Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes

Abstract

Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle.

Introduction

Tropomyosin (TPM) is an actin binding protein widely distributed from yeast to mammals (Bailey, 1948). TPM is a rod-like protein composed of 2 α-helical chains arranged as a coiled-coil structure and is localized along each of the grooves of F-actin (Ohtsuki et al., 1986; Xu et al., 1999; Perry, 2001). In striated muscle, TPM is an essential protein that, in association with troponin T, I and C components (TnT, TnI, and TnC, respectively), regulates the calcium-sensitive interaction of actin and myosin during muscle contraction (Ebashi et al., 1969; Endoh, 2008).

Four TPM genes (TPM1, TPM2, TPM3 and TPM4) have been characterized in vertebrates (Perry, 2001; Gunning et al., 2008; Geeves et al., 2015). TPM1-4 genes are regulated by alternative splicing and over 40 TPM isoforms are expressed in cardiac, skeletal, smooth, and non-muscle cells, and are controlled in a complicated manner during development in each of these tissues (Wieczorek et al., 1988; Lindquester et al., 1989; Fleenor et al., 1992; Perry, 2001; Wang et al., 2008; Hook et al., 2011). It has been shown that the TPM1 gene expresses 2 mRNA isoforms named TPM1α and TPM1κ in avian embryonic cardiac muscle but these were not expressed in adult cardiac muscle (Wang et al., 2008). Furthermore, it has been reported that knockout in mice of the TPM1 gene is embryonic lethal (Blanchard et al., 1997; Rethinasamy et al., 1998; Robbins, 1998). Thus the TPM1gene has been shown to play an important role for embryonic cardiac muscle during development.

In contrast, the TPM4 gene has been shown to express 3 mRNA isoforms, namely TPM4α, low molecular weight TPM and TPM4β. TPM4α is the only isoform that is expressed over the whole period of embryonic and adult cardiac muscle (Forry-Schaudies et al., 1990; Wang et al., 2008). However, the function of TPM4α is still incompletely understood. The only physiological relevance report­ed so far is that lack of cardiac TPM4 gene is responsible for heartbeat failure in zebra fish (Zhao et al., 2008) and Mexican axolotl mutants (Spinner et al., 2002).

The aim of this study is to examine the roles of TPM4α protein in the function of cytoskeleton and myofibrils in cardiomyocytes. RNAi was used for suppressing TPM4α. Small interference RNAs (siRNA) for TPM4α (SiTPM4) was designed with a nucleotide sequence corresponding to exon 8 in TPM4 gene and transfected to chicken cardiomyocytes. Several myofibril proteins, such as myosin, actin, TPM, troponin, connectin (also known as titin) and TnT, were examined by immunofluorescence microscopy. The TPM4α-suppressed cells displayed unique cytoskeletal and myofibril structures. The present results suggest that TPM4α plays significant roles in the assembly and maintenance of myofibril in cardiomyocytes.

Materials and Methods

Antibodies

The antibodies used are shown in Table I. These antibodies react­ed with chicken cardiac muscle.

Table I Antibodies used in this study

antigen	names	mono/poly	labeled bands	use for this study
Chicken cardiac muscle TPMa) (specific for TPM4α)	anti-CTPM	poly rabbit	I bands	immunoblotting, immunofluorescence
Myosin heavy chainb)	HV11	mono mouse	A bands	immunoblotting, immunofluorescence
Connectin (also called titin)c)	Me 1G1	mono mouse	A-I junction	immunofluorescence
Sarcomeric α-actinind)	EA-53	mono mouse	Z discs	immunoblotting, immunofluorescence
Chicken pectoral muscle TnTe)	G5G4	mono mouse	I bands	immunoblotting, immunofluorescence
Chicken cardiac muscle TnTf)	anti-CTnT	poly rabbit	I bands	immunofluorescence
Rhodamine-phalloidind)			I bands (F-actin)	immunofluorescence

The antibodies were purchased or obtained as gift, as indicated below:

a) Chicken cardiac muscle TPM was prepared from adult ventricles according to Bailey (1948), and anti-CTPM antibody was prepared according to Toyota and Shimada (1981). Adult cardiac muscle expresses only TPM4α protein. The antibody against chicken cardiac muscle TPM is specific for TPM4α.

b) HV11 were from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa, IA, USA).

c) Me 1G1 was supplied by Dr. S. Kimura from the Dept. of Biology, Chiba University.

d) EA-53 and rhodamine-phalloidin were from Sigma Aldrich (St. Louis, MO, USA).

e) G5G4 was the same antibody used previously (Shimizu and Shimada, 1985; Toyota et al., 2008).

f) Anti-CTnT was the same antibody used previously (Toyota and Shimada, 1981).

Immunoblot analyses

Culture of embryonic chicken ventricular muscle cells and siRNA transfection were carried out as described previously (Toyota et al., 2008). Ventricular muscle cells were prepared from 7 or 8-day-old chicken embryos according to DeHaan (1970). After all myocytes were dissociated completely, cells (2×10⁶) were plated in 100-mm dishes in 8 mL of culture medium. Cells were maintained in growth culture medium 818B supplemented with 5% (v/v) horse serum as described by DeHaan (1970). Hereinafter, the first 24 h after plating is defined as day 1 of culture.

