Modulation of L-type Calcium Channel Expression during Retinoic Acid-induced Differentiation of H9C2 Cardiac Cells

Abstract

The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca2+ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (α1S) and cardiac (α1C) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nm all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca2+ channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca2+ channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (α1C) L-type Ca2+ channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca2+ channels in mature cardiac cells. The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca2+ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (α1S) and cardiac (α1C) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nm all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca2+ channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca2+ channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (α1C) L-type Ca2+ channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca2+ channels in mature cardiac cells. voltage-dependent calcium channel retinoic acid phosphate-buffered saline fetal calf serum reverse transcription polymerase chain reaction myosin light chain It is accepted that adult cardiac myocytes exhibit tissue-specific expression of L-type voltage-dependent calcium channels (VDCCs),1 i.e. the class C (α1C) channel subtype (1Hofmann F. Biel M. Flockerzi V. Annu. Rev. Neurosci. 1994; 17: 399-418Crossref PubMed Scopus (452) Google Scholar). However, despite the large body of information on the properties of these VDCCs in adult mammalian heart (1Hofmann F. Biel M. Flockerzi V. Annu. Rev. Neurosci. 1994; 17: 399-418Crossref PubMed Scopus (452) Google Scholar, 2McDonald T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (925) Google Scholar), little is known concerning the regulation of the functional expression of these channels in the developing heart. No triggering or regulatory mechanism for cardiac L-type VDCC expression has been clearly identified to date, although there is some evidence that cardiac embryonic differentiation may be controlled by specific inducers, such as transforming growth factor-β1 (3Eghbali M. Tomek R. Woods C. Bhambi B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 795-799Crossref PubMed Scopus (136) Google Scholar), triiodothyronine (4Rodriguez E.R. Tan C.D. Onwuta U.S., Yu, Z.X. Ferrans V.J. Parrillo J.E. Biochem. Biophys. Res. Commun. 1994; 205: 652-658Crossref PubMed Scopus (18) Google Scholar), and retinoic acid (5Kastner P. Grondona J.M. Mark M. Gansmuller A. LeMeur M. Decimo D. Vonesch J.L. Dolle P. Chambon P. Cell. 1994; 78: 987-1003Abstract Full Text PDF PubMed Scopus (608) Google Scholar). The skeletal muscle, in which myoblasts fuse to form mature multinucleated myotubes, is a good model to address basic mechanisms involved in muscle differentiation (6Olson E.N. Dev. Biol. 1992; 154: 261-272Crossref PubMed Scopus (377) Google Scholar). It is now clear that the expression of the skeletal L-type VDCC subtype specifically correlates with skeletal muscle differentiation (7Cognard C. Constantin B. Rivet-Bastide M. Imbert N. Besse C. Raymond G. Development. 1993; 117: 1153-1161Crossref PubMed Google Scholar). In the cardiac muscle lineage, cell proliferation and differentiation are not mutually exclusive events, and embryonic cardiac myocytes undergo maturation during development. The appearance of L-type VDCCs occurs at early stages of cardiogenesis (8Maltsev V.A. Wobus A.M. Rohwedel J. Bader M. Hescheler J. Circ. Res. 1994; 75: 233-244Crossref PubMed Scopus (405) Google Scholar, 9Kolossov E. Fleischmann B.K. Liu Q. Bloch W. Viatchenko-Karpinski S. Manzke O. Ji G.J. Bohlen H. Addicks K. Hescheler J. J. Cell Biol. 1998; 143: 2045-2056Crossref PubMed Scopus (159) Google Scholar). However, the study of L-type VDCC expression during cardiogenic differentiation is a difficult task, mainly because of the laboriousness of obtaining embryonic cardiac cells from early embryos. The development of in vitro models that recapitulate molecular events underlying the establishment of the cardiac muscle phenotype is an important step toward understanding the mechanisms involved in the regulation of cardiac ion channel gene expression (9Kolossov E. Fleischmann B.K. Liu Q. Bloch W. Viatchenko-Karpinski S. Manzke O. Ji G.J. Bohlen H. Addicks K. Hescheler J. J. Cell Biol. 1998; 143: 2045-2056Crossref PubMed Scopus (159) Google