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(Circulation Research. 2003;92:444.)
© 2003 American Heart Association, Inc.

Cellular Biology

Structural Adaptation of the Nuclear Pore Complex in Stem Cell–Derived Cardiomyocytes

Carmen Perez-Terzic, Atta Behfar, Annabelle Méry, Jan M.A. van Deursen, Andre Terzic, Michel Pucéat

From the Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics (C.P.-T., A.B., A.T., M.P.); Physical Medicine and Rehabilitation (C.P.-T.); and Pediatrics and Adolescent Medicine (J.M.A.v.D.), Mayo Clinic, Mayo Foundation, Rochester, Minn, and CNRS UPR1086 (C.P.-T., A.B., A.M., M.P.), Centre de Recherches de Biochimie Macromoléculaire, Montpellier, France.

Correspondence to Dr Michel Pucéat, CRBM, CNRS UPR 1086, 1919, route de Mende, 34293 Montpellier, France. E-mail puceat{at}crbm.cnrs-mop.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macromolecules are transported in and out of the nucleus through nuclear pores. It is poorly understood how these megadalton conduits support nucleocytoplasmic traffic during genetic reprogramming associated with cell commitment to a specific lineage. Murine embryonic stem cells were differentiated into cardiomyocytes within embryoid bodies, and contracting cells expressing myocardial-specific proteins were isolated from the mesodermal layer. Compared with postmitotic cardiac cells from heart muscle, these proliferative and differentiating stem cell–derived cardiomyocytes demonstrated a significantly lower density of nuclear pores. At nanoscale resolution, the pore channel was commonly unoccupied in heart muscle–isolated cardiac cells, yet a dense material, presumably the central transporter, protruded toward the cytosolic face of the nuclear pore complex in stem cell–derived cardiomyocytes. Stem cell–derived cardiac cells distributed the nuclear transport factor Ran in the nucleus, decreased the number of spare nuclear pore complexes from the cytosolic annulate lamellae reservoir, and expressed a set of nucleoporins, NUP214, NUP358, NUP153, and p62, involved in nuclear transport. Stem cell–derived cardiomyocytes secured transport of nuclear constitutive proteins, cardiogenic transcription factors, and cell cycle regulators, including the prototypic histone H1, myocyte enhancer binding factor 2, and p53. Thus, differentiating stem cell–derived cardiomyocytes undergo structural adaptation and mobilize nuclear transport regulators in support of nucleocytoplasmic communication during commitment to mature cardiac lineage.

Key Words: nucleus • nucleoporin • embryonic stem cells • differentiation • heart

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In eukaryotes, the nucleus confines genetic material in each cell. This compartmentalization obligates extensive nucleocytoplasmic exchange of constitutive and regulatory proteins to ensure regulation of gene expression and processing of genetic information.¹ Several steps in the exchange process are recognized, including targeting and movement to the nuclear surface, with translocation through the nuclear envelope ultimately mediating transfer of molecules.² Translocation occurs via nuclear pore complexes (NPCs), megadalton-large conduits that span the nuclear envelope.^3,4

Emerging evidence indicates that traffic through nuclear pores is a dynamic process determined by the state of a cell.⁵ In heart muscle under metabolic or hypertrophic stress, nuclear pores remodel to gate traffic across the nuclear envelope.^6–8 In this way, the NPC contributes to the homeostasis of terminally differentiated cardiomyocytes by adapting nuclear transport to demands for nucleocytoplasmic exchange.^6,7 However, little is known on nuclear pore structure and function early in cardiac differentiation.

Commitment of mesodermal cells to a cardiac lineage results from activation of intracellular signals by specific growth factors.⁹ Effectors of cardiogenic pathways comprise transcription factors, such as the myocyte enhancer binding factor 2 (MEF2C), which work in concert with other differentiating factors (eg, Nkx2.5 and GATA4) to transactivate promoters of cardiac restricted genes encoding constitutive proteins, including myocardial actin and myosin.^10–13 As the transcriptional activity of cardiogenic factors requires translocation from the cytoplasm into the nucleus, resolving the architectural and functional competence of nuclear pores in stem cell–derived cardiomyocytes is warranted. Indeed, early steps of cardiac differentiation can be reproduced in embryoid bodies generated from embryonic stem cells.^14–16

