rAAV6 harbouring FLAG-PLCβ1b or FLAG-PLCβ1a was delivered by IV (tail-vein) into 6-week old 57Bl/6 .... the PKCα inhibitor Ro-31-8220 (6) (6mg/g/day).
Contractile dysfunction in the mouse heart caused by phospholipase C1b mediated activation of protein kinase C. David R. Grubb, Yi Ma, Jieting Luo, Bryony Crook, Nicola Cooley, Helen Kiriazis, Paul Gregorevic, Xiao-Jun Du and Elizabeth A. Woodcock Molecular Cardiology Laboratory, BakerIDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, VIC 3004 Australia.
Introduction. Phospholipase C (PLC1) subtypes are early signaling enzymes that respond to activation of Gq-coupled receptors by hydrolysing the plasma membrane lipid, phosphatidylinositol(4,5)bisphosphate (PIP2) to inositol(1,4,5)trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG).
PIP2
Gq
PLC1b
Other receptors
PKC
Stretch
PLC1a has a C-terminal PDZ interacting motif, does not target to the plasma membrane in cardiomyocytes, and is therefore not active.
aa1142
NH2-
Mechanism The effect of PLC1b expression in vivo is reminiscent of previous findings regarding a role for PKC in contractile dysfunction(5-8).
ETA-R
1-AR
PLC1 exists as two splice variants (PLC1a and PLC1b) that differ only in the extreme C-terminal sequence.
PLC1b possesses Pro-motifs that are responsible for its targeting to the plasma membrane by association with the scaffolding protein, Shank3 (1, 2). PLC1b is the immediate down stream effector of Gq in cardiomyocytes (3).
IP3 + DAG
PH
EF
Catalytic
C2
PLC1a
Gq
1. PLC1b-induced contractile depression is reversed by PKC inhibition in vivo.
2. PLC1b increases the membrane localization of PKC in cardiomyocytes.
16 weeks post rAAV6 vector delivery, mice were treated for 5 days with the PKC inhibitor Ro-31-8220 (6) (6mg/g/day). Echocardiography was performed before (-Ro) and after (+Ro) treatment. FLAG-PLC1b
Neonatal rat ventricular myocytes (NRVM) were treated with adenoviruses expressing FLAG-PLC1b or FLAG-PLC1a, soluble (s) and membrane (m) fractions prepared. and PKC PKC and PKC content assessed by western blot. Ca2+ (2mM) was added to extracts as a positive control. B. Average of 3 experiments. * p
