Developmental expression and differential cellular localization of obscurin and obscurin-associated kinase in cardiac muscle cells

Abstract

Obscurin and obscurin-associated kinase are two products of the obscurin transcriptional unit that encodes a recently identified giant muscle-specific protein obscurin. In this study, we characterized the developmental expression and cellular localization of obscurin and obscurin-associated kinase in cardiac muscle cells. We cloned murine obscurin-associated kinase and found that it is abundantly expressed in the heart as two isotypes encoded by 2.2 and 4.9 kb sequences. The 2.2 kb isotype of the kinase was more prominently expressed than the 4.9 kb isotype. Both obscurin and the kinase-like domains were progressively upregulated since the early stages of cardiac development. Obscurin-associated kinase was expressed at higher levels than obscurin at early stages of cardiomyogenesis. Increasing intensity of obscurin expression in the developing heart positively correlated with progressive cell differentiation and was higher in the ventricles compared to the atria. These data were supported by the results of experiments with primary cardiac cell cultures. Obscurin localization changed from a weakly immunopositive diffuse pattern in poorly differentiated cells to an intensely immunolabeled cross-striated distribution at the level of mid-A-bands and Z-disks during the assembly of the myofibrillar contractile apparatus. In dividing myocytes, unlike the interphase cells, obscurin translocated from disassembling myofibrils into a diffuse granulated pattern segregated separately from alpha-actinin-immunopositive aggregates. Obscurin-associated kinase was localized mainly to cell nuclei with increasing incorporation into the Z-disks during differentiation. Our results suggest that these two novel proteins are involved in the progression of cardiac myogenesis during the transition to advanced stages of heart development.

Fig. 1

A: Schematic representation of the obscurin-associated MLCK-like single and dual kinase isoforms. Putative functional domains identified by PROSITE search include one full and one partial serine threonine kinase (SKI and SKII, respectively), one immunoglobulin-like (Ig), and one fibronectin-like (Fn) domains. Antisense RNA and cDNA probes, for in situ and Northern analysis respectively, were prepared using cDNA sequence from the underlined regions. A polyclonal antibody (link 7) generated to human obscurin-associated MLCK-like kinase recognizes epitopes in the SKI domain (indicated by the pink square and short line on the right). B: Northern blot analysis of obscurin-MLCK mRNA expression in adult murine tissues. The single kinase isoform, detected with the SKI probe, is selectively expressed in the heart (H) and not detectable in other tissues including brain (Br), spleen (S), lung (Lu), liver (Li), skeletal muscle (SM), kidney (K), and testis (T). The dual kinase isoform (top of panel A), detected with the SKII probe, was expressed in the heart and skeletal muscle (SM). Note the 4.6 kb band in the SKII blot. Larger transcripts may reflect inclusion of the kinase domains at the end of the obscurin transcript. C: Western blot analysis using the anti-obscurin-MLCK-like terminal kinase antibody. The antibody detected the native obscurin-MLCK single kinase isoform in lysates from adult rat cardiac myocytes in culture and in lysates of Cos7 cells transfected with an obscurin-MLCK expression construct (a positive control, marked with +) but not with a control plasmid (a negative control, marked with −).

Fig. 2

Expression of obscurin and obscurin-associated kinase during mouse cardiac development. Whole mount RNA in situ hybridization of obscurin (A) and obscurin-associated kinase (C). Note weak to moderate level of obscurin expression in the ventricles (A) and the prominent ventricular expression of obscurin-associated kinase (C) 12 days post-conception. Also, note that the atria visible on the upper part of the photos show a considerably lower level of reactivity than the ventricles. A,B show the lateral views of the heart, and C,D show the frontal views of the heart. The letter a marks the atria. B,D show a negative control incubated without the specific probe demonstrating the specificity of the in situ hybridization reaction, 21×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3

Expression of obscurin in the developing myocardium. Immunofluorescent labeling for this protein in the developing free left ventricular wall on days 11, 13, 14.5, and 16-day post-conception is illustrated in A–C,F, respectively. Note the increasing levels of obscurin expression during cardiac development and its shift from the diffused pattern to more intensely labeled structured dotted and periodic fibrillar distribution in the tissue. D: Negative control: the section of the heart on day 13 post-conception incubated only with the secondary antibody (compare to panel B). V marks the ventricular cavity. Asterisks in B indicate the non-muscle tissue immunonegative for obscurin and located close to epicardial surface of the ventricular wall. The arrow in B indicates the area shown in panel E at a high magnification. C: The ventricular wall following trabecular fusion located closely to the apical area of the heart near the interventricular septum. Arrowheads in D show the boundaries of the immunonegative cardiac tissue in a negative control sample. E: The area marked with the large arrow in panel B presented at a higher magnification as a single confocal optical section. The arrows in B and E show the same group of cells. At this stage of obscurin is visualized both as the diffuse labeling pattern and as the developing elements of the striated myofibrillar pattern. Small arrows in E show the elements of sarcomeric cross-striation. A–D, F: 280×; E: 630×.

