AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 287: H2528-H2534, 2004; doi:10.1152/ajpheart.00553.2004
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Titin isoform-dependent effect of calcium on passive myocardial tension

Hideaki Fujita,1 Dietmar Labeit,2 Brenda Gerull,3 Siegfried Labeit,2 and Henk L. Granzier1

1Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99164; 2Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Mannheim 68167, and 3Max-Delbrueck Center for Molecular Medicine, Berlin 13092, Germany

Submitted 8 June 2004 ; accepted in final form 28 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied the effects of Ca2+ on titin (connectin)-based passive tension in skinned myocardium expressing either predominantly N2B titin (rat right ventricle, RRV) or predominantly N2BA titin (bovine left atrium, BLA). Actomyosin-based tension was abolished to undetectably low levels by selectively removing the thin filaments with a Ca2+-insensitive gelsolin fragment (FX-45). Myocardium was stretched in the presence and absence of Ca2+, and passive tension was measured. Ca2+ significantly increased passive tension during and after stretch in the BLA. The increase was insensitive to the actomyosin inhibitor 2,3-butanedione 2-monoxime, supporting the conclusion that the effect is titin based. Passive tension did not respond to calcium in the RRV, indicating that passive tension developed by N2B titin is calcium insensitive. Western blot analysis and immunofluorescence studies indicated that N2BA titin expresses E-rich PEVK motifs, whereas they are absent from N2B titin, supporting earlier single molecule studies that reported that E-rich motifs are required for calcium sensitivity. We conclude that calcium affects passive myocardial tension in a titin isoform-dependent manner.

connectin; diastole; passive stiffness; proline-glutamate-valine-lysine residue

TITIN [also known as connectin (29)] is a giant protein with a molecular mass as large as ~3.5 MDa and single molecules spanning from the Z-line to M-line region of the sarcomere. Titin is inextensible in, and near, the Z line as well as in the A-band region of the sarcomere, whereas the majority of the I-band region of the molecule functions as a molecular spring that develops passive force (for a recent review with original citations, see Ref. 16). The molecular spring region comprises 1) tandem Ig segments (serially linked Ig-like domains); 2) the PEVK segment [a sequence rich in proline (P), glutamate (E), valine (V), and lysine (K) residues]; 3) the N2A element found in skeletal muscle titins as well as in the cardiac N2BA isoform; and 4) the cardiac-specific N2B element, which contains the extensible unique sequence (N2B-Us) (8, 25). At physiological sarcomere lengths (SLs), the passive force of sarcomeres in skeletal muscle is largely determined by extension of the PEVK segment (27, 38) and in the myocardium by extension of both the PEVK and N2B-Us sequences (28, 35). The force of titin is important in maintaining the central location of the A band in the sarcomere (22), preventing overextension of the sarcomere (41), limiting SL inhomogeneity along the myofibrils (14), sensing stretch and modulating actomyosin interaction (5, 6, 10, 11, 13), and determining the passive stiffness of the myocardium that is important for diastolic filling of the heart (45). When sarcomeres shorten to lengths below the slack length, titin extends in a direction opposite of that seen during stretch, producing the so-called restoring force. This force pushes the sarcomere back toward slack upon relaxation and is likely to play a role in early diastolic filling of the heart (20).

Passive properties of striated muscle can be adjusted long term via altering expression of titin isoforms with length variants of the PEVK and tandem Ig segments, or short term via modulating the mechanical properties of the extensible region of titin (13). Recent single molecule mechanical experiments indicate that calcium lowers the bending rigidity of the PEVK segment and that the effect requires the presence of E-rich motifs (24). E-rich motifs are conserved domains with ~30–50% of their amino acid residues as glutamate (E). Several lines of evidence support the notion that skeletal muscle titin increases its stiffness on Ca2+ binding. Increase in passive stiffness occurs in activated intact frog muscle fibers, well ahead of active tension development (1, 2), whereas stretch during tetani of intact cat muscle results in passive force enhancement (21). Evidence was also obtained by us in skinned mouse soleus muscle fibers from which thin filaments had been extracted and that when stretched showed a calcium-dependent passive force response (24).