Scholar). An interesting experimental model is the clonal cardiac cell line H9C2 derived from embryonic rat heart (10Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 367-381Crossref PubMed Scopus (461) Google Scholar). This cell line has been studied for its electrophysiological properties, and it was demonstrated that H9C2 cells exhibit L-type VDCCs with cardiac-specific characteristics (11Hescheler J. Meyer R. Plant S. Krautwurst D. Rosenthal W. Schultz G. Circ. Res. 1991; 69: 1476-1486Crossref PubMed Scopus (409) Google Scholar), although concomitant expression of skeletal L-type VDCCs occurs in H9C2 cells (12Mejia-Alvarez R. Tomaselli G.F. Marban E. J. Physiol. 1994; 478: 315-329Crossref PubMed Scopus (43) Google Scholar). Here, we present evidence that the H9C2 cell line is valuable for the investigation of the ontogenic properties of these two Ca2+channel isotypes. We have conducted experiments to characterize the changes in the expression of VDCCs during differentiation of H9C2 cells. We show that upon a chronic treatment with 10 nmall-trans-retinoic acid (all-trans-RA), H9C2 cells exhibit enhanced cardiac Ca2+ channel expression as a consequence of the maintenance of their cardiac phenotype. By contrast, myogenic transdifferentiation is inhibited. The data unambiguously demonstrate that the functional expression of muscle L type VDCC isotypes (α1C versus α1S) correlates well with specific differentiation processes, cardiogenesisversus myogenesis. H9C2 cells obtained from the European Collection of Cell Cultures were grown as a stock of cells in culture flasks in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (Eurobio), glutamine (2 mm), penicillin (100 IU/ml), and streptomycin (100 μg/ml) under standard culture conditions (37 °C, 5% CO2, and water-saturated atmosphere). Before confluence, cells were split, plated at a lower density in Petri dishes in 10% FCS culture medium and cultured for 1 day. Cells were then cultured in Dulbecco's modified Eagle's medium supplemented with 1% FCS. The culture medium was replaced every 2 days. Stimulation with retinoic acid analogs was performed daily. All-trans-RA, as well as 9-cis-RA (Sigma) were diluted in the dark in Me2SO (1 mm stock solution) and stored at −20 °C. Aliquots were used only once and diluted extemporaneously in 1% FCS culture medium in the dark. Calcium channel currents were recorded by means of the whole cell configuration of the patch clamp technique using an Axopatch 200A amplifier (Axon Instruments, CA) and Ba2+ (40 mm) as the charge carrier. The bath solution was 40 mm Ba(OH)2, 80 mm N-methyl-d-glutamine, 40 mmglutamate, 10 mm Hepes, 2 mm MgCl2, pH 7.4, with CH3SO3H. The intracellular solution was 140 mm N-methyl-d-glutamine, 20 mm EGTA, 15 mm Hepes, 2 mm MgCl2, pH 7.4, with CH3SO3H. Electrophysiological recordings were performed as described elsewhere (13Lory P. Varadi G. Slish D.F. Varadi M. Schwartz A. FEBS Lett. 1993; 315: 167-172Crossref PubMed Scopus (50) Google Scholar). Multiplex RT-PCR detection of transcripts encoding α1C and α1S was chosen for its high sensitivity allowing adequate semiquantitation of these transcripts. Total RNAs were prepared by using the guanidinium thiocyanate-phenol-chloroform procedure (14Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar, 15Lory P. Rassendren F.A. Richard S. Tiaho F. Nargeot J. J. Physiol. 1990; 429: PubMed Scopus Google and stored in was for 1 at in of 1 of and reverse to the The was then at that were to (12Mejia-Alvarez R. Tomaselli G.F. Marban E. J. Physiol. 1994; 478: 315-329Crossref PubMed Scopus (43) Google to transcripts encoding α1C and α1S were using of these by P. and B. as Multiplex RT-PCR conditions allowing the of a to α1C and a to α1S were to using a reverse of was on and with were then and a was performed using were using and on a were by by and Full Text PDF PubMed Scopus (43) Google that an of the α1S was used at a for indirect immunofluorescence experiments. the α1C was in It was a from the intracellular to of the serum for immunofluorescence experiments were in the of to a myogenic we used a specific of rat the by The was used In addition, a specific of skeletal muscle was In this the was used at a in our immunofluorescence experiments. a cardiac we used a that specifically myosin light chain by S. and K. for immunofluorescence experiments were in the of that specifically the by J. M. A. A. J. A. J.M. PubMed Scopus (18) Google Scholar), was used as control in experiments. were