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Sustained cardiomyocyte DNA synthesis in whole embryo cultures lacking the TSC2 gene product
1Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202-4800, USA.
Abstract:
Tuberous sclerosis complex (TSC) is characterized by the appearance of nonmalignant tumors that affect a wide spectrum of organs, including the heart. TSC disease-causing genes have been identified on chromosomes 9 (TSC1) and 16 (TSC2). This study examined the impact of the TSC2 gene product on cardiomyocyte proliferation and terminal differentiation. We took advantage of the observation that Eker rats carry a germ-line TSC2 mutation. Rats heterozygous for the mutation (TSC2EK/+) are predisposed to renal carcinoma, whereas animals homozygous for the mutation (TSC2EK/EK) die in utero during midgestation. Spontaneously contractile cardiomyocytes were observed after multiple passages of whole embryo cultures prepared from embryonic day 12.5 TSC2EK/EK fetuses but not from TSC2EK/+ or wild-type fetuses. The TSC2EK/EK cardiomyocytes continued to actively synthesize DNA after as many as eight passages. Cytological, ultrastructural, and molecular analyses indicated that the TSC2EK/EK cardiomyocytes retained a highly differentiated phenotype similar to that observed for normal rat cardiomyocytes during late embryonic and early neonatal life. These results suggested that the TSC2 gene product is required for normal cardiomyocyte cell-cycle withdrawal and terminal differentiation.
Insights
The TSC2 gene is crucial for heart cell development. Loss of the TSC2 gene in Eker rats prevents normal cardiomyocyte cell cycle withdrawal, leading to continued proliferation in differentiated heart cells.
Area of Science:
- Cardiovascular Biology
- Developmental Biology
- Genetics
Background:
- Tuberous sclerosis complex (TSC) involves nonmalignant tumors affecting organs like the heart.
- Gene mutations in TSC1 (chromosome 9) and TSC2 (chromosome 16) cause TSC.
- The role of the TSC2 gene product in cardiomyocyte development is not fully understood.
Purpose of the Study:
- To investigate the impact of the TSC2 gene product on cardiomyocyte proliferation and terminal differentiation.
- To utilize Eker rats, which carry a germ-line TSC2 mutation, to study TSC2's function.
Main Methods:
- Culturing whole embryos from Eker rats (TSC2EK/EK, TSC2EK/+, and wild-type) at embryonic day 12.5.
- Passaging spontaneously contractile cardiomyocytes derived from TSC2EK/EK fetal cultures.
- Conducting cytological, ultrastructural, and molecular analyses on the cultured cardiomyocytes.
Main Results:
- Spontaneously contractile cardiomyocytes were observed in TSC2EK/EK embryo cultures but not in TSC2EK/+ or wild-type.
- TSC2EK/EK cardiomyocytes continued DNA synthesis after multiple passages.
- These cardiomyocytes maintained a differentiated phenotype similar to late embryonic/early neonatal normal cardiomyocytes.
Conclusions:
- The TSC2 gene product is essential for normal cardiomyocyte cell-cycle withdrawal.
- Deficiency in TSC2 function impairs terminal differentiation of heart cells.
- This study highlights TSC2's critical role in regulating cardiomyocyte development and cell cycle exit.