| To whom correspondence should be addressed: Naoji Toyota, Department of Environmental Biology, Kumamoto Gakuen University, Oe 2-5-1, Kumamoto, Kumamoto 862-8680, Japan. Tel: +81–96–364–9214, Fax: +81–96–372–0702 |
Skeletal and cardiac muscle cells contain myofibrils consisted of sarcomeres which are their contractile units, and the myofibrils show a striated pattern due to the repeating sarcomeres. In addition to these sarcomeric structures, the cardiac muscle has a unique membrane structure within interconnected individual cells, joined end to end by specific cellular interfaces called intercalated disks. Incorporation of distinctive proteins into these structures is considered to be important for constructing the myofibrils, sarcomeres and cell-to-cell interfaces of cardiomyocytes. It is certain that lack of one or more of sarcomeric proteins may affect the sarcomeric organization as well as its structure, and even more, the cell function and shape. It has been shown that truncation of key domains of the giant elastic protein, connectin/titin (MW: >3,000 kDa), spanning from Z line to the tip of the actin filament, leads to impaired myofibrillogenesis with poorly organized sarcomeres in cardiac muscle cells (Miller et al., 2003). However, it is not well-understood whether the regulatory or associated proteins such as tropomyosin, troponins, and myosin light chains participate in the morphogenesis of sarcomere and/or the myofibrillar structure, and also, how they influence the cell function and shape.
In this report, we focus on troponin T (TnT), a component of the troponin complex (Tn complex), which is a regulatory protein involved in the contraction of striated muscles. The Tn complex, which is located on the thin filaments of striated muscles, is consisted of three components: troponin I (TnI), troponin C (TnC), and TnT. Regulation of striated muscle contraction depends on binding of Ca2+ to the Ca2+-binding protein, TnC. TnI is an inhibitory component of muscle contraction. TnT has a high affinity for tropomyosin and is particularly important for localizing the Tn complex along the myofibril (Ebashi, 1961; reviewed by Perry, 1998). Binding of Ca2+ to TnC results in a conformational change to the thin filament, then leading to actin-myosin interaction and finally, muscle contraction (Ebashi, 1961). Further, TnT in cardiac muscle (CTnT) is one of the key proteins related to heart disease. It has been reported that genetic defects of CTnT are associated with hypertrophic cardiomyopathy (reviewed by Gomes et al., 2004; Lombardi et al., 2008).
In the present study, the expression of CTnT was exhaustively suppressed in primary cultures of cardiomyocytes by introducing small interference RNA (siRNA) with a nucleotide sequence homologous to CTnT (CTnT-siRNA). Suppression of CTnT in cultured cardiomyocytes treated with siRNA was monitored by immunoblot analyses and immunocytochemistry. Our data show that suppression of CTnT affected beating, and to a lesser extent, myofibril structure and cell-to-cell adhesion in cultured cardiomyocytes.
Reagents for transfection were obtained from Invitrogen (Carlsbad, CA). Monoclonal antibodies against ventricular myosin heavy chain (HV11), tropomyosin (CHI) and connectin/titin (9D10) were purchased from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa, IA), and that against α-actinin (EA-53), from Sigma Aldrich (St. Louis, MO). A previously described monoclonal antibody (G5G4) which recognizes both cardiac and fast skeletal muscle TnTs (Shimizu and Shimada, 1985), and rabbit polyclonal antibodies against CTnT and CTnI (Toyota and Shimada, 1981 and 1983) were also used. Anti-mouse or anti-rabbit IgG labeled with biotin were used as secondary antibodies, and avidin-labeled horseradish peroxidase (Vecstain; Vector Laboratories, Burlingame, CA) was used to visualize the immunoreactive bands on immunoblots. Fluorescein isothiocyanate (FITC)-labeled or tetramethylrhodamine-labeled antibody against mouse IgG, or FITC-conjugated antibody against rabbit IgG (all from Cappel Laboratories, Aurora, OH) were used as secondary antibodies for immunocytochemistry.