Whether passive force of cardiac muscle is calcium sensitive has not been investigated. Because of differential splicing, the PEVK segment contains only ~180 residues in N2B cardiac titin and ~800 residues in N2BA cardiac titin (8). Thus it is well possible that effects that are present in skeletal muscle [where the PEVK can be as large as ~2,200 residues (8)] are spliced out in cardiac titins. To test whether the passive force of titin is calcium sensitive in cardiac muscle, we studied the effect of Ca2+ on passive stiffness in the bovine left atrial (BLA) muscle, which expresses predominantly N2BA titin, and the rat right ventriclar (RRV) muscle, which expresses predominantly N2B titin (4, 39). Thin filaments were extracted with gelsolin so that active tension was completely abolished and the effect of Ca2+ on just titin could be studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscle, proteins, and solutions. Bovine hearts (animals were ~18 mo old and weighed ~1,100 lbs.) were obtained from local slaughterhouses. The hearts were excised typically within ~20 min following death. Muscle strips of ~2 mm in diameter and 1 cm in length were dissected in HEPES buffer solution containing (in mM) 133.5 NaCl, 5 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 10 HEPES (pH 7.4 by NaOH), and 30 2,3-butanedione 2-monoxime (BDM), with 2 g/l glucose at 2°C. Solutions were bubbled with 95% O2-5% CO2. Muscles were then skinned overnight at ~2°C in relaxing solution {in mM: 6.03 ATP, 40 N-bis[2 hydroxyethyl]-2-aminoethanesulfonic acid (BES) (pH 7.0), 10 EGTA, 6.49 MgCl2, 45 K-propionate, 15 phosphocreatine, 1 diothreitol (DTT), and 30 BDM, with 15 U/ml creatine phosphokinase}. Muscles were thoroughly washed with relaxing solution and transferred to relaxing solution containing 50% (vol/vol) glycerol and stored at –20°C. On the day of use, muscle strips with a diameter of ~100–250 µm were dissected. The obtained preparations produced normal active tension levels and had a normal calcium sensitivity that was length dependent (see also Ref. 11). To obtain rat trabeculae, LBNF1 rats were anesthetized with 50 mg/kg of pentobarbital sodium, and the hearts were quickly removed and then perfused with HEPES buffer solution (bubbled with 95% O2-5% CO2), and trabeculae were carefully excised from the right ventricle. This resulted in healthy preparations as revealed by their normal twitch force-SL relation and normal {beta}-adrenergic response (Fukuda et al., unpublished observations). Trabeculae were transferred to relaxing solution containing 1% (wt/vol) Triton X-100 for 2 h at 2°C and used within the same day. All procedures were approved by the Institutional Animal Care and Use Committee of Washington State University.

For activating muscle we used the following solutions: 1) preactivating solution: (in mM) 6.03 ATP, 40 BES (pH 7.0), 0.5 EGTA, 9.5 hexamethylenediaminetetraacetic acid (HDTA), 6.49 MgCl2, 45 K-propionate, 15 phosphocreatine, and 1 DTT, with 15 U/ml creatine phosphokinase; and 2) activating solution: (in mM) 6.32 ATP, 40 BES (pH 7.0), 10 CaCO3-EGTA, 6.29 MgCl2, 44 K-propionate, 15 phosphocreatine, and 1 DTT, with 15 U/ml creatine phosphokinase. All solutions contained a protease inhibitor cocktail (in mM: 0.5 phenylmethylsulfonyl fluoride, 0.05 leupeptin, and 0.01 E64). To remove thin filaments from relaxed skinned cardiac muscle, we used a calcium-independent gelsolin fragment (FX-45) that was purified according to the method described previously (12, 36).

