Abstract
The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca(2+) channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (alpha(1S)) and cardiac (alpha(1C)) 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 Ca(2+) 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 Ca(2+) channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (alpha(1C)) L-type Ca(2+) channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca(2+) channels in mature cardiac cells.
Highlights
It is accepted that adult cardiac myocytes exhibit tissuespecific expression of L-type voltage-dependent calcium channels (VDCCs),1 i.e. the class C (␣1C) channel subtype [1]
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 [11], concomitant expression of skeletal L-type VDCCs occurs in H9C2 cells [12]
The important finding of this study is that expression of cardiac (␣1C) L-type voltage-dependent Ca2ϩ channels is upregulated during retinoic acid-induced differentiation of embryonic cardiac H9C2 cells
Summary
It is accepted that adult cardiac myocytes exhibit tissuespecific expression of L-type voltage-dependent calcium channels (VDCCs),1 i.e. the class C (␣1C) channel subtype [1]. Mab427 (1/300) and pA1C (1/500) antibodies reacted with a 170-kDa protein (Fig. 4A) and a 195kDa protein (Fig. 4B), respectively, when used in Western blotting experiments with skeletal muscle and heart membranes purified from a 2-month-old rat.
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