Ventricular muscle cells were prepared from 7 or 8 day-old chicken embryos and cultured for 6 days according to DeHaan (1970). For fluorescence immunocytochemistry, cardiomyocytes were plated at a concentration of 2×105 cells in 1.5 mL of culture medium in 35-mm plastic culture dishes. For immunoblot analyses, the cells were plated in 100-mm dishes at a concentration of 1, 2 or 3×106 cells in 8 mL of culture medium. Herein, the first 24 hours after plating is defined as day 1 of culture. Cardiomyocytes were maintained in growth medium 818B supplemented with 5% (v/v) fetal horse serum, as described by DeHaan (1970).
CTnT-siRNA was designed based on chicken CTnT cDNA (GenBank accession number NM_205449), and purchased from Qiagen (Hilden, Germany). We used the AA(N17)CUU (N=any nucleotide) sequence from the open reading frame of CTnT cDNA. The sequences of CTnT-siRNA were GCUUUCUCCAACAUGCUGCTT and GCAGCAUGUUGGAGAAAGCTT. Cultured cardiomyocytes were transiently transfected with CTnT-siRNA, as described below. In order to estimate transfection efficiency, siRNA labeled with Alexa Fluor 546 (Neg-RNA; Qiagen) was introduced to the cells. To test whether CTnT expression changes upon transfection with DNA, cells were also transfected with cmyc-CTnI, which is an expression vector that includes a c-myc tag and CTnI cDNA, as described previously (Toyota et al., 1998). Transfection with siRNAs or cmyc-CTnI was carried out on day 1 or day 2 of culture using Oligofectamine (Invitrogen) according to the manufacturer’s protocols.
For immunocytochemistry, culture medium 818B was replaced with 0.8 mL of Opti-MEM I medium (Invitrogen) in 35-mm dishes 20 min prior to transfection. Then, the cells were transfected with 0.4 nmol of CTnT-siRNA dissolved in 190 μL of Opti-MEM I medium plus 4 μL of Oligofectamine. Four hours after addition of siRNA, 0.5 mL of culture medium 818B, in which the concentration of fetal horse serum was increased to 15%, was added to the dishes. The mock control cells were treated with Oligofectamine without any siRNA. Otherwise mentioned, the cells were transfected on day 2 and cultured afterwards for 3 days before immunocytochemistry. Transfection efficiency was higher than 80%.
For immunoblot analyses, culture medium was replaced with 5 mL of Opti-MEM I medium in 100-mm dishes 20 min prior to transfection. Varying amounts of CTnT-siRNA were introduced into the cells, by addition of mixtures A, B and C, which contained 0.5 nmol, 1 nmol and 2 nmol of CTnT-siRNA, respectively, dissolved in the following volumes of Opti-MEM I medium plus Oligofectamine (A: 460 μL+8 μl; B: 920 μL+15 μL; and C: 1820 μl+30 μL). These mixtures were added to culture plates having initial cell counts of 1, 2 or 3×106. In some cases, either 2 nmol of Neg-RNA or 12 μg of cmyc-CTnI in Opti-MEM I medium and Oligofectamine, equivalent to mixture C, were used to serve as controls. Four hours after addition of the CTnT-siRNA mixtures, 5 mL of culture medium 818B containing 15% of horse serum was added to the dishes. Cells cultured for 2–4 days after transfection were used for immunoblot analyses.
Actomyosin was extracted from homogenates of adult chicken ventricular muscle in KCl (final concentration: 0.6 M), and precipitated by adding 10 volumes of MiliQ grade water as described previously (Toyota et al., 1998). The cells grown as described above in 100-mm culture dishes were washed with phosphate-buffered saline (PBS), and collected with a scraper before lysis with SDS sample buffer (final volume: 200 μL; final SDS concentration: 2%). Fifteen μL of the lysates were subjected to 10% polyacrylamide gel electrophoresis in the presence of 0.1% SDS, according to Laemmli’s method as described previously (Toyota et al., 1998). Immunoblot analyses were carried out as described previously (Toyota et al., 1998). After electrophoresis, proteins were transferred to nitrocellulose membranes and reacted with the appropriate antibodies indicated in the legends. Bound primary antibodies were reacted with biotinylated anti-rabbit or -mouse secondary antibody, and thereafter detected by avidin-biotin-horseradish peroxidase method. Bands were visualized by reaction with hydrogen peroxide in the presence of 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan).
