Comparison of mRNA expression of transcriptional factors and intercalated disk constituent proteins between in vivo and cultured cardiomyocytes

Abstract

The weak contractile force exerted by engineered cardiac muscle is a big problem in cardiac muscle tissue engineering, even though the field has made great progress over the past decade. We believe that one major reason for the weak contractile force is that the expression of genes regulating cardiomyocyte differentiation and cardiac tissue syncytium may be different for in vivo and cultured cells. In the present study, we investigated the difference of mRNA expression under in vivo and culture conditions in order to seek a target for further gene transfer treatment in the process of cardiac tissue construction. To this end, mRNA expression of four major transcriptional factors (SRF, p300, Nkx2.5, and myocardin) and two intercalated disk constituent proteins (N-cadherin and connexin43) in rat cardiomyocytes was measured by means of ratiometric reverse-transcription polymerase chain reaction. Cardiomyocytes were harvested from the hearts of 18-day (about 3 days before birth) Wistar-rat embryos (embryonic cells), 12-day neonatal rat hearts (neonatal cells), or 14-day successive dish culture of the embryonic cells harvested from 18-day embryos (cultured cells). The results indicated that, except for SRF, the mRNAs had a lower expression tendency in cultured cells than in embryonic and in neonatal cells; in particular, the mRNA expression of myocardin, N-cadherin, and connexin43 of cultured cardiomyocytes was significantly lower than that of neonatal cells. Therefore, myocardin is a candidate for forced gene up-expression during the construction of engineered cardiac tissue; in addition, a plausible reason for the weak contractile force of engineered cardiac tissue is the weak constitution of intercalated disk, because it was elucidated that mRNA expression of proteins related to intercalated disk were lower in culture.

Controllable Expansion of Primary Cardiomyocytes by Reversible Immortalization

“…The TAg clone8 cells exhibited normal patterns of Cx-43 expression in cell junctions, while Bmi-1/hTERT clone4 cells did not exhibit these cardiac specific patterns. Both clones had low expression of troponin I, a regulatory protein for cardiac muscle contraction, low expression of Nkx2.5, transcription factor related to cardiac differentiation, and high expression of vimentin, which was consistent with the de-differentiation of cells during transformation (Nakamura et al, 2008; LaFramboise et al, 2007). Both clones maintained Cx-43 and α-actin expression, however only the TAg clone8 exhibited localized Cx-43 expression in cell-cell junctions.…”

Effects of docosahexaenoic acid or arachidonic acid supplementation on gene expression and contractile force of rat cardiomyocytes in primary culture

“…In particular, the effect of AA supplementation on the cardiomyocyte connection was manifest. It has been reported by our previous study that the expressions of Cx43 and Cdh2 in cultured cardiomyocytes were significantly lower than that in fetal and neonatal myocardium (Nakamura et al, 2008). In this paper, we firstly reported that DHA or AA could prevent the decline and further promote the expressions of the two vital adhesion molecules for cardiac function.…”

“…In view of the above analysis and our previous research (Nakamura et al, 2008), which investigated the mRNA expression of SRF, p300, Nkx2.5, myocardin, Cdh2, and Cx43 in fetal cardiomyocytes under conventional monolayer culture and found the tendency of cardiac de-differentiation in terms of the attenuation of the mRNA expressions; so that the role of the PUFA supplementation may be at first suggested as preventing the cardiomyocytes under culture from the de-differentiation.…”

“…It is required for the terminal differentiation of cardiomyocytes to establish and maintain a ventricular gene expression program and it is used as cardiac differentiation marker in numerous studies (Bruneau, 2002). Although Srf is not a myocardial-specific gene, it is known as a cofactor gene expressed in myocardium to regulate the expression of α-actin, α-heavy chain, and β-myosin heavy chain and used to assess cardiac differentiation (Parlakian et al, 2004;Psichari et al, 2002;Nakamura et al, 2008). P300 is a cofactor for myocardial transcription factors (Backs and Olson, 2006;Shen et al, 2016).…”