Different actions of salt and pyrophosphate on protein extraction from myofibrils reveal the mechanism controlling myosin dissociation.

BACKGROUND: Myosin is the major functional protein in muscle foods for water retention, protein binding/gelation and fat holding/emulsification. To maximize its functionality, myosin needs to be released from thick filaments. Understanding of the mechanism controlling myosin extraction will help improve quality traits of meat products. RESULTS: The data obtained show that actomyosin binding is the rate-limiting constraint for myosin release in rigor condition. Magnesium pyrophosphate (MgPPi) increased myosin extraction by weakening actomyosin interaction and maximized myosin extraction at 0.4 mol L(-1) NaCl, which was not attained at 1.0 mol L(-1) NaCl in the absence of PPi. Interaction between myosin rod domains is another critical constraint for myosin extraction, which is, rather than PPi, salt dependent. Further, our data suggest that MyBP-C (myosin binding protein C) and M-line might not be of significance in the process of NaCl-induced myosin extraction, though further study was needed. CONCLUSION: Our study provides new insight into the mechanism that controls myosin extraction from intact sarcomere, which could be applied to maximize myosin function and to improve meat quality in practice.

Effect of hypertrophic cardiomyopathy-linked troponin C mutations on the response of reconstituted thin filaments to calcium upon troponin I phosphorylation.

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudophosphorylation on the rate of dissociation of calcium from reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of dissociation of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was weakened. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was weakened after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted a minor to no effect on the rate of dissociation of calcium from reconstituted thin filaments or on the calcium sensitivity of actomyosin ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study enhances our understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.

A kinetic model of troponin dissociation in relation to thin filament regulation in striated muscle.

The apparent rate of troponin (Tn) dissociation from myofibrils has been used as a method to study thin filament regulation in striated muscle. The rate is dependent upon calcium and strong crossbridges and supports the three-state model for thin filament regulation. The dissociation rate of Tn is extremely low so it is not intuitively clear that such a slow process would probe thin filament regulation. We have investigated this issue by developing a simple kinetic model to explain the Tn dissociation rate measured by labeled Tn exchange in the myofibrils. Tn is composed of three interacting subunits, TnC, TnI and TnT. In our model, TnI's regulatory domain switches from actin-tropomyosin to TnC followed by TnT dissociation from actin-tropomyosin. This TnI regulatory domain switching is linked to the transition of the thin filament from the blocked state to the closed state. It is calcium dependent and several orders of magnitude faster than TnT dissociation from actin-tropomyosin. By integrating the dimensionless rate equations of this model, we have computed the time course of each of the various components. In our numerical simulations, the rate constant for TnI switching from actin-tropomyosin to TnC was varied from 10 s⁻¹ to 1000 s⁻¹ to simulate the low calcium, blocked state to high calcium, closed state. The computed progress curves for labeled Tn exchange into the myofibrils and the derived intensity ratio between the non-overlap and overlap regions well explains the intensity ratio progress curves observed experimentally. These numerical simulations and experimental observations reveal that the apparent rate of Tn dissociation probes the blocked state to closed state equilibrium of the myofibrillar thin filament.

Measurement of calcium dissociation rates from troponin C in rigor skeletal myofibrils.

Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca(2+) dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca(2+) dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin C(IAEDANS), reported Ca(2+) dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca(2+) dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin [Formula: see text] reported Ca(2+) dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin [Formula: see text] reported a biphasic fluorescence change upon Ca(2+) dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin [Formula: see text] and troponin C(IAEDANS) at all levels of the troponin C systems. Furthermore, the rate of Ca(2+) dissociation from troponin C in the myofibrils was similar to the rate of Ca(2+) dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca(2+) dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca(2+) dissociation from troponin C may influence relaxation.

Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment.

To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.

Tripolyphosphate hydrolysis by bovine fast and slow myosin subfragment 1 isoforms.

Polyphosphates are used in the meat industry to increase the water holding capacity of meat products. Tripolyphosphate (TPP) is a commonly used polyphosphate and it is metabolized into pyrophosphate and monophosphate in meat. The enzymes responsible for its metabolism have not been fully characterized. The motor domain of myosin (subfragment 1 or S1) is a likely candidate. The objectives of this study were to determine if bovine S1 hydrolyzes TPP, to characterize the TPPase activity of the fast (cutaneous trunci) and slow (masseter) isoforms, and to determine the influence of pH on S1 TPPase activity. S1 hydrolyzed TPP and in comparison with ATP as substrate, it hydrolyzed TPP 16-32% more slowly. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The V(max) was 0.94 and 5.0 nmol Pi/mg S1 protein/min while the K(m) was 0.38 and 0.90 mM TPP for slow and fast S1, respectively. Pyrophosphate was a strong inhibitor of TPPase activity with a K(i) of 88 and 8.3 microM PPi for fast and slow S1 isoforms, respectively. Both ATPase and TPPase activities were influenced by pH with the activity being higher at low pH for both fast and slow S1 isoforms. The activity at pH 5.4 was 1.5 to 4-fold higher than that at pH 7.6 for the different isoforms and substrates. These data show that myosin S1 readily hydrolyzes TPP and suggest that it is a major TPPase in meat.

