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. 2024 Jan 26;244(6):1030–1039. doi: 10.1111/joa.14008

Temporal involvement of phosphatidylinositol 4‐phosphate 5‐kinase γ in differentiation of Z‐bands and myofilament bundles as well as intercalated discs in mouse heart at mid‐gestation

A Ratchatasunthorn 1, H Sakagami 2, H Kondo 1,3, W Hipkaeo 1, S Chomphoo 1,✉
PMCID: PMC11095301  PMID: 38275211

Abstract

Considering the occurrence of serious heart failure in a gene knockout mouse of PIP5Kγ and in congenital abnormal cases in humans in which the gene was defective as reported by others, the present study attempted to localize PIP5Kγ in the heart during prenatal stages. It was done on the basis of the supposition that phenotypes caused by gene mutation of a given molecule are owed to the functional deterioration of selective cellular sites normally expressing it at significantly higher levels in wild mice. PIP5Kγ‐immunoreactivity was the highest in the heart at E10 in contrast to almost non‐significant levels of the immunoreactivity in surrounding organs and tissues such as liver. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non‐significant levels at newborn and postnatal stages. Six patterns in localization of distinct immunoreactivity for PIP5Kγ were recognized in cardiomyocytes: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils and (2) its localization on them as well as short myofibrils in association with them in cardiomyocytes of early differentiation at E10; (3) its spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrences of such spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z‐bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. No distinct localization of PIP5Kγ‐immunoreactivity of any patterns was seen in the heart at E18 and P1D. The present finding suggests that sites of PIP5Kγ‐appearance and probably of its high activity in cardiomyocytes are shifted from the plasma membranes through short myofibrils subjacent to the plasma membranes and long myofibrils, to Z‐bands as well as to the intercalated discs during the mid‐term gestation. It is further suggested that PIP5Kγ is involved in the differentiation of myofibrils as well as intercellular junctions including the intercalated discs at later stages of the mid‐term gestation. Failures in its involvement in the differentiation of these structural components are thus likely to cause the mid‐term gestation lethality of the mutant mice for PIP5Kγ.

Keywords: cardiomyocyte, intercalated discs, mid‐term gestation, myofibril, PIP5Kγ

PIP5Kγ‐immunoreactivity was the highest in the heart at E10. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non‐significant levels at newborn and postnatal stages. PIP5Kγ‐appearance high activity in cardiomyocytes is shifted from the plasma membranes through short and long myofibrils to Z‐bands during the mid‐term gestation.

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1. INTRODUCTION

Since Hokin conducted initial studies on phosphoinositide turnover/cycle in salivary glandular cells (1953, 1957), the idea emerged that receptor‐mediated changes in the intra‐membranous levels of phosphoinositides (PIs) represent an early step in the stimulus–response pathway. For the PI turnover to function effectively, individual PI molecules must be compartmentalized properly both spatially and temporally within various membranes of cells. This compartmentalization is probably the case for enzyme molecules responsible for synthesis of PIs, although most, if not all, of the enzymes are basically present in the cytosol and they move back and forth to the membranes on demand for synthesizing (Hipkaeo & Kondo, 2023).

Among the PI‐synthesizing enzyme molecules, phosphatidylinositol phosphate 5‐kinase (PIP5K) catalyzes the production of PI(4,5)P2 from PI(4)P, and it is composed of three isoforms; α, β, and γ (Kanaho et al., 2007). Its product PI(4,5)P2, abbreviated as PIP2 hereafter, plays multiple roles influencing a variety of cellular activities: (1) as a “lipid anchor” that attaches actin cytoskeleton to the plasma membrane, (2) as a participant in membrane trafficking, (3) as a stabilizer or activator of many intramembranous bioactive proteins, such as ion channels and transporters, (4) as a regulator of apoptosis and autophagy, (5) as a precursor of such second messengers as DAG, IP3, and PI(3,4,5)P3, and (6) as a player implicated in apoptosis and autophagy (Balla et al., 2009; Delage et al., 2013; Doughman et al., 2003; Oude Weernink et al., 2004). Furthermore, PIP2 regulates the interaction between scaffolding and signaling proteins comprising the cell adhesions such as integrin, talin and actin (Ling et al., 2003). Based on the general idea that individual isoforms of a given enzyme have overlapping, but not identical, functions, there have been studies generating murine lines lacking individual isoforms of this lipid kinase and analyzing their phenotypes (for its α, Sasaki et al., 2005; for its β, Yamazaki et al., 2013; for its γ, Wang et al., 2007).

