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 Ca 2؉ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (␣ 1S ) and cardiac (␣ 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 (␣ 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.

Cardiomyocyte differentiation: Experience and observations from 2 laboratories

“…Potential reasons for the discrepancy between our laboratories and the work published by Menard, et al (19) and Pereira, et al (20) are unknown. Two potential confounding variables must be kept in mind, namely (i) the passage number and history of the cells used in the differentiation experiments and (ii) the handling of RA.…”

“…Menard, et al (1999) stated that the H9c2 cells are easy to manipulate, and when differentiated, provide a suitable cardiac cellular model with a maintained cardiac phenotype for a range of investigations commonly challenging to perform on primary cardiomyocytes. (19) It is therefore tempting to pursue differentiation of these cells as a more relevant experimental model than the undifferentiated cardiomyoblast cell line. This paper will describe the efforts of 2 laboratories, working independently from one another, to differentiate H9c2 cells.…”

“…(8) In 1976, Kimes as daily supplementation with RA, exhibit features indicative of cardiac specific differentiation. (19,20) These include an increased expression of cardiac specific proteins such as Myosin Light chain-2v (MLC-2v) and Cardiac Troponin T (cTnT), (19,20) as well as a shift in metabolic preference from primarily glycolytic to oxidative metabolism. (20) Lopashuk, et al (2010) identified that a switch from glycolysis to oxidative phosphorylation is a fundamental characteristic of differentiated cardiac cells, since cardiomyotubes prefer fatty acids to glucose as a source of fuel.…”

“…Each laboratory independently investigated the capacity of the H9c2 cell line to differentiate into a more cardiac-specific cell line upon daily treatment with RA and/or reduced serum conditions as described by Pereira, et al and Menard, et al respectively. (19,20) Lab 1 made use of bright field microscopy to investigate morphology, Western blotting to investigate the expression of the cardiac specific marker MLC-2v, and immunocytochemistry to assess the expression of a second cardiac specific marker, cTnT. Oxidative phosphorylation analysis was carried out to establish whether any changes in oxygen consumption had occurred following differentiation.…”

“…(28) A third variable to investigate is the time allowed for differentiation. Although both Menard, et al (19) and Pereira, et al (20) cultured their cells for 5 -7 days, it is noteworthy that for the original description of the H9c2 cells by Kimes and Brandt (9) cells started showing pronounced myotube formation after 15 days of culturing and much less so after 7 days. It is therefore conceivable that an extended period of culturing, in the range of 15 -20 days, might produce a more pronounced degree of differentiation.…”