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Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes¹

Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan; and
^* Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan

²Correspondence: Department of Cardiovascular Science and Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 Japan. E-mail: komuro-tky{at}umin.ac.jp

SPECIFIC AIMS

Skeletal myoblasts are thought to be a good cell source for autologous transplantation to myocardium, but the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses a stem cell population that is distinct from myoblasts. The present study was performed to elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes and develop electromechanical coupling with neighboring cardiomyocytes.

PRINCIPAL FINDINGS

1. Skeletal muscle-derived cells expressed cardiac TnT and ANP in coculture with cardiomyocytes
Skeletal muscle cells were prepared from green fluorescent protein (GFP) -expressed transgenic mice and cultured with cardiac myocytes of neonatal rats. Expression of cardiac-specific proteins such as cardiac troponin T (TnT) and atrial natriuretic peptide (ANP) was examined by immunostaining. Although the majority of GFP-positive cells did not express cardiac TnT and ANP, a few GFP-positive cells (0.02%) expressed cardiac TnT and ANP. Expression of cardiac TnT was first observed at day 2 from the start of the coculture. At day 4, GFP-positive cells expressing cardiac TnT showed a fine striated pattern, indicating sarcomere formation (Fig. 1 A, B). ANP staining was observed at perinuclear region at day 2 (Fig. 1C ). Complete colocalization of GFP signal and cardiac TnT was confirmed by merged picture of confocal image (Fig. 1D ). The expression of these two cardiac-specific proteins in GFP-positive cells suggests that skeletal muscle-derived cells transdifferentiate into cardiomyocytes.

View larger version (74K): [in this window] [in a new window]   Figure 1. Immunostaining of skeletal muscle-derived cells after cocultured with cardiomyocytes. Skeletal muscle-derived cells that were prepared from GFP-transgenic mice were cultured with contracting cardimyocytes for 5 days. Skeletal muscle-derived cells expressed GFP (A, D, green). Cells were double-stained by anti-cardiac TnT antibody visualized with Cy3-conjugated secondary antibody (B, D, red), and anti-ANP antibody visualized by Cy5-conjugated secondary antibody (C, D, blue). A GFP-positive skeletal muscle-derived, cell-expressed cardiac TnT and ANP. The skeletal muscle-derived cells that expressed cardiac TnT and ANP showed fine striated pattern (D).

2. Skeletal muscle-derived cells expressed Nkx2E and GATA4 in coculture with cardiomyocytes before expression of cardiac TnT and ANP
We next examined the expression of cardiac transcription factors Nkx2E (formerly named as Csx/Nkx2.5) and GATA4 in skeletal muscle-derived cells using the coculture system. Expression of Nkx2E was examined by double staining with anti-Nkx2E antibody and anti-cardiac TnT antibody. Expression of GATA4 was examined by double staining with anti-GATA4 antibody and anti-ANP antibody. At day 1 from starting the coculture, expression of Nkx2E and GATA4 was observed in GFP-positive cells. On day 2 some skeletal muscle-derived cells expressed both Nkx2E and cardiac TnT, and GATA4 and ANP. These results suggest that cardiac transcription factors Nkx2E and GATA4 were expressed in skeletal muscle-derived cells 1 day earlier than cardiac TnT and ANP.

3. Skeletal muscle-derived cells expressed connexin 43 and cadherin at the junction of neighboring cardiomyocytes
To investigate whether transdifferentiated skeletal muscle-derived cells contact with cardiomyocytes via gap and adherence junctions, expression of connexin 43 and cadherin was examined. Cells were double stained by anti-connexin 43 antibody and cardiac TnT or anti-pan-cadherin antibody and anti-ANP antibody. Expression of connexin 43 was observed at the junction of cardiac TnT-expressing skeletal muscle-derived cells and neighboring cardiomyocytes. Expression of cadherin was observed at the junction of ANP-expressing, skeletal muscle-derived cells and neighboring cardiomyocytes. Skeletal myoblasts and skeletal myotubes expressed little connexin43 and cadherin at the junction of neighboring cardiomyocytes as reported previously. These results suggest that skeletal muscle-derived cells contact with cardiomyocytes through gap junctions and adherence junctions after transdifferentiation into cardiomyocytes.

4. Skeletal muscle-derived cells showed cardiomyocyte-like action potential in coculture with cardiomyocytes
An electrophysiological study was performed on the GFP-positive skeletal muscle-derived cells that contract synchronously with neighboring cardiomyocytes. The contracting GFP-positive cell demonstrated cardiomyocyte-like action potentials. This action potential was characterized by 1) a relatively long action potential duration and 2) a relatively shallow resting membrane potential. These properties are consistent with the action potential observed in cardiomyocytes of early developmental stage.

5. Direct contact was necessary for transdifferentiation of skeletal muscle-derived cells
All these transdifferentiated cells were adjacent to cardiomyocytes, suggesting that cell–cell contact is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes. To prove this hypothesis, skeletal muscle-derived cells were cultured in three different ways: monoculture, coculture, and double chamber system, which has cell culture inserts. In the double chamber system, cardiomyocytes and skeletal muscle cells were cultured separately but in the same culture media. Skeletal muscle-derived cells expressed cardiac TnT and ANP in the coculture condition but not in monoculture condition or double chamber condition. These results suggest that humoral factors are not sufficient but direct cell–cell contact is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes.