Two siRNA (SiTPM4 and SiTPM5) were designed based on the avian TPM4 gene (Fleenor et al., 1992; Wang et al., 2008) and were purchased from Qiagen (Hilden, Germany). SiTPM4 sequence was dTdTAAGATTCTTTCTGACAAGCTC, and SiTPM5 sequence was dTdTAAGCTGAAGTACAAAGCAA­TC. SiTPM4 was the most effective sequence suppressing TPM4 protein as shown in Fig. 1A–C. SiTPM4 was used in subsequent analyses. Cultured cardiomyocytes were transiently suppressed using SiTPM4 and Oligofectamine (Invitrogen, Carlsbad, CA, USA) on day 1 as follows. The culture medium was replaced with 5 μL of Opti-MEM I medium prior to transfection. Then, a mixture consisting of 500 pmol of SiTPM4, 460 μL of Opti-MEM I medium, and 8 μL of Oligofectamine was added per dish. Four hours after addition of the SiTPM4 mixture, 5 mL of culture medium containing 15% of horse serum was added per dish. Cardiomyocytes on days 1–5 were used for immunoblotting. Parallel control cells were treated with Oligofectamine without SiTPM4. The cells grown as described above were washed with PBS and collected with a scraper before lysis with SDS sample buffer (final volume: 150 μL; final SDS concentration: 2% w/v). SDS samples were homogenized with Physcotron NS-310EII (Micdotec Co., Ltd, Tokyo Japan). These lysates (12 μL) were subjected to SDS-polyacrylamide gel electrophoresis (10% gel) in the presence of 0.1% SDS according to Laemmli’s method as described previously (Toyota et al., 1998). After electrophoresis, proteins were transferred to nitrocellulose sheets and reacted with the appropriate antibodies. Bound primary antibodies were incubated with biotinylated anti-mouse or anti-rabbit IgG and then with horseradish peroxidase linked to avidin (Vectastain; Vector Laboratories, Burlingame, CA). Protein bands were visualized by reaction with hydrogen peroxide in the presence of 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan). Cardiac actomyosin was extracted from homogenates of adult ventricular muscle in KCl (final concentration: 0.6 M) as described previously (Toyota et al., 1998, 2008).

Fig. 1

Suppression of TPM4α protein on cultured chicken ventricular muscle cells. Immunoblot analysis of TPM4α was carried out with cells on days 1–5 of culture, using anti-CTPM and EA-53 (A and B). Panel A shows controls of cardiac TPM4α expression with Oligofectamine without siRNA (b–f). Panel B shows RNAi effects in cultured cells transfected on day 1 of culture with SiTPM4 (c–f). Between 2–5 days of culture, the expression of TPM4α was gradually suppressed but α-actinin was expressed at a constant level (Panel B, c–f). Lane a in panels A and B shows α-actinin and TPM4α in actomyosin prepared from adult ventricular muscle. Lane b in panel B shows cells before SiTPM4 transfection. The arrowhead in panel B shows SiTPM4 transfection. Panel C shows expression of myofibrillar proteins in cultured ventricular muscle cells on day 5. Control cells (b, d, f and h) and cells with substantially suppressed TPM4α (c, e, g, and i) were electrophoresed side-by-side, and analyzed for expression of myosin (b, c), α-actinin (d, e), TPM4α (f, g) and CTnT (h, i) using the following respective antibodies: HV11, EA-53, anti-CTPM and G5G4. Lanes c, e, g and i were loaded with the same sample as those of Panel B, f. Lane a in panel C is a bench marker protein ladder (Invitrogen, Carlsbad, CA, USA) showing molecular weights indicated on the left side. Control cells with Oligofectamine without siRNA expressed myofibrillar proteins, and their bands were detected respectively corresponding to each protein position (b, d, f and h). TPM4α-suppressed cells also expressed myosin (c), α-actinin (e) and CTnT (i), but TPM4α protein was suppressed (g).

Immunofluorescence microscopy

Preparation of embryonic chicken ventricular muscle cells was performed as described above. Cells (2×10⁵) were plated in 35-mm plastic culture dishes with 1.5 mL of culture medium and transiently transfected with SiTPM4 on day 1. The culture medium was replaced with 0.8 mL of Opti-MEM I medium prior to transfection. Then, a mixture consisting of 400 pmol of SiTPM4, 190 μL of Opti-MEM I medium, and 4 μL of Oligofectamine was added to each dish. Four hours after addition of SiTPM4, 0.5 mL of culture medium 818B containing 15% horse serum was added per dish. SiTPM4-transfected cardiomyocytes on days 5 were used for immunofluorescence microscopy. Parallel control cells were treated with Oligofectamine without SiTPM4 and used for immunofluorescence microscopy. Cells of both groups were fixed with ethanol at 4°C for 1 min and treated with antibodies to the sarcomeric proteins mentioned above.