prepared from and skeletal were were at in the prepared extemporaneously mm pH 1 using a were for at and were and for in a solution and were then by by on were (40 were at in mm 2 pH supplemented with 5% and were then for 1 at in diluted in and 5% and for 10 with were for at with diluted at and the using used to the the detection of α1S in skeletal muscle with were obtained using to were then in mm pH at for and to the A. A. J. A. J.M. PubMed Scopus (18) Google Scholar). Cells were with at in a solution in and then for with of was performed by cells in a serum solution for at °C. cell was performed by with in for Cells were then for 1 at or at with the at the in serum Cells were with and for at with or were then with and by a treatment with were in and in a in on a using a were with or a treatment on a with the and or were using a the patch clamp we have the Ca2+ channel properties in H9C2 cells using 40 mm Ba2+ as the charge carrier. plated cells 1 and an L-type Ba2+ 1 as described for cardiac myocytes T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (925) Google Scholar). The density of this was 1 in their This was to and and was by the as described (11Hescheler J. Meyer R. Plant S. Krautwurst D. Rosenthal W. Schultz G. Circ. Res. 1991; 69: 1476-1486Crossref PubMed Scopus (409) Google Scholar). Ba2+ in cells (2 of was not characterized H9C2 cells confluence, were to a 1% FCS and and multinucleated cells were cells a specific for as well as for and P. S. C. Nargeot J. Biophys. J. and be identified as skeletal muscle H9C2 myotubes a and L-type Ba2+ and This the recorded in skeletal muscle myotubes and in a of differentiated skeletal muscle cell and cultures J. Physiol. PubMed Scopus Google Scholar). The of and to cells that exhibit the two 1 as well as to their we that in myotubes was with and two despite their in were clearly high and In addition, we identified Ba2+ in such myotubes In myotubes with to 1 was of density and was data that the functional expression of skeletal muscle L-type Ca2+ channels occurs with the of the electrophysiological conducted on a large of cells to have that the of is exclusive of However, it is important to that was still in cells present in with an Ba2+ density of The H9C2 cell line was derived from rat cardiac embryonic (10Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 367-381Crossref PubMed Scopus (461) Google Scholar). plated cells were as from by We of this of of the L-type Ca2+ to that muscle differentiation with Ca2+ channel We the of a chronic of H9C2 cells to RA of H9C2 cells with RA analogs nm or for as from the lower of of the cells to this cells changes with large and cells from the myotubes present in control cultures 2 or in or cultures cells in cultures 2 and the myotubes 2 were the phenotype of H9C2 which a FCS differentiation of H9C2 cells was characterized using skeletal and cardiac RA treatment of H9C2 cells myogenic differentiation because of the multinucleated cells were for the expression of 2 and 2 By contrast, myotubes present in control differentiated cultures 1% 1% and 1 for 2 and of culture in 1% of cells be H9C2 cells were then for the of the a well characterized of cardiac differentiation E. J. Biol. 1994; Full Text PDF PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that H9C2 cells with RA an for of that obtained with cultured cardiac cells. By contrast, H9C2 cells in myogenic differentiation conditions only a for 2 the H9C2 cells were for and cardiac markers an that is to that for rat cardiac myocytes in culture Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In contrast, and as P. S. C. Nargeot J. Biophys. J. Scholar), these cells were for myogenic differentiation such as and we have identified a in Ca2+channel α1C and α1S at a Multiplex performed with a for the of α1C and α1S specific under was conducted by using from at and 10 in control and 10 nm were obtained from of that the changes in of in the of RA experiments that the of encoding α1S present in control cultures that large myotubes was we that the of was enhanced at in By contrast, the of α1C was in cultures that of during cardiogenic differentiation of H9C2 cells. the the α1S are the of the cardiac and skeletal L-type channel expression, respectively. In our a that α1C and a α1S were used under and specifically with a and a used in experiments with skeletal muscle and heart from a The which as a be by the with the of for of α1S and α1C with and was performed using with from