Here, embryonic stem cells were allowed to differentiate into a cardiac lineage, and nuclear pore structure and function were probed. Compared with heart-isolated cardiac cells, stem cell–derived cardiomyocytes exhibited NPCs with a decreased density and depth, along with a different distribution of nuclear transport factors. The observed plasticity in components of nucleocytoplasmic communication may accommodate nuclear transport as stem cell–derived cardiomyocytes progress to a mature state.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stem Cell Differentiation Into Embryoid Bodies
The pluripotent murine embryonic stem cell line CGR8 was propagated in BHK21 medium with pyruvate, nonessential amino acids, mercaptoethanol, 7.5% fetal calf serum, and leukemia inhibitory factor (LIF).^17,18 Cells were split every 2 days to keep undifferentiated status or were differentiated using the "hanging drop" method.^17,19 Drops of differentiation medium (BHK21 with selenium, pyruvate, nonessential amino acids, mercaptoethanol, and 20% FCS, without LIF), containing 500 CGR8 cells, were placed for 2 days on a plate lid to allow embryoid body organization. Embryoid bodies, suspended for 3 days, were plated on gelatin-coated dishes for 7 days. Formation of contracting areas in the mesoderm was monitored with microscopy, and cardiomyocytes were revealed in 12- to 14-day-old embryoid bodies using monoclonal antibodies against cardiac sarcomeric -actinin (Sigma) on fixation with 3% paraformaldehyde and permeabilization with 1% Triton X-100.¹⁷ Images were acquired on a Leica microscope with a 63x objective-mounted piezo-electric controller (Physik Instrumente) driven by Metamorph software (Visitron Universal Imaging), and recorded with a 1300YHS charge-coupled device (CCD) camera (Micromax). Three-dimensional reconstruction was carried out in 0.1-mm steps in the z axis and analyzed by Huygens (Scientific Volume Imaging) and Imaris (Bitplane) software on an Octane workstation (Silicon Graphics).

Cardiomyocyte Isolation and Characterization
Embryoid bodies, at day 12 of differentiation, were detached from dishes with 0.05% trypsin and dissociated using 1 mg/mL collagenase (CLSII, Worthington) and 0.25 mg/mL pancreatin in (in mmol/L) NaCl 117, HEPES 20, NaH₂PO₄ 1.2, KCl 5.4, MgSO₄ 1, and glucose 5 (pH 7.35). After centrifugation through a discontinuous two-layer Percoll gradient, stem cell–derived cardiomyocytes, which were present at the interface between the two layers, were collected and plated on gelatin/laminin–coated dishes. To monitor cell division, cells were cultured in a temperature- and gas-controlled chamber (37°C, 5% CO₂) and set on a stage of a CCD camera–coupled microscope, and phase-contrast images were acquired every 15 minutes and digitized online with the Metamorph software. Forty-eight hours after isolation, cells were immunostained with -actinin, ventricular and atrial myosin light chain, or ß-myosin heavy chain antibodies.¹⁷ Embryoid body–derived cells were also patched in the voltage-clamp and current-clamp mode using Axopatch 200 or Axopatch 1C amplifiers, and current-voltage relationships and action potential profiles analyzed with pClamp 7.0 or Bioquest software. Separately, cardiomyocytes were isolated from collagenase/pancreatin–digested 2-day-old heart and were cultured as described.^6,7 Cells were used 48 hours after isolation.

Transmitted and Field-Emission Scanning Electron Microscopy
Cells were fixed in 0.1 mol/L PBS with 1% glutaraldehyde and 4% formaldehyde (pH 7.2). For transmitted electron microscopy, cells were processed in phosphate-buffered 1% OsO₄, stained with 2% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in epoxy resin. Thin (90-nm) sections were placed on copper grids and stained with lead citrate, and micrographs were taken with a JEOL electron microscope. For field-emission scanning electron microscopy (FESEM), sarcolemma was stripped using a hypotonic solution and 1% Triton X-100.^6,7 Sarcolemma-stripped cells or whole embryoid bodies were fixed with 1% glutaraldehyde and 4% formaldehyde in PBS. Specimens were rinsed in PBS with 1% osmium, dehydrated with ethanol, and dried in a critical point dryer (Ted Pella). On coating with platinum, samples were examined on a Hitachi scanning microscope.^6,7