According to the study by Wang et al. (2007), artificially PIP5Kγ‐null embryos of mice have myocardial developmental defects and neural tube closure defects as well as defects in the erythropoiesis, resulting in lethality at embryonic day 11.5 (E11.5). In their histological analysis of the distribution of β‐gal in the PIP5Kγ+/− hearts of mice at E10.5, β‐gal‐positive cells were confined to the interior of cardiac walls and trabeculae, predicting cardiomyocytes to express PIP5Kγ. Their electron microscopy of PIP5Kγ‐null cardiomyocytes showed disorganization of actin cables within the sarcomeres of PIP5Kγ+/− and a loss of association of the actin cables with the fascia adherens seen in mature cardiomyocytes. The disturbance in the fascia adherens is well expected and understood from the known information on the involvement of PIP2 in the maintenance of intercellular junctions as noted above. On the other hand, it is known that embryonic hearts are composed of cardiomyocytes at various levels of differentiation even at a given embryonic stage (Bani et al., 2010; Chacko, 1976; Hirschy et al., 2006; Kastner et al., 1997; Niederreither et al., 2001; Westfall et al., 1997; Zhang & Pasumarthi, 2007). This information leads one to question if the localization of PIP5Kγ is simply confined to such specific subcellular sites as intercellular junctions throughout the embryonic stages or if it is changeable subcellularly at different levels of the cell differentiation at individual embryonic stages of wild mice. It is also reasonable to consider that phenotypes caused by gene mutation of a given molecule are owed to its deletion in selective cell species and subcellular components that distinctly express/localize PIP5Kγ at significantly higher levels than others. No studies, however, have been reported on detailed cellular and intracellular localization of PIP5Kγ in embryonic cardiomyocytes throughout the prenatal stages of wild mice. In addition to the phenotypes of the artificial mouse mutant, regarding the possible importance of PIP5Kγ in normal development of the heart, a severe form of congenital arthrogryposis termed lethal contractual syndrome type 3 (LCCS3) of humans should be noted. It is because this syndrome has been disclosed to be caused by a mutation in PIP5K1C, the same as PIP5Kγ, and its phenotypes of LCCS3 include dilated cardiomyopathy as well as musculoskeletal disorders represented by the arthrogryposis (Narkis et al., 2007).

In view of the information described above and the general principle that information on the localization of a given molecule in cells in situ presents important clues to its function, the present immunohistochemical study was attempted to examine the cellular and subcellular localization of PIP5Kγ in the heart of wild mice because of ontogeny, with special attention to the prenatal stages. This present group of investigators has so far performed localization studies of this signal‐related molecule in the brain and various peripheral tissues by immunohistochemistry and in situ hybridization histochemistry (Akiba et al., 2002; Chomphoo et al., 2020, 2021; Hipkaeo & Kondo, 2023; Khrongyut et al., 2019; Pakkarato et al., 2022, 2023; Sirisin et al., 2021), and the present analysis represents one of the series of these localization studies of this PIP5K isoforms.

2. MATERIALS AND METHODS

2.1. Animals and tissues preparation

ICR mice were obtained from the Northeastern Laboratory Animal Center KKU. A total of six pregnant mice was used to collect all of the prenatal samples at embryonic days 10 (E10), E14D, E18D pregnant mice n = 2 each, embryos/fetuses n ≥ 5 from each mother by cesarean section. Mice at newborn P1D n = 5 and young mice at postnatal 1 (P1W), 8 weeks (P8W) n = 5 each were selected as postnatal samples. Mice were given ad libitum access to food and water and were kept under the condition of a 12 h‐dark/12 h‐light cycle. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Northeast Laboratory Animal Center, Khon Kaen University, Thailand. The study was reviewed and approved by the Animal Ethics Committee of Khon Kaen University based on the Ethical Principles and Guidelines for the Use of Animals by the National Research Council of Thailand with the ethics number IACUC‐KKU‐99/65. Mice at P8W, P1W, P1D, and E18 were all male. The sexes of mouse embryos at E14 and E10 were not identified because of the technical difficulty. Under pentobarbital sodium anesthesia (60 mg/kg body weight), all postnatal mice were transcardially perfused with 25 mL of 4% paraformaldehyde/0.1 M phosphate buffered saline (PBS). Hearts were extirpated from the embryonic mice as well as the perfused postnatal mice, and fixed by immersion with the same fixative overnight for immunohistochemical analyses.

