INTRODUCTION

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THE STRIATED MUSCLE SARCOMERE consists of thin filaments attached to Z lines, thick filaments located in the center of the sarcomere, and titin filaments that span from the Z line to the middle of the sarcomere (8). The thin and thick filaments develop active force, whereas titin filaments develop passive force (6). The force of titin is derived from the I-band region of the molecule that extends while the sarcomeres are stretched (17). The I-band region of cardiac titin has a complex sequence with three distinct elements that extend along the physiological sarcomere length (SL) range of the heart: 1) the PEVK segment, rich in proline (P), glutamic acid (E), valine (V), and lysine (K) residues; 2) serially linked Ig-like domains that make up tandem Ig segments; and 3) the N2B element (4, 10-12).

Most of the thin and thick filament-based muscle proteins exist as multiple isoforms, either derived from different genes or from differential splicing of the same gene that are expressed in a developmental- and disease-specific manner (13). Recent work (4) indicates that titin is derived from a single gene on chromosome 2 (region 2q31) and that extensive exon shuffling in the I-band region of the molecule results in a wide range of isoforms. In the heart, titin transcripts are processed by two distinct splice routes, which vary within their extensible I-band regions: 1) the so-called N2B titins containing the N2B element and 2) N2BA titins containing both the N2B and N2A elements (2, 4) (see Fig. 1). N2BA titins contain a much longer PEVK segment than do N2B titins (600-800 vs. 163 residues), as well as an additional tandem Ig segment (4).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Domain structure of I-band regions of cardiac N2B and N2BA titins and location of binding sites of used antibodies (4) [T12, N2BN, N2BC, N2A, I84-86, and I109-111]. Two N2BA splice pathways are shown, representing the pathway with the lowest (12) and highest (25) number of Ig domains identified so far in the titin gene sequence (4). Red, Ig domains; white, fibronectin domains; yellow, PEVK; blue, unique sequence.

Recent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies (2) of myocardium isolated from various species indicate that the expression level of N2B and N2BA cardiac titins varies from predominantly N2B (rat and mouse) to predominantly N2BA (bovine atrium), with many species (including human) coexpressing isoforms at intermediate levels. Changes in titin isoform expression may be important during heart disease because alterations in the titin isoform expression level have been observed in pacing tachycardia heart failure in the dog (1). It is not known whether sarcomeres are isoform pure and whether coexpression is the result of mixtures of sarcomeres that express different isoforms or whether coexpression occurs at the level of the half-sarcomere. If the latter were to be the case, the question arises of whether different isoforms contained within the half-sarcomere interact. Solving these issues is important for understanding the functional significance of coexpressing isoforms and its alterations in heart disease. Immunofluorescence and differential staining of the sarcomere with isoform-specific antibodies cannot be used to study coexpression because the full N2B sequence is contained within N2BA titin (4). Here we used immunoelectron microscopy (IEM) with the N2B antibody, raised against a sequence found in both isoform types, that labels closer to the Z line in N2BA titin than in N2B titin (see RESULTS). Thus the N2B_C antibody and IEM are ideal for the study of cardiac titin isoform expression at the sarcomeric level. Our results show that coexpression of N2B and N2BA isoforms takes place at the level of the half-sarcomere and that different isoforms expressed in the half-sarcomere extend independently. We propose that coexpression of isoforms at the level of the half-sarcomere allows for tuning of the passive properties of cardiac muscle.

	METHODS

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Muscles. Hearts were excised from ~9-wk-old BALB/C male mice, mongrel dogs (~20 kg), and cows (~18 mo old) obtained from slaughterhouses. The free wall of the left atrium and left ventricle were cut into pieces and placed into oxygenated Krebs solution (19). Muscle strips were dissected in the direction of the fibers, while stretch of the preparations was minimized. Muscles were skinned in relaxing solution (6) containing 1% wt/vol Triton X-100.

SDS-PAGE. Muscles were quick-frozen in liquid nitrogen, pulverized to a fine powder, rapidly solubilized, and then analyzed with the use of SDS-PAGE (6). The gels were scanned as described in an earlier study (6).

Titin antibodies. Antibodies that were used and the locations of their epitopes in titin are shown in Fig. 1. Titin cDNA fragments coding for the I-band epitopes I24/I25 to I109-I111 were isolated by polymerase chain reaction from total human cDNAs. All primer sequences were derived from European Molecular Biology Laboratory data library accessions X90568/X90569 and are described in detail elsewhere (4). The amplified cDNA fragments were subcloned into modified pET9D vectors and fusion peptides with NH₂ terminal His⁶-tags were purified from the soluble fractions by nickel chelate affinity chromatography. Antibodies to the respective peptides were raised in rabbits and were affinity purified (4). The titin antibody T12 recognizes the I2/I3 repeats (14) and was purchased from Boehringer (Indianapolis, IN).

