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Am J Physiol Heart Circ Physiol 287: H1756-H1761, 2004. First published May 27, 2004; doi:10.1152/ajpheart.00172.2004
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Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

Julian E. Stelzer,1 Jitandrakumar R. Patel,1 M. Charlotte Olsson,1 Daniel P. Fitzsimons,1 Leslie A. Leinwand,2 and Richard L. Moss1

1Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706; and 2Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309

Submitted 23 February 2004 ; accepted in final form 25 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnTtrunc) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnTtrunc (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 Ca2+ sensitivity of force development [pCa for half-maximal tension generation (pCa50)] and the rate constant of force redevelopment (ktr) in cTnTtrunc 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, cTnTtrunc mice exhibited greater pCa50, reduced steepness of the force-pCa relationship [Hill coefficient (nH)], and faster ktr at submaximal Ca2+ concentration ([Ca2+]), i.e., reduced activation dependence of ktr. Treatment with NEM-S1 elicited similar increases in pCa50 and similar reductions in nH in WT and cTnTtrunc myocardium but elicited greater increases in ktr at submaximal activation in cTnTtrunc myocardium. Contrary to our initial hypothesis, cTnTtrunc appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca2+-activated force and the rate of cross-bridge attachment at submaximal [Ca2+]. Although these mechanisms would not be expected to depress systolic function per se in cTnTtrunc hearts, they would account for slowed rates of myocardial relaxation during early diastole.

calcium ion sensitivity of force; kinetics of actin-myosin interaction; skinned myocardium

IT SEEMS LIKELY that familial hypertrophic cardiomyopathy (FHC) mutations in the T2 domain of cardiac troponin T (cTnT) would alter thin filament activation by perturbing its interactions with other thin filament proteins. This idea can be tested experimentally by assessing the effects on contraction due to incorporation of mutant cTnT into the thin filament. One FHC mutation, cTnT truncation (cTnTtrunc), involves a splice-site donor mutation in the cTnT gene, which results in a shift in the reading frame and premature termination (20). The resulting RNA transcript, lacking exons 15 and 16, encodes a truncated protein that is missing the COOH-terminal 28 amino acid residues. Deletion of the COOH-terminal residues of TnT has been shown to have important functional effects, presumably due to its interactions with the troponin I (TnI)-troponin C (TnC) complex (8) and with tropomyosin (Tm) (17). Deletion of residues 216–263 of the T2 domain of TnT also results in reduced binding of TnI-TnC to the thin filament (8). The severity of the cTnTtrunc mutation is evident in studies in which even low levels (8–10%) of expression in mice result in stillbirth or death within 24 h after birth (19). The cTnTtrunc mutation has been shown to have a range of effects on thin filament activation, including an increase in Ca2+ sensitivity of force (10), reduced cTnT-induced inhibition of actin-Tm-activated myosin ATPase activity, and an inability to inhibit Tm/actin thin filaments in the in vitro motility assay (14). Other studies have shown that cTnTtrunc destabilizes the binding of Tn to the thin filament, which affects the transitions between states that regulate myosin binding (3). This mutation has also been shown to cause severe diastolic dysfunction and mild hypocontractile systolic dysfunction in whole heart preparations (19). Such findings are consistent with the idea that cTnT influences the extent and possibly the kinetics of cross-bridge binding and thereby contributes to the regulation of force development.

In the present study we examined the effects of the cTnTtrunc mutation on the kinetics of cross-bridge binding to the thin filament and thin filament responsiveness to the activating effects of strong-binding cross bridges. We show that cTnTtrunc directly affects thin filament activation by accelerating the rate of cross-bridge binding under basal conditions and enhances the sensitivity of the thin filament to the activating effects of Ca2+ and strong-binding cross bridges.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice expressing truncated cTnT. Transgenic mice expressing truncated cTnT (cTnTtrunc) were generated as described previously (19). Expression of the transgene was driven by the murine {alpha}-myosin heavy chain promoter, which restricts expression of cTnTtrunc to cardiac muscle. Previous studies documented that transgenic mice expressing >10% of total cTnT as cTnTtrunc die within hours of birth (19). To ensure survival for mechanical measurements, transgenic mice that expressed relatively low levels of cTnTtrunc, i.e., <5% total TnT, were studied (19). Analysis of global ventricular function has documented that mice expressing cTnTtrunc at levels comparable to those in the current study exhibit significant diastolic dysfunction (19). Compared with wild-type (WT) littermate controls, transgenic mice expressing low levels of cTnTtrunc exhibited no gross phenotypic abnormalities, no change in mortality, and no apparent ventricular hypertrophy. Transgenic animal production and animal usage were conducted under strict guidelines established by animal use committees at the University of Colorado and the University of Wisconsin.