2.2. Immuno‐light microscopy

The specimens were subsequently immersed into 30% sucrose/PBS for cryoprotection. Sections of 20 μm thickness were made on the cryostat and mounted on glass slides and treated with 0.1% TritonX‐100/phosphate‐buffered saline (PBS) for 30 min at room temperature, then with 0.3% H2O2/methanol for 10 min, followed by 10% normal goat serum/PBS for 30 min. The sections were incubated overnight at 4°C with a rabbit antibody against mouse PIP5Kγ (1 μg/mL) (RRID: AB_2819240). The PIP5Kγ‐antibody was generated and its specificity was reported by one (HS) of the present authors (Hara et al., 2013). The sections were subsequently incubated for 1 h at room temperature with biotinylated anti‐rabbit IgG (Cat. #ab64256, RRID: AB_2661852, Abcam). They were then treated for the DAB (diaminobenzidine) reaction using an Elite ABC kit (Cat. #PK‐6100, RRID: AB_2336819, Vector Laboratories). As a control for the immunoreactivity, omission of the primary antibodies was applied to tissue sections.

2.3. Immuno‐electron microscopy

For immuno‐DAB electron microscopy, some of the sections immunostained with the PIP5Kγ‐antibody on polyl‐lysine‐coated plastic slides were postfixed with 0.5% OsO4 for 30 min and embedded in Epon (Cat. #14120, Electron Microscopy Sciences) after en bloc staining with 1% uranyl acetate. Ultrathin sections were observed under a transmission electron microscope (JEM1010, JEOL).

3. RESULTS

In lower magnification immuno‐light microscopy of the mouse embryos at E10, PIP5Kγ immunoreactivity was remarkably intense in the heart, while it was at almost negligible levels in any other tissues including adjacent liver and alimentary tracts except for the brain and spinal cord which showed it at weak to moderate levels (Figure 1a, d, g). At E14, the immunoreactivity in the heart was evident, but not distinctly high any more unlike at E10 in comparison with the adjacent organs which remained at almost negligible levels. Different from the previous stage, thin muscularis layers of the alimentary tracts were weakly to moderately immunoreactive for PIP5Kγ (Figure 1b, e, h). At E18, no distinct differences in the immunoreactive intensity were discerned between the heart and surrounding liver and lung which remained at negligible levels of the immunoreactivity. PIP5Kγ immunoreactivity was discerned intensely in the muscular layers of the tubular organs such as the alimentary tracts (Figure 1c, f, i). The brain and spinal cord remained weakly to moderately immunoreactive at E14 and E18. At P1D and P8W, no significant immunoreactivity for PIP5Kγ was discerned in the heart (data not shown). When sections from the three embryonic stages were mounted on the same glass slide and immunostained with the PIP5Kγ‐antibody, the relative differences in the immunoreaction intensity of the hearts at the three prenatal stages with the highest at E10 were confirmed (Figure 1a, c).

FIGURE 1.

FIGURE 1

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(a–i) Lower magnification immuno‐light micrographs for PIP5Kγ of the heart (Ha: atrium, Hv: ventricle) and surrounding tissues in embryos of mice at E10 (a), E14 (1b) and E18 (c). Note selectively intense immunoreactivity in the heart at E10 in contrast to its negligible levels in almost all surrounding organs and tissues including liver (d‐f) and alimentary tract (g–i). Also note the immunoreactivity in the heart at E14 at levels higher than that of surrounding organs and tissues, but not at such levels distinctly different from them unlike at E10. Further note a marked decrease in the immunoreaction intensity of the heart at E18 without distinct differences in its immunoreaction from surrounding organs and tissues. In contrast, the smooth muscle layers of the alimentary tract (A) show an increase in the immunoreaction intensity starting at E14 and especially remarkable at E18. An area corresponding to that enclosed by a rectangle in (a) is shown at higher magnification in Figure 2. A, Alimentary tract; B, Brain; E, Esophagus; Hv, heart ventricle; Ha, heart atrium; L, lung; Li, Liver; R, Rib; SC, Spinal cord. Bars represent 1 mm (a–c), 25 μm (d–i) and 50 μm (g–i).

