Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation.

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction. Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation. Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activated the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development.

Smads, TAK1, and their common target ATF-2 play a critical role in cardiomyocyte differentiation.

We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1. Transcription factors Smads mediate transforming growth factor-beta signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to act as a common target of the Smad and the TAK1 pathways. We here examined the role of Smads and ATF-2 in cardiomyocyte differentiation of P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Cooverexpression of Smad1, a ligand-specific Smad, and Smad4, a common Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated the betaMHC promoter activity by the synergistic manner with Smad1/4 and TAK1 and promoted terminal cardiomyocyte differentiation of P19CL6noggin, whereas overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation.

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Calcineurin inhibitor attenuates the development and induces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension.

BACKGROUND: It remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. METHODS AND RESULTS: In the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg twice daily) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg twice daily but not with 0.01 mg/kg twice daily of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. CONCLUSIONS: These results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

[Calcineurin inhibitor attenuates the development and induces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension].

BACKGROUND: It remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. METHODS AND RESULTS: In the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg every second day) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg every second day but not with 0.01 mg/kg every second day of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. CONCLUSIONS: These results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

A novel myocyte-specific gene Midori promotes the differentiation of P19CL6 cells into cardiomyocytes.

Although several cardiac-specific transcription factors have been shown to play vital roles in various steps during the heart formation, the precise mechanism of the early stage of cardiogenesis has yet to be elucidated. By differential display technique, we tried to identify molecules that are expressed earlier than cardiac transcription factors such as CSX/NKX2-5 and GATA-4 and are involved in cardiomyocyte differentiation using the P19CL6 cell line, which efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide. We isolated a novel gene designated Midori. Its deduced amino acid sequence contained an ATP/GTP-binding site, Ig-like domain, and Kringle-like domain. Northern blot analysis revealed that expression of Midori was restricted to the fetal and adult heart and adult skeletal muscle in mice. In whole mount in situ hybridization, Midori was expressed in cardiac crescent and developing heart but not in somites. The MIDORI protein was localized in the nucleus and overexpression of Midori induced expression of endogenous Midori itself, suggesting that MIDORI may act as a transcriptional regulator. Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes.

Fibroblast growth factor plays a critical role in SM22alpha expression during Xenopus embryogenesis.

Although smooth muscle cells (SMCs) are critical components of the circulatory system, the regulatory mechanisms of SMC differentiation remain largely unknown. In the present study, we examined the mechanism of SMC differentiation by using Xenopus laevis SM22alpha (XSM22alpha) as an SMC-specific marker. XSM22alpha cDNA contained a 600-bp open reading frame, and the predicted amino acid sequences were highly conserved in evolution. XSM22alpha transcripts were first detected in heart anlage, head mesenchyme, and the dorsal side of the lateral plate mesoderm at the tail-bud stage, possibly representing the precursors of muscle lineage. At the tadpole stage, XSM22alpha transcripts were restricted to the vascular and visceral SMCs. XSM22alpha was strongly induced by basic fibroblast growth factor (FGF) in animal caps. Although expressions of Xenopus cardiac actin were not affected by the expression of a dominant-negative FGF receptor, its injection dramatically suppressed the XSM22alpha expression. These results suggest that XSM22alpha is a useful molecular marker for the SMC lineage in Xenopus and that FGF signaling plays an important role in the induction of XSM22alpha and in the differentiation of SMCs.

Context-dependent transcriptional cooperation mediated by cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Although the cardiac homeobox gene Csx/Nkx-2.5 is essential for normal heart development, little is known about its regulatory mechanisms. In a search for the downstream target genes of Csx/Nkx-2. 5, we found that the atrial natriuretic peptide (ANP) gene promoter was strongly transactivated by Csx/Nkx-2.5. Deletion and mutational analyses of the ANP promoter revealed that the Csx/Nkx-2.5-binding element (NKE2) located at -240 was required for high level transactivation by Csx/Nkx-2.5. We also found that Csx/Nkx-2.5 and GATA-4 displayed synergistic transcriptional activation of the ANP promoter, and in contrast to previous reports (Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and Nemer, M. (1997) EMBO J. 16, 5687-5696; Lee, Y., Shioi, T., Kasahara, H., Jobe, S. M., Wiese, R. J., Markham, B., and Izumo, S (1998) Mol. Cell. Biol. 18, 3120-3129), this synergism was dependent on binding of Csx/Nkx-2.5 to NKE2, but not on GATA-4-DNA interactions. Although GATA-4 also potentiated the Csx/Nkx-2.5-induced transactivation of the artificial promoter that contains multimerized Csx/Nkx-2.5-binding sites, Csx/Nkx-2.5 reduced the GATA-4-induced transactivation of the GATA-4-dependent promoters. These findings indicate that the cooperative transcriptional regulation mediated by Csx/Nkx-2.5 and GATA-4 is promoter context-dependent and suggest that the complex cis-trans interactions may fine-tune gene expression in cardiac myocytes.