Atomic Force Microscopy (AFM)
Contact-mode AFM was performed with silicon nitride NP-S tips (spring constant, 0.58 Newton/meter) using a Nanoscope III controller (Digital Instruments).^6,7 Sarcolemma-stripped cells were fixed in situ as described above, rinsed with nanopure water, and air-dried.^6,7 An E-type scanner, with linear scanning frequencies (5 to 15 Hz), was used to build 512x512-pixel AFM images.^6,7 Data were analyzed with the Nanoscope IIIa software, and three-dimensional images generated from topographical height information.⁶

Calcium Transients and Contractions
Stem cell–derived cardiomyocytes, loaded with the Ca²⁺ probe fluo-3-acetoxymethyl ester (AM) (5 µmol/L), were transferred to a Leica epifluorescence microscope and illuminated with a mercury lamp at 400±20 nm. Fluorescence was recorded at 37°C with a 1300YHS CCD camera using an X114-2 CFP Leica filter cube (dichroic mirror DM 455 and emission filter 480±30 nm). Cell shortening was tracked at a region of interest set to overlap the border of a fluo-3–loaded cell and the surrounding dark field, and intracellular Ca²⁺ spike intensity was plotted as a function of time and analyzed with Metamorph software.

Nucleoporins and Ran
Cells were fixed in 3% paraformaldehyde; permeabilized with 0.5% Triton X-100; and immunostained with polyclonal antibodies against nucleoporins NUP214, NUP153, and NUP98, and with the monoclonal antibody mAb414, which recognizes p62 and to a lesser extent NUP214 and NUP358.²⁰ Annulate lamellae revealed by mAb414 were counted in the cytosol using Metamorph, which computes regions with predefined pixel intensity. To localize the small GTPase Ran, a regulator required for nuclear transport, cells were fixed, permeabilized, and labeled with an anti-Ran monoclonal antibody (from Dr Y. Yoneda, Osaka University, Japan), and the DNA fluorescent probe Hoechst.^7,21 Images were collected on a Carl Zeiss confocal or a Leica fluorescence microscope and analyzed with Metamorph.

Nuclear Transport
Nuclear import was monitored in stem cell–derived cardiomyocytes for 24 to 36 hours on Effectene (Qiagen) transfection of plasmids encoding the classic nuclear localization signal (NLS), the myocardial transcription factor MEF2C,¹⁰ or the cardiac cell cycle regulator p53.²² NLS from the SV40 large T antigen (PKKKRKV) was fused to the yellow fluorescent protein (YFP; from Dr E. Bertrand, Institute of Molecular Genetics [IGMM], Montpellier, France). MEF2C cDNA (from Dr J. Han, Scripps Institute, La Jolla, Calif) was subcloned in the pEGFP-C2 plasmid (Clontech) using HindIII and ApaI restriction sites. p53 cDNA was fused to green fluorescent protein (GFP) cDNA (from Dr S.H. Liang, University of Michigan).²³ Nuclear import of chromosomal histone H1 was monitored in stem cell–derived or heart-isolated cardiomyocytes transferred to DMEM with 0.5% BSA, 10 mmol/L HEPES (pH 7.5), and 20 mmol/L 2,3-butanedione monoxime (Sigma), and microinjected (injection buffer in mmol/L: KCl 150, PIPES 1, EDTA 0.1, and EGTA 0.025 [pH 7.2]) into the cytosol with 0.07 mg/mL fluorescein-coupled H1 (fl-H1) using a microinjector unit (Eppendorf 5242) coupled to a micromanipulator (Eppendorf 5171).⁶ Steady-state transport was analyzed 5 minutes after microinjection. Nuclear transport of expressed NLS-YFP, MEF2C-GFP, or p53-GFP or microinjected fl-H1 was captured with a CCD camera on a Leica microscope, and nuclear fluorescence intensity determined 24 hours after transfection with Metamorph software.

Statistical Analysis
Results are expressed as mean±SE. Statistical analysis was carried out by the Student t test. Significant difference was accepted at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Commitment of Stem Cells Into Cardiac Lineage
The embryonic CGR8 stem cell clone gives rise to specialized cell types, including cardiomyocytes.¹⁷ In the presence of the cytokine LIF, CGR8 cells grow as compact colonies typical of undifferentiated, pluripotent stem cells (Figure 1A, inset). On removal of LIF, CGR8 colonies were induced to differentiate and aggregated into three-layered embryoid bodies (Figure 1A). In the mesodermal layer (Figure 1B), foci with spontaneous contractions were observed 6 to 7 days after differentiation, mirroring mouse cardiogenesis characterized by initiation of beating at 6.5 days after coitum.²⁴ In contracting clusters within embryoid bodies, cells that committed to cardiac lineage were distinguished by immunostaining with the myocardial sarcomeric -actinin antibody. At day 12 of differentiation, myofibrillar bundles in these cells were organized in sarcomeric units (Figure 1C), which is typical for cardiomyocytes.²⁵ Thus, CGR8 stem cells undergo a cardiac differentiation program underscoring their propensity to recapitulate early stages in cardiac differentiation.¹⁴