control and H9C2 cells. experiments were conducted and are in C In a the with was with control a in α1C expression, α1S a at the were obtained with two a that α1C by and a skeletal muscle Ca2+ channels that α1S not data demonstrated that H9C2 cells α1S and of present in rat skeletal and cardiac and that RA treatment of cells enhanced α1C expression the of α1S patterns of α1S and in were obtained using the indirect immunofluorescence technique performed with the and from a of differentiated control and H9C2 the of cardiac and skeletal markers and were The described in this were in of α1S and α1C in cells. and using were to with and and then with and and were performed on H9C2 cells differentiation nm for for and In experiments performed with channel in we that and specifically α1C and H9C2 myoblasts a with in good with electrophysiological data described The specific with was upon with the α1C By contrast, the only a of in H9C2 that there was expression, or of the α1S at the be with in myotubes that α1S was to were then performed on differentiated control and cells myotubes in control cultures for to in medium were with to that described for cultured myotubes with Full Text PDF PubMed Scopus (43) Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). myotubes a with for α1S for α1S be at the of the of the myotubes as well as in the of the In α1S was to the in although a of these cells show a This was in and from the described for myotubes because it was the in these cells. this is in good with the it is from a functional of because of the cells present in cultures that we by a Ba2+ to in control myotubes In we a with the with control H9C2 cells H9C2 cells in control large myotubes, a However, we have evidence that the of large myotubes is specific that in cells because serum of these cells H9C2 cells were in the of RA during was a in α1C expression that was in and multinucleated cells the of α1C in cells was from the of in for α1C is and be the with some of characterize the α1C and we performed experiments with the and in differentiated H9C2 cells. in show that α1C and α1S are mutually exclusive in differentiated under and culture In a control and for α1S only a of be In contrast, in cells present in cultures with 10 nm a α1S was an with a of α1C Ba2+ currents were recorded in multinucleated and cells present in H9C2 cultures with RA analogs In multinucleated properties of a cardiac L-type the was for Ba2+ currents recorded on multinucleated cells at 10 with cells In multinucleated and in was by as by the of 1 and two of cells as in C. No was in multinucleated cells in cultures multinucleated cells in control cultures or cultures with Me2SO or (1 that density for we report that the cardiac density was in multinucleated cells in cells from cultures or in myoblasts and myotubes in control conditions were obtained using nm 9-cis-RA of 10 nm of cardiac or skeletal L-type Ca2+ channels by RA be in our experiments because in L-type properties were in cells with The important of this study is that expression of cardiac (α1C) L-type voltage-dependent Ca2+ channels is during retinoic differentiation of embryonic cardiac H9C2 cells. The data provide evidence that myogenic transdifferentiation of H9C2 cells is by RA supporting the that RA establishment of the cardiac phenotype in H9C2 cells. Altogether, our of Ca2+ channel in H9C2 cells the hypothesis that α1C and α1S channel are markers of cardiogenic and myogenic respectively. their cardiac by the expression of cardiac markers such as and at the E. J. Biol. 1994; Full Text PDF PubMed Google Scholar), H9C2 cells in a of serum and a skeletal muscle phenotype. for skeletal muscle differentiation of H9C2 cells is on expression of specific such as and and (10Kimes B.W. Brandt B.L. Exp. Cell Res. 1976; 98: 367-381Crossref PubMed Scopus (461) Google Scholar). of and two specific markers of skeletal muscle has not been described in cardiac cells E.N. Development. 1994; PubMed Google Scholar). In contrast, skeletal muscle differentiation be in cardiac cells myogenic are Development. 1992; Google Scholar). differentiation of H9C2 cells is not by a expression of myogenic from a transdifferentiation for a from cardiac to skeletal treatment with RA transdifferentiation of H9C2 cells because myotubes be in these We the of multinucleated cells in we have evidence to that occurs in H9C2 and we that an mechanism is It has been that by cell occurs in cardiac myocytes to adult and multinucleated cultured Dev. Biol. PubMed Scopus Google Scholar). 