In higher magnification immuno‐light microscopy of the heart at E10, the immunoreactivity was distinctly intense in forms of curved or straight lines in the cardiac walls and trabeculae (Figure 2). The immunoreactive curved lines were likely to represent the periphery of individual cells and they were found more frequently in the walls than in the trabeculae, while the immunoreactive straight lines were likely to represent fibrous bundles in cells, and they appeared commonly in both walls and trabeculae. No striated features were clearly discerned along either of the immunoreactive lines.

FIGURE 2.

FIGURE 2

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Higher magnification immuno‐light micrograph of the heart at E10. Note curve‐lined (white arrows) and straight‐lined (yellow arrows) patterns of intense PIP5Kγ‐immunoreactivity in cardiac walls (W) and trabeculae (T). Ultrastructural features of areas corresponding to those enclosed by white and yellow rectangles are shown in Figures 3, 4. IS: interstitial space, Lu: cardiac lumen. Bar represents 25 μm.

In immuno‐electron microscopy of the E10 hearts, all the intensely immunoreactive cells in the walls and trabeculae of the heart were revealed to contain more or less myofibrils (Figures 3a–c, 4a–h), while endocardial/endothelial cells and epicardial cells covering the walls and trabeculae were immunonegative (data not shown). The immunoreactive cells were classified into two types: polygonal or oblong cells and elongated cells. In the former cells, the immunoreactivity was mainly localized discontinuously along the plasma membranes and in their subjacent cytoplasm (Figure 3a, c). No myofibrils but a few vesicles were discerned in some portions of the subjacent cytoplasm, while short myofibrils were embedded in other portions of the subjacent cytoplasm. In the latter elongated cells, on the other hand, the immunoreactive deposits mainly appeared in forms of small spots randomly along myofibrils, more frequently in immaturely organized A‐bands than I‐bands of longer and wider myofibrils (Figure 4a, c). In addition, the immunoreactive deposits were found at some, but not all, of Z‐bands and intercalated discs. The immunoreactivity along the plasma membranes was much less frequent in the elongated cells than in the former cells (Figure 4b). In cross‐sections of myofibrils, distinct differences in electron density and thickness were evident between myofilaments with and without association with the immunoreactivity (Figures 4e–h).

FIGURE 3.

FIGURE 3

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(a–c) Immuno‐electron micrographs of polygonal/oblong cardiomyocytes of early differentiation showing a curve‐lined pattern of the intense immunoreactivity at E10. Note polygonal shape of the cells (a) having the intense immunoreactivity along the plasma membranes at focal sites (white arrows) and in their subjacent cytoplasm. At higher magnification of an area enclosed by a rectangle in Figure 3(a), the immunoreactive cytoplasm contains neither myofibrils nor distinct organelles except for a few vesicles (white arrowheads), and short myofibrils (f) are located away from the immunoreactive cytoplasm (b). In other areas of the immunoreactive cytoplasm, cross‐sectioned myofibrils (cf) are seen to be embedded, in addition to myofibrils (f) away from the immunoreactive sites (c). Arrowheads indicate vesicles and vacuoles embedded in the immunoreactive cytoplasm. ID(−), immature intercalated disc without the immunoreactivity. IS, interstitial space, Lu, cardiac lumen, N, nucleus. Bars represent 1 mm (a), 300 nm (c) and 600 nm (b).

FIGURE 4.

