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Agonist activated PKC?II translocation and modulation of cardiac myocyte contractile function.

ABSTRACT: Elevated protein kinase C ?II (PKC?II) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKC?II activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKC?II-expressing myocytes. PHE also caused PKC?II translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKC?II-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKC?II-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKC?II.

SUBMITTER: Hwang H

PROVIDER: S-EPMC3679501 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Agonist activated PKCβII translocation and modulation of cardiac myocyte contractile function.

Hwang Hyosook H Robinson Dustin D Rogers Julie B JB Stevenson Tamara K TK Lang Sarah E SE Sadayappan Sakthivel S Day Sharlene M SM Sivaramakrishnan Sivaraj S Westfall Margaret V MV

Scientific reports 20130101

Elevated protein kinase C βII (PKCβII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCβII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCβII-expressing myocytes. PHE also caused PKC ...[more]

PMID: 23756828

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Project description:RationaleLoss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation.ObjectiveTo develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs.Methods and resultsWe developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus-transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor.ConclusionsCRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Dataset Information

Agonist activated PKC?II translocation and modulation of cardiac myocyte contractile function.

Publications

Agonist activated PKCβII translocation and modulation of cardiac myocyte contractile function.

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