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Published on: November 3, 2011
Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes
Koshiro Monzen1, Weidong Zhu, Hiroki Kasai
1Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan.
Abstract:
A homeobox-containing transcription factor Csx/Nkx2-5 is an important regulator of cardiac development. Many different human CSX/NKX2-5 mutations have been reported to cause congenital heart disease. We here examined the effects of three representative CSX/NKX2-5 mutations on cardiomyocyte differentiation and death with the use of the P19CL6 cardiomyogenic cell lines. Stable overexpression of wild-type CSX/NKX2-5 enhanced expression of cardiac-specific genes such as MEF2C and MLC2v, the promoter activity of the atrial natriuretic peptide gene, and the terminal differentiation of P19CL6 into cardiomyocytes, while all CSX/NKX2-5 mutants attenuated them by different degrees. When exposed to H(2)O(2) or cultured without change of the medium, many differentiated P19CL6 cells overexpressing the mutants, especially the mutant which lacks the carboxyl terminal region just after the homeodomain, were dead, while most of the cells overexpressing wild-type CSX/NKX2-5 survived. Overexpression of the carboxyl terminus-deleted mutant down-regulated expression of an anti-apoptotic protein Bcl-x(L) and up-regulated that of a pro-apoptotic protein CAS, while in the cells overexpressing wild-type CSX/NKX2-5, expression of a pro-apoptotic protein RIP was reduced. Furthermore, overexpression of wild-type CSX/NKX2-5 decreased the number of H(2)O(2)-induced TUNEL-positive cultured cardiomyocytes of neonatal rats, whereas overexpression of the mutants enhanced it. These results suggest that Csx/Nkx2-5 not only regulates expression of cardiac-specific genes but protects cardiomyocytes from stresses and that cell death may be another cause for the cardiac defects induced by human CSX/NKX2-5 mutations.
Insights
Mutations in the cardiac transcription factor Csx/Nkx2-5 impair cardiomyocyte differentiation and survival. This suggests that cell death contributes to congenital heart disease caused by CSX/NKX2-5 mutations.
Area of Science:
- Cardiovascular Biology
- Molecular Genetics
- Developmental Biology
Background:
- The homeobox-containing transcription factor Csx/Nkx2-5 is crucial for cardiac development.
- Mutations in human CSX/NKX2-5 are linked to congenital heart disease.
Purpose of the Study:
- To investigate the impact of representative CSX/NKX2-5 mutations on cardiomyocyte differentiation and death.
- To elucidate the role of Csx/Nkx2-5 in protecting cardiomyocytes from stress.
Main Methods:
- Utilized P19CL6 cardiomyogenic cell lines for stable overexpression of wild-type and mutant CSX/NKX2-5.
- Assessed gene expression (MEF2C, MLC2v, Bcl-x(L), CAS, RIP), promoter activity, and cell viability under oxidative stress (H2O2).
- Evaluated TUNEL-positive cardiomyocytes in neonatal rat cultures.
Main Results:
- Wild-type CSX/NKX2-5 enhanced cardiac-specific gene expression and cardiomyocyte differentiation.
- CSX/NKX2-5 mutants attenuated differentiation and significantly increased cell death under stress, particularly the carboxyl terminus-deleted mutant.
- Mutant overexpression altered apoptotic protein expression (down-regulated Bcl-x(L), up-regulated CAS), while wild-type reduced RIP expression.
- In vivo, CSX/NKX2-5 overexpression protected neonatal rat cardiomyocytes from H2O2-induced death, whereas mutants exacerbated it.
Conclusions:
- Csx/Nkx2-5 regulates cardiac-specific gene expression and promotes cardiomyocyte survival.
- Cell death, in addition to differentiation defects, is a significant contributor to cardiac anomalies associated with CSX/NKX2-5 mutations.
- These findings highlight a dual role for Csx/Nkx2-5 in cardiac development and protection.
