Krp1 Localization in Primary Mouse Embryonic Cardiomyocytes
Krp1 localization in primary cultures of mouse embryonic cardiomyocytes was determined by immunostaining and confocal microscopy. Optical slices of a typical cardiomyocyte double stained with antibodies against Krp1 and sarcomeric α-actinin are shown in (left 3 columns), and LSM software was used to generate orthogonal views of the Z stack (right panels). Sarcomeric α-actinin staining (middle row) shows that cultured cardiomyocytes contain organized striations corresponding to mature myofibrils, narrow myofibrils that appear to be fusing with mature structures (arrowhead), and closely spaced α-actinin dots (Z-bodies) that characterize premyofibrils (arrow). Krp1 is present as punctate staining throughout the cell, with most located above the plane of the myofibrils as shown in the software-generated orthogonal views (, right hand panels). Therefore, most of the Krp1 staining is not associated with α-actinin. However, punctate patches of Krp1 are also observed in the plane of the myofibrils, concentrated in premyofibril areas (arrow) and alongside narrow myofibrils fusing into more mature structures (arrowhead). In these areas, Krp1 staining is adjacent to developing myofibrillar structures, but is not colocalized with α-actinin.
Figure 1 Krp1 (green, lower row) and α-actinin (red, middle row) localization in a single cultured mouse cardiomyocyte by confocal microscopy. DAPI labeling of the nucleus is also shown in the blue channel of the composite images (top row). Confocal imaging (more ...)
To better assess Krp1 localization with respect to the cell boundaries, cardiomyocytes were double stained with antibodies against Krp1 and caveolin-3 (). Caveolin-3 is an integral membrane protein linked with the dystrophin-associated membrane cytoskeleton [30
], and its staining marks the ventral and dorsal surfaces of the cell, demonstrating that the spread cardiomyocytes are flattened, with a thicker area around the nucleus (). Punctate patches of Krp1 are clearly localized within the cytoplasm (arrows), between the cell boundaries defined by caveolin-3. Immunostaining is specific, as only low levels of background staining are seen in neighboring fibroblasts (, arrowheads).
Figure 2 Krp1 (green, lower row) and caveolin-3 (red, middle row) localization in a single cultured mouse cardiomyocyte by confocal microscopy. DAPI labeling of the nucleus is also shown in the blue channel of the composite images (top row). Confocal imaging at (more ...)
Krp1 association with cellular compartments was further explored by separation of extracted and insoluble fractions following treatment with Nonidet P-40. Cytosolic components were completely extracted, as indicated by the presence of all of the GAPDH in the extracted cytosolic fraction (). Some integral membrane proteins, such as ß1-integrin, were almost completely extracted, while others, such as caveolin-3, remained insoluble. The insoluble pellet fraction was enriched for cytoskeletal elements such as myosin, actin, and N-RAP. A large proportion of Krp1 (42%) remained in this insoluble pellet, suggesting that a sizable pool of Krp1 is strongly associated with the cytoskeleton.
Figure 3 Immunoblot analysis of Nonidet P-40 extracted and insoluble fractions. Equal volumes of total cell lysate, extracted and insoluble fractions were analyzed by immunoblot (left) and densitometric analysis (right). Representative immunoblots are shown. The (more ...)
We were unable to determine by immunofluorescence whether unextracted Krp1 is co-localized with actin since the cultured cells detached from the substrate during detergent extraction. A more gentle detergent treatment that preserves attachment failed to completely extract the cytosol as determined by GAPDH immunostaining (data not shown).
