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Published on: September 17, 2015
Phosphorylation of contractile proteins in response to alpha- and beta-adrenergic stimulation in neonatal
Robert S Decker1, Amy K Rines, Sakie Nakamura
1Feinberg Cardiovascular Research Institute, Northwestern University Medical Center, Chicago, Ill 60611, USA.
Abstract:
alpha- and beta-Adrenergic receptor agonists induce an inotropic response in the adult heart by promoting the phosphorylation of several regulatory proteins, including myosin-binding protein C (MyBP-C), cardiac troponin I (cTnI), and phospholamban (PLB). However, the adrenergic-induced phosphorylation of these proteins has not been characterized in the developing heart. Accordingly, we evaluated MyBP-C, cTnI, and PLB phosphorylation in cultured neonatal rat cardiomyocytes (NRCMs) after alpha- and beta-receptor activation with phenylephrine and isoproterenol. alpha-Receptor stimulation increased, whereas beta-receptor activation reduced MyBP-C phosphorylation. Isoelectric-focusing experiments indicated that the amount of monophosphorylated MyBP-C was sensitive to alpha-adrenergic activation, but diphosphorylated and triphosphorylated MyBP-C levels were largely unaffected. The phosphorylation of cTnI and PLB was consistent with the mechanism observed in adult hearts: alpha- and beta-Receptor stimulation phosphorylated both proteins. For cTnI, the greatest difference associated with beta-receptor activation was observed in the diphosphorylated state, whereas alpha-receptor activation was associated with a marked increase in the tetraphosphorylated protein and absence of the unphosphorylated state. Despite these apparent changes in cTnI and PLB phosphorylation, beta-receptor activation failed to alter calcium transients in NRCMs. Collectively, these findings suggest that, unlike cTnI and PLB, MyBP-C and inotropy are not coupled to beta-adrenergic stimulation in NRCMs. Therefore, cTnI and PLB probably play a more central role in modulating contractile function in NRCMs in response to catecholamines than does MyBP-C, and MyBP-C may have a structural role in stabilizing thick filament assembly rather than influencing cross-bridge formation in developing hearts.
Insights
Adrenergic receptor activation affects protein phosphorylation differently in developing hearts. While cardiac troponin I and phospholamban phosphorylation changed, myosin-binding protein C did not correlate with beta-adrenergic inotropy in neonatal rat cardiomyocytes.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Developmental Biology
Background:
- Adrenergic receptor agonists stimulate adult heart inotropy via regulatory protein phosphorylation.
- Phosphorylation of myosin-binding protein C (MyBP-C), cardiac troponin I (cTnI), and phospholamban (PLB) is key in adult hearts.
- Adrenergic-induced phosphorylation patterns in developing hearts remain largely uncharacterized.
Purpose of the Study:
- To investigate alpha- and beta-adrenergic receptor-mediated phosphorylation of MyBP-C, cTnI, and PLB in neonatal rat cardiomyocytes (NRCMs).
- To determine the relationship between adrenergic signaling, protein phosphorylation, and contractile function in the developing heart.
Main Methods:
- Primary cultures of neonatal rat cardiomyocytes (NRCMs) were treated with alpha-adrenergic agonist phenylephrine and beta-adrenergic agonist isoproterenol.
- Phosphorylation levels of MyBP-C, cTnI, and PLB were analyzed using isoelectric focusing.
- Calcium transients were measured to assess functional responses to beta-adrenergic stimulation.
Main Results:
- Alpha-adrenergic stimulation increased MyBP-C phosphorylation, primarily affecting the monophosphorylated state.
- Beta-adrenergic stimulation reduced MyBP-C phosphorylation but did not alter calcium transients.
- Both alpha- and beta-adrenergic stimulation increased cTnI and PLB phosphorylation, consistent with adult heart mechanisms, but beta-adrenergic stimulation failed to alter calcium transients.
Conclusions:
- MyBP-C phosphorylation is not directly coupled to beta-adrenergic inotropy in NRCMs, suggesting a potential structural role in developing hearts.
- cTnI and PLB phosphorylation are more likely to play a central role in modulating contractile function in response to catecholamines in neonatal rat cardiomyocytes.
- These findings highlight developmental differences in adrenergic signaling pathways regulating cardiac contractility.
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