Skinned myocardial preparations. The methods used for preparing skinned myocardium for mechanical measurements under isometric conditions were described previously (13). After intraperitoneal injection of 5,000 U heparin/kg body wt, WT and cTnTtrunc mice (8–12 mo old) were anesthetized with inhaled isoflurane. Their hearts were excised, and right and left ventricles were dissected in Ringer solution (in mM: 118 NaCl, 4.8 KCl, 2 NaH2PO4, 1.2 MgCl2, 25 HEPES, 11 glucose, and 0.5 CaCl2; pH 7.4, 22°C). Both ventricles were then rapidly frozen in liquid nitrogen, a step that was essential for obtaining high-quality preparations (13). To prepare skinned myocardium, pieces of frozen ventricle were thawed and homogenized for ~4 s in ice-cold relaxing solution (in mM: 100 KCl, 20 imidazole, 7 MgCl2, 2 EGTA, and 4 ATP; pH 7.0) with a Polytron homogenizer, which yielded multicellular preparations of 100–250 µm x 600–900 µm. The homogenate was centrifuged at 120 g for 1 min, and the resulting pellet was washed with fresh relaxing solution and resuspended in relaxing solution containing 250 µg/ml saponin and 1% Triton X-100. After 30 min, the skinned preparations were washed three times with fresh relaxing solution and were dispersed in ~50 ml of relaxing solution in a glass petri dish. The dish was kept on ice at all times except during the selection of individual preparations for mechanical experiments.

Experimental solutions, apparatus, and protocol. Solution compositions were calculated with the computer program of Fabiato (4) and stability constants (corrected to pH 7.0 and 15°C) listed by Godt and Lindley (7). All solutions contained (in mM) 100 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 14.5 creatine phosphate, and 5 DTT. In addition, 1) pCa 9.0 solution contained (in mM) 7 EGTA, 0.02 CaCl2, 5.42 MgCl2, and 4.74 ATP; 2) pCa 4.5 solution contained (in mM) 7 EGTA, 7.01 CaCl2, 5.26 MgCl2, and 4.81 ATP; and 3) preactivating solution contained (in mM) 0.07 EGTA, 5.42 MgCl2, and 4.74 ATP. The ionic strength of all solutions was adjusted to 180 mM with potassium propionate. A range of solutions containing different free Ca2+ concentrations ([Ca2+]free) (i.e., pCa 6.5–5.5) were prepared by mixing appropriate volumes of solutions of pCa 9.0 and pCa 4.5.

Skinned preparations with well-defined edges and no free ends evident in the middle region were transferred from the petri dish to a stainless steel experimental chamber (11) containing relaxing solution. The ends of a preparations were attached to the arms of a motor (model 312B, Aurora Scientific) and a force transducer (model 403; Aurora Scientific) as described previously (11). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a x40 objective and a CCTV camera (model WV-BL600; Panasonic). Light from a halogen lamp was passed through a cut-off filter (transmission >620 nm) and was used to illuminate the skinned preparations. Bitmap images of the preparations were acquired with an AGP 4X/2X graphics card and associated software (ATI Technologies) and were used to assess mean sarcomere length (SL) during the course of each experiment. Changes in force and motor position were sampled (16-bit resolution, DAP5216a; Microstar Laboratories, Bellevue, WA) at 2.0 kHz with SLControl software developed in our laboratory (http://www.slcontrol.com) and saved to computer files for later analysis. Changes in force were also recorded on a chart recorder with a slow time base.

At the start of each experiment, the preparation was stretched to a mean SL of ~2.20 µm for measurements of steady-state Ca2+-activated force and the rate constant of force redevelopment (ktr) after a release and restretch maneuver (2). The force recorded in a solution of pCa 9.0 was taken as resting force and then subtracted from the total force in activating solutions to yield the Ca2+-activated force (P). The values of ktr reported here were determined as the rate constant of force redevelopment after the release and restretch of Ca2+-activated preparations (Fig. 1). Mechanical measurements of steady-state Ca2+-activated force and the rate of force redevelopment were repeated after 15-min incubations of the preparations in pCa 9.0 solution containing either 3 µM or 6 µM non-force-generating derivative of myosin subfragment-1 (NEM-S1) (5). At the end of each experiment, the preparation was cut free, placed in SDS sample buffer, and stored at –80°C until subsequent analysis of contractile protein content analysis by 12% SDS-PAGE (5). Gels were stained with silver and scanned with a densitometer (Molecular Analyst; Bio-Rad).