Double staining

Cells were incubated with anti-CTnT polyclonal antibody and then tetramethyl-rhodamine-isothiocyanate (TRITC)-labeled antibody against rabbit IgG. Subsequently, cells were incubated with α-actinin (EA-53) antibody and fluorescein isothiocyanate (FITC)-labeled antibody against rabbit or mouse IgG, respectively. In some cases, G5G4 was used as first antibody followed by TRITC-labeled antibody against mouse IgG. Subsequently, cells were incubated with anti-CTPM and FITC-labeled antibody against rabbit IgG. For actin visualization, cells were fixed with 0.1% (v/v) formalin in PBS, stained with G5G4 and FITC-labeled antibody against mouse IgG, and treated with rhodamine-phalloidin. Antibody incubation was performed for 45 min at room temperature, and subsequently washings were carried out appropriately.

Triple staining

Cells were stained with anti-CTnT polyclonal antibody and TRITC-labeled antibody against rabbit IgG, then reacted with monoclonal antibody against α-actinin (EA-53) and Alexa Fluor 647-labeled antibody against mouse IgG. Next, the cells were stained with monoclonal antibody against myosin (HV11) and FITC-labeled antibody against mouse IgG. In some cases, monoclonal antibody against connectin/titin (Me1G1) was used instead of EA-53. Antibody incubation and washing were performed as mentioned above. A Zeiss Axio Imager A1 immunofluorescence microscope equipped with a phase contrast system, a scanning digital camera (AxioCam MRm, Carl Zeiss Co., Ltd.), and 100× objective was used for observation of the cells as described previously (Toyota and Shimada, 1983).

Results

The general pattern of differentiation of cultured cardiac muscle cells has been reported elsewhere (DeHaan and Hirakow, 1972; Schultheiss et al., 1990; Ono, 2010; Sanger et al., 2010) and will not be reported here.

Effects of SiTPM4 transfection on the expression of TPM4α

SiTPM4 was transfected into the cardiomyocytes in order to suppress TPM4α-expression. Cellular lysates were subjected to immunoblot analyses with a polyclonal antibody specific for TPM4α (anti-CTPM) and EA-53 which was a monoclonal antibody against α-actinin (Fig. 1A and B).

To analyze suppression of TPM4α, immunobloting of α-actinin and TPM4α was examined with the anti-CTPM and EA-53 (Fig. 1A, B). Bands of α-actinin and TPM4α were detected in the control cells (Fig. 1A, a–f) and TPM4α-suppressed cells (Fig. 1B, a–f). The mobility of the α-actinin and TPM4α bands were the same as that of respective proteins in actomyosin extracted from adult ven‍tricular muscle (Fig. 1A and B, a). Similar levels of each α-actinin and TPM4α expression were observed in the control cells cultured for 1–5 days (Fig. 1A, b–f). Then, the duration and intensity of the effect of RNAi in cultures of transfected cardiomyocytes were examined (Fig. 1B). SiTPM4 transfection was performed on day 1 (Fig. 1B, arrowhead), and the cells were cultured for the following 4 days. Suppression of TPM4α was gradually detected from day 3 to day 5 (Fig. 1B, c–f), showing the lowest level on day 5 (Fig. 1B, f). However, the α-actinin band was unaffected with TPM4α suppression, and similar levels of α-actinin were detected between cells before and after transfection (Fig. 1B, b–f). Thus, the expression of TPM4α was progressively suppressed in the SiTPM4-transfected cells.

Next, the expression of myofibrillar proteins of TPM4α-suppressed cells on day 5 of culture which showed maximum TPM4α suppression (Fig. 1B, f) was examined by immunoblot analyses (Fig. 1C), and compared with control cells. Control cells (Fig. 1C, b, d, f, h) were electrophoresed parallel to TPM4α-suppressed cells (Fig. 1C, c, e, g, i), and expression of myosin (Fig. 1C, b, c), α-actinin (d, e), TPM4α (f, g) and CTnT (h, i) were observed. Each protein was detected at the respective molecular size expected (Fig. 1C, a–i), and all protein bands were detected in both control and TPM4α-suppressed cells, excepting TPM4α, which was undetectable in the TPM4α-suppressed cells (Fig. 1C, g). The results suggest that the suppression of TPM4α with SiTPM4 transfection does not significantly affect the expression of the other sarcomeric proteins.

Analysis of myofibril structure of TPM4α-suppressed cardiomyocytes

As shown in Fig. 1B, sufficient TPM4α-suppression was detected in cells of day 4–5 transfected with SiTPM4. Therefore, cells cultured according to this procedure were stained with antibodies against striated muscle for evaluation of sarcomeric structure in their myofibrils.

Fig. 2 shows the control (a, b, c) and the TPM4α-suppressed cells (d–i) double labeled with monoclonal antibodies G5G4 (a, d, g), anti-CTPM (b, e) and rhodamine-phalloidin (h) on day 4. Regular striations of I bands were observed on myofibrils in control cells with both G5G4 (Fig. 2a, c) and anti-CTPM (Fig. 2b, c) antibodies. However, the staining patterns of TPM4α-suppressed cells were different from that of control cells (Fig. 2d–i). In the TPM4α-suppressed cells, linear bundles were labeled with G5G4 (Fig. 2d, f, triple arrowheads) but bundles were undetectable with anti-CTPM (Fig. 2e). Several aggregates were detected, and labeled with G5G4 antibody (Fig. 2d, f, double arrowhead). The aggregates began to appear in the TPM4α-suppressed cells on day 4. The aggregates were branched from linear bundles labeled with rhodamine-phalloidin (g–i). Regular myofibril striation could not be detected in the TPM4α-suppressed cells (Fig. 2d, e, f). It suggested that SiTPM4 effectively suppressed TPM4α and that it affects structure of myofibrils in cardiomyocytes.