IEM. Skinned muscles while in relaxing solution were stretched to different lengths, fixed, immunolabeled, embedded, and processed for IEM essentially as described (5). The mid-Z-line to mid-epitope distances were measured from IEM negatives after high-resolution scanning (model UC-1260, UMAX) and digital image processing using custom-written macros for the NIH Image 1.6. For spatial calibration, the magnification of the microscope was used.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Gel electrophoresis of left ventricular myocardium (Fig. 2A) revealed that the mouse expressed predominately N2B titin and that large mammals (dog and bovine) coexpressed N2B and N2BA titins, whereas the bovine atrium is nearly N2BA pure. These findings are consistent with earlier results (2). To study the coexpression of titin, we used IEM with an antibody against the COOH-terminal end of the N₂B element (N₂B_C). This element is found in both isoforms but is closer to the A band in N2B than N2BA titin (see Fig. 1 and DISCUSSION). Immunoelectron micrographs of cardiac sarcomeres from a range of species labeled with N2B_C are shown in Fig. 2B. At a given SL, sarcomeres from myocardium that expresses predominately N2B titin contain a single epitope toward the A band (Fig. 2B, top) and sarcomeres from myocardium that expresses predominately N2BA titin contain a single epitope closer to the Z line (Fig. 2B, bottom). Thus the N2B_C antibody can be used to study coexpression of isoforms in the I band because coexpression will result in two epitopes, one near the A band derived from N2B titin and one closer to the Z line derived from N2BA titin. When myocardium of species that coexpress isoforms (as deduced from SDS-PAGE) was labeled with N2B_C, two I-band epitopes were typically found (see Fig. 2B, top middle and bottom middle; the near Z line and near A-band epitopes are referred to as A and B epitopes, respectively). The finding of two epitopes is consistent with the notion that the sarcomere of ventricular myocardium of large mammals coexpresses isoforms of titin.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   A: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myocardial tissues from left ventricle (V) of mouse, dog, and bovine and left atrium (A) of bovine. Mouse ventricle contains predominantly N2B titin. Dog and bovine ventricle (BV) express N2B and N2BA2 titins, whereas bovine atrium expresses predominately N2BA titin. T2 is a degradation product of titin. MHC: myosin heavy chain. B: examples of sarcomeres labeled with the N2BC antibody. Note that dog and BV sarcomeres contain two N2BC epitopes: one near the Z line (A) and the other closer to the A band (B), whereas the other specimen contains a single epitope (see Fig. 3). Asterisk denotes labeling with I109-111 (stains at A-band edge). See text for details. Scale bar, 1.0 µm.

We studied the location of the two N2B_C epitopes in the bovine ventricular sarcomere and determined the dependence of the epitope-Z-line distance on SL. The two epitopes are merged at a SL of ~1.8 µm but they separate as SL increases and are ~0.2 µm apart at a SL of 3.0 µm (Fig. 3). Figure 3 also shows results (16, 18) with the N2B_C antibody in rat ventricle and bovine atrium. The N2B_C(B) epitope of bovine ventricle superimposes with rat ventricular results and the N2B_C(A) epitope with the results of the bovine atrium. These findings suggest that the bovine ventricle coexpresses two isoforms, one that behaves like the N2B isoform of the rat and the other like the N2BA isoform of the bovine atrium.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Distance between N2BC epitopes and middle of Z line in BV sarcomeres. Also shown are the N2BC results of rat ventricle (RV) and bovine atrium (BA) (see Refs. 16 and 18). Results of these earlier studies were pooled in 0.1-µm sarcomere length (SL) bins, and means ± SD of SL and epitope distances are plotted. Lines are second-order polynomial fits of the BV data. , N2BC antibody in RV; , N2BC antibody in BA.

We also determined in the bovine ventricle the lengths of the PEVK and N2B unique sequences, by using antibodies that demarcate the PEVK and N2B unique sequence domains in the extensible region of N2B and N2BA titins (Fig. 1). Representative micrographs are shown in Fig. 4, A-D, and a graph of epitope to Z-line distance is shown in Fig. 4E. The various epitopes in the bovine ventricle were assumed to result from coexpressed N2B and N2BA isoforms. The epitopes that demarcate the PEVK segment and N2B unique sequence of N2B titin are shown in Fig. 4F and those of N2BA titin in Fig. 4G. With the use of these epitopes, we determined the lengths of the PEVK segments and N2B unique sequences. Our results show that the two PEVK segments that can be discerned in the bovine ventricle sarcomere respond differentially to sarcomere stretch (red and green symbols in Fig. 5A), as do the two N2B sequence elements (red and green symbols in Fig. 5B).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   A-D: example of BV sarcomeres labeled with indicated anti-titin antibodies. (Scale bar, 1.0 µm.) E: scattergram of epitope to Z-line distance vs. SL. Labeling patterns are assumed to be derived from N2B and N2BA isoforms that are coexpressed; their PEVK and N2B unique sequence segments are shown in 4F (N2B titin) and G (N2BA titin). Lines in F and G are the second-order polynomial fits of the data in E.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   SL dependence of end-to-end lengths of PEVK (A) and N2B unique sequence (B) of the BV, superimposed with results of RV and BA. Earlier RV and BA data (16, 18) calculated in Fig. 3. Lines are second-order polynomial fits of the BV data.