6. Nifedipine treatment inhibited the transdifferentiation of skeletal muscle-derived cells and cyclic stretch restored the inhibitory effect of nifedipine
The cultured cardiomyocytes of neonatal rats were rhythmically beating. To access whether contraction of cardiomyocytes is necessary for transdifferentiation of skeletal muscle-derived cells, cells were cultured in the presence or absence of nifedipine. Nifedipine (5 µM) inhibited contraction of half of cardiomyocytes and 20 µM nifedipine abolished the beating. Nifedipine treatment suppressed transdifferentiation of skeletal muscle-derived cells in a dose-dependent manner (Fig. 2 ). To further examine the effect of mechanical stretch on the transdifferentiation of skeletal muscle-derived cells, cells were cultured on the silicone dish and passive cyclic stretch (60 cycles/min) was applied to the skeletal muscle-derived cells cocultured with cardiomyocytes whose spontaneous beating was inhibited with 5 µM nifedipine. After 48 h treatment, cells were stained by anti-cardiac TnT antibody and anti-ANP antibody. Treatment of 5 µM nifedipine markedly reduced the number of cardiac TnT-positive cells compared with control; cyclic stretch completely restored this inhibition (Fig. 2) . These results suggest that mechanical load on the skeletal muscle-derived cells is important for the transdifferentiation of skeletal muscle-derived cells.

View larger version (19K): [in this window] [in a new window]   Figure 2. Role of contraction. Skeletal muscle cells and cardiomyocytes were cocultured in silicone dishes in the absence (Control) or presence of nifedipine (nifedipine 5 µM, nifedipine 20 µM). Cyclic stretch was applied in the presence of 5 µM nifedipine (nifedipine 5 µM +stretch). (*P<0.05 **P<0.01) The number of cells that are double positive for cardiac TnT and GFP was counted and normalized by total number of GFP-positive cells in each silicone dish.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we demonstrate that skeletal muscle-derived cells can transdifferentiate into cardiomyocytes when cocultured with contracting cardiomyocytes. This is demonstrated by the expression of 1) cardiac-specific proteins (cardiac TnT and ANP), 2) cardiac transcription factors (Nkx2E and GATA4), and 3) adhesion and gap junction proteins (cadherin and connexin43) in the skeletal muscle-derived cells. The anti-cardiac TnT antibody does not react with adult skeletal muscle and stained specifically cardiomyocytes. ANP expression is known to be restricted to the heart but not to skeletal muscle. Expression of these two cardiac-specific proteins suggests that skeletal muscle-derived cells transdifferentiated into cardiomyocytes. We also demonstrated the expression of two cardiac transcription factors (Nkx2E and GATA4) in skeletal muscle-derived cells. ANP gene expression is activated by Nkx2E and GATA4 synergistically. Cardiac TnT also contains potential Nkx2E binding site and GATA binding site in its promoter region. Expression of Nkx2E and GATA4 was recognized 1 day earlier than cardiac TnT and ANP in skeletal muscle-derived cells, suggesting that the skeletal muscle-derived cells acquire phenotype of cardiomyocytes by the transcriptional regulation of cardiac-specific genes.

N-Cadherin is a major adhesion molecule of the adherence junction and connexin 43 is a gap junction protein. They are located at the intercalated disc of myocardium. Gap junction forms low resistance pathway of cardiac action potential. In our study, these two proteins were clearly expressed at the border of the transdifferentiated skeletal muscle-derived cells and cardiomyocytes. Action potentials recorded from contracting GFP-positive skeletal muscle cells had cardiomyocyte-like properties clearly different from action potentials of the skeletal muscle. These results suggest that skeletal muscle-derived cells not only express cardiac-specific proteins but also show cardiac electrical properties.

To investigate the mechanisms of transdifferentiation, we examined whether direct cell–cell contact and beating of cardiomyocytes are needed to transdifferentiation. The double chamber experiment revealed that humoral factors are not sufficient but direct cell–cell contact is needed for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes. Treatment of nifedipine, an L-type calcium channel antagonist, and Ca²⁺-free culture media inhibit spontaneous beating of cardiomyocytes. Both treatments clearly reduced the number of transdifferentiated cells. Moreover, the cyclic stretching restored this inhibition by 12%, suggesting that mechanical stress is important for the transdifferentiation. Mechanical load has been reported to activate various signaling pathways through autocrine/paracrine secretable factors, Ca²⁺-dependent signaling, and adhesion molecules, including integrins. Further investigation is necessary to clarify how mechanical load is connected to transdifferentiation of skeletal muscle-derived cells.

Skeletal muscle has been reported to contain stem cell populations besides satellite cells. We enriched the stem cell population of skeletal muscle cells by collecting the SP fraction. Cells of the SP fraction showed much higher rate (10-fold) of transdifferentiation than unfractionated cells when cocultured with cardiomyocytes (unpublished data). Although this is not direct evidence, it suggests that multipotent muscle-derived stem cells differentiate into cardiomyocytes.

In conclusion, we demonstrated that skeletal muscle-derived cells could transdifferentiate into cardiomyocytes and that direct cell–cell contact and mechanical force of beating cardiomyocytes were important for transdifferentiation.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1048fje; doi: 10.1096/fj.02-1048fje

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