FIGURE 4

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(a–h) Immuno‐electron micrographs of elongated cardiomyocytes of advanced differentiation showing a straight‐lined pattern of the intense immunoreactivity at E10. Note the immunoreactivity on individual myofilaments of long myofibril bundles (F) in spot‐forms (arrowheads) and in some immature intercalated discs (ID) and Z‐bands (Z) as well as on plasma membranes in focal sites (yellow arrows) (a–c). Levels of intensities of the immunoreactivity on myofibrils, ID and Z‐bands are represented by such marks as –, +, ++. Also note portions of long myofibril bundles (f) and a Z‐band without association of the immunoreaction (d). Further note cut‐views of myofibrils enclosed by rectangles marked by F and f in (e, g) and their higher magnification micrographs in (f, h). Thicker myofilaments owing to the immunoreactive deposits in (f) in comparison with thinner ones without association of the immunoreaction in (h). IF, interstitial space; m, mitochondria. Bars represent 200 nm (a–d), 300 nm (f, h) and 600 nm (e, g).

On the other hand, in higher magnification immuno‐light microscopy of the heart at E14, the immunoreactivity in straight forms was dominant in both cardiac walls and trabeculae, and distinct immunoreactive striations with regular intervals were often seen at right angles to the longitudinal axis of the straight immunoreactive structures (Figures 5a, b).

FIGURE 5.

FIGURE 5

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(a, b) Immuno‐light micrographs of the hearts of E14 mice. Note frequent occurrence of straight patterns of the intense immunoreaction with rare curved patterns of the intense immunoreaction in the walls (w) and trabeculae (T) (a). Note distinct striations at right angles to the trajectory of some immunoreactive straight fibers as indicated by an oval circle in (a), and those at higher magnification are shown in (b). Lu: cardiac lumen, P: immunonegative pericardial cell. Bars represent 25 μm (a) and 12.5 μm (b).

In immuno‐electron microscopy of the E14 hearts, most of the immunoreactive cells were of the elongated type in cardiac walls and trabeculae. Unlike the hearts at E10, however, no deposits of the distinct immunoreactivity in forms of small spots were seen along the myofibrils, nor along the plasma membranes in the elongated cells. In contrast, Z‐bands and intercalated discs were frequently seen to be immunoreactive moderately or intensely, although some of them were not immunoreactive (Figure 6a, c).

FIGURE 6.

FIGURE 6

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(a, b, c) Immuno‐electron micrographs of the heart at E14. Note intensely immunoreactive deposits in association with most Z‐bands (Z) and some intercalated discs (ID) with varieties of the immunoreaction intensity whose levels are indicated by –, +, ++, +++. Also note, rare occurrence of the immunoreaction in spot forms on myofilaments (f) unlike at E10. m: mitochondria. Bars represent 1 mm (c) and 200 nm (a, b).

With the omission of the primary antibody, no significant immunoreactivities were found in any portions of the hearts and any tissues throughout the specimens in all the prenatal and postnatal stages (data not shown).

4. DISCUSSION

Judging from the distinctly higher immunoreactivity for PIP5Kγ in the heart than any other organs and tissues in the embryo at E10, and a progressive decrease in the immunoreactivity of the heart thereafter and finally to a level similar to its surrounding organs and tissues such as the liver at E18 as well as PD1 and P8W, it is likely that the exertion of major roles of PIP5Kγ and its product PIP2 in the heart is confined to such mid‐term gestation stages (Figure 7).

FIGURE 7.

FIGURE 7

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Schematic drawing of localization of intense PIP5Kγ‐immunoreactivity in cardiomyocytes of early and advanced differentiating and differentiated stages, and immunonegative precursor mesenchymal cells. Note multiple, but not single, populations of these cells of different differentiation levels in hearts at each of E10, E14 and E18 which show individually characteristic patterns of intracellular localization. The peak of the immunoreaction intensity at E10 is shown by a bottom arrow with graded color tones.

Since all the intensely PIP5Kγ‐immunoreactive cardiac cells at E10 and E14 in the present study contained more or less myofibrils, it is now evident that PIP5Kγ was expressed and localized in cardiomyocytes at various levels of differentiation, but not undifferentiated cells containing no myofilaments whose presence has been known (Bani et al., 2010; Chacko, 1976; Hirschy et al., 2006; Kastner et al., 1997; Niederreither et al., 2001; Westfall et al., 1997; Zhang & Pasumarthi, 2007). This confirms the previous prediction by Wang et al. (2007) based on their finding that β‐gal‐positive cells in PIP5Kγ+/− hearts of embryonic mice were confined to the interior of cardiac walls and trabeculae.