Fig. 2

Detection of CTnT, TPM4α and actin in control and SiTPM4-transfected cardiomyocytes on day 4. Control cells were double stained with both anti-CTnT (G5G4) monoclonal (a), and anti-CTPM polyclonal (b) antibodies. Panel c shows their merged image. Regular I bands were detected (a, b, c). Panels d-i show TPM4α-suppressed cells, labeled with G5G4 (d, g), and anti-CTPM polyclonal (e) antibodies, and rhodamine-phalloidin (h). In the TPM4α suppressed cells, aggregates deviating from linear bundles were detected (d, f, g, h, i), however bundles labeled with anti-CTPM were undetectable (e). Panels f and i show merged images. Triple arrowheads indicate linear bundle. Double arrowheads indicate aggregate. Scale bar=10 μm.

Fig. 3 shows control cells (a, b, c) and TPM4α-suppressed cells (d–h) on day 5, stained with anti-CTnT and α-actinin (EA-53) antibodies. In the control cells, regular striation of I bands (Fig. 3a, c) and Z disks (b, c) were confirmed with anti-CTnT (a, c) and α-actinin (b, c) antibodies on myofibrils. In the TPM4α-suppressed cells, however, structures different from normal cells were observed (Fig. 3d, rectangles). Myofibril differentiation in the SiTPM4-transfected cells was slower than that in normal cells, as judged by the fact that regular striation was undetectable in TPM4α-suppressed cells (Fig. 3, c versus d). The non-striated linear bundles were labeled with both α-actinin and anti-CTnT antibodies (Fig. 3d, triple arrowheads). However, a few striations corresponding to Z-like lines were labeled with EA-53, but tended to be unreactive with anti-CTnT antibody (Fig. 3d). This suggests that the linear bundles (Fig. 3d, triple arrowheads) correspond to underdeveloped myofibrils extending along the longitudinal axis of the cell.

Fig. 3

Localization of CTnT and α-actinin examined in myofibrils of control and TPM4α-suppressed cardiomyocytes on day 5. Control cells were double stained with anti-CTnT (a) and EA-53 (b) antibodies, and the images were merged in c. I bands and the Z disks were observed at each regular positions of myofibrils (a, b, c). Panels d–h show TPM4α-suppressed cells double stained with anti-CTnT (e, g) and α-actinin antibodies (d, f, h). A unique structure different from normal cardiomyocytes was detected in TPM4α-suppressed cells (d, rectangles: e–h). Many curved fibers (d–h, arrows) extended from the aggregates (double arrowheads), and constructed the large structure. Panels e and f are magnified views corresponding to the smaller rectangle in panel d. Panels g and h are magnified figures corresponding to the larger rectangle in panel d. In panels e-h, staining with anti-CTnT (e, g), and both anti-CTnT and α-actinin (f, h) antibodies were distinguished by fluorescent filters. In panels d, f and h, red indicates CTnT and green indicates α-actinin. Arrowheads indicate small nicks of non-striated linear bundles (f, h). Double arrowheads show the aggregates. Triple arrowheads indicate linear bundles. Arrows show curved bundles extending from the aggregate. Asterisks indicate a few tiny bundles between large structure and nicks on linear bundles. Scale bar=10 μm.

In addition to the linear bundles mentioned above, the unique large structure including aggregates and curved bun‍dles were detected (Fig. 3d–h, double arrowheads and arrows, respectively) in the TPM4α-suppressed cells. The large structure reacted with anti-CTnT antibody, but was unreactive with EA-53 (Fig. 3e–h, arrows and double arrowheads). In the magnified views, the large aggregate seemed to protrude from nicks on the linear bundles unreactive with α-actinin antibody (Fig. 3f, h, arrowheads), and many curved filaments spread bilaterally along linear bundles (Fig. 3e–h, arrows). A few tiny CTnT positive bundles were observed between the nicks on the linear bundles and the aggregate (Fig. 3e, f, asterisk), suggesting that the large structure originated from extremely small fissures of the linear bundles (Fig. 3f, h, arrowheads). The large structure was 10–20 μm in length and 1.4–2.5 μm in width.

The distribution of myosin was examined, and compared with that of CTnT and α-actinin by triple staining with anti-CTnT (Fig. 4a, d), α-actinin (b, e) and myosin antibodies (c, f). In control cells, regular striation of I bands, Z disks and A bands was detected on myofibrils with antibodies against CTnT, α-actinin and myosin (Fig. 4a, b, c, respectively).