Transfection with Krp1 siRNA Specifically Reduces Krp1 Expression and Myofibril Accumulation
An RNA interference approach was used to gain insight into Krp1 function. shows a schematic diagram of the Krp1 mRNA, domain organization, and siRNA target areas. Cardiomyocytes were transfected with one of the Krp1 siRNAs 24 hours after plating, while replicate wells were mock-transfected or transfected with control siRNA. After 48 hours, total RNA was prepared and used for cDNA synthesis and quantitative RT-PCR analysis. Both of the Krp1 siRNAs significantly reduced Krp1 transcript levels (siRNA1: 55 ± 2% and siRNA2: 73 ± 2% decrease compared to mock transfected), while transfection with control siRNA had no effect (, Krp1). The reduction of Krp1 expression is specific, as transcript levels of another kelch repeat protein, Keap1, and levels of muscle specific proteins N-RAP and α-actinin, were unchanged compared to mock-transfected controls (). Unless otherwise noted, data reported in the following sections are from experiments using Krp1 siRNA 2, and the term “Krp1 siRNA” is used for simplicity; similar experimental results were obtained using Krp1 siRNA 1 (data not shown).
Figure 4 Transfection with siRNA against Krp1 sequences specifically reduces Krp1 expression. (A) Krp1 protein is comprised of an N-terminal BTB-BACK domain and a C-terminal kelch repeat region. In the nucleotide sequence, Krp1 siRNA 1 targets an area within the (more ...)
To determine siRNA effects on protein levels, lysates were prepared at various times following transfection. Proteins were detected by SDS-PAGE and immunoblotting, and levels were quantitated by densitometric analysis. Three days following transfection, Krp1 protein levels were reduced 49 ± 3% compared to the mock-transfected control (). Krp1 protein continued to decrease through day 5 and remained low over the period of study (reduced 68 ± 8% compared to mock at day 7). Again, the reduction of Krp1 expression is specific, as muscle myosin and sarcomeric actin levels were unchanged compared to mock transfected controls (). At later time points, slight decreases in Krp1, myosin, and actin levels were observed in both mock and siRNA-transfected samples, likely due to cardiac fibroblast proliferation in the primary cultures.
To assess the effects of Krp1 knockdown on myofibrillogenesis, cardiomyocytes were fixed five days after transfection and analyzed by confocal microscopy following immunostaining for Krp1 and sarcomeric α-actinin. Similar to untransfected cardiomyocytes (), control siRNA-transfected cardiomyocytes displayed bright punctate Krp1 staining throughout the cytoplasm (, panel 1). These cells are frequently filled with mature myofibrils as indicated by α-actinin organization into wide Z-lines (, panel 2). Fibroblasts in the culture exhibit background levels of Krp1 staining and do not contain sarcomeric α-actinin (, panels 1-3, arrowheads). In Krp1 siRNA-transfected cardiomyocytes, Krp1 immunostaining is usually dim and similar in intensity to background staining of fibroblasts (, panel 4, filled versus open arrows). Sarcomeric α-actinin staining is still present but rarely organized into wide Z-lines typical of mature myofibrils (, panel 5, filled arrow). The correlation between Krp1 levels and mature myofibril content is typically observed within the population of siRNA treated cells. Cardiomyocytes with bright Krp1 staining frequently contain mature myofibrils, similar to control cells (, panels 4 & 5, arrowheads) whereas cells with dim Krp1 staining often lack large areas of mature myofibrils (, panels 4 & 5, filled arrows). These observations demonstrate a strong link between Krp1 levels and myofibril content.
Figure 5 Krp1 knockdown decreases mature myofibril content. (A) Cells were fixed and stained for Krp1 and sarcomeric α-actinin 5 days after transfection with control siRNA (top row) or Krp1 siRNA (bottom row). Nuclei were counterstained with DAPI. Images (more ...)
The effect of Krp1 knockdown on mature myofibril content was measured in cardiomyocytes fixed at various time points following transfection. Cells were stained with antibody against sarcomeric α-actinin and randomly chosen cardiomyocytes were imaged by confocal microscopy. Mature myofibril content was assessed by measuring areas containing α-actinin organized into wide Z-lines. At 2 days following transfection, the mean total area of mock-transfected cardiomyocytes was 3050 ± 183 μm2, increasing to 7953 ± 619 μm2 at 7 days post-transfection. Similar results were obtained from cardiomyocytes transfected with Krp1 siRNA (p-value not significant at any time point), indicating normal cell growth in cardiomyocytes with reduced Krp1 expression (, left panel). In mock-transfected cells, mature myofibril content increased from 1373 ± 110 μm2 at 2 days after transfection to 4647 ± 330 μm2 at 7 days post-transfection. In contrast, the mature myofibril area in cells transfected with Krp1 siRNA was 1358 ± 132 μm2 at day 2, but remained low (1873 ± 225 μm2) at day 7 after transfection (, right panel). Mature myofibril area was significantly reduced compared to both mock transfected and control siRNA treated cardiomyocytes at days 3, 5, and 7 after transfection (p-value < 0.001 for all). Thus, Krp1 is not required for normal cell spreading but is essential for accumulation of mature myofibrils in primary mouse embryonic cardiomyocytes. Furthermore, spontaneous beating was observed in both control and Krp1 siRNA treated cultures, suggesting that although accumulation of addiitonal myofibrils was halted by Krp1 knockdown, the existing myofibrils remained functional.