Based on all the present immuno‐light and electron microscopic features of PIP5Kγ‐immunoreactive structures in the cardiomyocytes, it is likely that the light microscopic appearance of the immunoreactivity in forms of curved lines represents the plasma membranes and subjacent cytoplasm in the immunoreactive polygonal/oblong cells, and that the light microscopic appearance of the immunoreactivity in forms of straight lines represents long myofibrils in the immunoreactive elongated cells.

Judging from the relatively infrequent appearance of the immunoreactive polygonal/oblong cells having shorter myofibrils at E14 as compared with E10, and from the longer and wider myofibrils in the immunoreactive elongated cells at E10 and E14, the polygonal/oblong cells are likely to be cardiomyocytes of early differentiation, and the elongated cells to be cardiomyocytes of advanced differentiation. This interpretation is compatible with the ultrastructural criteria of differentiating cardiomyocytes in conventional electron microscopy by Hirschy et al. (2006).

From the present findings, six patterns of PIP5Kγ‐localization can be recognized to occur differentially in the cardiomyocytes of early and advanced differentiation: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils, and (2) its localization on the plasma membranes and subjacent cytoplasm as well as short myofibrils in association with them. Patterns 1 and 2 occur in cardiomyocytes of early differentiation at E10; (3) its spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrence of such spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z‐bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs. Patterns 5 and 6 occur in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. It is thus possible to conclude that cardiomyocytes exhibit serial changes in the six patterns of localization of PIP5Kγ during the prenatal stages of E10 and E14.

It is generally considered that PIP5K is essentially of the soluble nature and moves back and forth on demand between the plasma membrane and the cytoplasm through interaction with its binding proteins such as AP2 and talin for catabolism of its substrate PIP on the plasma membranes including the intercellular junctions, one of its known working sites (Doughman et al., 2003; Ishihara et al., 1998; Ling et al., 2003; Mao & Yin, 2007; Nakano‐Kobayashi et al., 2007). Therefore, it is reasonable to find the patterns 1 and 2 of PIP5Kγ‐immunoreaction localization on the plasma membranes and subjacent cytoplasm in cardiomyocytes of early differentiation regardless of association with or not short myofibrils. It is also the case for the pattern 6, the localization on intercellular junction sites, because the sites represent special forms of the plasma membranes. Therefore, following the supposition that phenotype(s) caused by gene mutation of a given molecule are owed to the functional deteriorations at selective cellular sites normally expressing it at significantly higher levels, the present finding further confirms the impaired formation of the adherens junctions in PIP5Kγ‐mutant hearts by Wang et al. (2007) as an authentic result.

On the other hand, the significance of the patterns 3 ~ 5, PIP5Kγ‐localization on short and long myofibrils and Z‐bands in cardiomyocytes and its rare occurrence on long myofibrils, are difficult to interpret at present. It is because of lack of information about any molecular species to which PIP5Kγ binds in these non‐membranous components and about how PIP, PIP5K substrate, is supplied there. In this regard, the present authors previous study on embryonic skeletal muscle cells of mice should be noted (Chomphoo et al., 2020). In that study, PIP5Kγ‐immunoreactivity was first associated with the plasma membranes of skeletal myoblasts on E14, while it was then associated with I‐band myofibrils including Z‐bands in skeletal myotubes at E18, and it was finally at negligible levels in postnatal skeletal muscle fibers. That study also showed on skeletal myofibrils at E18, distinct immunoreactivity for Arf6 which is known to be involved in actin remodeling via activation of PIP5Kγ (Donaldson & Jackson, 2011; D'Souza‐Schorey & Chavrier, 2006). That study thus suggested the possible involvement of PIP5Kγ, together with its activator Arf6, in maturation of myofibrils as well as its movement from the plasma membrane of myoblasts to the myofibrils of myotubes, despite the enigma of its localization in non‐membranous components. Although the co‐localization with Arf6 remains to be examined, the suggestion in the embryonic skeletal muscles of that study may be applicable to the present finding: the transient appearance of PIP5Kγ‐immunoreactivity in myofibrils of differentiating cardiomyocytes also suggests the significance of PIP5Kγ in the maturation of cardiac myofibril organization, although the appearance/expression peak is different between the two muscle cells.