Mechanics. Active and passive tensions were measured as described previously (15). Briefly, muscles were connected to a force transducer and servomotor via aluminum T-clips that were attached to the ends of the preparations. Experiments were performed at room temperature (20–22°C). SL was measured online with laser diffraction. Muscles were activated at slack length (SL ~1.9 µm) by perfusing them with first preactivating solution and then activating solution (pCa 4), and active tension was measured, followed by perfusion with relaxing solution. Treatment with FX-45 (~0.8 mg/ml) was in relaxing solution for 4 h to remove thin filaments. We then measured active tension again (as above), and those preparations in which active tension was completely abolished by the FX-45 treatment were stretched at a chosen velocity to a predetermined SL, held for a predetermined duration, and then released. This protocol was carried out in relaxing solution (pCa 9), activating solution (pCa 4), and activating solution (pCa 4) with 30 mM BDM. In preliminary experiments, we also used pCa 9 + 30 mM BDM and, after finding no effect of BDM on passive tension at this pCa, we used in these reported experiments exclusively pCa 9 without BDM (this was done to eliminate the chance that during subsequent activation at pCa 4 actomyosin interaction was inhibited by residual BDM remaining in the muscle). Force, muscle length, and SL were digitized and stored for analysis. Conditions of interest (pCa 4 and pCa 4 + BDM) were sandwiched by control conditions (see Fig. 2A) so that if rundown in the mechanical properties of the preparations occurred, it could be accounted for by comparing results of the condition of interest with the average result under control conditions. Rundown was 1.4 ± 1.2% (n = 30). After the experiments were completed, myocardium was either saved for SDS-PAGE analysis or treated with relaxing solution containing 0.6 M KCl (30 min) and 1.0 M KI (30 min) to remove thick and thin filament and to measure collagen-based passive tension and determine from this the titin-based tension (total tension minus collagen-based tension) (45).



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  		Fig. 2. A: experimental protocol. Muscles were stretched at 10 slack length/s from the slack sarcomere length (SL) of ~1.9 µm to a SL of 2.25 µm, were held for 5 min, and then released back to the slack length. Muscles were kept for 25 min in relaxing solution followed by a change in solution [in the shown examples pCa 4 + 2,3-butanedione 2-monoxime (BDM) and pCa 9] and imposition of another identical stretch, hold, release protocol. Examples of tension records of actin-extracted BLA muscle are shown. B: tension difference between pCa 4 + 30 mM BDM and pCa 9 for BLA (top) and RRV (bottom). Average data of 7 muscles. C and D: left, peak tension at end of stretch; right, steady-state tension after 5 min hold. Average results in RRV (C) and BLA (D). Error bars indicate SE for 7 muscle fibers, and asterisks show significant difference between pCa 9 (P < 0.05; repeated-measures ANOVA). There is no significant effect of BDM on tension (P = 0.63 for peak tension and 0.86 for steady-state tension).

 
Gel electrophoresis and Western blot analysis. Myocardium was solubilized and electrophoresed on 2–8% acrylamide gradient gels that were stained with silver. Gels were digitized (EPSON 800) and analyzed using One-D-Scan v.2.03 software (Scanalytics; Fairfax, VA). For quantification, different amounts of the same sample were loaded on a gel, and the slopes of the linear range of peak area of protein bands versus loading curves were determined (for details, see Ref. 17). For Western blot analysis, samples were run on 2.5–5% acrylamide gel and transferred to polyvinylidene fluoride membrane. We used the 9D10 antibody and antibodies against E-rich exons (X156 and X122–129). For detection of PEVK repeats, we used the 9D10 antibody (42). For the detection of the E-rich exons 156 and 129, we expressed exon 156 and exon group 126–133 (exon 129 alone was too small) in Escherichia coli. Specific antisera were raised in rabbits (for details, see Ref. 8) and affinity-purified by Biogenes (Berlin) (for Western blot analysis details, see Ref. 12).

Immunofluorescence microscopy. Skinned myocardium was fixed in relaxing solution containing 3.7% freshly prepared formaldehyde for 1 h, followed by extensive washing with relaxing solution. The specimens were then labeled with primary antibody against {alpha}-actinin [Sigma no. A-7811, monoclonal anti-{alpha}-actinin (sarcomeric), clone EA-53], and E-rich exon 156 all were diluted to ~20 µg/ml in phosphate-buffered saline (PBS) overnight at 2°C. The samples were then thoroughly washed with PBS and immersed in PBS containing 40 µg/ml secondary antibody (for exon 156 mouse anti-rabbit; for {alpha}-actinin goat anti-mouse) conjugated with Alexa488 (mouse anti-rabbit) and Alexa594 (goat anti-mouse; Molecular Probes; Eugene, OR) overnight at 2°C, followed by washing. Specimens were then studied by confocal microscopy.