Immunocytochemistry was performed as described previously (Toyota et al., 1998). The cells cultured as described above were fixed with ethanol at 4°C for 1 min. The fixed cells were treated with antibodies against the sarcomeric proteins listed above, diluted with 1% bovine serum albumin in PBS, for 45 min at room temperature. Reacted antibodies were then visualized with fluorescence-labeled secondary antibodies. For actin visualization, cells were fixed with 1% of formalin in PBS previous to staining with rhodamine-phalloidin. In experiments with glycerol-permeabilized cells, the cells were treated with 50% glycerol in 10 mM potassium phosphate buffer (pH 7.5) containing 50 mM KCl overnight at 4°C, and then kept for 2 weeks at –20°C. EDTA (final 5 mM) was added to the cells immersed in the above buffer to allow relaxation of myofibrils, where mentioned. The stained cells along culture were observed under a Zeiss Axio Imager A1 immunofluorescence microscope equipped with a scanning digital camera (AxioCam Zeiss, Carl Zeiss Co., Ltd.) and 100× and 40× objectives.
Monoclonal antibodies G5G4 (striated muscle TnT), HV11 (ventricular myosin heavy chain), CHI (tropomyosin), EA-53 (α-actinin) and 9D10 (connectin/titin) stain I bands, A bands, I bands, Z lines and I bands near the A-I junction of myofibrils, respectively. The polyclonal antibodies against CTnT and CTnI both stain I bands of cardiac myofibrils.
Eight 35-mm plastic culture dishes were plated with 2×105 cells. Half of the dishes were transfected with 0.4 nmol of CTnT-siRNA on day 2, whereas the other half was treated with Oligofectamine as a control. To count beating cells, four 1-mm diameter circles were randomly marked with a sharpened metal tube on the bottom of each dish. The number of beating cells was assessed using an inverted phase-contrast microscope equipped with an enclosed stage maintained at 37°C in a humidified environment of 5% CO2 and 95% air. Cell counts were determined at a final magnification of 200×. The total number of single beating cells and of individual cells in synchronously-beating groups consisting of 2–10 cells in contact with one another were recorded in the marked areas of all dishes from both groups. Only cells that showed unequivocal beating were counted. On day 2 (i.e., transfection day), 60–100 beating cells were present in each marked area. The number of beating cells in the same marked areas was then assessed for the following four days, for comparison with results of day 2. Ratios of beating cells were determined as a percentage of total cells (non-beating +beating) and normalized to the ratios on day 2. The same procedure was repeated in three independent experiments, and the results expressed as mean±SD. Presented results are from a total of ~47,000 beating cells counted in both groups. Statistical significance was analyzed by Student’s t-test, which is a test for two parametric and unpaired groups with homogeneous variance. Homogeneity of variance between the two groups was demonstrated with Bartlett’s test. Values were considered statistically significant at P<0.01.
CTnT-siRNA was transfected into the cardiomyocytes in order to suppress CTnT expression. Cellular lysates were subjected to immunoblot analyses with the G5G4 antibody against striated muscle TnT (Fig. 1). CTnT was detected in control cells: control cells without any treatment (lane b), Oligofectamine treatment alone (lane c), as well as cells with Neg-RNA and cmyc-CTnI transfection (lanes d and e, respectively). The mobility of these bands was the same as that of CTnT in actomyosin prepared from adult ventricular muscle (lane a). However, CTnT was undetectable in cells on day 5 transfected with 0.5, 1 or 2 nmol of CTnT-siRNA on day 2 (lanes g–o), in cultures started with different cell numbers (1, 2 or 3×106 cells). Thus, the expression of CTnT was suppressed at all above mentioned CTnT-siRNA/cell ratios, with transfection on day 2. However, when the CTnT-siRNA transfection was done on day 1 instead of day 2, suppression of CTnT is negligible as shown by a detectable band on immnunoblots (Fig. 1, lane f).