It is worth repeatedly saying, no information is so far available concerning the mechanism on association of PIP5Kγ with the non‐membranous myofibrils and Z‐bands and on the supply of PIP to the non‐membranous components, and about how PIP5Kγ is involved in the differentiation/maturation of myofibrils at molecular levels in cardiac as well as skeletal muscles of the mouse embryo. As for the association targets of PIP5Kγ, it is possible to speculate that it is not myofilaments of actin and myosin themselves, but titin, a third filament in the myofibrils, which extends over the entire course of the myofibrils by spanning sarcomeres from the M‐line to Z‐band. It is because phosphorylation of tandemly arranged Ser‐Pro repeats in the Z‐bands and M‐line titin may control integration of the titin filament into Z discs and M‐lines during myogenesis (Gautel et al., 1993). On the other hand, since the level of phosphorylation of PIP5Kγ has been shown to be critical for its interaction with its binding/associated counterpart, talin, and AP2 (Ling et al., 2003; Unoki et al., 2012), it may be a key to examine the phosphorylation level of PIP5Kγ during embryonic development when searching for its binding/associated counterpart molecule(s) in the embryonic heart.

Regardless of the mechanism for PIP5Kγ to work on such non‐membranous structures as myofibrils and Z‐bands, the present finding on the six patterns of localization of PIP5Kγ‐immunoreactivity makes it possible to assume as follows: During the differentiation course of cardiomyocytes, PIP5Kγ and its product PIP2 in differentiating myocardial cells are possible to exert their functions intensely at differing subcellular sites in a way that is progressively shifted from the plasma membranes to the Z‐band through the myofilaments depending on levels of differentiation. If this possibility is confirmed by further analyses, it is likely that the cardiac failure owing to the deletion of PIP5Kγ is caused not only by impairment of formation of intercellular junctions including the intercalated discs, but also by disturbance of the developmental shift of localization and functional exertion of PIP5Kγ, resulting in impaired maturation of myofilament organization.

The fact that PIP5Kγ‐immunoreactivity in the heart, after progressive decrease during the late prenatal stages, reached non‐significant levels in the heart at newborn and postnatal stages may raise a question if other isoforms of PIP5K take over the enzymatic reaction in postnatal hearts. It is known, however, that individual isoforms of a given enzyme play rather discrete roles (van den Bout & Divecha, 2009). In fact, the expression of α and β isoforms of PIP5K in postnatal hearts was examined in western blots by us (data not shown) and either isoforms including its γ was similarly low in the heart. Therefore, the distinct expression confined to cardiomyocytes at E10 and E14 is likely to indicate a specific requirement of PIP5Kγ in the cardiomyocytes for their development and maturation.

AUTHOR CONTRIBUTIONS

Ratchatasunthorn Arissara prepared and analyzed the data. Hiroyuki Sakagami synthesized antibody and characterized it. Surang Chomphoo and Wphawi Hipkaeo performed immunohistochemical analyses and wrote the core of manuscript. Hisatake Kondo conceived and designed the research and edited the manuscript. All authors read and approved the final manuscript.

FUNDING INFORMATION

Rutchasunthorn Arissara was supported by a Postgraduate Study Support Grant of Medicine Khon Kaen University, Thailand. The authors are grateful for research grants from the Faculty of Medicine, Khon Kaen University, Thailand (No. IN66062 to SC). This research was partially supported by the Fundamental Fund of Khon Kaen University, and the National Science, Research and Innovation Fund, Research and Graduated Studies Khon Kaen University (grant number RP65‐2‐002 to WH).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS

The authors extend sincerely thanks to Ms. Y Polsan and Mr. D Hipkaeo for his technical supports. We would like to acknowledge Professor James A Will for editing the manuscript via the Publication Clinic, Khon Kaen University, Thailand.

Ratchatasunthorn, A. , Sakagami, H. , Kondo, H. , Hipkaeo, W. & Chomphoo, S. (2024) Temporal involvement of phosphatidylinositol 4‐phosphate 5‐kinase γ in differentiation of Z‐bands and myofilament bundles as well as intercalated discs in mouse heart at mid‐gestation. Journal of Anatomy, 244, 1030–1039. Available from: 10.1111/joa.14008

DATA AVAILABILITY STATEMENT

All data of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data of this study are available from the corresponding author upon reasonable request.

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