Unloaded shortening of skinned muscle fibers from rabbit activated with and without Ca2+.

Unloaded shortening velocity (VUS) was determined by the slack method and measured at both maximal and submaximal levels of activation in glycerinated fibers from rabbit psoas muscle. Graded activation was achieved by two methods. First, [Ca2+] was varied in fibers with endogenous skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified form of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (VE) followed by a slower late phase (VL). In fibers with endogenous sTnC, both VE and VL varied with [Ca2+], but VE was less affected than VL. Similar results were obtained after extraction of TnC and reconstitution with either sTnC or cTnC, except for a small increase in the apparent activation dependence of VE. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, VE and VL were similar to those obtained in fibers reconstituted with sTnC or cTnC at equivalent force levels. In these fibers, which contained aTnC and cTnC, VE and VL increased with isometric force when [Ca2+] was increased from pCa 9.2 to 4.0. Fibers that contained a mixture of a TnC and cTnC were then extracted a second time to selectively remove cTnC. In fibers containing aTnC only, VE and VL were proportional to the resulting submaximal isometric force compared with maximum Ca(2+)-activated control. With aTnC alone, force, VE, and VL were not affected by changes in [Ca2+]. The similarity of activation dependence of VUS whether fibers were activated in a Ca(2+)-sensitive or -insensitive manners implies that VUS is determined by the average level of thin filament activation and that, with sTnC or cTnC, VUS is affected by Ca2+ binding to TnC only.     

Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].     

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle

Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5'ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca2+], while rigor cross-bridges caused dichroism to increase more with 5'ATR-labeled cTnC(C84) than cTnC(C98). The pCa50 values and n(H) from Hill analysis of the Ca2+-dependence of force and dichroism were 6.4 (+/-0.02) and 1.08 (+/-0.04) for force and 6.3 (+/-0.04) and 1.02 (+/-0.09) (n = 5) for dichroism in cTnC(C84) reconstituted trabeculae. Corresponding data from cTnC(C98) reconstituted trabeculae were 5.53 (+/-0.03) and 3.1 (+/-0.17) for force, and 5.39 (+/-0.03) and 1.87 (+/-0.17) (n = 5) for dichroism. The contribution of active cycling cross-bridges to changes in cTnC structure was determined by inhibition of force to 6% of pCa 4.0 controls with 1.0 mM sodium vanadate (Vi). With 5'ATR-labeled cTnC(C84) Vi caused both the pCa50)of dichroism and the maximum value at pCa 4.0 to decrease, while with 5'ATR-labeled cTnC(C98) the pCa50 of dichroism decreased with no change of dichroism at pCa 4.0. The dichroism of 5'ATR-labeled cTnC(C35) was insensitive to either Ca2+ or strong cross-bridges. These data suggest that both Ca2+ and cycling cross-bridges perturb the N-terminal structure of cTnC at Cys-84, while C-terminal structure is altered by site II Ca2+-binding, but not cross-bridges.     

Faster force transient kinetics at submaximal Ca2+ activation of skinned psoas fibers from rabbit.

The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.     

Nitric oxide impairs Ca2+ activation and slows cross-bridge cycling kinetics in skeletal muscle.

The effects of the nitric oxide (NO) donor spermine NONOate (Sp-NO, 1.0 mM) on cross-bridge recruitment and cross-bridge cycling kinetics were studied in permeabilized rabbit psoas muscle fibers. Fibers were activated at various Ca2+ concentrations (pCa, negative logarithm of Ca2+ concentration), and the pCa at which force was maximal (pCa 4.0) and approximately 50% of maximal (pCa50 5.6) were determined. Fiber stiffness was determined using 1-kHz sinusoidal length perturbations, and the fraction of cross bridges in the force-generating state was estimated by the ratio of stiffness during maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+ activation to stiffness during rigor (at pCa 4.0). Cross-bridge cycling kinetics were evaluated by measuring the rate constant for force redevelopment after quick release (by 15% of optimal fiber length, L(o)) and restretch of the fiber to L(o). Exposing fibers to Sp-NO for 10 min reduced force and the fraction of cross bridges in the force-generating state at maximal and submaximal (pCa50) Ca2+ activation. However, the effects of Sp-NO were more pronounced during submaximal Ca2+ activation. Sp-NO also reduced the rate constant for force redevelopment but only during submaximal Ca2+ activation. We conclude that Sp-NO reduces Ca2+ sensitivity by decreasing the number of cross bridges in the strongly bound state and also impairs cross-bridge cycling kinetics during submaximal activation.     

Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tension recovery in permeabilized rat soleus muscle fibers after rapid shortening and restretch

Permeabilized rat soleus muscle fibers were subjected to rapid shortening/restretch protocols (20% muscle length, 20 ms duration) in solutions with pCa values ranging from 6.5 to 4.5. Force redeveloped after each restretch but temporarily exceeded the steady-state isometric tension reaching a maximum value approximately 2.5 s after relengthening. The relative size of the overshoot was <5% in pCa 6.5 and pCa 4.5 solutions but equaled 17% +/- 4% at pCa 6.0 (approximately half-maximal Ca2+ activation). Muscle stiffness was estimated during pCa 6.0 activations by imposing length steps at different time intervals after repeated shortening/restretch perturbations. Relative stiffness and relative tension were correlated (p < 0.001) during recovery, suggesting that tension overshoots reflect a temporary increase in the number of attached cross-bridges. Rates of tension recovery (k(tr)) correlated (p < 0.001) with the relative residual force prevailing immediately after restretch. Force also recovered to the isometric value more quickly at 5.7 < or = pCa < or = 5.9 than at pCa 4.5 (ANOVA, p < 0.05). These results show that k(tr) measurements underestimate the rate of isometric force development during submaximal Ca2+ activations and suggest that the rate of tension recovery is limited primarily by the availability of actin binding sites.     

Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T

The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy in humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity of detergent-skinned fiber bundles from a transgenic (TG) mouse model in which 67% of the total cTnT in the heart was replaced by the R92Q mutant cTnT. TG fibers were more sensitive to Ca(2+) than nontransgenic (NTG) fibers [negative logarithm of half maximally activating molar Ca(2+) (pCa(50)) = 5.84 +/- 0.01 and 6.12 +/- 0.01 for NTG and TG fibers, respectively]. The shift in pCa(50) caused by increasing the sarcomere length from 1.9 to 2.3 microm was significantly higher for TG than for NTG fibers (DeltapCa(50) = 0.13 +/- 0.01 and 0.29 +/- 0.02 for NTG and TG fibers, respectively). The relationships between rate of ATP consumption and steady-state isometric tension were linear, and the slopes were the same in NTG and TG fibers. Rate of tension redevelopment was more sensitive to Ca(2+) in TG than in NTG fibers (pCa(50) = 5.71 +/- 0.02 and 6.07 +/- 0.02 for NTG and TG fibers, respectively). We concluded that overall cross-bridge cycling kinetics are not altered by the R92Q mutation but that altered troponin-tropomyosin interactions could be responsible for the increase in myofilament Ca(2+) sensitivity in TG myofilaments.     

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle.

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.     

An improved method for exchanging troponin subunits in detergent skinned rat cardiac fiber bundles.

We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca(2+)-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI(33-211), which was used to aid in the visualization of the troponin exchange, decreased to 85-90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI(33-211) and the cTnT(77-289)-cTnI(33-211) treated fiber bundles demonstrated that 70-80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80-85% of the Ca(2+)-regulated force was restored in cTnT-cTnI/cTnI(33-211) treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.     

Force regulation by Ca2+ in skinned single cardiac myocytes of frog.

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.     

Influence of temperature on the calcium sensitivity of the myofilaments of skinned ventricular muscle from the rabbit

The steady-state myofilament Ca sensitivity was determined in skinned cardiac trabeculae from the rabbit right ventricle (diameter, 0.13-0.34 mm) at 36, 29, 22, 15, 8, and 1 degree C. Muscles were stimulated to 0.5 Hz and stretched to a length at which maximum twitch tension was generated. The preparation was then skinned with 1% vol/vol Triton X-100 in a relaxing medium (10 mM EGTA, pCa 9.0). Each preparation was exposed to a series of Ca-containing solutions (pCa 6.3-4.0) at two of the six temperatures studied (temperature was regulated to +/- 0.1 degree C). The pCa values (mean +/- SD, n = 6) corresponding to half maximal tension at 36, 29, 22, 15, 8, and 1 degree C were 5.47 +/- 0.07, 5.49 +/- 0.07, 5.34 +/- 0.05, 5.26 +/- 0.09, 4.93 +/- 0.06, and 4.73 +/- 0.04, respectively. Mean (+/- SD) maximum tension (Cmax) developed by the preparation as a percentage of that at 22 degrees C was 118 +/- 10, 108 +/- 5, 74 +/- 6, 57 +/- 7, and 29 +/- 5% at 36, 29, 15, 8, and 1 degree C, respectively. As cooling led to a shift of Ca sensitivity towards higher [Ca2+] and a reduction of Cmax, the Ca sensitivity curves over this range of temperatures do not cross over as has been described for canine Purkinje fibers (Fabiato 1985). Since tension is decreased by cooling at all levels of [Ca2+] it is unlikely that changes in myofilament Ca sensitivity play a role in the large hypothermic inotropy seen in rabbit ventricular muscle. The increase in sensitivity of the myofilaments to Ca on warming from 1 to 29 degrees C might be related to the increase in force seen on rewarming from a rapid cooling contracture in intact rabbit ventricular muscle.     

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca(2+) sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 +/- 2.5 vs. 33.4 +/- 2.6 mN/mm(2)) reduction in maximum tension in the nontransgenic (NTG) preparations (n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations (n = 11). TPA induced a small but significant (P < 0.02) increase in Ca(2+) sensitivity (untreated pCa(50) = 5.63 +/- 0.01 vs. treated pCa(50) = 5.72 +/- 0.01) only in TG preparations. In TG preparations, (32)P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.     

Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.