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  		Fig. 1. Experimental protocol for determination of Ca2+-activated force and rate constant of force redevelopment (ktr) in skinned myocardium. Bottom: changes in force recorded before, during, and after a step change in length (top) of the skinned myocardium. Once Ca2+-activated force reached steady state in a solution of pCa 4.5, muscle length was rapidly reduced by 20%. The amplitude of Ca2+-activated force was determined by subtracting the resting force measured at pCa 9.0 from the total force generated at pCa 4.5. After ~20 ms of unloaded shortening, the preparation was restretched to its original length. ktr was calculated as the apparent rate constant of force redevelopment after restretch of the muscle to its original length.

 
Data analysis. Cross-sectional areas of skinned preparations were calculated by assuming that the preparations were cylindrical and by measuring width of the mounted preparation. Submaximal Ca2+-activated force (P) was expressed as a fraction of the maximum Ca2+-activated force (Po) generated at pCa 4.5, i.e., P/Po. Force-pCa data were fitted with the Hill equation: P/Po = [Ca2+]nH/(knH + [Ca2+]nH), where nH is the slope (Hill coefficient) and k is the [Ca2+] required for half-maximal activation. Rate constants of force redevelopment (ktr) were determined by linear transformation of the half-time of force recovery [ktr = –ln 0.5 x (t1/2)–1] (5, 13).

All data are reported as means ± SE. Comparisons between WT and cTnTtrunc data in the absence of NEM-S1 were made with an unpaired t-test and, when appropriate (nonparametric data), with the Wilcoxon Mann-Whitney test. To evaluate the effects of NEM-S1 on WT or cTnTtrunc myocardium, ANOVA with a Tukey post hoc test was used or, when appropriate, the Wilcoxon Mann-Whitney test. Significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mechanical properties of skinned WT and TnTtrunc myocardium. TnTtrunc and WT myocardium generated comparable resting forces and maximum Ca2+-activated forces (Table 1). In both cases, the force-pCa relationship was sigmoidal and was well fit with the Hill equation (Fig. 2). In the absence of NEM-S1, mean pCa50 was 5.81 ± 0.01 in WT myocardium and 5.92 ± 0.02 in TnTtrunc myocardium, indicating that the Ca2+ sensitivity of force was greater in TnTtrunc myocardium ({Delta}pCa50 = 0.11 ± 0.01; P < 0.01). Furthermore, the steepness of the force-pCa relationship was markedly reduced in TnTtrunc compared with WT myocardium (nH was 3.6 ± 0.1 in TnTtrunc vs. 4.3 ± 0.2 in WT; P < 0.001), indicating that low levels of TnTtrunc expression reduce the apparent cooperativity of force development.


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  		Table 1. Steady-state mechanical measurements in skinned myocardium isolated from nontransgenic and transgenic mice in absence and presence of NEM-S1

 


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  		Fig. 2. Effects of non-force-generating derivative of myosin subfragment-1 (NEM-S1; 3 µM) on force-pCa relationships in skinned wild-type and transgenic myocardium. Data points are means and the error bars are SE. Smooth lines were generated by fitting the mean data with the Hill equation described in MATERIALS AND METHODS. Values for Ca2+ sensitivity of force (i.e., pCa50) in the absence (open symbols) and the presence (closed symbols) of 3 µM NEM-S1 were as follows: wild type: {circ}, 5.81 ± 0.01 (n = 10) and {bullet}, 5.90 ± 0.03 (n = 7); cardiac troponin T with COOH-terminal truncation (cTnTtrunc): {square}, 5.92 ± 0.02 (n = 10) and {blacksquare}, 6.03 ± 0.01 (n = 5). P/P0, force normalized to maximum Ca2+-activated force.