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We have that H9C2 cell line is a cardiac model to RA, and our study is an important that the experimental for the maintenance of the cardiac phenotype of this cell line. we that H9C2 cells as a model for a of that are difficult to on cardiac myocytes or to cardiac An of L-type Ca2+channel expression is in with RA The in α1C expression is a consequence of the cardiac phenotype because it correlates with expression of functional cardiac L-type Ca2+ channels was in developing derived from (9Kolossov E. Fleischmann B.K. Liu Q. Bloch W. Viatchenko-Karpinski S. Manzke O. Ji G.J. Bohlen H. Addicks K. Hescheler J. J. Cell Biol. 1998; 143: 2045-2056Crossref PubMed Scopus (159) Google Scholar), that this channel is an of early We have demonstrated that an important consequence of myogenic and cardiogenic differentiation is the triggering and of the expression of functional L-type channel isotypes. H9C2 myoblasts or cells exhibit cardiac L-type Ca2+ In contrast, H9C2 cells that have transdifferentiation to a myogenic phenotype skeletal muscle L-type Ca2+ our data some of the studies, which the of cardiac and skeletal of the L-type Ca2+ channel in this cell line (11Hescheler J. Meyer R. Plant S. Krautwurst D. Rosenthal W. Schultz G. Circ. Res. 1991; 69: 1476-1486Crossref PubMed Scopus (409) Google Scholar, R. Tomaselli G.F. Marban E. J. Physiol. 1994; 478: 315-329Crossref PubMed Scopus (43) Google Scholar). and patch-clamp experiments were to the expression patterns of these channel isotypes during Altogether, our data to that the electrophysiological properties of these two channel isotypes in H9C2 cells are to in T.F. Pelzer S. Trautwein W. Pelzer D.J. Physiol. Rev. 1994; 74: 365-507Crossref PubMed Scopus (925) Google Scholar, Annu. Rev. Biochem. PubMed Scopus Google Scholar). H9C2 cells therefore be for channel such as and regulation known to cardiac or skeletal Ca2+ In addition, it be important to Ca2+ channel expression, using or the differentiation of H9C2 cells. The of a transdifferentiation in vitro an to study the of cardiac and skeletal muscle differentiation development from the of derived as for skeletal The of embryonic to adult is by changes in the expression patterns of a of fetal skeletal muscle B. Physiol. Rev. PubMed Scopus Google Scholar), are identified as skeletal muscle maturation is by changes in Ca2+ channel or expression has not been developing that from (9Kolossov E. Fleischmann B.K. Liu Q. Bloch W. Viatchenko-Karpinski S. Manzke O. Ji G.J. Bohlen H. Addicks K. Hescheler J. J. Cell Biol. 1998; 143: 2045-2056Crossref PubMed Scopus (159) Google have the to undergo a myogenic transdifferentiation isotypes of Ca2+ channels is and important to In this it is important to that cardiac myocytes in culture functional α1S that H. G. Flockerzi V. W. F. 1994; Google Scholar). skeletal muscle cells in as well as developing muscle cells in cardiac α1C N. Dev. Biol. 1993; PubMed Scopus Google Scholar, Raymond G. C. J. Cell Sci. 1998; Google Scholar). Altogether, our data provide the tissue-specific expression of L-type Ca2+ channel isotypes and that the expression of functional skeletal L-type Ca2+ channels the cardiac cell a demonstrated in a cardiac cell line It is now important to for the Ca2+ channel that are in early stages of cardiac development. study in such as in models that have been for their pathway E. F.A. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Development. 1998; PubMed Google Scholar), to Ca2+ channel expression is that occurs during cardiac might expression of channel such as Ca2+ channels Circ. Res. 1993; PubMed Scopus Google and channels E. R. F. G. M. M. G. A. PubMed Scopus Google Scholar). experiments the of a skeletal L-type Ca2+ channel in obtained from because expression of the α1S be identified at and C. P. Richard S. Nargeot J. J. Cell. 1998; Scholar). the of expression of skeletal L-type Ca2+ channels in heart is a channel has not been described to in models J. Physiol. 1998; Google Scholar). However, functional expression of Ca2+ channels that mainly as a is to be difficult to in cardiac myocytes, and and Ca2+ experiments be to this In addition, it be now important to the RA treatment of Ca2+ properties identified in cardiac such as and heart We are to K. and S. of at CA) for and and We E. of for J. M. for and M. for and for skeletal muscle We J. B. for with and A. N. J. G. S. A. and D. for and of the