To determine if apoptosis is induced in cells lacking Krp1, cells were transfected with control or Krp1 siRNA and compared to those in replicate wells in which apoptosis was induced by incubation in serum-free, low glucose medium containing 20 mM 2-deoxy-glucose. Protein lysates were prepared and analyzed by SDS-PAGE and immunoblotting. Krp1 knockdown was verified, with reduced expression evident at three days and a further decrease observed seven days following transfection with Krp1 siRNA (). Cleaved caspase-3, a positive marker for apoptosis [31
], was easily detected in the glucose-deprived cells (, apoptosis+ lane), but was not detected in cardiomyocytes transfected with control or Krp1 siRNA at either time point (). Further analysis was performed on fixed cells stained for sarcomeric α-actinin and cleaved caspase-3. Nuclei were counterstained with DAPI. Apoptotic cardiomyocytes are smaller than normal, have fragmented nuclei with condensed chromatin, and cleaved caspase-3 immunostaining is bright and punctate (, right panels). In contrast, nuclei of control and Krp1 siRNA-transfected cardiomyocytes are round and intact, and cleaved caspase-3 immunostaining is dim and diffuse (, left and center panels).
Figure 6 Apoptosis is not induced after Krp1 knockdown. Cardiomyocytes were transfected with the indicated siRNA; as a positive control, apoptosis was induced in untreated cells by incubating for 1 day in serum-free, low glucose medium containing 20 mM 2-deoxy-glucose. (more ...)
Characterization of Krp1 Knockdown Phenotypes
Although primary cardiomyocytes in culture are generally heterogeneous in size, shape, and myofibrillar organization, particular patterns of α-actinin organization were observed with increased frequency after Krp1 knockdown. The predominant patterns observed are illustrated in representative images of control and Krp1 siRNA-transfected cardiomyocytes that were fixed and stained with antibodies against sarcomeric α-actinin and Krp1. Patterns of sarcomeric α-actinin staining identified multiple structures in untransfected cardiomyocytes such as wide Z-lines of mature myofibrils, stress fiber-like structures (SFLS) with nearly continuous α-actinin staining, and periodically spaced Z-bodies characteristic of newly forming myofibrils ( and ). These patterns of α-actinin organization were also present in Krp1 siRNA-transfected cardiomyocytes. However, fewer cells were filled with mature myofibrils (), and a larger proportion of cells were filled with periodically spaced Z-bodies or narrow Z-lines () and more randomly arranged α-actinin positive dots (). Lack of Krp1 immunostaining confirmed Krp1 knockdown in these cells (data not shown).
Figure 7 Classification of cardiomyocytes according to α-actinin organization. Each cardiomyocyte was classified into one of four categories of α-actinin organization corresponding to the dominant phenotype observed by visual inspection. (A) Prototypical (more ...)