 
The ktr after rapid release and restretch of myocardium is frequently used to estimate the kinetics of cross-bridge cycling in contracting muscle. Representative traces showing the Ca2+ dependence of the kinetics of force redevelopment in WT and TnTtrunc myocardium are shown in Fig. 3. At pCa 4.5, the mean values of maximal ktr recorded in WT myocardium did not differ from those recorded in TnTtrunc myocardium (Table 1). In both WT and TnTtrunc myocardium, ktr varied with the level of Ca2+ activation (Fig. 4A), although expression of TnTtrunc significantly reduced the Ca2+-dependent increase in ktr. The ktr increased more than fourfold in WT myocardium (from 4.10 ± 0.16 s–1 at pCa 6.0 to 17.21 ± 0.60 s–1 at pCa 4.5), which is consistent with the results of earlier studies (1, 6, 15, 25), but only threefold in TnTtrunc myocardium (from 5.29 ± 0.22 s–1 at pCa 6.1 to 17.24 ± 0.50 s–1 at pCa 4.5).



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  		Fig. 3. Force records obtained during measurements of ktr in skinned wild-type and transgenic myocardium. The force records have been scaled to the peak steady-state force attained after the step change in muscle length to facilitate direct visualization of differences in rates of force recovery in skinned myocardium from wild-type (A) and TnTtrunc (B) mice.

 


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  		Fig. 4. Effects of 3 µM NEM-S1 on the activation dependence of ktr in wild-type and transgenic myocardium. Force redevelopment after rapid release and restretch was measured in the absence (open symbols) and the presence (closed symbols) of 3 µM NEM-S1 in wild-type (circles) and TnTtrunc (squares) myocardium. Data points are means, and the error bars are SE. A: Ca2+ dependence of ktr. B: ktr as a function of force (expressed as a fraction of maximal Ca2+-activated force) measured at each pCa.

 
The ktr was also plotted as a function of steady-state isometric force at each pCa to assess the variation in ktr with a variable that reflects the overall level of thin filament activation. In both WT and TnTtrunc myocardium, ktr increased as a function of increasing isometric force, gradually at lower forces and more steeply at high forces (Fig. 4B). However, at low forces expression of TnTtrunc resulted in values of ktr that were greater than WT values. These data suggest that expression of TnTtrunc enhances the cooperativity of thin filament activation.

Effects of NEM-S1 on mechanical properties of skinned WT and TnTtrunc myocardium. The effects of 3 and 6 µM NEM-S1 on resting force, maximum Ca2+-activated force, Ca2+ sensitivity of force (pCa50), and the steepness of the force-pCa relationship are summarized in Table 1. In both WT and TnTtrunc myocardium, NEM-S1 increased both resting force and submaximal Ca2+-activated forces in a concentration-dependent manner. Mean pCa50 increased significantly in both WT and TnTtrunc myocardium after treatment with 3 µM NEM-S1, indicating an increase in the Ca2+ sensitivity of force. Qualitatively similar increases in mean pCa50 were observed after treatment with 6 µM NEM-S1. In addition, treatment with 3 or 6 µM NEM-S1 significantly reduced the steepness of the force-pCa relationships in both WT and TnTtrunc myocardium, an observation consistent with results reported previously in skinned ventricular myocardium (6).

Treatment with either 3 or 6 µM NEM-S1 had no appreciable effect on the kinetics of force redevelopment in maximally activated preparations. However, at low levels of Ca2+ activation, NEM-S1 had concentration-dependent effects on ktr. In both WT and TnTtrunc myocardium, treatment with 3 or 6 µM NEM-S1 increased ktr at low levels of activation to values identical to ktr obtained in maximally activated preparations. At intermediate levels of activation, NEM-S1 increased ktr to greater than control values, but ktr was still less than maximal. Also, the ktr values obtained at intermediate levels of activation in the presence of NEM-S1 were significantly greater in TnTtrunc myocardium than in WT myocardium.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Summary. Earlier functional studies on whole hearts isolated from cTnTtrunc transgenic mice indicated that the mutation induced severe diastolic dysfunction and mild hypocontractile systolic dysfunction (19), whereas studies in reduced systems found that the cTnTtrunc mutation increased the Ca2+ sensitivity of force (10), reduced the inhibition of actin-activated myosin ATPase activity in regulated thin filaments, and reduced inhibition of sliding in an in vitro motility assay comprised of regulated thin filaments (14). Although the mechanisms underlying this range of phenotypes are likely to be complex, an hypothesis that is consistent with these findings is that cTnTtrunc directly affects cross-bridge transitional rate constants such that the rate of force development is slowed by reductions in the apparent rate constants of both attachment and detachment of cross bridges from actin. In fact, our results showed that the opposite was the case: at submaximal activations cTnTtrunc myocardium exhibited faster rates of force development (Fig. 4A), implying that the rates of cross-bridge attachment or detachment or both were accelerated compared with WT myocardium. Our results further show that at least some of these effects of the cTnTtrunc mutation were a consequence of greater cross-bridge recruitment and increased thin filament responsiveness to the activating effects of Ca2+ and strong-binding cross bridges.