Statistics. Data are presented as means ± SE, with n representing the number of experiments. Significant differences were assigned using Student's t-test or repeated measures ANOVA, as indicated. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Removal of thin filament by FX-45. BLA muscle strips and RRV trabeculae were treated for 4 h with FX-45 (calcium-independent F-actin severing domain of gelsolin) in relaxing solution. Before FX-45 treatment, active tension at pCa 4.0 was 24.19 ± 1.62 mN/mm2 (n = 12) and 54.10 ± 5.59 mN/mm2 (n = 11) for BLA and RRV, respectively (SL: 1.9 µm). After treatment, active tension was undetectably low. SDS-PAGE analysis revealed that after treatment ~76% and 83% of actin was removed from BLA and RRV, respectively (Fig. 1 and Table 1). The remaining low level is consistent with previous results (9) and is presumably due to extensive cross-linking of actin in and near the Z disk to other proteins that prevents diffusion of severed actin fragments out of these regions of the sarcomere (36). As for titin content, both BLA and RRV had two bands in the titin region of the gel: an upper T1 band (intact molecule) and a bottom T2 band (degradation product of T1). The T1 band of BLA had a lower mobility than that of RRV, consistent with our previous Western blot analysis results (39) that typed BLA T1 as N2BA titin (~3.3 MDA) and T1 of RRV as N2B titin (~3.0 MDa). Gelsolin treatment resulted in a T1 decrease of ~28% and ~15% and T2 increase of ~31% and ~17% in BLA and RRV, respectively, revealing 15–30% titin degradation. There was no significant change in {alpha}-actinin or C-protein content (Table 1). The degradation of titin without significant effects on other proteins may be explained by contaminating proteases and the high sensitivity of titin to proteolysis.



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  		Fig. 1. SDS-PAGE of skinned myocardium before and after FX-45 treatment. Lane 1: bovine left atrium (BLA) control; lane 2: BLA after gelsolin treatment; lane 3: rat right ventricle (RRV) control; lane 4: RRV after FX-45 treatment. Note that FX-45 largely extracts actin, with minimal effects on other proteins. MHC, myosin heavy chain.

 

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  		Table 1. Effect of gelsolin treatment on protein content

 
Effect of Ca2+ on passive tension. To assess the effect of calcium on passive tension, actin-extracted muscles were stretched at 10 slack length/s to SL 2.25 µm and held for 5 min to measure both peak tension and (near) steady-state tension (Fig. 2A). High calcium significantly increased both peak and steady-state passive tension of BLA (Fig. 2B, top, and D) but had no effect in RRV (Fig. 2B, bottom, and C). Although active tension at slack length was undetectable after gelsolin treatment, we nevertheless tested whether residual cycling cross bridges contributed to the tension response following stretch by studying the calcium effect in the presence of BDM (inhibitor of cross-bridge cycling). Peak and steady-state tensions in the presence of calcium were not significantly affected by BDM (Fig. 2D), suggesting that cross-bridge cycling does not contribute to the calcium-sensitive tension of thin-filament extracted BLA myocardium. It seems also unlikely that rigor cross bridges had formed during the preceding exposure to pCa 4 solution and that had subsequently persisted throughout the pCa 4 + BDM exposure (rigor bridges are insensitive to BDM), because measurements in the presence of BDM were made after the preparations had been extensively washed with relaxing solution. We also tested the effect of 30 mM BDM on tension at pCa 9 and also found no significant effect under these conditions (results not shown). Thus it is likely that both cycling cross bridges and rigor cross bridges are absent in the thin-filament extracted BLA muscles. We conclude that the effect of calcium on tension is likely due to titin.

