David R. Grubb

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

The extreme C-terminal region of phospholipase Cβ1 determines subcellular localization and function; the “b” splice variant mediates α1-adrenergic receptor responses in cardiomyocytes

Phospholipase Cβ1 (PLCβ1) exists as two splice variants, PLCβ1a (150 kDa) and PLCβ1b (140 kDa), which differ only in their C‐terminal sequences of 64 and 31 amino acids, respectively. The 3 C‐terminal amino acid residues of PLCβ1a comprise a PDZ‐interacting domain, whereas the PLCβ1b sequence has no PDZ‐interacting domain but contains unique proline‐rich domain 5 residues from the C terminus. PLCβ1a is localized in the cytoplasm, whereas PLCβ1b targets to the sarcolemma and is enriched in caveolae. Deletion of 3 amino acids from the C terminus of PLCβ1b did not alter its sarcolemmal localization, but deletion of the entire unique 31 amino acid sequence caused cytosolic localization. A myristoylated 10 amino acid peptide from the C terminus of PLCβ1b selectively dissociated N‐terminally GFP‐tagged PLCβ1b from the sarcolemma and inhibited PLC responses to α1‐adrenergic agonists, with a half maximal effective concentration of 12 ± 1.6 µΜ (mean±SE, n=3). A similar peptide from PLCβ1a was without effect at concentrations below 100 µΜ. Thus, the extreme C‐terminal sequences of the PLCβ1 splice variants determine localization and, thus, function. In cardiomyocytes, responses initiated by α1‐adrenergic receptor activation involve only PLCβ1b, and the selective targeting of this splice variant to the sarcolemma provides a potential therapeutic target to reduce hypertrophy, apoptosis, and arrhythmias.—Grubb, D. R., Vasilevski, O., Huynh, H., and Woodcock, E. A. The extreme C‐terminal region of phospholipase Cβ1 determines subcellular localization and function; the “b” splice variant mediates α1‐adrenergic receptor responses in cardiomyocytes. FASEB J. 22, 2768–2774 (2008)

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 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.

Novel Therapeutic Targets in Heart Failure: The Phospholipase Cβ1b–Shank3 Interface

Inotropic agents are often used to improve the contractile performance of the failing myocardium, but this is often at a cost of increased myocardial ischemia and arrhythmia. Myocyte contractility depends on the release of Ca²⁺ from the sarcoplasmic reticulum, and this Ca²⁺ is subject to regulation by the phosphorylation status of phospholamban (PLN). Many currently used inotropic agents function by increasing the phosphorylation of PLN, but these also heighten the risk of ischemia. Another approach is to reduce the dephosphorylation of PLN, which can be achieved by inhibiting pathways upstream or downstream of the protein kinase Cα. Phospholipase Cβ1b is responsible for activating protein kinase Cα, and its activity is substantially heightened in failing myocardium. We propose phospholipase Cβ1b, a cardiac-specific enzyme, as a promising target for the development of a new class of inotropic agents. By reversing changes that accompany the transition to heart failure, it may be possible to provide well-tolerated improvement in pump performance.

Chronic Contractile Dysfunction without Hypertrophy Does Not Provoke a Compensatory Transcriptional Response in Mouse Hearts.

Diseased myocardium from humans and experimental animal models shows heightened expression and activity of a specific subtype of phospholipase C (PLC), the splice variant PLCβ1b. Previous studies from our group showed that increasing PLCβ1b expression in adult mouse hearts by viral transduction was sufficient to cause sustained contractile dysfunction of rapid onset, which was maintained indefinitely in the absence of other pathological changes in the myocardium. We hypothesized that impaired contractility alone would be sufficient to induce a compensatory transcriptional response. Unbiased, comprehensive mRNA-sequencing was performed on 6 biological replicates of rAAV6-treated blank, PLCβ1b and PLCβ1a (closely related but inactive splice variant) hearts 8 weeks after injection, when reduced contractility was manifest in PLCβ1b hearts without evidence of induced hypertrophy. Expression of PLCβ1b resulted in expression changes in only 9 genes at FDR<0.1 when compared with control and these genes appeared unrelated to contractility. Importantly, PLCβ1a caused similar mild expression changes to PLCβ1b, despite a complete lack of effect of this isoform on cardiac contractility. We conclude that contractile depression caused by PLCβ1b activation is largely independent of changes in the transcriptome, and thus that lowered contractility is not sufficient in itself to provoke measurable transcriptomic alterations. In addition, our data stress the importance of a stringent control group to filter out transcriptional changes unrelated to cardiac function.