Altered Ca2+ sensitivity of force and apparent cooperativity in cTnTtrunc myocardium. Previous studies reported that expression of cTnTtrunc reduced maximal Ca2+-activated force compared with controls (12, 24). Our results did not reproduce this effect, and because the maximal rates of force redevelopment (ktr) did not differ between cTnTtrunc and WT myocardium, a reduction in maximal Ca2+-activated force is not likely to contribute to the changes in cross-bridge kinetics observed in cTnTtrunc myocardium at submaximal activations.

Our data also show that transgenic expression of small amounts (<5%) of cTnTtrunc produced dramatic effects on Ca2+ sensitivity of force and the apparent cooperativity of thin filament activation, as indicated by changes in nH. The pCa required at half-maximal activation (pCa50) was shifted to lower [Ca2+] by 0.11 units (Fig. 2) in transgenic myocardium compared with WT myocardium, indicating that less Ca2+ is required to produce the same amount of force as controls. This finding agrees well with results of Montgomery et al. (10), who reported a 0.14-pCa unit shift in the tension-pCa relationship in the same cTnTtrunc mice compared with WT myocardium.

Another notable finding is that nH, an index of apparent cooperativity in the activation of force, was reduced in cTnTtrunc myocardium as evidenced by the shallower slope of the force-pCa relationship (Fig. 2). This phenomenon was observed earlier by other investigators (3) and may manifest a diminished capacity of the Tm-Tn complex to inhibit cross-bridge binding. Impaired suppression of myosin binding at low levels of Ca2+ is consistent with slowed and incomplete diastolic relaxation and may indirectly give rise to diminished systolic function in cTnTtrunc mice (19). It has been suggested that the TnT truncation destabilizes the blocked conformation of the thin filament regulatory strand, which enhances the transition from the blocked to the active state. Thus, in cTnTtrunc myocardium, there is greater likelihood that the thin filament will assume the "switched-on" state even at low [Ca2+] and thereby reduce cooperativity in myosin binding to actin (3, 23).

Accelerated cross-bridge kinetics in cTnTtrunc myocardium. Although cTnTtrunc had no effect on the maximal rate of force redevelopment compared with control, the mutation increased ktr at submaximal [Ca2+] (Fig. 4). Such increases could be due to a direct effect of the mutation on cross-bridge cycling kinetics or an indirect effect of increased cross-bridge binding to the thin filament. Both mechanisms appear to be operational in cTnTtrunc myocardium, because some of the difference in rates between cTnTtrunc and WT disappeared when results were plotted against relative force as a measure of Ca2+ activation; however, at P/Po less than ~0.4, ktr was still faster in cTnTtrunc myocardium. The reduction in difference between cTnTtrunc and WT myocardium when ktr was plotted against force implies that some of the difference (Fig. 4A) is due to acceleration of ktr in cTnTtrunc myocardium secondary to the activating effects of greater numbers of cross bridges bound to the thin filament. The observation that some of the difference persists at low levels of activation suggests that cTnTtrunc also has direct effects to accelerate cycling kinetics. In this regard, it is important to reiterate that the acceleration of ktr at low levels of Ca2+ activation was observed despite low levels of cTnTtrunc expression (19). Incorporation of <5% cTnTtrunc into the thin filament was sufficient to elicit a significant increase in the rate of force redevelopment in the absence of NEM-S1 (Fig. 4B).

Earlier measurement of ATPase activity is consistent with these results. Montgomery et al. (10) reported that maximal ATPase cycling rates did not differ between WT and cTnTtrunc; however, the ATPase activity-pCa relationship was shifted to lower [Ca2+] in cTnTtrunc, indicating that cross-bridge cycling rates were increased at submaximal [Ca2+]. Reports of increased sliding speed of actin filaments in in vitro motility assays (14, 22), decreased inhibition of ATPase activity in the absence of Ca2+ (18), and the accelerated ktr at low Ca2+ observed in the present study are consistent with the idea that cTnTtrunc attenuates inhibition of thin filaments at low Ca2+.