We overlaid the bovine ventricular results with those obtained earlier in the rat ventricle and bovine atrium (16, 18). The bovine ventricular PEVK segment that extends most steeply with SL (Fig. 5A, red symbols) behaves like the PEVK of the bovine atrium (Fig. 5A), whereas the other PEVK segment (Fig. 5A, green symbols) extends like the PEVK of the rat ventricle (Fig. 5A). The bovine ventricular N2B unique sequence that extends most steeply with SL (Fig. 5B, red symbols) behaves like the unique sequence of the rat ventricle (Fig. 5B) and the one that extends less steeply behaves like the unique sequence of the bovine atrium (Fig. 5B). As further explained below, these findings indicate that the bovine ventricle coexpresses, at the level of the half-sarcomere, N2B and N2BA isoforms that extend independently of one another.

Although staining with the N2B_C antibody typically revealed two epitopes of similar intensity in the I band of bovine ventricular sarcomeres, on occasion, one of the epitopes dominated. Figure 6A, bottom, shows an example of such atypical labeling pattern (seen in <1% of the examined sarcomeres). Variation in staining was also found in other species. For example, in the rat ventricle, the overwhelming majority of sarcomeres (>99%) contained a single epitope near the A band, but on occasion there was a second epitope more toward the Z line (Fig. 6B, bottom). Because both epitopes are derived from a single antibody, variation in diffusion of the antibody into the tissue is unlikely to underlie the variable staining patterns. Instead, they may derive from variation in the expression patterns of N2B and N2BA isoforms.

View larger version (79K): [in this window] [in a new window]   Fig. 6.   Unusual N2BC staining patterns. A, top: typical labeling pattern with two I-band epitopes (A and B) of BV sarcomeres; bottom, atypical sarcomere with strong N2BC(A) epitope and barely visible N2BC(B) epitope (arrow). B, top: typical labeling pattern with one epitope in I band for N2BC-labeled rat sarcomeres; bottom, unusual pattern with two epitopes. Asterisk denotes labeling with I109-111. C: example of bovine atrial sarcomeres labeled with the N2A antibody. Note two closely spaced epitopes at bottom right corner. D: SDS-PAGE of BA, BV, and RV. Note in BA two closely spaced bands in the N2BA region of gel. E: densitometry of gel revealing two N2BA peaks in BA (solid lines), BV (dashed lines), and RV (gray solid lines). The lower mobility N2BA peak of BA comigrates with the single N2BA of BV. Scale bars in A-C, 1.0 µm.

We also studied variation in isoform expression in the bovine atrium and were unable to find clearly separated N2B_C epitopes, suggesting that bovine atrium expresses predominantly N2BA titins. When labeling bovine atrium with the N2A antibody we noted that long sarcomeres often contained closely spaced double epitopes (Fig. 6C). The separation of the epitopes did not vary markedly along the studied SL range (~2.0-2.5 µm), with a mean separation of 68 ± 13 nm (n = 24). These findings can be explained by assuming that bovine atrium coexpresses N2BA isoforms and that the two N2A epitopes result from differences in the middle tandem Ig segment contained within these isoforms (Fig. 1). To test whether different N2BA bands can be detected on gels, we performed high-resolution electrophoresis. This revealed a doublet in the N2BA region (Fig. 6D), the bottom band of which comigrated with the single N2BA band of the bovine ventricle (Fig. 6E). We conclude that the bovine atrium coexpresses N2BA isoforms that vary considerably in molecular mass.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Work at the mRNA and protein levels has revealed that cardiac muscle contains two classes of titin isoforms, the N2B and N2BA titins (2, 4). Our goal was to investigate whether coexpression occurs at the level of the smallest functional unit of muscle, the half-sarcomere, and how coexpression affects the extensibility of the isoforms. We used the N2B_C antibody, raised against a sequence found in both isoform types, that labels closer to the Z line in N2BA titin than in N2B titin. This different sarcomeric location results from the large number of additional Ig domains and PEVK residues that are present in N2BA titin, COOH-terminal of the N₂B_C epitope (4). These extra sequences greatly increase the length of the extensible region of N2BA titin, and a given SL can therefore be reached with a fractional extension of the extensible region that is lower in N2BA than in N2B titin. Hence, the N2B_C epitope will be closer to the Z line in N2BA than in N2B titin, and the N2B_C antibody can be used to study coexpression of titin isoforms within the sarcomere (Fig. 7A).