Sarcomere length dependence of Ca2+ effect on passive tension. We studied the effect of SL on the Ca2+-induced increase in passive tension of thin-filament extracted muscles (Fig. 3). The protocol used for the experiments in Fig. 2 (a rapid stretch, followed by a long hold) was too demanding to be used at a range of amplitudes, and instead we measured tension during a slow stretch (see MATERIALS AND METHODS). Obtained tensions were ~30% larger than the steady-state tension (Fig. 2) revealing that they were partially viscoelastic in origin. In BLA, the effect of Ca2+ on passive tension had no significant SL dependence, as revealed by the slope of the linear regression line of the data (Fig. 3A), which is not significantly different from 0. The increase in tension at all SLs was 11.3 ± 3.3%. We did not observe a significant increase in passive tension at any SL that was tested in RRV (Fig. 3B).



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  		Fig. 3. Effect of Ca2+ on passive tension at a range of sarcomere lengths in BLA (n = 5) (A) and in RRV (n = 4) (B). Muscles were stretched at 0.1 slack length/s, and passive tension was measured during stretch. Shown lines are linear regression lines (slopes are not significantly different from 0). B: inset, mean and SE of the results at all SLs. *Significant difference from 0 using a one-sample t-test and a population mean of 0. (Passive tension at pCa 9 was subtracted from passive tension at pCa4 + 30 mM BDM, and the result is expressed as a percentage of the former.)

 
Western blot analysis and immunofluorescence microscopy. Previous single molecule mechanical studies indicated that E-rich motifs in PEVK segment of titin may be responsible for the effect of Ca2+ on passive tension (24). E-rich motifs are conserved motifs with ~30–50% of their amino acid residues as glutamate (E); in the PEVK segment, these motifs are interspersed with ~28 residues containing PEVK repeats (3). Differential splicing of titin results in a PEVK segment that is short in N2B cardiac titin, intermediate in N2BA cardiac titin, and longest in soleus skeletal muscle titin (8). To test whether E-rich motifs are differentially expressed in cardiac isoforms, Western blot analysis and immunofluorescence studies were performed using various anti-PEVK antibodies. We used 9D10, a monoclonal antibody that reacts all along the PEVK sequence (18), in both soleus skeletal and rat cardiac muscle (37), and that is likely to bind a PEVK repeat sequence (Fig. 4A, lanes 2). We also used an antibody against an E-rich motif encoded by exon 156 (Fig. 4A, lanes 3) and an antibody against a sequence that contains both an E-rich motif encoded by exon 129 and PEVK repeats encoded by exons 126–128 and 130–133 (Fig. 4A, lanes 4). We studied mouse soleus (MS) titin, which expresses a N2A titin isoform, rat left ventricle (RLV), which expresses predominately N2B cardiac titin, and bovine left ventricle (BLV), which expresses both N2BA and N2B cardiac titins (Fig. 4, top and bottom T1 bands on gels, respectively). Results showed that both N2A and N2BA titins react with all three anti-PEVK antibodies, whereas N2B titin is positive for 9D10 but negative for the anti-E-rich antibodies (Fig. 4A). Consistent with these findings, immunofluorescence with the anti-exon 156 antibody revealed strong I-band labeling in mouse skeletal muscle fibers (Fig. 4B,a) as well as in the predominantly N2BA titin-expressing bovine left atrium (Fig. 4B,b) but absence of labeling in rat left ventricle (Fig. 4B,c).



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  		Fig. 4. A: Western blot analysis of proline, glutamate, valine, and lysine (PEVK) composition. Lane 1: membrane stained with India Ink (stains all proteins); lane 2: 9D10; lane 3: anti-exon 156; lane 4: anti-exon 122–129. Note that all antibodies label N2A titin (mouse soleus, MS) and N2BA titin [top band in bovine left ventricle, bovine left ventricle (BLV)]. However, N2B titin [rat left ventricle (RLV) and bottom band in BLV] is positive for only 9D10. B: immunofluorescence confocal microscopy of MS (a), BLA (b), and RLV muscles (c) labeled with anti E-rich motif (exon 156) antibody. Bottom right insets, double labeling with the Z disk staining {alpha}-actinin (in red) and anti-exon 156 (in green). Note that anti-exon 156 stains the I band in MS and BLA, but is negative in RLV. (Scale bar: 5 µm.)

