Jieting Luo

Gq-initiated cardiomyocyte hypertrophy is mediated by phospholipase Cβ1b

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell culture models. Hypertrophic responses induced by pressure or volume overload are exacerbated by increased Gq activity and ameliorated by Gq inhibition. Gq activates phospholipase Cβ (PLCβ) subtypes, resulting in generation of the intracellular messengers inositol(1,4,5)tris‐phosphate [Ins(1,4,5)P3] and sn‐1,2‐diacylglycerol (DAG), which regulate intracellular Ca2+ and conventional protein kinase C subtypes, respectively. Gq can also signal independently of PLCβ, and the involvement of either Ins(1,4,5)P3 or DAG in cardiomyocyte hypertrophy has not been unequivocally established. Overexpression of one splice variant of PLCβ1, specifically PLCβ1b, in neonatal rat cardiomyocytes causes increased cell size, elevated protein/ DNA ratio, and heightened expression of the hypertrophy‐related marker gene, atrial natriuretic peptide. The other splice variant, PLCβ1a, had no effect. Expression of a 32‐aa C‐terminal PLCβ1b peptide, which competes with PLCβ1b for sarcolemmal association, prevented PLC activation and eliminated hypertrophic responses initiated by Gq or Gq‐coupled α1‐adrenergic receptors. In contrast, a PLCβ1a C‐terminal peptide altered neither PLC activity nor cellular hypertrophy. We conclude that hypertrophic responses initiated by Gq are mediated specifically by PLCβ1b. Preventing PLCβ1b association with the sarcolemma may provide a useful therapeutic target to limit hypertrophy.—Filtz, T. M., Grubb, D. R., McLeod‐Dryden, T. J., Luo, J., Woodcock, E. A. Gq‐initiated cardiomyocyte hypertrophy is mediated by phospholipase Cβ1b. FASEB J. 23, 3564–3570 (2009). www.fasebj.org

Selective activation of the “b” splice variant of phospholipase Cβ1 in chronically dilated human and mouse atria

Atrial fibrillation (AF) is commonly associated with chronic dilatation of the left atrium, both in human disease and animal models. The immediate signaling enzyme phospholipase C (PLC) is activated by mechanical stretch to generate the Ca2+-releasing messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol (DAG), an activator of protein kinase C subtypes. There is also evidence that heightened activity of PLC, caused by the receptor coupling protein Gq, can contribute to atrial remodelling. We examined PLC activation in right and left atrial appendage from patients with mitral valve disease (VHD) and in a mouse model of dilated cardiomyopathy caused by transgenic overexpression of the stress-activated protein kinase, mammalian sterile 20 like kinase 1 (Mst1) (Mst1-TG). PLC activation was heightened 6- to 10-fold in atria from VHD patients compared with right atrial tissue from patients undergoing coronary artery bypass surgery (CABG) and was also heightened in the dilated atria from Mst1-TG. PLC activation in human left atrial appendage and in mouse left atria correlated with left atrial size, implying a relationship between PLC activation and chronic dilatation. Dilated atria from human and mouse showed heightened expression of PLCbeta1b, but not of other PLC subtypes. PLCbeta1b, but not PLCbeta1a, caused apoptosis when overexpressed in neonatal rat cardiomyocytes, suggesting that PLCbeta1b may contribute to chamber dilatation. The activation of PLCbeta1b is a possible therapeutic target to limit atrial remodelling in VHD patients.

Phospholipase Cβ1b associates with a Shank3 complex at the cardiac sarcolemma

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell models. Our previous studies have shown that responses to activated Gq in cardiomyocytes are mediated exclusively by phospholipase Cβ1b (PLCβ1b), because only this PLCβ subtype localizes at the cardiac sarcolemma. In the current study, we investigated the proteins involved in targeting PLCβ 1b to the sarcolemma in neonatal rat cardiomyocytes. PLCβ 1b, but not PLCβ1a, coimmunoprecipitated with the high‐MW scaffolding protein SH3 and ankyrin repeat protein 3 (Shank3), as well as the known Shank3‐interacting protein α‐fodrin. The 32‐aa splice‐variant‐specific C‐terminal tail of PLCβ 1b also associated with Shank3 and α‐fodrin, indicating that PLCβ 1b binds via the C‐terminal sequence. Shank3 colocalized with PLCβ 1b at the sarcolemma, and both proteins were enriched in the light membrane fractions. Knockdown of Shank3 using siRNA reduced PLC activation and downstream hypertrophic responses, demonstrating the importance of sarcolemmal localization for PLC signaling. These data indicate that PLCβ 1b associates with a Shank3 complex at the cardiac sarcolemma via its splice‐variant‐specific C‐terminal tail. Sarcolemmmal localization is central to PLC activation and subsequent downstream signaling following Gq‐coupled receptor activation.—Grubb, D. R., Iliades, P., Cooley, N., Yu, Y. L., Luo, J., Filtz, T. M., Woodcock, E. A. Phospholipase Cβ1b associates with a Shank3 complex at the cardiac sarcolemma. FASEB J. 25, 1040–1047 (2011). www.fasebj.org