Fig. 4

Localization of CTnT, α-actinin and myosin examined in myofibrils of control and TPM4α-suppressed cardiomyocytes on day 5. Control and TPM4α-suppressed cells were stained with anti-CTnT (a, d), EA-53 (b, e) and HV11 (c, f) antibodies. Regular striation of I bands (a), Z disks (b) and A bands (c) was observed with each antibody in control cells. In TPM4α-suppressed cells, anti-CTnT antibody labeled large structure (d, double arrowheads) and linear bundle absent striation (d, triple arrowheads). However, α-actinin antibody labeled striated bundles (e, triple arrows) and non-striated linear bundles (e, triple arrowheads). Myosin was detected only in striated linear bundles diffusely (f, triple arrows), but was undetectable in large structure (e, double arrowheads) and linear bundle absent striation (e, triple arrowheads). Double arrowheads indicate aggregate. Triple arrowheads indicate irregular striation corresponding to Z disk precursors. Triple arrows indicate the linear bundles without striation. Scale bar=10 μm.

In TPM4α-suppressed cells, however, aggregates were detected with anti-CTnT antibody staining (Fig. 4d, double arrowheads). These aggregates were undetectable with α-actinin and myosin antibodies (Fig. 4e, f, double arrowheads). Non-striated linear bundles with irregular punctate striation were detected with α-actinin antibodies (Fig. 4e, triple arrows). These Z disk precursors were undetectable with anti-CTnT and myosin antibodies (Fig. 4d, f, triple arrows). Then the distribution of myosin was examined by the staining with the myosin antibody (Fig. 4f). Myosin was detected only in the Z disk precursor area (Fig. 4f, triple arrows), and was undetectable on non-striated linear bundle (f, triple arrowheads) and large aggregate (f, double arrowheads). The distribution of myosin was restricted to the Z disk precursor area.

Next, connectin was examined and compared with CTnT and myosin. Connectin is a sarcomeric giant protein bridging the Z disk and the M line, which plays an important role in maintaining the A band localization in the middle of sarcomere in normal myofibrils (Maruyama, 1976; Meyer and Wright, 2013). Control cells were stained with anti-CTnT, myosin (HV11) and connectin (Me 1G1) antibodies, and regularly ordered striations were confirmed at I bands, A bands and A-I junctions of myofibrils (Fig. 5a, b, c, respectively).

Fig. 5

Localization of CTnT, myosin and connectin (titin) examined in myofibrils of control and TPM4α-suppressed cardiomyocytes on day 5. Control cells were triple stained with anti-CTnT (a), HV11 (b), and Me 1G1 (c) antibodies as mentioned in Materials and Methods. Regular myofibril striation was observed on I bands, A bands and A-I junctions (a, b, c, respectively). M bands were also labeled with anti-connectin antibody with ethanol fixation (c, open arrowheads). TPM4α-suppressed cells were stained with anti-CTnT (d, red), HV11 (d, green) and Me 1G1 (d, blue) antibodies. Disorganized structures corresponding to linear bundles were weakly labeled with anti-CTnT antibody (e, triple arrowheads). Large structures were detectable between thick filaments with incomplete striation (e, f, g, double arrowheads). Thick filaments with striated bundles labeled with anti-myosin antibody were similar to those seen with anti-connectin antibody (f and g, respectively). Panels e–g are magnified views of the rectangle in panel d. Double arrowheads indicate large structure. Triple arrowheads indicate linear bundle. Scale bar=10 μm.

TPM4α-suppressed cells were examined with the same antibodies (Fig. 5d–g). The bundles shown in Fig. 5 (d–g) were the same as those in Fig. 3 (d–h) and Fig. 4 (d–f), but myofibril development was much progressed compared with those bundles. Myofibrils with blurred striation were detectable in the middle portion of the cell (Fig. 5d, rectangle). Aggregates and linear bundles were labeled with anti-CTnT antibody (Fig. 5e, double arrowheads and triple arrowheads, respectively), but curved bundles spreading from aggregates were undetectable. Thick filaments with blurred striation were examined with myosin (Fig. 5f) and connectin antibodies (Fig. 5g). Linear bundle and large structures were undetectable with myosin and connectin antibodies (Fig. 5f, g, triple arrowheads and double arrowheads). The staining pattern with connectin antibody was similar to that of myosin antibodies (Fig. 5f, g), but showed a complementary pattern with anti-CTnT antibody (Fig. 5e). This suggests that the aggregates and large structure were independent from thick filaments.

Fig. 6 shows SiTPM4-transfected cells labeled with G5G4 and anti-CTPM antibodies. The SiTPM4-transfected cells exhibited distinctive staining patterns with G5G4 and anti-CTPM antibodies (Fig. 6a and b, respectively). Myofibrils with regular striations corresponding to I bands were detected in lower cells with both G5G4 and anti-CTPM antibodies (Fig. 6a, b). However, some aggregates were observed in upper cells with G5G4 (Fig. 6a, double arrowheads), but they were undetectable with anti-CTPM (Fig. 6b). The micrograph suggested that TPM4α was suppressed with SiTPM4-transfection in the upper cells. It was possible that lower cells escaped SiTPM4-transfection (Fig. 6a, b).