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To study the effect of calcium on titin only, actomyosin interaction has to be completely abolished. In preliminary experiments, we used BDM and N-benzyl-p-toluene sulfonamide (BTS), membrane-permeable inhibitors of actomyosin interaction (7). Although they abolished the majority of active tension, a small level (~10%) of active tension remained even at the highest concentrations of these inhibitors that were practical. To rule out that a small number of cycling cross bridges contributed to measured tensions, we instead used gelsolin to fully eliminate actomyosin interaction by extracting thin filaments from skinned muscles. A side effect of this method is that in cardiac muscle it results in ~30% titin degradation (Table 1), which lowers titin-based tension. This drawback is offset, however, by being able to completely abolish active tension, as shown by the lack of an effect of BDM on stiffness in the presence of calcium. Thus actomyosin interaction can be fully eliminated by extracting thin filaments with gelsolin, making it possible to study the effect of calcium on only titin-based tension.

Effect of calcium on the stiffness of titin is isoform dependent. Previous work on skeletal muscle has shown that, in thin-filament extracted soleus fibers, calcium increases passive stiffness (24), and the present study is the first to address whether this phenomenon occurs in cardiac muscle. No calcium effect was found in RRV, whereas passive stiffness of BLA was significantly increased by calcium, revealing that the calcium sensitivity of titin is isoform dependent. Adult RRV expresses predominantly the cardiac N2B titin isoform and BLA high levels of N2BA titin (39). Sequences in common between these isoforms are unlikely to underlie calcium-sensitive passive stiffness (because in that case passive stiffness of rat trabeculae would also be calcium sensitive), making it unlikely that the N2B element and constitutively expressed Ig domains are involved.

The much longer extensible region of N2BA titin is due to 1) additional Ig domains that make up the so-called middle tandem Ig segment, 2) the N2A element (4 Ig domains and three short unique sequences), and 3) a much longer PEVK segment (~800 residues for N2BA titin and ~180 residues for N2B titin) (8). As for the Ig domains in the N2A element and middle tandem Ig segment, if some of them were to unfold during stretch at pCa 9 but not at pCa 4, a calcium-dependent force response would ensue. However, no evidence for a calcium effect on unfolding force has been found (43) and, furthermore, unfolding at pCa 9 is unlikely to take place at physiological force levels (40). Instead, it is likely that extension of tandem Ig segments is solely due to straightening of linker sequences that align the folded domains. Thus Ig domains could give rise to calcium-sensitive force if linker sequences of differentially expressed Ig domains were to bind calcium and thereby increase their resistance to straightening. Although this possibility can currently not be excluded, we consider it unlikely because of sequence similarity between differentially and constitutively expressed Ig domain linker sequences (44). Similarly, the unique sequences of the N2A element (25) could in principle be involved in rendering force of titin to be calcium dependent, for example, by adopting more compact structures in the presence of calcium or by a reduction in their persistence length, but supportive evidence for such mechanisms is lacking.

As for the PEVK, to explain our findings, the much longer PEVK segment of N2BA titin would have to contain calcium-sensitive elements that are absent in N2B titin. In recent single molecule experiments with recombinant PEVK fragments, we found that the mechanical properties of the PEVK are calcium sensitive and that this requires the presence of E-rich motifs (24). Greaser and colleagues (19) have shown that the N2B isoform expressed in the rat heart is composed solely of PEVK repeats (rat orthologous sequences corresponding to exons 219–225 of the human titin gene, accession no. AJ277892). Thus the absence of E-rich motifs in the PEVK sequence of rat N2B titin and the absence of a calcium-sensitive passive stiffness in rat trabeculae are consistent with the notion that E-rich PEVK motifs are required for calcium-sensitive passive stiffness. As for the PEVK of bovine N2BA titin, its sequence is unknown. However, the available N2BA PEVK sequences of the human (25) and dog (19) both reveal the presence of several E-rich motifs, making it likely that the same is true for the bovine sequence. Consistent with this are the Western blot and immunofluorescence studies with antibodies to E-rich motifs (Fig. 4) that were positive for bovine N2BA titin. Thus our findings support the conclusion that calcium-sensitive tension of BLA is due to E-rich motifs in the PEVK segment of N2BA titin and that the calcium-insensitivity of the rat is due to the absence of E-rich motifs in N2B titin.