 View Details | Fig. 1. Expression of CTnT protein upon transfection with siRNAs. Immunoblot analysis of CTnT in cultured chicken cardiac ventricular muscle cells was carried out with cells collected on day 5 of culture, using the G5G4 monoclonal antibody against striated muscle TnTs. Lanes b to e show controls (all cultures of 1×106 cells) of CTnT expressed in normal (lane b: without Oligofectamine and siRNA), no-siRNA (lane c: Oligofectamine alone), Neg-RNA (lane d: control siRNA) and cmyc-CTnI (lane e: CTnI reporter gene) cells, respectively. Lane f shows cells transfected with CTnT-siRNA on day 1, whereas those transfected on day 2 with the indicated siRNA amounts and cell numbers are shown in lanes g-o. Lane a shows CTnT in actomyosin prepared from adult ventricular cardiac muscle. |
Next, the duration and intensity of the effects of RNAi in cultures of transfected cardiomyocytes were examined with the monoclonal antibody against TnT (G5G4), as shown in Fig. 2. The cells were transfected with CTnT-siRNA on day 2, and were cultured for the following 4 days. The intensity of the CTnT protein bands gradually decreased from day 3 to day 5 (Fig. 2, lanes c–e), but started to increase again on day 6 (lane f). Almost complete suppression was seen on day 5 (lane e). Therefore, in order to assure an effective suppression of CTnT expression in cultured cardiomyocytes, it is important to transfect CTnT-siRNA into day 2 cells and with this protocol, the suppression could be confirmed 3 days after transfection (cells on day 5).
 View Details | Fig. 2. Duration of RNAi effects in cultured cardiomyocytes transfected with CTnT-siRNA. CTnT protein expression was examined along culture in cells transfected on day 2 of culture with CTnT-siRNA (0.5 nml/1×106 cells; lanes c–f) and compared to those in its absence (day 2, lane b; day 6, lane g), using G5G4 antibody against striated muscle TnTs. Between day 3 to day 5 of culture, the expression of CTnT is gradually decreased (lanes c–e), showing the lowest level on day 5 (lane e), but on day 6, its expression is partly recovered (lane f). Lane a shows CTnT in actomyosin as described in Fig. 1. |
The expression of sarcomeric proteins other than CTnT in cells with maximum suppressed CTnT (cells on day 5 transfected on day 2, shown in Fig. 2, lane e) was examined with respective antibodies, as shown in Fig. 3. CTnT was confirmed to be undetectable (lane e), but bands of expected sizes for myosin heavy chain (lane a), α-actinin (lane b), tropomyosin (lane c), and CTnI (lane d) were detected. Similar expression levels relative to control cells were found for myosin heavy chain, α-actinin, and tropomyosin, but a somewhat lower expression was seen for CTnI (data not shown). The present results suggest that absence of CTnT due to CTnT-siRNA transfection does not affect significantly the expressions of the other sarcomeric proteins.
 View Details | Fig. 3. Expression of myofibrillar proteins in CTnT-suppressed cultured cardiomyocytes. Cells with maximum suppressed CTnT protein (same as those of Fig. 2, lane e) were examined for expression of myosin heavy chain (lane a), α-actinin (lane b), tropomyosin (lane c) and CTnI (lane d) using the following respective antibodies: EA-11, EA-53, CH1 and anti-CTnI. The arrows on the left indicate the positions of proteins shown in lanes a–d ordered from the top, respectively. Lane e shows undetectable CTnT expression with G5G4 antibody against striated muscle TnTs. |
As shown above (Figs. 1 and 2), the maximum CTnT suppression was detected in day 5 cells transfected with CTnT-siRNA on day 2. Therefore, cells cultured according to this protocol were stained with the antibody against striated muscle TnTs for evaluation of sarcomeric structure in their myofibrils. In control cells, the antibody stained I bands of sarcomeres showing the striated pattern of myofibrils (Fig. 4, panel a). However, in CTnT-siRNA-transfected cells the fluorescence signal for CTnT in myofibrils was quite undetectable (panel b).