Fig. 6

Bundles with myofibril striation and aggregate in TPM4α-suppressed cardiomyocytes on day 6. TPM4α-suppressed cells were treated with G5G4 and TRITC-labeled antibody against mouse IgG (a). Subsequently cells were stained with anti-CTPM and FITC-labeled antibody against rabbit IgG (b). In this microscopic field of SiTPM4-transfected cells, the lower cells showed myofibril striation and the upper cells exhibited aggregates (double arrowheads). Double arrowheads indicate aggregates. Scale bar=10 μm

Although it is known that myofibrils tend to lose the striations along the time in culture, regular myofibril striation was maintained in some cells under the TPM4α-suppressed condition after 5 days. The result suggested that aggregates and lack of striation were caused by the TPM4α-suppression with SiTPM4 transfection but not due to long-term culture. These results were summarized in Table II.

Table II Summary of the expression of sarcomeric proteins in TPM4α-suppressed cells

Control cardiomyocyte		TPM4α-suppressed cardiomyocyte
myofibril (striations)		linear bundles (striations and non-striation)			large structure (non-striation)
CTM4α	+	CTM4α	regardless of striations	–	CTM4α	–
CTnT	+	CTnT	regardless of striations	+	CTnT	+
Actin	+	Actin	regardless of striations	+	Actin	+
α-actinin	+	α-actinin	regardless of striations	+	α-actinin	–
Myosin	+	Myosin	striation	+	Myosin	–
			non-striation	–
Connectin	+	Connectin	striation	+	Connectin	–
			non-striation	–

Discussion

The expression of TPM1-4 is known to be intricately controlled by alternative splicing, and regulated during development from muscle to non-muscle cells (Wieczorek et al., 1988; Lindquester et al., 1989; Fleenor et al., 1992; Perry, 2001; Wang et al., 2008; Hook et al., 2011). Several dozens of TPM RNAs are expressed in each tissue (Wieczorek et al., 1988; Perry, 2001). In chicken cardiac muscle, TPM1α, TPM1κ and TPM4α RNA isoforms are expressed (Wang et al., 2008).

In contrast to the study of RNA isoforms, it has been shown that only two protein isoforms, α and β TPMs are produced in cultured cardiac muscle in chicken by analyses with 2-dimensional gel electrophoresis (Izant and Lazarides, 1977; Montarras et al., 1981). The molecular weight of α TPM is almost identical to that of β TPM and their isoelectric points are different. It has been reported that β TPM is the phosphorylated protein of α TPM (Montarras et al., 1981). Therefore, only α TPM is synthesized in chicken cardiac muscle in vitro. The number of isoforms of TPM RNA is inconsistent with that of TPM proteins. Probably, translational regulation plays an important role in producing TPM in cardiac muscle.

In addition, the above view suggests that α TPM corresponds to TPM4α in chicken cardiac muscle. Consequently, a polyclonal antibody against TPM prepared from adult ventricular muscle, anti-CTPM, was specific for TPM4α. Our result shows that the TPM4α band decreased with SiTPM4 transfection as analyzed by immunoblotting using anti-CTPM. It reveals that TPM4α is suppressed with SiTPM4.

TPM4α-suppressed cells exhibit significant changes of myofibril structures and antibody reactivity (Table II). In linear bundles, striated and non-striated regions are observed. The region with several myofibril striations exists between non-striated regions. The striated domain contains CTnT, actin, α-actinin, myosin and connectin, suggesting that basal myofibril proteins exist in the area, and that the striated region is the underdeveloped myofibril including several immature Z discs. In contrast, in the non-striated region, CTnT, actin and α-actinin are present but myosin and connectin are absent, indicating that the non-striated domain consists of essentially thin filament.

A unique large structure, constructed from the aggregate and some curved bundles, was produced on linear bundles. Striations are undetectable on the large structure. The aggregate rises by a few extremely short tiny filaments found at narrow places without α-actinin on linear bundles, and many curved filaments extend from aggregates bilaterally along the bundles. CTnT and actin are present but α-actinin, myosin and connectin are absent in the large structure. The large structure is a concentrated actin bundle lacking α-actinin, myosin and connectin. It is considered that TPM4α suppression results in a lack of α-actinin and striations in the large structure. This suggests that TPM4α plays an important role in constructing Z disks on actin filaments and maintaining myofibrils during development.

Although the large structure is formed in the TPM4α-suppressed cells, it is unclear whether the large structure is consisted of newly synthesized actin filaments being incorporated into linear bundles or free actin filaments leaving from incomplete linear bundles at nicks lacking α-actinin. The aggregate begins to appear in the TPM4α-suppressed cells on day 4, and the aggregate on day 5 is larger than that of day 4. It is expected that the aggregate develops gradually in the TPM4α-suppressed cells, and that myofibrils degrade their filament structure in cytoplasm. The large structure seems to be free actin bundles without α-actinin. It is possible that actin filaments containing diminished TPM4α are selected and excluded before constructing nor‍mal and functional thin filaments within the cell. The sorting of the incomplete actin filaments arises in the cytoplasm of the TPM4α-suppressed cell.

It has been shown that connectin and myosin are tightly linked, and that connectin plays an important role for myosin filaments to integrate into preformed Z disks for making regular sarcomeres of myofibrils (Wang et al., 1979; Komiyama et al., 1999; Hill et al., 1986; Noguchi et al., 2007). Normal myofibrillogenesis may be inapplicable to TPM4α-suppressed cells, but our results also showed that connectin co-existed with myosin. Our results are consistent with those reports that connectin and myosin are strongly associated in myofibrils and striated linear filaments. However, in the large structure, connectin and myosin are undetectable. The large structure was independent from myosin and connectin. It shows that the large structure has no relationship with connectin and myosin in the TPM4α-suppressed cells.

