-Starling mechanism of the heart

The basis of the Frank-Starling mechanism of the heart is the increase in active force when muscle is stretched. Various findings have shown that muscle length, i.e., sarcomere length (SL), modulates activation of cardiac myofilaments at a given concentration of Ca²⁺ ([Ca²⁺]). This augmented Ca²⁺ activation with SL, commonly known as “length-dependent activation”, is manifested as the leftward shift of the force-pCa (=−log [Ca²⁺]) relation as well as by the increase in maximal Ca²⁺-activated force. Despite the numerous studies that have been undertaken, the molecular mechanism(s) of length-dependent activation is (are) still not fully understood. The giant sarcomere protein titin/connectin is the largest protein known to date. Titin/connectin is responsible for most passive force in vertebrate striated muscle and also functions as a molecular scaffold during myofibrillogenesis. Recent studies suggest that titin/connectin plays an important role in length-dependent activation by sensing stretch and promoting actomyosin interaction. Here we review and extend this previous work and focus on the mechanism by which titin/connectin might modulate actomyosin interaction.     

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca²⁺-activated force and reduces the effectiveness Ca²⁺ as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca²⁺-activated force and increase [Ca²⁺] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca²⁺ and RXB activated muscle contraction. Because maximum force can be reduced with no change—or even an increase—in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.     

Titin-based modulation of active tension and interfilament lattice spacing in skinned rat cardiac muscle

The effect of titin-based passive tension on Ca²⁺ sensitivity of active tension and interfilament lattice spacing was studied in skinned rat ventricular trabeculae by measuring the sarcomere length (SL)-dependent change in Ca²⁺ sensitivity and performing small angle X-ray diffraction studies. To vary passive tension, preparations were treated with trypsin at a low concentration (0.31 g/ml) for a short period (13 min) at 20°C, that resulted in ~40% degradation of the I-band region of titin, with a minimal effect on A-band titin. We found that the effect of trypsin on titin-based passive tension was significantly more pronounced immediately after stretch than at steady state, 30 min after stretch (i.e., trypsin has a greater effect on viscosity than on elasticity of passive cardiac muscle). Ca²⁺ sensitivity was decreased by trypsin treatment at SL 2.25 m, but not at SL 1.9 m, resulting in marked attenuation of the SL-dependent increase in Ca²⁺ sensitivity. The SL-dependent change in Ca²⁺ sensitivity was significantly correlated with titin-based passive tension. Small-angle X-ray diffraction experiments revealed that the lattice spacing expands after trypsin treatment, especially at SL 2.25 m, providing an inverse linear relationship between the lattice spacing and Ca²⁺ sensitivity. These results support the view that titin-based passive tension promotes actomyosin interaction and that the mechanism includes interfilament lattice spacing modulation.     

Effect of Ca<Superscript>2+</Superscript> gradient on the structure of sarcoplasmic reticulum membranes

The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca²⁺ gradient corresponding to the status of Ca²⁺ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca²⁺-ATPase, ryanodine receptor, and lipids). The transmembrane Ca²⁺ gradient modulates the protein-lipid interactions and structural characteristics of the membrane. The proposed model can be used for studies of the effects of pharmacologically active substances and endogenous regulators. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 517–521, November, 2007