Regulation of autophagy in cardiomyocytes by Ins(1,4,5)P3 and IP3-receptors

Autophagy is a process that removes damaged proteins and organelles and is of particular importance in terminally differentiated cells such as cardiomyocytes, where it has primarily a protective role. We investigated the involvement of inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) and its receptors in autophagic responses in neonatal rat ventricular myocytes (NRVM). Treatment with the IP(3)-receptor (IP(3)-R) antagonist 2-aminoethoxydiphenyl borate (2-APB) at 5 or 20 μmol/L resulted in an increase in autophagosome content, defined as puncta labeled by antibody to microtubule associated light chain 3 (LC3). 2-APB also increased autophagic flux, indicated by heightened LC3II accumulation, which was further enhanced by bafilomycin (10nmol/L). Expression of Ins(1,4,5)P(3) 5-phosphatase (IP(3)-5-Pase) to deplete Ins(1,4,5)P(3) also increased LC3-labeled puncta and LC3II content, suggesting that Ins(1,4,5)P(3) inhibits autophagy. The IP(3)-R can act as an inhibitory scaffold sequestering the autophagic effector, beclin-1 to its ligand binding domain (LBD). Expression of GFP-IP(3)-R-LBD inhibited autophagic signaling and furthermore, beclin-1 co-immunoprecipitated with the IP(3)-R-LBD. A mutant GFP-IP(3)-R-LBD with reduced ability to bind Ins(1,4,5)P(3) bound beclin-1 and inhibited autophagy similarly to the wild type sequence. These data provide evidence that Ins(1,4,5)P(3) and IP(3)-R act as inhibitors of autophagic responses in cardiomyocytes. By suppressing autophagy, IP(3)-R may contribute to cardiac pathology.

Scaffolding protein Homer 1c mediates hypertrophic responses downstream of Gq in cardiomyocytes

Activation of the heterotrimeric G protein, Gq, causes cardiomyocyte hypertrophy in vivo and in cell models. Responses to activated Gq in cardiomyocytes are mediated exclusively by phospholipase Cβ1b (PLCβ1b), because it localizes at the sarcolemma by binding to Shank3, a high‐molecular‐weight (MW) scaffolding protein. Shank3 can bind to the Homer family of low‐MW scaffolding proteins that fine tune Ca2+ signaling by facilitating crosstalk between Ca2+ channels at the cell surface with those on intracellular Ca2+ stores. Activation of α1‐adrenergic receptors, expression of constitutively active Gαq (GαqQL), or PLCβ1b initiated cardiomyocyte hypertrophy and increased Homer 1c mRNA expression, by 1.6 ± 0.18‐, 1.9 ± 0.17‐, and 1.5 ± 0.07‐fold, respectively (means ± SE, 6 independent experiments, P<0.05). Expression of Homer 1c induced an increase in cardiomyocyte area from 853 ± 27 to 1146 ± 31 μm2 (P<0.05); furthermore, expression of dominant‐negative Homer (Homer 1a) reversed the increase in cell size caused by α1‐adrenergic agonist or PLCβ1b treatment (1503±48 to 996±28 and 1626±48 to 828±31 (μm2, respectively, P<0.05). Homer proteins were localized near the sarcolemma, associated with Shank3 and phospholipase Cβ1b. We conclude that Gq‐mediated hypertrophy involves activation of PLCβ1b scaffolded onto a Shank3/Homer complex. Signaling downstream of Homer 1c is necessary and sufficient for Gq‐initiated hypertrophy.—Grubb, D. R., Luo, J., Yu, Y. L., Woodcock, E. A. Scaffolding protein Homer 1c mediates hypertrophic responses downstream of Gq in cardiomyocytes. FASEB J. 26, 596–603 (2012). www.fasebj.org

Ins(1,4,5)P3 regulates phospholipase Cβ1 expression in cardiomyocytes

The functional significance of the Ca2+-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 phosphatidylinositol(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 alpha(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 PLCbeta1, that served to normalise Ins(1,4,5)P(3) content. Similar increases in PLC activity and PLCbeta1 expression were observed when Ins(1,4,5)P(3) was sequestered onto the PH domain of PLCdelta1, 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 PLCbeta1. 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 PLCbeta1 expression. We conclude that Ins(1,4,5)P(3) and IP(3)-receptor (type 2) regulate PLCbeta1 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.