 View Details | Fig. 4. Myofibrillar structure in cultured cardiomyocytes transfected with CTnT-siRNA. The localization of sarcomeric proteins in myofibrils of cardiomyocytes transfected with CTnT-siRNA (+) and of control cells (–) were examined by fluorescence microscopy as described in Materials and Methods, upon reaction with specific antibodies: TnT (panels a and b; G5G4), myosin heavy chain (panel c; HV11), connectin/titin (panel d; 9D10), tropomyosin (panel e; CHI), and CTnI (panels f and g; polyclonal anti-CTnI antibody). The rectangles in panels c-g depict sarcomere length in selected myofibrils. The arrowheads indicate the positions corresponding to Z lines in the sarcomeres enclosed in the rectangles. Scale bar=10 μm. |
To examine whether CTnT-siRNA transfection affects the localization of sarcomeric myofibrillar proteins other than CTnT, transfected cells were stained with antibodies against myosin heavy chain, connectin/titin, tropomyosin and CTnI (Fig. 4, panels c, d, e and f, respectively). In transfected cells, the four sarcomeric proteins were found localized accordingly to the profile expected for myofibrils. Micrographs of transfected and control cells showed similar fluorescence intensities for myosin heavy chain, connectin/titin and tropomyosin (data not shown). In the case of CTnI, however, a lower intensity was observed in myofibrils of transfected cells when compared to control cells (panel f versus g).
These results were consistent with those of immunoblot analyses (Figs. 1, 2 and 3). Our results suggest that myosin, connectin/titin, tropomyosin and CTnI are all present in myofibrils in muscle cells with suppressed CTnT expression.
Next, the structure of myofibrils was examined focusing on Z-Z intervals. Myofibrils in control cells are shown in Fig. 4 (panels a and g). In most CTnT-siRNA-transfected cells, the Z-Z interval was similar to that in control cells (data not shown). However, unusually long sarcomeres with longer Z-Z intervals were observed in the transfected cells (Fig. 4, panels c–f). The number of cells with longer sarcomeres was low in cells transfected with 0.4 nmol of CTnT-siRNA (0.01±0.008%), but increased when 2 nmol was used (1.0±0.5%), both tested with 2×105 cells at culture start. The rectangles in Fig. 4 show sarcomere length in transfected and control cells (panels c–g, identical magnifications). It was clear that the myofibrils in the CTnT-siRNA-transfected cells had 6–7 sarcomeres per rectangles (panels c–f, arrowheads), in contrast with 8–9 sarcomeres per rectangle in the control cells (in panel g, an example with 8 sarcomeres is shown by arrowheads). In other words, fewer sarcomeres were observed in transfected cells (panels c–f). The Z-Z interval in transfected cells was 2.6–3.0 μm (n=50), while those in control cells was 2.0–2.2 μm (n=80).
In order to verify the dimension of this unusually long Z-Z interval seen in transfected cells, we next compared with that of myofibrils of control cells (which were previously permeabilized with glycerol) in the presence of EDTA, when the maximal relaxation takes place due to Ca2+ chelation. Fig. 5, panel b shows relaxed myofibrils in control cells. The Z-Z interval in CTnT-suppressed cells was longer than that of the relaxed myofibrils (panel a versus b, as stained with the antibody against CTnI). Although cells with unusually long sarcomeres were a minority in transfected cultures, the suppression of CTnT appears to be causing this phenomenon.
 View Details | Fig. 5. Comparison of Z-Z intervals in myofibrils of control and CTnT-suppressed cardiomyocytes in culture. Cardiomyocytes transfected with CTnT-siRNA (panel a), and two representative lots of glycerol-permeabilized control cells (i.e., expressing CTnT) relaxed with EDTA (panel b) are shown for comparison of Z-Z intervals in their myofibrils, as revealed by staining with the antibody against CTnI. Images are from cells on day 5 of culture, which were transfected or not on day 2. Arrowheads indicate the positions corresponding to Z lines in the sarcomeres. Scale bar=10 μm. |
Double stainings with antibodies against CTnT and α-actinin (Fig. 6, panels a–d) revealed structural changes in myofibrils of the transfected cells (3 days after transfection), as follows. A regular striation pattern was always observed in the control cells with anti-CTnT and anti-α-actinin antibodies (panels a and b). On the other hand, in the transfected cells, the antibody against CTnT was unable to show the striations (Fig. 6. panel c, in agreement with Fig. 4, panel b). Staining with the antibody against α-actinin showed irregular small striations (Fig. 6, panels d and f, dotted lines with arrowheads). These irregular small striations were also visible with actin staining (panel e, dotted lines with arrowheads), indicating that both actin and α-actinin were co-localized within the striations (panel e versus f).
