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Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart
1Cellular and Molecular Biology Laboratory, Rayne Institute, St. Thomas' Hospital, London, UK.
Objective:
Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts.
Methods:
Poly A/ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42 day cardiac myocytes or from whole ventricular tissue at 2, 5-11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of gamma 32 P-ATP transferred to a specific peptide substrate.
Results:
RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6-8 day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then; followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood.
Conclusions:
Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6-8 day development period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.
Insights
The expression of 80K/MARCKS protein in cardiac myocytes changes significantly during development. This transient loss of protein may be due to phosphorylation and down-regulation linked to increased protein kinase C (PKC) activity.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Developmental Biology
Background:
- Protein kinase C (PKC) is crucial for cardiac myocyte growth and differentiation.
- The downstream molecules of PKC in the heart remain largely uncharacterized.
- 80K/MARCKS is a major substrate of PKC with a known role in cellular processes.
Purpose of the Study:
- To investigate the expression of 80K/MARCKS in developing rat cardiac myocytes and ventricles.
- To understand the relationship between 80K/MARCKS expression and PKC activity during cardiac development.
Main Methods:
- RT-PCR was used to determine mRNA expression of 80K/MARCKS.
- Immunoblotting was employed to assess 80K/MARCKS protein levels.
- PKC activity was measured by quantifying substrate phosphorylation.
Main Results:
- 80K/MARCKS mRNA and protein were detected in neonatal cardiac myocytes, but not in adult cells.
- 80K/MARCKS protein expression peaked at 5 days and transiently decreased between 6-8 days.
- PKC activity increased at 8 days, coinciding with the decrease in 80K/MARCKS protein.
Conclusions:
- 80K/MARCKS expression in cardiac myocytes is developmentally regulated.
- The transient decrease in 80K/MARCKS protein likely results from PKC-mediated phosphorylation and subsequent down-regulation.
- These findings elucidate a novel PKC-regulated pathway in cardiac development.