Concerning the TPM4 gene, it has been shown that the abnormal organization of cardiac thin filaments and heartbeat failure occur in mutant zebrafish (Zhao et al., 2008). In cardiac lethal mutant Mexican axolotl, lack of TPM4 gene was responsible for the heartbeat failure (Spinner et al., 2002). It is unknown whether the TPM4α-suppressed cells beat or not. Since TPM4α is the main regulatory protein for rhythmic contraction in chicken cardiac muscle, it is not expected that beating occurs in the TPM4α-suppressed cardiomyocyte. Instead, the unique large structure, distinct from the collapsed structure of the mutant zebrafish, was assembled on thin filament. This suggests that TPM4α plays an important role in constructing and maintaining actin filaments, sarcomeres, myofibrils and heartbeat in chicken cardiac muscle.

It has been shown that a number of myopathies are asso‍ciated with mutations in TPM genes (Perry, 2001; Wieczorek et al., 2008; Bai et al., 2013). The suppression of TPM4α focused in this study might be associated with myocardial disorder.

Acknowledgments

This study was supported by a grant from the Kumamoto Gakuen University Foundation (to N.T.) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H. T-O.).

References

•  Bai,  F.,  Wang,  L., and  Kawai,  M. 2013. A study of tropomyosin’s role in cardiac function and disease using thin-filament reconstituted myocardium. J. Muscle Res. Cell Motil., 34: 295–310.
•  Bailey,  K. 1948. Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochem. J., 43: 271–279.
•  Blanchard,  E.M.,  Iizuka,  K.,  Christe,  M.,  Conner,  D.A.,  Geisterfer-Lowrance,  A.,  Schoen,  F.J.,  Maughan,  D.W.,  Seidman,  C.E., and  Sedman,  J.G. 1997. Targeted ablation of the murine alpha-tropomyosin gene. Circ. Res., 81: 1005–1010.
•  DeHaan,  R.L. 1970. The potassium-sensitivity of isolated embryonic heart cells increases with development. Dev. Biol., 23: 226–240.
•  DeHaan,  R.L. and  Hirakow,  R. 1972. Synchronization of pulsation rates in isolated cardiac myocytes. Exp. Cell Res., 70: 217–220.
•  Ebashi,  S.,  Endo,  M., and  Otsuki,  I. 1969. Control of muscle contraction. Q. Rev. Biophys., 2: 351–384.
•  Endoh,  M. 2008. Cardiac Ca²⁺ signaling and Ca²⁺ sensitizers. Circ. J., 72: 1915–1925.
•  Fleenor,  D.E.,  Hickman,  K.H.,  Lindquester,  G.J., and  Devlin,  R.B. 1992. Avian cardiac tropomyosin gene produces tissue-specific isoforms through alternative RNA splicing. J. Muscle Res. Cell Motil., 13: 55–63.
•  Forry-Schaudies,  S.,  Gruber,  C.E., and  Hughes,  S.H. 1990. Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing. Cell Growth Differ., 10: 473–481.
•  Geeves,  M.A.,  Hitchcock-DeGregori,  S.E., and  Gunning,  P.W. 2015. A systematic nomenclature for mammalian tropomyosin isoforms. J. Mus‍cle Res. Cell Motil., 36: 147–153.
•  Gunning,  P.,  O’Neill,  G., and  Hardeman,  E. 2008. Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiol. Rev., 88: 1–35.
•  Hill,  C.S.,  Duran,  S.,  Ling,  Z.,  Weber,  K., and  Holtzer,  H. 1986. Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myofibrils. J. Cell Biol., 103: 2185–2196.
•  Hook,  J.,  Lemckert,  F.,  Schevzov,  G.,  Fath,  T., and  Gunning,  P. 2011. Functional identity of the gamma tropomyosin gene: Implications for embryonic development, reproduction and cell viability. Bioarchitecture, 1: 49–59.
•  Izant,  J.G. and  Lazarides,  E. 1977. Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis. Proc. Natl. Acad. Sci. USA, 74: 1450–1454.
•  Komiyama,  M.,  Maruyama,  K., and  Shimada,  Y. 1999. Assembly of con‍nectin (titin) in relation to myosin and a-actinin in cultured cardiac muscle. J. Muscle Res. Cell Motil., 11: 419–428.
•  Lindquester,  G.J.,  Flach,  J.E.,  Fleenor,  D.E.,  Hickman,  K.H., and  Devlin,  R.B. 1989. Avian tropomyosin gene expression. Nucleic Acids Res., 17: 2099–2118.
•  Maruyama,  K. 1976. Connectin, an elastic protein from myofibrils. J. Biochem., 180: 405–407.
•  Meyer,  L.C. and  Wright,  N.T. 2013. Structure of giant muscle proteins. Front Physiol., 4: 368–380.