Effect of calcium-sensitizing mutations on calcium binding and exchange with troponin C in increasingly complex biochemical systems.

The calcium-dependent interactions between troponin C (TnC) and other thin and thick filament proteins play a key role in the regulation of cardiac muscle contraction. Five hydrophobic residues (Phe(20), Val(44), Met(45), Leu(48), and Met(81)) in the regulatory domain of TnC were individually substituted with polar Gln, to examine the effect of these mutations that sensitized isolated TnC to calcium on (1) the calcium binding and exchange with TnC in increasingly complex biochemical systems and (2) the calcium sensitivity of actomyosin ATPase. The hydrophobic residue mutations drastically affected calcium binding and exchange with TnC in increasingly complex biochemical systems, indicating that side chain intra- and intermolecular interactions of these residues play a crucial role in determining how TnC responds to calcium. However, the mutations that sensitized isolated TnC to calcium did not necessarily increase the calcium sensitivity of the troponin (Tn) complex or reconstituted thin filaments with or without myosin S1. Furthermore, the calcium sensitivity of reconstituted thin filaments (in the absence of myosin S1) was a better predictor of the calcium dependence of actomyosin ATPase activity than that of TnC or the Tn complex. Thus, both the intrinsic properties of TnC and its interactions with the other contractile proteins play a crucial role in modulating the binding of calcium to TnC in increasingly complex biochemical systems.

Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin.

The kinetics of myosin dissociation from actin was investigated and also the impact of salt, MgPPi, and myosin heavy chain isoform on myosin subfragment 1 (S1) dissociation from actin using purified proteins and fluorescence spectroscopy. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentrations increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine masseter (slow) and cutaneous trunci (fast) muscle increased 38 and 78 fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin. With the addition of MgPPi to the mixture of pyrene actin and S1, the fluorescence increased about 85% within the dead time of the mixing approach.. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin. The results reveal that salt and MgPPi increase myosin extraction and functionality in meat by weakening the actomyosin interaction and that some of the difference in the functionality of red and white muscle may be related to actomyosin dissociation.

Differences between cardiac and skeletal troponin interaction with the thin filament probed by troponin exchange in skeletal myofibrils.

Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa-ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa50 = 6.14, nH = 1.46) than sTn myofibrils (pCa50= 5.90, nH = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were approximately 2-10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 x 10(-3) min(-1), 150 x 10(-3) min(-1), and 260 x 10(-3) min(-1), whereas for sTn they were 0.6 x 10(-3) min(-1), 88 x 10(-3) min(-1), and 68 x 10(-3) min(-1) for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.

Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W.

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.

Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C.

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells.

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments.

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.

Smooth muscle adherens junctions associated proteins are stable at the cell periphery during relaxation and activation.

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca2+-free PSS or regular PSS or were activated in 10 muM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane.

Unloaded shortening velocity in single permeabilized vascular smooth muscle cells is independent of microtubule status.

Microtubules may influence smooth muscle contraction either via involvement in signal transduction processes or by serving as an internal load that opposes contraction. To test the latter hypothesis, microtubule distribution and the unloaded shortening velocity were investigated in freshly isolated single vascular smooth muscle cells (VSMCs) treated with microtubule modulating drugs. Immunocytochemical studies showed that microtubules run mainly longitudinally in relaxed VSMCs. They are oriented more obliquely, almost transversely to the long axis of the cells after contraction, suggesting that microtubules are compressed during shortening, and thus might impart an internal passive load. Quantitative immunocytochemical analysis revealed that, relative to the control group, colchicine (15 microM) decreased the microtubule density by 40% while taxol (10 microM) increased the microtubule density by 46%. However, alteration of microtubule polymerization status by these microtubule-modulating drugs did not have a significant effect on unloaded shortening velocity in alpha-toxin permeabilized VSMCs under maximal activating conditions or submaximal activating conditions (about 36% of maximal velocity). These results suggest that microtubules do not present an appreciable internal load to dampen single VSMCs shortening in the present experimental system, and that their influence on smooth muscle contraction is primarily via signal transduction mechanisms.