View larger version (68K): [in this window] [in a new window]   Figure 1. Stem cells differentiate into cardiomyocytes in embryoid bodies. A, Phase-contrast microscopy of 7-day-old embryoid body formed by aggregation of embryonic CGR8 stem cells. Square indicates area in mesoderm with spontaneous contractions. Bar=100 µm. Inset, Undifferentiated CGR8 stem cells in culture form colonies. B, Mesodermal area under FESEM. Bar=10 µm. C, Sarcomeric organization in mesoderm of 12-day-old embryoid body, immunostained with sarcomeric -actinin antibody. Bar=20 µm.

Stem Cell–Derived Cardiomyocytes Divide and Contract
Dissociation of embryoid bodies at day 12 of differentiation released, in addition to quiescent cells, beating cells that continued to divide. For the next 48 to 96 hours of follow-up, these cells underwent vigorous karyokinesis (Figure 2A), indicative of sustained mitotic activity. When loaded with the calcium probe fluo-3–AM, rhythmic intracellular calcium transients (1.2±0.1 oscillations per second, n=6) were observed (Figure 2B, top panel) and correlated with cell shortening (Figure 2B, bottom panel). Cardiac phenotype was confirmed by positive immunoreactivity for the myocardial-specific -actinin sarcomeric protein (Figure 2C, left panel), as well as for the atrial and ventricular myosin light chain isoform, MLC2a (Figure 2C, middle panel) and MLC2v (Figure 2C, right panel), respectively, along with positive staining for the ß-myosin heavy chain (ßMHC; not illustrated). Indeed, sarcomeric units, visualized at high resolution with AFM, had an average length of 1.9±0.3 µm (n=18; Figure 2D), characteristic of cardiac cells.²⁵ Under patch clamp, stem cell–derived cardiomyocytes displayed spontaneous (Figure 2E) as well as triggered action potentials and current-voltage relationships with inward Na⁺ and Ca²⁺ components along with outward I_K but with no apparent inwardly rectifying I_K1 current (not illustrated). Such electrophysiological characteristics indicate that embryonic stem cell–derived cardiomyocytes have advanced in differentiation and transition through the "intermediate stage" on their way to full terminal differentiation.^15,26

View larger version (74K): [in this window] [in a new window]   Figure 2. Cardiac phenotype in embryoid body–derived cells. A, On isolation at day 12 from embryoid bodies, a dividing cell at time 0 (top panel) and 3 hours later (bottom panel). N indicates nuclei. B, Top, Intracellular Ca2+ transients from embryoid body–derived cell loaded with fluo 3-AM. Line scan image represents changes in fluorescence along a line crossing the cell (see insert) as a function of time. Bottom, Corresponding cell shortening in same cell. Images are representative of 6 experiments. Horizontal bar=1 second. Vertical bar=30 µm. a.u. indicates arbitrary units. C, Positive immunostaining with antibodies against myocardial -actinin (left panel), atrial myosin light chain 2 (MLC2a, middle panel), and ventricular myosin light chain 2 (MLC2v, right panel). Bars=10 µm. D, High-resolution AFM of two adjacent stem cell–derived cardiomyocytes with respective nuclei (N) and myofibrils organized into sarcomeric units (arrowheads). E, Current-clamp recordings of spontaneous action potential activity recorded, at 34±2°C, from CGR8-derived cells 48 hours after isolation from 12-day-old embryoid bodies. Em indicates membrane potential. Horizontal bar=150 ms.

