Original article Ins(1,4,5)P 3 regulates phospholipase Cβ1 expression in cardiomyocytes Oliver Vasilevski a , David R. Grubb a , Theresa M. Filtz a , Sundy Yang a , Tiffany J. McLeod-Dryden a , Jieting Luo a , Divya Karna a , Ju Chen b , Elizabeth A. Woodcock a, ⁎ a Cellular Biochemistry Laboratory, Baker Heart Research Institute, 75 Commercial Road, Melbourne, 3004, Victoria, Australia b Department of Medicine, University of California, San Diego, La Jolla, CA 92093-4276, USA abstract article info Article history: Received 14 April 2008 Received in revised form 18 June 2008 Accepted 7 July 2008 Available online 23 July 2008 Keywords: IP 3 5-phosphatase PLCδ1-PH domain IP 3 -R(2)-/- mouse Phospholipase C Neonatal rat ventricular myocytes The functional significance of the Ca 2+ -releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3 , IP 3 ) in the heart has been controversial. Ins(1,4,5)P 3 is generated from the precursor lipid phosphatidyli- nositol(4,5)bisphosphate (PIP 2 ) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P 3 in cardiomyocytes (NRVM), we overexpressed IP 3 5-phosphatase to increase degradation. Overexpression of IP 3 5-phosphatase reduced Ins (1,4,5)P 3 responses to α 1 -adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCβ1, that served to normalise Ins(1,4,5)P 3 content. Similar increases in PLC activity and PLCβ1 expression were observed when Ins(1,4,5)P 3 was sequestered onto the PH domain of PLCδ1, a high affinity selective Ins(1,4,5) P 3 -binding motif. These findings suggested that the available level of Ins(1,4,5)P 3 selectively regulates the expression of PLCβ1. Cardiac responses to Ins(1,4,5)P 3 are mediated by type 2 IP 3 -receptors. Hearts from IP 3 - receptor (type 2) knock-out mice showed heightened PLCβ1 expression. We conclude that Ins(1,4,5)P 3 and IP 3 -receptor (type 2) regulate PLCβ1 and thereby maintain levels of Ins(1,4,5)P 3 . This implies some functional significance for Ins(1,4,5)P 3 in the heart. © 2008 Elsevier Inc. All rights reserved. 1. Introduction The regulation of Ca 2+ in cardiomyocytes is orchestrated princi- pally by depolarization of the sarcolemma leading to Ca 2+ entry and subsequent Ca 2+ release from the sarcoplasmic reticulum (SR) by a process of Ca 2+ -induced Ca 2+ release mediated by ryanodine receptors [1]. Despite this, cardiomyocytes also respond to activation of a number of receptor types by generating Ins(1,4,5)P 3 [2,3], an intracellular molecule capable of causing Ca 2+ release by binding its own receptors on appropriate Ca 2+ stores [4]. However, whether any Ins(1,4,5)P 3 generated is of functional importance in cardiomyocytes has been the subject of some debate. Currently, there is little evidence that Ins(1,4,5)P 3 -induced Ca 2+ responses contribute substantially to excitation–contraction (EC) coupling, and the factors that initiate Ins (1,4,5)P 3 responses in the heart, α 1 -adrenergic agonists, endothelin and purine nucleotides, are not major beat-to-beat regulators of contractility [5]. However, Ca 2+ deriving from Ins(1,4,5)P 3 -sensitive stores may contribute to hypertrophic growth responses by activating either calcineurin [6] or CaMKIIδ [7], thereby initiating nuclear events culminating in activation of hypertrophic gene transcription. Also, there is suggestive evidence from a number of laboratories that Ins (1,4,5)P 3 can initiate arrhythmias under some conditions [8–11]. Ins (1,4,5)P 3 presents an attractive target for anti-hypertrophic or anti- arrhythmic therapy because it appears to be of only limited importance for the normal functioning of the heart under physiolo- gical conditions. However, regulating the available Ins(1,4,5)P 3 within cells is difficult. In theory this could be achieved by manipulating either the enzymes responsible for its generation or those involved in its metabolism. Ins(1,4,5)P 3 is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP 2 ) by phospholipase C enzymes and is produced along with sn-1,2-diacylglycerol (DAG) [12]. PIP 2 is an important cardiac effector in its own right [13] and DAG is an activator of conventional protein kinase C isoforms [14] and TrpC channels [15]. Any change in Ins(1,4,5)P 3 caused by altered PLC activity will also be associated with altered DAG and PIP 2 . Interpretation of any alterations in cardiomyocyte responses, therefore, could not be easily related to changes in Ins(1,4,5)P 3 . For this reason, we attempted to manipulate Ins(1,4,5)P 3 content in cardiomyocytes by increasing its metabolism. Instead of observing altered cardiomyocyte responses, we found that Ins(1,4,5)P 3 levels were sustained by heightened PLC activity, associated with increased expression of PLCβ1. This finding implies, first, that the content of Ins(1,4,5)P 3 is important for cardiomyocyte functions, second, that Ins(1,4,5)P 3 regulates PLCβ1 expression and, third, that any attempt to manipulate the content of Ins(1,4,5)P 3 in cardiomyocytes may be problematic. Journal of Molecular and Cellular Cardiology 45 (2008) 679–684 ⁎ Corresponding author. Baker Heart Research Institute, PO Box 6492, St. Kilda Road Central,Melbourne, 8008, Victoria, Australia. Tel.: +613 85321255; fax: +613 85321100. E-mail address: liz.woodcock@baker.edu.au (E.A. Woodcock). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.07.006 Contents lists available at ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc