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Biochemical aspects of cardiac muscle differentiation
Abstract:
Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase alpha) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3'-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase alpha are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase alpha under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3'-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3'-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3'-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases.
Insights
DNA synthesis stops in differentiating cardiac muscle because the key DNA polymerase alpha enzyme loses activity. This enzyme loss, not the cessation of DNA replication, causes terminal differentiation in rat heart cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Terminally differentiating cardiac muscle in rats exhibits decreasing DNA synthesis and DNA polymerase alpha activity.
- The relationship between DNA synthesis cessation and DNA polymerase activity loss in this process remains unclear.
Purpose of the Study:
- To investigate the causal relationship between DNA synthesis cessation and DNA polymerase alpha activity loss during cardiac muscle differentiation.
- To determine whether DNA polymerase alpha activity loss precedes or follows the halt in DNA replication.
Main Methods:
- Assessed DNA-template availability and 3'-hydroxyl termini in isolated cardiac muscle nuclei and chromatin using various DNA polymerases.
- Employed density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis.
- Utilized electron microscopy to examine nuclear morphology and chromatin structure.
Main Results:
- DNA chain elongation by exogenous polymerases continued in isolated nuclei, indicating intact replicative templates.
- Template availability and 3'-hydroxyl termini did not change during differentiation or with induced DNA synthesis inhibition.
- Adult rat cardiac muscle nuclei showed increased template availability and fragmented DNA, without altered nuclease activity or nuclear morphology.
Conclusions:
- DNA synthesis ceases in terminally differentiating cardiac muscle due to the loss of replicative DNA polymerase activity.
- The loss of DNA polymerase alpha activity is the cause, not the consequence, of DNA synthesis cessation in these cells.