Structure of Nuclear Pores in Stem Cell–Derived Cardiomyocytes
Cell differentiation requires programmed DNA replication and transcription, maintained by intense nucleocytoplasmic communication through NPCs.^1,5,7 Nuclei, in stem cell–derived cardiomyocytes, were exposed by stripping the sarcolemma (Figures 3A and 3B), and FESEM (Figure 3C) or AFM (Figure 3D) revealed toroid-shaped structures through the nuclear envelope, characteristic of NPCs. The central channel of each NPC was encircled by a distinctive structure corresponding to the cytoplasmic ring of the nuclear pore (Figure 3C, inset). Ultrastructural analysis captured NPCs spanning the nuclear envelope in specialized domains formed by fusion of the inner and outer nuclear membrane (Figure 3E). Resolving the structure of NPCs at nanoscale resolution identified that the central channel was commonly occupied by dense material (Figure 3F). This material has been proposed to represent the central transporter of NPCs.^27–29 Thus, stem cell–derived cardiomyocytes display physical components of the nuclear pore required for nuclear transport.

View larger version (96K): [in this window] [in a new window]   Figure 3. Nuclear pores in nuclear envelope of stem cell–derived cardiomyocytes. A, Cell, isolated from 12-day-old embryoid body, was stripped of sarcolemma to expose nucleus for FESEM scanning 48 hours after isolation. Bar=10 µm. B, AFM of exposed nucleus surrounded by remnants of cytoskeleton. C, Patch of nuclear envelope, where multiple nuclear pores are observed by FESEM. Inset, Close-up of two nuclear pores displaying typical cytosolic rings. Bar=500 nm. D, High-resolution AFM of cytosolic surface of the nuclear envelope revealed the toroid structure of NPCs. E, Cross section of a single nucleus with individual NPCs spanning the nuclear envelope (arrowheads) in transmission electron microscopy. N and C indicate nucleus and cytoplasm, respectively. Bar=200 nm. F, Nanoscale AFM resolution of two NPCs with central channel occupied by prominent material connected to the cytosolic ring.

Density and Configuration of Nuclear Pores
The NPC is a dynamic structure that adapts to cellular demands for nucleocytoplasmic exchange.^{6,7,27,28,30–32} In the nuclear envelope of stem cell–derived cardiomyocytes, 48 hours after isolation from 12-day-old embryoid bodies, 14.8±0.8 NPCs/µm² (n=12) were detected (Figure 4A), a density that persisted throughout the 17-day postdifferentiation follow-up. This is significantly lower than in postmitotic heart–isolated cardiomyocytes that displayed a 46% higher density of NPCs, ie, at 27.6±1.3 NPCs/µm² (n=8; P<0.05; Figure 4A). Yet the overall NPC diameter (168.5±2.1 versus 166.4±11.2 nm) and the NPC height (5.2±0.2 versus 5.2±0.3 nm) were comparable in stem cell–derived (n=65) and heart muscle–isolated (n=45) cardiomyocytes (P>0.05), in line with typical NPCs.^6,29 However, the nuclear pore was significantly shallower in stem cell–derived than in heart muscle–isolated cardiomyocytes, ie, with the depth of the central channel at 1.7±0.1 (n=65) versus 3.7±0.6 (n=45) nm, respectively (Figure 4C). In stem cell–derived cardiomyocytes (n=5), 81±3% of nuclear pores adopted a configuration with the dense material in the central channel displaced toward the cytoplasmic face of the NPC. Such a position of the dense material was observed only in 27±4% of NPCs in heart muscle–isolated cardiac cells (n=5). Thus, stem cell–derived cardiomyocytes possess a low density of NPCs, which adopt a distinct configuration.

View larger version (25K): [in this window] [in a new window]   Figure 4. NPC density and depth. A, NPC density in nuclear envelope of heart-isolated (n=45) versus stem cell–derived (n=12) cardiomyocytes. *Significant difference (P=0.025). B, High-resolution two-dimensional images and height profiles of NPCs. Left, In heart-isolated cardiomyocytes, cross section through central pore (top panel) shows an open central channel with height profile indicating a central depression below the cytoplasmic surface (bottom panel). Right, In stem cell–derived cardiomyocytes, the NPC cross section shows the pore occupied by dense material (top panel), and on height profile (bottom panel) a shallow central depression. C, Depth of central channel in heart-isolated (n=45) versus stem cell–derived (n=64) cardiomyocytes. *Significant difference (P=0.002).