 View Details | Fig. 6. Structural changes in the CTnT-suppressed cardiomyocytes in culture. Horizontally paired panels represent double-stainings of the same cell cultures with different antibodies or rhodamine-phalloidin, as below. Cardiomyocytes transfected on day 2 with CTnT-siRNA (+) and control cells (–) were examined after 3 days by fluorescence microscopy upon reaction with antibodies against CTnT (panels a and c; polyclonal) and α-actinin (panels b, d and f; EA-53). In panel e, cells were stained with rhodamine-phalloidin, to show actin filaments. Secondary antibodies were labeled with FITC (panels a, c and f) and tetramethylrhodamine (panels b and d), respectively. Continuous and regular striations of myofibrils are shown in control cells (panels a, b), in contrast with irregular small striations in the transfected cells (panels d–f; dotted lines with arrowheads). These cells also present jagged cell interfaces with multiple sharp-pointed projections, separated at the cell-to-cell adhesion sites (panels d–f; arrows). The projections at the periphery of the cells show strong reactivity for α-actinin (panels d and f; arrows), and also for actin filament (panel e, arrows). Scale bar=10 μm. |
Moreover, transfected cultures presented cardiomyocytes facing each other but separated by a narrow space between their adhesion interfaces (Fig. 6, panels d–f; short arrows). The profile of such interfaces was often complementary to the opposing side. Both sides showed jagged cell interfaces with multiple sharp-pointed projections stained for α-actinin (panels d and f, arrows). The jagged cell interfaces and the irregular small striations were simultaneous events observed in the affected cells. Upon CTnT suppression, the population of such cells amounted to 5.0±0.5% of transfected cells (for cultures of 4 nmol CTnT-siRNA/2×105 cells) while in control cultures they remained at 0.1±0.5%. The cell population with this morphological abnormality was greater than that of cells showing increased Z-Z interval, but cells showing both morphological changes were undetectable.
We then examined whether CTnT suppression affects beating ability of cultured cardiomyocytes. Fig. 7 shows the ratio of beating cells upon transfection. The population of beating cells tended to decrease along culture even in control cells. However, the decrease in the beating cell population is more drastic upon CTnT-siRNA transfection compared to that of day-matched controls: decreases by 29%, 45% and 85% for the first, second, and third days after transfection, respectively (Fig. 7). On the fourth day after transfection, no beating cells were detectable in both cultures, in spite of the fact that cells were alive.
 View Details | Fig. 7. Changes in the ratio of beating cardiomyocytes in culture upon CTnT-siRNA transfection. Beating was evaluated as described in Materials and Methods for cultured cardiomyocytes (2×105 cells) after transfection with 0.4 nmol of CTnT-siRNA. Transfection was performed on day 2 of culture. The number of beating cells is expressed as a percentage of total cells in day-matched transfected and control cultures. The number of beating cells on day 2 (before transfection) was considered as 100%. *: Significantly different from day-matched control cells at P<0.01. |
Taken together, these results suggest that cardiac contractility was affected significantly in the cultured chicken cardiomyocytes with suppressed CTnT.
Herein, we succeeded in the transient, but almost complete inhibition of CTnT expression in embryonic cardiac ventricular muscle cells in primary culture by the use of CTnT-siRNA. Immunoblot analyses indicated that the suppression of CTnT is complete three days after transfection, and then is followed by a somewhat recovered CTnT expression on the fourth day. However, the almost negligible CTnT three days after transfection had no effect on the expression of other myofibrillar proteins such as myosin heavy chain, tropomyosin, connectin/titin, and α-actinin. On the other hand, although CTnI was still expressed, it was decreased relative to control cells (Fig. 4, panel f versus g). The presently observed tendency of simultaneous decrease of CTnI with CTnT has been noted previously, as a more drastic phenotype, in the silent heart-presenting zebrafish bearing mutations in the CTnT gene (Sehnert et al., 2002): affection of the CTnT gene prevented the expression of CTnI in mutant hearts examined by immunocytochemistry. Because CTnT plays an important role in the assembly of the Tn complex, its absence may cause CTnI to dissociate from thin filaments (Ebashi, 1961; reviewed by Perry, 1998). Furthermore, CTnT-siRNA transfection provoked an increase in the number of cells compromised functionally. The beating ability decreased by 85% relative to day-matched controls after three days of transfection, concomitantly with suppression of CTnT. This coincides with the time at which maximal suppression of CTnT occurs (Fig. 2, lane e), suggesting that the severe suppression of CTnT leads to impaired beating function of the cells. Thus, CTnT is an essential protein for stable contraction of myofibrils in cardiomyocytes.