Fig. 4

Cellular localization of obscurin in differentiating rat cardiac muscle cells. A: Localization of obscurin in a poorly differentiated rat early neonatal cardiac myocyte after 4 days in culture; (B) localization of sarcomeric α-actinin in the same field; (C) a merged image showing the localization of obscurin (red fluorescence) and α-actinin (green fluorescence) in the same cell. D: Structural interrelations of obscurin and sarcomeric α-actinin localization in the area marked with the arrowhead in C shown at a higher magnification. Note the topographical association of intense immunolabeling for obscurin with the nascent myofibrillar system and its localization predominantly in the middle of the A-band at the time of formation of mature myofibrillar clusters. Also note that most of premyofibrillar structures immunopositive for sarcomeric α-actinin are only weakly immunopositive for obscurin. Bar, 20 mm (A–C), 6.4 mm (D). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

Localization of obscurin in cardiac myocytes during mitosis. Double immunofluorescent labeling for obscurin (red fluorescence) and sarcomeric α-actinin (green fluorescence) in a rat neonatal cardiac muscle cell at the stage of early telophase of mitosis. Two daughter nuclei (arrows) are visible in the central area of the cytoplasm. Staining for nuclear DNA with Hoechst dye (blue fluorescence) shows that the nuclei contain highly condensed chromatin typical of the telophase of mitosis. Note that obscurin is segregated in diffusely located granulated bodies that are separated from the protein aggregates containing sarcomeric α-actinin. The fragmented remnants of the contractile system disassembled during mitosis are seen at the cell periphery. Bar, 10 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6

Immunofluorescent labeling for obscurin-associated protein kinase in the permanent line of H9c2 cells transfected with a non-coding control construct (A) and transfected with His-tagged construct encoding obscurin-associated kinase (B). Note intense fluorescent labeling of anti-His n the nucleus of a transfected cell in the center of the field in panel B. Arrowheads in B show the location of weakly and moderately positive nuclei. Bar, 30 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 7

Muscle-specific expression of obscurin-associated kinase in cell cultures of differentiating rat neonatal cardiac muscle cells as demonstrated by double immunolabeling for the sarcomeric isoform of α-actinin (green fluorescence) and obscurin-associated kinase (red fluorescence) on day 4 in culture. A: Immunofluorescent labeling for sarcomeric α-actinin; (B) immunofluorescent labeling for obscurin-associated kinase; the insert shows the intense reactivity of cell nuclei and moderate reactivity of the cytoplasm in the myocardial tissue section of 16-day old rat embryo (C) nuclear staining with Hoechst; (D) combined image of A–C. Note the presence of immunoreactivity for obscurin-associated kinase only in a sarcomeric α-actinin positive muscle cell (the arrowhead in B,C) and the absence of obscurin-associated kinase expression in fibroblasts whose nuclei are seen on the right side of the photo. Bar, 30 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 8

Localization of obscurin-associated kinase at advanced stages of cardiomyogenic differentiation in culture. A: Immunofluorescent localization of sarcomeric α-actinin in primary culture of rat neonatal cardiac muscle cells on day 7 in vitro. B: Immunofluorescent localization of obscurin-kinase in the same field. Note the presence of immunolabeling for this protein in myonuclei and in Z-disks of myofibrils. C: A merged image of immunolabeled sarcomeric α-actinin (green fluorescence) and staining for nuclear DNA (blue fluorescence) in the same field shows that obscurin-kinase is localized in the myonuclei. The arrow shows a nucleus of a non-muscle, fibroblastic cell in the upper left corner. This cell is immunonegative for both sarcomeric α-actinin and obscurin-associated kinase; (D) a combined image of the cells immunolabeled for sarcomeric α-actinin and obscurin-associated kinase shows the association of obscurinkinase label with the Z-disks. 400×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]