Nucleoporins in Nuclear Envelope and Annulate Lamellae
NPCs are composed of nucleoporins, assembled protein units that include NUP98, NUP153, NUP214, NUP358, and p62 responsible for nuclear transport (Figure 5A).^1,20,32,33 Staining of the nuclear envelope for NUP214, NUP153, p62, and NUP358 proteins already assembled in nuclear pores of undifferentiated embryonic stem cells (data not shown) was comparable in stem cell–derived and heart muscle–isolated cardiomyocytes (Figure 5B). In contrast, NUP98, a nucleoporin dispensable for cell viability and growth,²⁰ was apparently lower in the nuclear envelope of stem cell–derived cardiomyocytes compared with heart muscle–isolated cardiac cells (Figure 5B). Nucleoporins also aggregate within annulate lamellae, a cytosolic formation that serves as a reservoir of spare NPC proteins.^20,34 Double staining with mAb414 and an antibody raised against NUP214, which recognize nucleoporins associated with annulate lamellae,^34,35 demonstrated fluorescence colocalized not only in the nucleus, but also in the cytosol (Figure 6A). Abundant annulate lamellae were observed in heart muscle–isolated cardiomyocytes (Figure 6A, top panel), but were scarce in stem cell–derived cardiomyocytes (Figure 6A, bottom panel). Immunostaining with antibodies against NUP153, a nucleoporin that does not label annulate lamellae,^31,32 did not show cytoplasmic dot-like fluorescence in any of the cell types (not shown). Using mAb414 to reveal annulate lamellae (Figure 6B, insets), their number was estimated at 46±3 and 14±2 in heart-isolated (n=14) versus stem cell–derived (n=14) cardiomyocytes (P<0.05; Figure 6B), suggesting a different intracellular distribution of spare NPC proteins.

View larger version (35K): [in this window] [in a new window]   Figure 5. Nucleoporins in the nuclear envelope. A, Schematic NPC with individual nucleoporins (NUP). NUP214 and NUP358 are at cytoplasmic filaments. p62 complex (ie, p62, p58, p54, and p45) is in the cytoplasmic and nucleoplasmic face of the central channel and within the nuclear basket. NUP153 and NUP98 are in the nuclear basket. B, Confocal images of heart-isolated and stem cell–derived cardiomyocytes immunostained with nucleoporin antibodies NUP214, mAb141, NUP153, and NUP98. Bars=10 µm.

View larger version (40K): [in this window] [in a new window]   Figure 6. Density of annulate lamellae (AL). A, Proteins labeled with mAb414 and NUP214 in heart-isolated and stem cell–derived cardiomyocytes with colocalization of fluorescence revealed in merge images. Bars=10 µm. B, Number of AL per cell in heart-isolated (n=14) and stem cell–derived (n=14) cardiomyocytes. *Significant difference (P=0.002). Inset, Fluorescence images of heart-isolated and stem cell–derived cardiomyocytes, immunostained with mAb414 antibody to reveal AL distribution.

Ran
Nuclear transport requires a number of soluble cofactors,¹ in particular the GTP binding protein Ran, a key regulator of macromolecular translocation.^21,36 In terminally differentiated cardiomyocytes isolated from heart muscle, immunostaining with the anti-Ran monoclonal antibody demonstrated a dominant cytosolic and lesser nuclear localization of Ran (Figure 7A). A rather diffuse cellular distribution of Ran is characteristic of cells with limited demand of nucleocytoplasmic exchange.⁷ In contrast, in differentiating stem cell–derived cardiomyocytes Ran was primarily confined to the nucleus (Figure 7B). A nuclear over cytosolic arrangement of Ran is consistent with vigorous nuclear transport.⁷

View larger version (141K): [in this window] [in a new window]   Figure 7. Distribution of Ran. Three-dimensional images show Ran predominantly in cytosol in heart-isolated (A) versus nucleus in stem cell–derived (B) cardiomyocytes. Ran immunofluorescence alone (left panels) and overlay of Ran with Hoechst fluorescence delineating the nucleus (right panels). Bars=10 µm.

Nuclear Transport in Stem Cell–Derived Cardiomyocytes
Nuclear transport proceeds through distinct import pathways.¹ The nuclear protein H1 linker histone, a component of the nucleosome, plays an important role in transcriptional activation.³⁷ The abundant nuclear import of histone H1 during the S phase of the cell cycle requires the heterodimer importin ß/importin 7 receptor for translocation.³⁷ Histone H1 was readily imported into nuclei of stem cell–derived cardiomyocytes. On cytosolic injection of fluorescein-tagged H1 (fl-H1), a marker of nuclear transport in heart-isolated cardiomyocytes (n=22, Figure 8A, inset),^6,7 13 of 13 microinjected stem cell–derived cardiomyocytes demonstrated nuclear fluorescence indicative of intact nuclear transport through the importin ß/7 heterodimer–dependent pathway (Figure 8A).

View larger version (93K): [in this window] [in a new window]   Figure 8. Nuclear transport imaged by fluorescence microscopy. After cytosolic microinjection, fluorescein-tagged histone (H1-FITC) transports into the nucleus of stem cell–derived (A) and heart-isolated (inset) cardiomyocytes. Upon expression in stem cell–derived cardiomyocytes, NLS-YFP (B), MEF2C-GFP (C), and p53-GFP (D) also accumulate in nuclei. Bars=10 µm.

Importin ß, in conjunction with the importin adapter, also supports classical NLS-directed import.¹ NLS-directed transport is responsible for import of >500 proteins, including TAFII 250, a regulator of gene expression in the heart,³⁸ and the bone morphogenetic superfamily of proteins, inductors of cardiac differentiation.³⁹ In stem cell–derived cardiomyocytes, transfection of the construct encoding a classical NLS fused to the fluorescent marker YFP, resulting in nuclear accumulation of the NLS-YFP protein (n=20; Figure 8B), which indicates that the canonical NLS-directed, importin /ß–dependent, nuclear import pathway is functional.

The myocardial transcription factor MEF2C, involved in cardiac morphogenesis¹⁰ (n=10; Figure 8C) or the cardiac cell cycle regulator p53²² (n=17; Figure 8D), each of which does not contain classical but bipartite NLS,^23,40 also readily accumulated into the nucleus. This was visualized after transfection of cDNA encoding MEF2C or p53, fused to the fluorescent marker GFP, suggesting that principal nuclear transport pathways are also operational in stem cell–derived cardiomyocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study describes the structural organization and transport competence of NPCs in proliferative cardiomyocytes isolated from stem cell–derived embryonic bodies. This is the first report on the architecture and function of nuclear pores in a stem cell–derived lineage, underscoring the adaptive potential of the nuclear transport machinery in cellular differentiation.

In accord with the propensity of CGR8 embryonic stem cells to differentiate toward cardiac lineage,^17,41 embryoid bodies generated from these pluripotent cells displayed a network of myofibrils within contracting zones. Cells isolated from CGR8-derived embryoid bodies expressed ventricular- and atrial-specified markers with sarcomeres typical of the cardiac phenotype. With each contraction, CGR8-derived cardiomyocytes demonstrated Ca²⁺ spikes and displayed action potential activity in line with functional excitation-contraction coupling.⁴² Indeed, nodal-like, atrial-like, and ventricular-like phenotypes coexist at each stage of differentiation of embryonic stem cell–derived cardiomyocytes.^15,26 Although cells used here advanced in differentiation, they may not have reached full maturation as they lacked prominent inwardly rectifying I_K1 current, a characteristic of the "intermediate stage" of differentiation in stem cell–derived cardiomyocytes.^15,26 In fact, in contrast to terminally differentiated heart muscle cells, stem cell–derived cardiomyocytes continued to develop and proliferated after isolation.¹⁷ Thus, stem cell–derived cardiomyocytes provide a unique system to probe the nuclear pore in a cellular environment in which cardiogenic transcription factors are delivered across the nuclear envelope to secure differentiation of proliferative cardiac precursors into specialized differentiated cells.

Differentiation dictates intense communication with the nucleus.^31,43 Although in stem cell–derived cardiomyocytes nuclear pores were of regular size, their total number was halved compared with that of mature heart muscle.⁶ Formation of nuclear pores and nuclear transport itself are, at least in part, energy-dependent processes.^32,44–46 Thus, stem cell–derived cardiomyocytes with a low number of nuclear pores would more efficiently complete pore assembly/disassembly associated with nuclear envelope breakdown during mitosis.

Profile analysis and molecular topography of stem cell–derived cardiomyocytes revealed that the central channel of the nuclear pore was occupied by dense material. Although previously referred to as cargo in transit, such dense material has been widely accepted to represent a specialized component of the nuclear pore, known as the "central transporter."²⁹ The putative transporter is presumably responsible for gating and translocation of molecules through the pore.^1–3 Such a structure was here found to be suspended in a framework of radial spokes. This arrangement has been proposed to be critical in providing a spring mechanism for vertical displacement and exposure of the transporter to the cytosolic phase and/or securing opening to allow passage of molecules.^28,29,43 Thus, the prominent material observed here to protrude into the cytosolic face of the pore could represent displacement and/or elongation of the central transporter itself.^29,47 Such structural adjustment would, in principle, increase the probability of NPCs to be captured while "engaged" in transport, thus securing access and translocation of molecules despite their low density. This is in contrast to normal heart, in which more abundant nuclear pores display a less prominent transporter,⁶ suggesting that a limited number of active pores is sufficient for regular nuclear transport. Heart cells may adjust the position of the central transporter under increased demand for nucleocytoplasmic communication, as during re-expression of embryonic genes in cardiac hypertrophy.⁷ Thus, the structural plasticity of the NPC could provide a mechanistic basis to accommodate cellular requirements for nuclear transport under normal conditions, such as during differentiation or in the setting of pathological remodeling.

Nuclear pore composition in stem cell–derived cardiomyocytes was largely comparable to that of heart muscle-isolated cardiac cells, with positive staining for nucleoporins NUP214, NUP153, p62, and NUP358. Although the immunofluorescence of NUP98 was lower in stem cell–derived cardiomyocytes, this apparent deficiency may not produce significant functional deficit as involvement of this protein in transport can be compensated by other nucleoporins, such as NUP214 and NUP358.²⁰ Heart-isolated and stem cell–derived cardiomyocytes harbored in the cytosol aggregates labeled by mAb414 and NUP214 antibodies, which likely represent annulate lamellae. These fine, parallel-stacked membranes contain spare NPCs,³⁴ and coimmunostaining with these antibodies has been shown to correlate with annulate lamellae visualized by electron microscopy.²⁰ Annulate lamellae were present only at low frequency in stem cell–derived cardiomyocytes, indicating high turnover of this reserve nucleoporin pool. Intense recruitment of nucleoporins would leave a low population of unutilized nuclear pore proteins in the cytosol, but would ensure maximal availability at the nuclear envelope for nuclear transport.

To be operational, nuclear transport requires a set of regulatory molecules, in particular the GTPase Ran.^1,36 The asymmetry in Ran distribution determines the efficiency and direction of nucleocytoplasmic transport,^36,48 with intranuclear accumulation of Ran reported under conditions of increased demand for nucleocytoplasmic exchange.⁷ In stem cell–derived cardiomyocytes, Ran was primarily confined to the nucleus, suggesting that a nuclear gradient of this small GTPase is maintained across the nuclear envelope to favor nuclear transport. Accordingly, histone H1, NLS-YFP, and the cardiac transcription factor MEF2C or the regulatory p53 protein were all readily transported into nuclei of stem cell–derived cardiomyocytes, demonstrating that the main nuclear transport pathways are functional. Thus, the machinery required for nuclear transport, a critical process in determining the proliferative capacity of cardiac cells⁴⁹ and the overall pattern of gene expression,^1,5 is in place in stem cell–derived cardiomyocytes, where it contributes to nuclear transport of constitutive and DNA-regulatory proteins.

In summary, stem cell–derived cardiomyocytes exhibit a distinct architectural organization of the NPC with mobilization of nuclear transport regulators required for nucleocytoplasmic exchange. NPCs undergo a structural adaptation along with favorable nuclear factor distribution to accommodate nuclear transport demand in proliferation and differentiation required in cardiogenesis. Demonstrating such plasticity in mediators of nucleocytoplasmic communication identifies a mechanistic basis supporting differentiation of stem cell–derived cardiomyocytes into mature cardiac cells.

	Acknowledgments

 
This study was supported by the Fondation de France (Grant 2000003470 to M.P.), the Association Francaise contre les Myopathies (Grants 7778 and 8524 to M.P.), the Mayo Clinician-Investigator Program (to C.P.-T.), the American Heart Association (Grant 0140057N to A.T.), and the NIH (Grant HL-64822 to A.T.). A.T. is an Established Investigator of the American Heart Association. M.P. is an established scientist of INSERM. We thank Dr P. Travo (Integrated Imaging Facility, Centre de Recherches de Biochimie Macromoléculaire, Montpellier [CRBM Montpellier]), and W. Wessels (Mayo Clinic) for assistance in cell imaging and AFM, and Drs P. Charnet (CRBM Montpellier) and A.E. Alekseev (Mayo Clinic) for electrophysiological characterization.

Received June 17, 2002; revision received January 17, 2003; accepted January 21, 2003.

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