Previous work on N2B titin-expressing myocardium has revealed that the N2B PEVK and actin interact (23, 46), enhancing passive stiffness. Evidence has been obtained that suggests that this interaction can be inhibited by S100, provided that calcium is present (47). This finding possibly explains why passive stiffness in rat trabeculae (predominately N2B titin) increases during the diastolic interval when the calcium level progressively decreases (34). Whether the PEVK segment of N2BA titin also interacts with actin has not been studied. Considering the different composition of N2B and N2BA PEVKs (with E-rich motifs in only the latter), differences in actin-binding properties are to be expected. Indeed, a recombinant PEVK fragment from skeletal muscle titin consisting of PEVK repeats and an E-rich motif does not bind actin under physiological conditions (46), and the ability to interact with actin may be most pronounced in sarcomeres that express high levels of N2B titin. Thus, by splicing in certain PEVK exons and excluding others, unique molecules can be constructed, some of which interact with F-actin (N2B PEVK) and others that have mechanical properties that are calcium sensitive (N2BA titin and skeletal muscle titin isoforms).

Physiological significance. Calcium-sensitive passive tension is expected only in myocardium that expresses relatively high levels of N2BA titin, such as in large mammals that express nearly pure N2BA titin in their atria and N2BA and N2B titin at similar levels in their ventricles. It has been proposed that the calcium-sensitive passive tension in skeletal muscle helps to maintain the structural integrity of the contracting sarcomere (1, 21, 24), and it is possible that a similar role is played in the myocardium. At long SLs where active tension is low, additional titin-based tension will aid in preventing sarcomere overstretch (for example, by shorter sarcomeres in the end regions of the fibers). Furthermore, at the beginning of contraction when an imbalance in active force is likely to exist within sarcomeres (due to slight differences in the degree of thin-thick filament overlap of the two halves of the sarcomere), additional titin-based tension might play a role in keeping the A band centered in the middle of the sarcomere. Although N2B titin is not calcium sensitive, it has a higher intrinsic stiffness than N2BA titin (45), and N2B-expressing myocardium might therefore not require a calcium-sensitive passive force to maintain its structural integrity (sarcomere length homogeneity and central A-band location).

Titin's calcium sensitivity of N2BA expressing myocardium might impact systolic and diastolic function of the heart. The elastic recoil of titin during early systole has been suggested to play a role in accelerating active contraction speed (32), and the calcium-sensitive tension of titin will augment this role. During early diastole, the restoring force of titin is likely to contribute to the suction force (20), and early diastolic calcium levels [pCa ~6.5 (34)] might be high enough to enhance the restoring force of titin [pCa50 of PL reduction is ~6.5 (24)], augmenting early diastolic filling. As diastole proceeds, diastolic calcium falls and titin-based passive tension will decrease (relative to the value in the presence of high calcium), increasing myocardial compliance and promoting late diastolic filling. To experimentally address the physiological significance of the calcium sensitivity of the tension of titin, future work could take advantage of the unusually high levels of N2BA titin that have recently been reported in fetal and neonatal myocardium (26, 33), as well as in the myocardium of patients with heart failure that have dilated cardiomyopathy (30) and coronary artery disease (31). In summary, our work revealed that passive tension developed by N2BA titin is calcium sensitive, and we propose that calcium-dependent passive tension plays roles during both systole and diastole.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-61497/62881 (to H. L. Granzier), Deutsche Forschungsgemeinschaft Ge 1222/1-1 (to B. Gerull), and La 668/7-1 (to S. Labeit), and a postdoctoral fellowship from American Heart Association, Northwest affiliate (to H. Fujita).


   ACKNOWLEDGMENTS
 
We thank Geetha Ramaprian, Xiuju Luo, and Mark McNabb for technical assistance. We thank Drs. Norio Fukuda and Yiming Wu for scientific discussion and providing muscle specimen and training.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Henk L. Granzier, Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State Univ., Pullman, WA 99164 (E-mail: granzier{at}wsunix.wsu.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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