To assess the prevalence of these phenotypes in control and Krp1 knockdown cells, we assigned cardiomyocytes to one of four categories depending on the dominant pattern of α-actinin organization assessed by visual inspection: Wide Z-lines filling the cell (, wide Z-lines), patches of wide Z-lines interspersed with SFLS (, SFLS and wide Z-lines), long series of periodic Z-bodies with very few wide Z-lines (, periodic Z-bodies), and more randomly oriented or short series of α-actinin dots (, randomly spaced dots). As expected, most untransfected cardiomyocytes were filled with wide Z-lines, and about a third were filled with patches of wide Z-lines interspersed with SFLS. Although periodic Z-bodies and randomly spaced α-actinin dots were frequently observed, they were typically present in small areas of the cell. Thus, few untransfected cardiomyocytes are scored as being predominantly filled with these structures (). Similar results were obtained for mock-transfected samples (). In control siRNA-transfected cultures, the relative proportions in each category were similar to untransfected and mock-transfected cells at day 2 following transfection, while at later times the percentage of cells filled with wide Z-lines exhibited a modest decrease. This was accompanied by a small increase in the proportion of cells with SFLS and wide Z-lines (). The proportion of cells filled with Z-bodies and/or α-actinin dots remained low, accounting for less than 9% of cardiomyocytes at any time point ().
In Krp1 siRNA-transfected samples 2 days after transfection, the relative proportions of each phenotype were similar to control samples. However, the percentage of cells filled with wide Z-lines dropped sharply by day 3 after transfection (), in agreement with the morphometric data shown in . Instead, a larger proportion of cells were filled with periodically spaced Z-bodies or narrow Z-lines, suggesting accumulation of myofibrillogenesis intermediates upon Krp1 knockdown. Interestingly, the proportion of cells with these structures peaked at day 3 and decreased at days 5 and 7, while the proportion of cells with more randomly spaced dots was greatest at days 5 and 7 following transfection (). These data suggest that the structures containing periodically spaced Z-bodies or narrow Z-lines disassembled into the structures represented by the more randomly spaced dots.
To characterize the structures that accumulate upon Krp1 knockdown, transfected cardiomyocytes were fixed and stained with antibodies against α-actinin, myomesin, or muscle myosin in combination with phalloidin for F-actin localization. In control cells, double staining revealed well-aligned myofibrils with the expected sarcomeric organizations of α-actinin at the Z-line, phalloidin marking F-actin in the I-band, myosin at the A-band, and myomesin at the central M-line (, left panels). After Krp1 knockdown, double staining demonstrated that α-actinin organized in long series of periodic Z-bodies or thin Z-lines corresponded to thin, separated fibrils containing actin (, center panel). The structures that appear as isolated or small groups of dots of α-actinin in random orientations are very short, sparse fibrils containing actin (, right panel). Sarcomeric myosin is also associated with the long separated and the short sparse actin fibrils (, center and right panels). Myomesin appears less organized than the other components, and is often absent from the shorter structures (, center and right panels). The data demonstrate that the major sarcomeric components retain their normal longitudinal organization in very thin myofibrils that accumulate after Krp1 knockdown.
Figure 8 Organization of sarcomeric components after Krp1 knockdown. Cardiomyocytes were fixed five days post-transfection and double stained with phalloidin to visualize actin filaments (green) and antibodies for either sarcomeric α-actinin (A), muscle (more ...)
Ultrastructural characterization by transmission electron microscopy supports this conclusion. Laterally aligned, wide myofibrils are abundant in untransfected and control siRNA transfected cardiomyocytes (). In cells transfected with Krp1 siRNA, thin fibrils containing Z-lines and thick myosin and thin actin filaments are commonly observed (), corresponding to the double-stained images obtained by confocal microscopy. These thin myofibrils are sparse and the cytoplasm between them contains organelles, such as mitochondria and endoplasmic reticulum, which are indistinguishable from those in control cells. In addition, some cardiomyocytes contain many 100-200 nm diameter electron dense granules (). These membrane bound organelles are numerous in approximately 10% of the cardiomyocytes examined, and their frequency was not significantly changed by Krp1 knockdown (data not shown). They resemble atrial natriuretic peptide secretory granules commonly found in the atrial cells of adult murine hearts and in both atrial and ventricular cardiomyocytes during embryonic development [32
Figure 9 Electron micrographs showing examples of myofibrillar structure and organization in control siRNA transfected (A), untransfected (B), and Krp1 siRNA transfected (C-F) cardiomyocytes. Cells were fixed 3 days (A, C, E) or 7 days (B, D, F) after transfection. (more ...)