The phosphatidylinositol(4,5)bisphosphate-binding sequence of transient receptor potential channel canonical 4α is critical for its contribution to cardiomyocyte hypertrophy

Cardiomyocyte hypertrophy requires a source of Ca2+ distinct from the Ca2+ that regulates contraction. The canonical transient receptor potential channel (TrpC) family, a family of cation channels regulated by activation of phospholipase C (PLC), has been implicated in this response. Cardiomyocyte hypertrophy downstream of Gq-coupled receptors is mediated specifically by PLCβ1b that is scaffolded onto a SH3 and ankyrin repeat protein 3 (Shank3) complex at the sarcolemma. TrpC4 exists as two splice variants (TrpC4α and TrpC4β) that differ only in an 84-residue sequence that binds to phosphatidylinositol(4,5)bisphosphate (PIP2), the substrate of PLCβ1b. In neonatal rat cardiomyocytes, TrpC4α, but not TrpC4β, coimmunoprecipitated with both PLCβ1b and Shank3. Heightened PLCβ1b expression caused TrpC4α, but not TrpC4β, translocation to the sarcolemma, where it colocalized with PLCβ1b. When overexpressed in cardiomyocytes, TrpC4α, but not TrpC4β, increased cell area (893 ± 18 to 1497 ± 29 mm2, P < 0.01) and marker gene expression (atrial natriuretic peptide increased by 409 ± 32%, and modulatory calcineurin inhibitory protein 1 by 315 ± 28%, P < 0.01). Dominant-negative TrpC4 reduced hypertrophy initiated by PLCβ1b, or PLCβ1b-coupled receptor activation, by 72 ± 8% and 39 ± 5 %, respectively. We conclude that TrpC4α is selectively involved in mechanisms downstream of PLCβ1b culminating in cardiomyocyte hypertrophy, and that the hypertrophic response is dependent on the TrpC4α splice variant-specific sequence that binds to PIP2.

The atypical 'b' splice variant of phospholipase Cβ1 promotes cardiac contractile dysfunction

The activity of the early signaling enzyme, phospholipase Cβ1b (PLCβ1b), is selectively elevated in diseased myocardium and activity increases with disease progression. We aimed to establish the contribution of heightened PLCβ1b activity to cardiac pathology. PLCβ1b, the alternative splice variant, PLCβ1a, and a blank virus were expressed in mouse hearts using adeno-associated viral vectors (rAAV6-FLAG-PLCβ1b, rAAV6-FLAG-PLCβ1a, or rAAV6-blank) delivered intravenously (IV). Following viral delivery, FLAG-PLCβ1b was expressed in all of the chambers of the mouse heart and was localized to the sarcolemma. Heightened PLCβ1b expression caused a rapid loss of contractility, 4-6 weeks, that was fully reversed, within 5 days, by inhibition of protein kinase Cα (PKCα). PLCβ1a did not localize to the sarcolemma and did not affect contractile function. Expression of PLCβ1b, but not PLCβ1a, caused downstream dephosphorylation of phospholamban and depletion of the Ca(2+) stores of the sarcoplasmic reticulum. We conclude that heightened PLCβ1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCβ1b is a cardiac-specific signaling system, and thus provides a potential therapeutic target for the development of well-tolerated inotropic agents for use in failing myocardium.

Phospholipase Cβ1b directly binds the SH3 domain of Shank3 for targeting and activation in cardiomyocytes

Phospholipase Cβ1b (PLCβ1b) is an atypical splice variant of PLCβ1 that has a C-terminal proline-rich sequence instead of the PDZ-interacting motif common to other PLCβ subtypes. PLCβ1b targets to the cardiomyocyte sarcolemma through an undefined association with the scaffolding protein Shank3. The C-terminal splice variant specific sequence of PLCβ1b bound to deletion mutants of Shank3 that included the SH3 domain, but not to constructs lacking this domain. Mutating proline residues in the extreme C-terminal region of PLCβ1b prevented the interaction between PLCβ1b and Shank3 resulting in reduced sarcolemmal localization and downstream signalling responses. We conclude that PLCβ1b activation and downstream signalling require the association of a previously unidentified C-terminal proline-rich motif with the SH3 domain of Shank3. PLCβ1b is the first confirmed protein ligand for the SH3 domain of Shank3.