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Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

Winitsky SO, Gopal TV, Hassanzadeh S, Takahashi H, Gryder D, Rogawski MA, Takeda K, Yu ZX, Xu YH, Epstein ND - PLoS Biol. (2005)

Bottom Line: Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion.Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials.By 6 d they begin to differentiate without fusing to recipient cardiac cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology Section, Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.

ABSTRACT
It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

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CPS Cells Stain Positive for Cardiac-Specific Proteins(A)							GATA-4 in day 7 CPS cells.						(B) Nuclear staining with DAPI.(C) Overlay of (A) and (B).(D) Nkx-2.5 is detected in the nuclei of round, day 21 beating cells (green).(E) Noncardiac cells (red arrowheads) do not show nuclear staining for Nkx-2.5.(F) Overlay of (D) and (E).(G) Beating cells, after 28 d in culture, stain positive for cardiac L-type Ca++ channel.(H) Connexin 43 (green) in cluster of uninucleate day 21 beating cells in culture.(I) Nomarski light micrograph (differential interference contrast) of cell cluster in (H).
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pbio-0030087-g003: CPS Cells Stain Positive for Cardiac-Specific Proteins(A) GATA-4 in day 7 CPS cells. (B) Nuclear staining with DAPI.(C) Overlay of (A) and (B).(D) Nkx-2.5 is detected in the nuclei of round, day 21 beating cells (green).(E) Noncardiac cells (red arrowheads) do not show nuclear staining for Nkx-2.5.(F) Overlay of (D) and (E).(G) Beating cells, after 28 d in culture, stain positive for cardiac L-type Ca++ channel.(H) Connexin 43 (green) in cluster of uninucleate day 21 beating cells in culture.(I) Nomarski light micrograph (differential interference contrast) of cell cluster in (H).

Mentions: During the first 7 d in medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF), Spoc cells undergo several rounds of division, begin to express GATA-4 (a mostly cardiac-specific transcription factor), and become clusters of floating round cardiac precursors from Spoc (CPS) cells with an increased diameter of 10–14 μm. The pattern of GATA-4 staining in these cells is unusual in that it is predominantly cytoplasmic, which is distinct from the expected nuclear staining of a transcription factor. Although rare, many transcription factors are well known to have a cytoplasmic phase. GATA-4 has been described similarly and in this case has been shown to move into the nucleus after the addition of the beta-adrenergic drug isoproterenol [24,25]. For these reasons, isoproterenol was added to a culture of CPS cells for 1 h, after which GATA-4 staining was observed in the nucleus of many of the cells (Figure 2). These cells go on to express other cardiac-specific markers, including cardiac troponin-T, Nkx-2.5, MLC-2v, and a cardiac L-type calcium channel, detected either by immunostaining or real-time PCR (Figure 3A–3G; real-time PCR data not shown). Although Nkx-2.5 may be present in vascular smooth muscle, neither alpha- nor beta-myosin is present in smooth muscle. Beating cardiomyocytes derived from Spoc cells express both alpha- and beta-myosin at day 28, as shown by staining with polyclonal antibodies against alpha- and beta-myosin (Figure S1). In later stages, connexin 43 is expressed in cell clusters (Figure 3H–3I). Even before they become adherent, some isolated cells begin to beat (Video S1).


Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

Winitsky SO, Gopal TV, Hassanzadeh S, Takahashi H, Gryder D, Rogawski MA, Takeda K, Yu ZX, Xu YH, Epstein ND - PLoS Biol. (2005)

CPS Cells Stain Positive for Cardiac-Specific Proteins(A)							GATA-4 in day 7 CPS cells.						(B) Nuclear staining with DAPI.(C) Overlay of (A) and (B).(D) Nkx-2.5 is detected in the nuclei of round, day 21 beating cells (green).(E) Noncardiac cells (red arrowheads) do not show nuclear staining for Nkx-2.5.(F) Overlay of (D) and (E).(G) Beating cells, after 28 d in culture, stain positive for cardiac L-type Ca++ channel.(H) Connexin 43 (green) in cluster of uninucleate day 21 beating cells in culture.(I) Nomarski light micrograph (differential interference contrast) of cell cluster in (H).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064849&req=5

pbio-0030087-g003: CPS Cells Stain Positive for Cardiac-Specific Proteins(A) GATA-4 in day 7 CPS cells. (B) Nuclear staining with DAPI.(C) Overlay of (A) and (B).(D) Nkx-2.5 is detected in the nuclei of round, day 21 beating cells (green).(E) Noncardiac cells (red arrowheads) do not show nuclear staining for Nkx-2.5.(F) Overlay of (D) and (E).(G) Beating cells, after 28 d in culture, stain positive for cardiac L-type Ca++ channel.(H) Connexin 43 (green) in cluster of uninucleate day 21 beating cells in culture.(I) Nomarski light micrograph (differential interference contrast) of cell cluster in (H).
Mentions: During the first 7 d in medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF), Spoc cells undergo several rounds of division, begin to express GATA-4 (a mostly cardiac-specific transcription factor), and become clusters of floating round cardiac precursors from Spoc (CPS) cells with an increased diameter of 10–14 μm. The pattern of GATA-4 staining in these cells is unusual in that it is predominantly cytoplasmic, which is distinct from the expected nuclear staining of a transcription factor. Although rare, many transcription factors are well known to have a cytoplasmic phase. GATA-4 has been described similarly and in this case has been shown to move into the nucleus after the addition of the beta-adrenergic drug isoproterenol [24,25]. For these reasons, isoproterenol was added to a culture of CPS cells for 1 h, after which GATA-4 staining was observed in the nucleus of many of the cells (Figure 2). These cells go on to express other cardiac-specific markers, including cardiac troponin-T, Nkx-2.5, MLC-2v, and a cardiac L-type calcium channel, detected either by immunostaining or real-time PCR (Figure 3A–3G; real-time PCR data not shown). Although Nkx-2.5 may be present in vascular smooth muscle, neither alpha- nor beta-myosin is present in smooth muscle. Beating cardiomyocytes derived from Spoc cells express both alpha- and beta-myosin at day 28, as shown by staining with polyclonal antibodies against alpha- and beta-myosin (Figure S1). In later stages, connexin 43 is expressed in cell clusters (Figure 3H–3I). Even before they become adherent, some isolated cells begin to beat (Video S1).

Bottom Line: Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion.Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials.By 6 d they begin to differentiate without fusing to recipient cardiac cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology Section, Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.

ABSTRACT
It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

Show MeSH
Related in: MedlinePlus