This phenomenon can also be interpreted in terms of a three-state model of thin filament activation (9) in which the thin filament exists in a blocked state (unable to bind myosin) in the absence of Ca2+ but rapidly transitions to a closed state on Ca2+ binding to TnC, with subsequent transition to an open state when cross bridges bind to actin. When Ca2+ binds to TnC, alterations in interactions among thin filament proteins are thought to release the thin filament from the blocked state and to promote the transition of weakly bound cross bridges to a strongly bound, force-generating state. The impairment in function caused by a low level of expression of TnTtrunc can be explained as an inability of those regions of the thin filament containing cTnTtrunc to completely assume the blocked state in the absence of Ca2+, which could result in incomplete relaxation. An increased number of strongly bound cross bridges at low levels of Ca2+ activation would also be expected to accelerate the rate of force redevelopment because of the greater activating affects of cross bridges already bound to the thin filament. This model implies that incomplete inactivation of cTnTtrunc thin filaments is most likely due to an incomplete return of Tm to the fully blocked position in the absence of Ca2+.

Effects of NEM-S1 on Ca2+ sensitivity of force and cooperativity in activation of force. In this study, NEM-S1 was used to assess the effects of strong-binding cross bridges on thin filament activation in WT and cTnTtrunc myocardium. As previously shown in rat myocardium (6), NEM-S1 slightly increases Ca2+ independent tension at pCa 9.0, a phenomenon that was similar in WT and cTnTtrunc myocardium. Furthermore, NEM-S1 markedly increased the Ca2+ sensitivity of force in both WT and cTnTtrunc myocardium, shifting the force-pCa relationships to lower [Ca2+] by similar amounts and having similar effects on WT and transgenic myocardium. Thus, although the cTnTtrunc mutation increased the Ca2+ sensitivity of force, it had no obvious effects on the cooperative activation of steady-state isometric force by strong-binding cross bridges.

Effects of NEM-S1 on rate constant of force redevelopment. Earlier studies from this laboratory (6) showed that NEM-S1 accelerates the rate of force development in rat skinned myocardium, presumably due to increased activation of the thin filament as a consequence of greater numbers of cross bridges bound to actin. In the present work, NEM-S1 increased ktr in both cTnTtrunc and WT myocardium but had greater effects in cTnTtrunc myocardium. In both WT and transgenic myocardium, the NEM-S1-induced acceleration of force development at submaximal [Ca2+] reduced the activation dependence of ktr. NEM-S1 had no effect on maximal ktr (measured at pCa 4.5) and increased ktr at very low [Ca2+] to maximal values (Fig. 4). The difference in NEM-S1-induced effects on ktr was observed only at intermediate levels of activation, where ktr was increased to values less than maximal with greater increases in ktr in cTnTtrunc myocardium.

In vitro studies of thin filament proteins in the presence of TnTtrunc suggest that Tm is held less tightly by Tn in its inhibitory position (3), thus reducing steric hindrance of myosin binding to actin. Results from the present study showing reduced cooperativity of activation and accelerated cross-bridge kinetics during submaximal activation of TnTtrunc myocardium support this view. Likewise, the greater activating effects of NEM-S1 on cross-bridge kinetics in TnTtrunc myocardium can be explained in terms of increased likelihood of cross-bridge binding at low levels of activation.

Functional significance of cTnTtrunc mutation. The primary function of the thin filament regulatory strand is to inhibit myosin binding in the absence of Ca2+ and to relieve this inhibition in the presence of Ca2+. Several published reports show that incorporation of cTnTtrunc into the thin filament impairs the ability of the regulatory strand to completely inhibit myosin cross-bridge cycling at very low Ca2+ or in its absence (3, 14, 18). Results from the present study support this view. Furthermore, incorporation of cTnTtrunc into the thin filament appears to accelerate cross-bridge cycling at submaximal Ca2+ both by increasing the responsiveness of thin filaments to the activating effects of Ca2+ and strong-binding cross bridges and by increasing the rate of cross-bridge interaction with actin. Together, our results could account for deficits in global ventricular function, including incomplete diastolic relaxation, which leads to decreased ventricular filling and a lower ejection fraction due to impaired systolic function (19).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. E. Stelzer, Dept. of Physiology, Univ. of Wisconsin School of Medicine, 1300 Univ. Ave., Madison, WI 53706 (E-mail: stelzer{at}physiology.wisc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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