Although CTnT is undetectable on immunoblots (Figs. 1 and 2), approximately 8% of cells still continue to beat three days after transfection (Fig. 7), suggesting that a small number of cardiomyocytes containing CTnT remain present in these cultured cells. In contrast, no beating cells can be seen four days after transfection (Fig. 7) even though CTnT expression becomes apparent again on immunoblots (Fig. 2, lane f). The fact that beating vanishes gradually along one week of culture even in control cells is an expected behavior of chicken cardiomyocytes in primary culture, since it is known that these cells naturally loose beating ability after a peak of maximum beating (DeHaan, 1970). Nevertheless, there is no doubt that CTnT suppression has a remarkable effect on the beating ability (Fig. 7).
The morphological changes such as cells with longer Z-Z interval in myofibrils (around 1%) and cells with jagged cell interfaces presenting irregular small striations in myofibrils (near 5%) were not in agreement with the remarkable decrease of beating cells (by 85%, 3 days after transfection). One possible explanation for the inhibition of beating might be that transfected cells loose Ca+2-regulated contractility because of a compromised assembly of the Tn complex caused by the suppression of CTnT: CTnI and CTnC are unable to incorporate into the Tn complex if CTnT is absent. The lowered CTnI in transfected cells relative to the control cells seen in Fig. 4 (panel f versus g) might suggest such a possibility.
It has been shown that α-actinin is one of the main proteins anchoring actin filaments to Z lines and to the plasma membrane of cardiomyocytes, at the region of the cell-to-cell adhesion known as the intercalated discs (Tokuyasu et al., 1983). Muir (1957) has shown that the intercalated discs are important for cardio-myofibrillogenesis. The irregular small striations labeled with the antibody against α-actinin in the transfected cardiomyocytes (Fig. 6, panels d and f) might be reflecting the structure of myofibrils corresponding to Z, A and I bands damaged at cell-to-cell adhesion interfaces. It has been shown that the intercalated disks are formed completely after 15 days in cultured cardiac myoblasts (Kostin and Schaper, 2001). Therefore, it is reasonable to assume that our 6 day-cultured cells have still incompletely developed intercalated disks. It is unknown whether CTnT exists in intercalated disks at an incomplete stage of development, but the observed increase in cells having irregular small striations and jagged interfaces in correlation with higher amounts of CTnT-siRNA suggested that such abnormalities in the developing intercalated disks occur due to suppression of CTnT.
The CTnT gene and protein are closely linked to cardiomyopathy (reviewed by Gomes et al., 2004) and it is known that certain amino acid substitutions and deletions in CTnT are responsible for impairment of cardiac diastolic and systolic function (Watkins at al., 1995; Tardiff et al., 1999; Kamisago et al., 2000). Our results also suggest that CTnT is responsible for the maintenance of beating in cultured cardiomyocytes. Troponins are vulnerable to proteases (Perry, 1998; Tanaka, et al., 2008), in addition to the fact that their damaged proteins lead to antibody production within the organism. Because of such properties, damage to Tns is thought to be linked to autoimmune diseases and myocardial infarction (Perry, 1998; Gomes et al., 2004).
Considering that targeting of the genes involved in cardiomyopathy in whole animals becomes often lethal (Gomes et al., 2004), the in vitro approach using RNAi for specific proteins might be advantageous for studying their individual roles in the structure and function of cardiac muscle cells. The present results suggest that the suppression of CTnT in myofibrils, at a stage when the structure of sarcomeres is pre-existent, has a drastic effect on the beating function of cultured cardiomyocytes. CTnT suppression also affected to some extent, the myofibrillar structure and cell-to-cell adhesion.
The authors are grateful to Dr. Elizabeth A. Howes for correcting English usage. This research was supported by a grant from the Kumamoto Gakuen University Foundation (to N.T.), by the “High-Tech Research Center” Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology of Japan, 2004–2008 (to H.T.-O.), and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (to M.A.).
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