•  Montarras,  D.,  Fiszman,  M.Y., and  Gros,  F. 1981. Characterization of the tropomyosin present in various chick embryo muscle types and in mus‍cle cells differentiated in vitro. J. Biol. Chem., 256: 4081–4086.
•  Noguchi,  H.,  Takemori,  S.,  Kajiwara,  J.,  Kimura,  M.,  Maruyama,  K., and  Kimura,  S. 2007. Chicken breast muscle connectin: passive tension and I-band region primary structure. J. Mol. Biol., 37: 213–219.
•  Ohtsuki,  I.,  Maruyama,  K., and  Ebashi,  S. 1986. Regulatory and cytoskel­etal proteins of vertebrate skeletal muscle. Adv. Protein Chem., 38: 1–67.
•  Ono,  S. 2010. Dynamic regulation of sarcomeric actin filaments in striated muscle. Cytoskeleton, 67: 677–692.
•  Perry,  S.V. 2001. Vertebrate tropomyosin: distribution, genetics, proteins and function. J. Muscle Res. Cell Motil., 22: 5–49.
•  Rethinasamy,  P.,  Muthuchamy,  M.,  Hewett,  T.,  Boivin,  G.,  Wolska,  B.M.,  Evans,  C.,  Solaro,  R.J., and  Wieczorek,  D.F. 1998. Molecular and physiological effects of alpha-tropomyosin ablation in the mouse. Circ. Res., 82: 116–123.
•  Robbins,  J. 1998. Alpha-tropomyosin knockouts: a blow against transcriptional chauvinism. Circ. Res., 82: 134–136.
•  Sanger,  J.W.,  Wang,  J.,  Fan,  Y.,  White,  J., and  Sanger,  J.M. 2010. Assembly and dynamics of myofibrils. J. Biomed. Biotechnol., 2010: 1–8.
•  Schultheiss,  T.,  Lin,  Z.X.,  Lu,  M.H.,  Murray,  J.,  Fischman,  D.A.,  Weber,  K.,  Masaki,  T.,  Imamura,  M., and  Holtzer,  H. 1990. Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and stri‍ated myofibrils. J. Cell Biol., 110: 1159–1172.
•  Shimizu,  N. and  Shimada,  Y. 1985. Immunochemical analysis of troponin T isoforms in adult, embryonic, regenerating and denervated chicken fast muscle. Dev. Biol., 111: 324–334.
•  Spinner,  B.J.,  Zajdel,  R.W.,  McLean,  M.D.,  Denz,  C.R.,  Dube,  S.,  Mehta,  S.,  Choudhury,  A.,  Nakatsugawa,  M.,  Dobbins,  N.,  Lemanski,  L.F., and  Dube,  D.K. 2002. Characterization of a TM-4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl. J. Cell Biochem., 85: 747–761.
•  Toyota,  N. and  Shimada,  Y. 1981. Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence. J. Cell Biol., 91: 487–504.
•  Toyota,  N. and  Shimada,  Y. 1983. Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves. Cell, 33: 297–304.
•  Toyota,  N.,  Uzawa,  H., and  Shimada,  Y. 1998. Assembly of force-expressed troponin I isoforms in myofibrils of cultured cardiac and fast skeletal muscle cells as studied by epitope tagging. J. Muscle Res. Cell Motil., 19: 937–947.
•  Toyota,  N.,  Takano-Ohmuro,  H.,  Yoshida,  L.S.,  Araki,  M.,  Yoshinobu,  K., and  Suzuki-Toyota,  F. 2008. Suppression of cardiac troponin T induces reduction of contractility and structural disorganization in chicken car‍diomyocytes. Cell Struct. Funct., 33: 193–201.
•  Wang,  J.,  Thurston,  H.,  Essandoh,  E.,  Otoo,  M.,  Han,  M.,  Rajan,  A.,  Dube,  S.,  Zajdel,  R.W.,  Sanger,  J.M.,  Linask,  K.K.,  Dube,  D.K., and  Sanger,  J.W. 2008. Tropomyosin expression and dynamics in developing avian embryonic muscle. Cell Motil. Cytoskeleton, 65: 379–392.
•  Wang,  K.,  McClure,  J., and  Tu,  A. 1979. Titin: Major myofibrillar components of striated muscle. Proc. Natl. Acad. Sci. USA, 76: 3698–3702.
•  Wieczorek,  D.F.,  Smith,  C.W.J., and  Nadal-Ginard,  B. 1988. The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth and nonmuscle isoforms by alternative splicing. Mol. Cell Biol., 8: 679–694.
•  Wieczorek,  D.F.,  Jagatheesan,  G., and  Rajan,  S. 2008. The role of tropomyosin in heart disease. Adv. Exp. Med. Biol., 644: 132–142.
•  Xu,  C.,  Craig,  R.,  Tobacman,  L.,  Horowitz,  R., and  Lehman,  W. 1999. Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. Biophys. J., 77: 985–992.
•  Zhao,  L.,  Zhao,  X.,  Tian,  T.,  Lu,  Q.,  Skrbo-Larssen,  N.,  Wu,  D.,  Kuang,  Z.,  Zheng,  X.,  Han,  Y.,  Yang,  S.,  Zhang,  C., and  Meng,  A. 2008. Heart-specific isoform of tropomyosin4 is essential for heartbeat in zebrafish embryos. Cardiovasc. Res., 80: 200–208.