View larger version (33K): [in this window] [in a new window]   Fig. 7.   A: schematic representation of sarcomere coexpressing N2B and N2BA titins with PEVK and N2B unique sequences that extend independently. B: titin-based passive tension-SL relations of isoform pure myocardium [see (19)]. N2B expressing sarcomeres develop much higher passive tensions than N2BA-expressing muscle. By coexpressing isoforms at varying ratios, an intermediate passive tension level can be obtained. See text for details.

When myocardium that coexpresses N2B and N2BA isoforms was labeled with N2B_C, two epitopes per half-sarcomere were seen, one near the Z line in a location that is similar to that of N2BA expressing bovine atrium and another closer to the A band in a location that is similar to that of N2B-expressing rat sarcomeres (Fig. 3). For the first time, these findings provide evidence that the cardiac sarcomere of large mammals coexpresses isoforms of titin at the level of the half-sarcomere. Thus, not only is the I-band region of cardiac titin a complex molecular spring with three distinct subsegments (5, 8), an additional level of complexity results from isoforms that contain length variants of these spring segments that are coexpressed side by side.

Variation in coexpression patterns. We found that the expression level of titin isoforms in sarcomeres obtained by IEM was typically similar to that in muscle obtained by using SDS-PAGE and Western blotting (2). However, deviations were found as well. For example, the overwhelming majority of sarcomeres in rat myocardium contain a single N2B_C epitope (as expected from gel results), but on occasion, sarcomeres were found with two epitopes (Fig. 6B), suggesting that both isoform types were coexpressed. Furthermore, in the bovine ventricle, two N2B_C epitopes of similar intensity were typical (consistent with gel results), but on occasion, the near-Z-line epitope derived from N2BA titin dominated (Fig. 6A). Thus SDS-PAGE and Western blotting provide insights in the average expression level of the isoforms within muscle, whereas the IEM method shows that expression patterns can locally deviate from this average. Deviations in expression patterns are likely to reflect local differences in demands on the functions of titin that are different from the tissue average.

Coexpression of N2BA isoforms. At the mRNA level, seven N2BA isoforms have been characterized that differ in the length of the middle tandem Ig segment (4) (the extremes are shown in Fig. 1). The double N2A epitopes that we found (Fig. 6C) suggest coexpression of N2BA isoforms in the bovine atrium. Considering that the average Ig domain spacing of tandem Ig segments is ~5 nm (16, 18), the N2A epitope separation of 68 nm (see RESULTS) suggests a ~14-Ig domain difference between the large and the small N2BA isoforms expressed in bovine atrium. A 14-Ig difference is expected to result in a molecular mass difference of ~140 kDa (~10 kDa per Ig domain), a difference that in the MDa molecular mass range may be just detectable on gels (7). Consistent with this are SDS-PAGE results that showed closely spaced N2BA bands in the bovine atrium (Fig. 6, D and E). Thus both SDS-PAGE and IEM results support that members of the N2BA isoform class are coexpressed in the bovine atrium.

Is there interaction between coexpressed isoforms? Understanding the functional significance of coexpression of isoforms at the level of the sarcomere requires insights into whether or not the isoforms act independently of one another. Sequences contained within the extensible region of the isoforms may interact with those of other isoforms in their vicinity or, alternatively, titin-binding proteins may laterally link the different isoforms and influence the elastic properties of titin. Our results indicate that the extensibility of PEVK and N2B unique sequences in isoform-pure sarcomeres is indistinguishable from that in isoform-mixed sarcomeres (Fig. 5, A and B). Thus lateral interactions between the isoforms are either weak or absent and the isoforms extend independently of one another. Independent behavior is shown schematically in Fig. 7A.

Functional significance. Passive force levels intermediate between that of myocardium that expresses predominately N2BA or N2B titin (Fig. 7B) can, in theory, be achieved by varying the number of titin molecules per sarcomere. However, this would also influence functions performed by the inextensible regions of titin. These regions play roles in construction and maintenance of Z lines and M lines, thick filament length control, and binding of ligands [for a review, see Gregorio et al. (8)]. Thus varying the number of titin molecules to achieve a certain passive force level may be undesirable because other roles of titin would be impacted as well. This issue does not exist when passive force levels are tuned via coexpressing isoforms because the inextensible regions of the isoforms are the same. Any force level intermediate between that of isoform-pure sarcomeres may be obtained by varying the expression ratio of isoforms (see also Fig. 7B) without impacting other functions of titin.