To explore the mechanisms of phospholipase Cϵ (PLCϵ) signaling in cardiomyocyte hypertrophy, PLCϵ protein was depleted by siRNA in neonatal rat ventricular myocytes (NRVMs). Endothelin-1 (ET-1), norepinephrine (NE), isoproterenol (ISO), and insulin-like growth factor-1 (IGF-1) were chosen as hypertrophic agonists. Expression of Gαq in NRVMs was also testes as a hypertrophic stimulus. We found that depletion of PLCϵ with siRNA significantly inhibited hypertrophic growth and gene expression induced by all agonists tested as well as Gαq, demonstrating that PLCϵ plays a convergent role in regulating cardiomyocyte hypertrophy. Our data show that PLC catalytic activity is required for PLCϵ-dependent regulation of hypertrophy, although PLCϵ deletion did not change global agonist-induced total inositol phosphate generation, suggesting PLC activity was required locally for PLCϵ-dependent regulation of hypertrophy. Further, PLCϵ binds to muscle-specific A kinase anchoring protein (mAKAPβ)—a nuclear membrane scaffold protein in the heart. The binding sites on the interaction surface of PLCϵ and mAKAPβ were mapped and the domains were expressed in NRVMs to disrupt endogenous binding of PLCϵ to mAKAPβ, which resulted in significantly reduced agonist or Gαq-induced hypertrophy, proving that the perinuclear localization of PLCϵ was required for its hypertrophic function.
Furthermore, we explored downstream effectors of PLCϵ in regulation of hypertrophy. We found that depletion of PLCϵ inhibited protein kinase D (PKD) activity stimulated by ET-1 or NE in NRVMs, and disruption of PLCϵ binding to mAKAPβ also reduces it, demonstrating that nuclear PKD activation required PLCϵ. Similarly, our data show that PLCϵ depletion inhibits ET-1 stimulated nuclear Ca2+ release in NRVMs, suggesting that IP3 generated by PLCϵ-dependent hydrolysis contributed to nuclear calcium signals.
To further understand the functional role of PLCϵ found in cardiomyocyte hypertrophy, inducible and tissue specific PLCϵ knockout mice were generated. Conditional deletion of PLCϵ in mouse cardiac myocytes after development protected the heart from pressure overload-induced hypertrophy, which strongly supports our mechanistic data from NRVMs.
Collectively, our studies have unveiled a novel and important mechanism of regulating cardiac hypertrophy, which suggests PLCϵ may be a promising target for pharmacological manipulation.
|Advisor:||Smrcka, Alan V.|
|Commitee:||Alexis, Jeffrey D., Blaxall, Burns C., Dirksen, Robert T., Tank, A. William, Yan, Chen|
|School:||University of Rochester|
|Department:||School of Medicine and Dentistry|
|School Location:||United States -- New York|
|Source:||DAI-B 74/07(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Pharmacology, Biochemistry|
|Keywords:||Cardiomyocytes, Convergent, Hypertrophy, Makap, Phospholipase cepsilon, Pkd|
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