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 PLCß1b. Previous studies from our group showed that increasing PLCß1b 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, PLCß1b and PLCß1a (closely related but inactive splice variant) hearts 8 weeks after injection, when reduced contractility was manifest in PLCß1b hearts without evidence of induced hypertrophy. Expression of PLCß1b resulted in expression changes in only 9 genes at FDR<0.1 when compared with control and these genes appeared unrelated to contractility. Importantly, PLCß1a caused similar mild expression changes to PLCß1b, despite a complete lack of effect of this isoform on cardiac contractility. We conclude that contractile depression caused by PLCß1b 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.

Expressing an inhibitor of PLCß1b sustains contractile function following pressure overload.

The activity of phospholipase Cß1b (PLCß1b) is selectively elevated in failing myocardium and cardiac expression of PLCß1b causes contractile dysfunction. PLCß1b can be selectively inhibited by expressing a peptide inhibitor that prevents sarcolemmal localization. The inhibitory peptide, PLCß1b-CT was expressed in heart from a mini-gene using adeno-associated virus (rAAV6-PLCß1b-CT). rAAV6-PLCß1b-CT, or blank virus, was delivered IV (4×10(9)vg/g body weight) and trans-aortic-constriction (TAC) or sham-operation was performed 8weeks later. Expression of PLCß1b-CT prevented the loss of contractile function, eliminated lung congestion and improved survival following TAC with either a 'moderate' or 'severe' pressure gradient. Hypertrophy was attenuated but not eliminated. Expression of the PLCß1b-CT peptide 2-3weeks after TAC reduced contractile dysfunction and lung congestion, without limiting hypertrophy. PLCß1b inhibition ameliorates pathological responses following acute pressure overload. The targeting of PLCß1b to the sarcolemma provides the basis for the development of a new class of inotropic agent.

Phospholipase Cß1b directly binds the SH3 domain of Shank3 for targeting and activation in cardiomyocytes.

Phospholipase Cß1b (PLCß1b) is an atypical splice variant of PLCß1 that has a C-terminal proline-rich sequence instead of the PDZ-interacting motif common to other PLCß subtypes. PLCß1b targets to the cardiomyocyte sarcolemma through an undefined association with the scaffolding protein Shank3. The C-terminal splice variant specific sequence of PLCß1b 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 PLCß1b prevented the interaction between PLCß1b and Shank3 resulting in reduced sarcolemmal localization and downstream signalling responses. We conclude that PLCß1b activation and downstream signalling require the association of a previously unidentified C-terminal proline-rich motif with the SH3 domain of Shank3. PLCß1b is the first confirmed protein ligand for the SH3 domain of Shank3.

The atypical 'b' splice variant of phospholipase Cß1 promotes cardiac contractile dysfunction.

The activity of the early signaling enzyme, phospholipase Cß1b (PLCß1b), is selectively elevated in diseased myocardium and activity increases with disease progression. We aimed to establish the contribution of heightened PLCß1b activity to cardiac pathology. PLCß1b, the alternative splice variant, PLCß1a, and a blank virus were expressed in mouse hearts using adeno-associated viral vectors (rAAV6-FLAG-PLCß1b, rAAV6-FLAG-PLCß1a, or rAAV6-blank) delivered intravenously (IV). Following viral delivery, FLAG-PLCß1b was expressed in all of the chambers of the mouse heart and was localized to the sarcolemma. Heightened PLCß1b expression caused a rapid loss of contractility, 4-6 weeks, that was fully reversed, within 5 days, by inhibition of protein kinase Cα (PKCα). PLCß1a did not localize to the sarcolemma and did not affect contractile function. Expression of PLCß1b, but not PLCß1a, caused downstream dephosphorylation of phospholamban and depletion of the Ca(2+) stores of the sarcoplasmic reticulum. We conclude that heightened PLCß1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCß1b 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.

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 Ca(2+) distinct from the Ca(2+) 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 PLCß1b that is scaffolded onto a SH3 and ankyrin repeat protein 3 (Shank3) complex at the sarcolemma. TrpC4 exists as two splice variants (TrpC4α and TrpC4ß) that differ only in an 84-residue sequence that binds to phosphatidylinositol(4,5)bisphosphate (PIP2), the substrate of PLCß1b. In neonatal rat cardiomyocytes, TrpC4α, but not TrpC4ß, coimmunoprecipitated with both PLCß1b and Shank3. Heightened PLCß1b expression caused TrpC4α, but not TrpC4ß, translocation to the sarcolemma, where it colocalized with PLCß1b. When overexpressed in cardiomyocytes, TrpC4α, but not TrpC4ß, increased cell area (893 ± 18 to 1497 ± 29 mm(2), 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 PLCß1b, or PLCß1b-coupled receptor activation, by 72 ± 8% and 39 ± 5 %, respectively. We conclude that TrpC4α is selectively involved in mechanisms downstream of PLCß1b culminating in cardiomyocyte hypertrophy, and that the hypertrophic response is dependent on the TrpC4α splice variant-specific sequence that binds to PIP2.

Regulation of autophagy in cardiomyocytes by Ins(1,4,5)P(3) and IP(3)-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.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy.

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

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 Cß1b (PLCß1b), 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 Ca(2+) signaling by facilitating crosstalk between Ca(2+) channels at the cell surface with those on intracellular Ca(2+) stores. Activation of α(1)-adrenergic receptors, expression of constitutively active Gαq (GαqQL), or PLCß1b 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 µm(2) (P<0.05); furthermore, expression of dominant-negative Homer (Homer 1a) reversed the increase in cell size caused by α(1)-adrenergic agonist or PLCß1b treatment (1503±48 to 996±28 and 1626±48 to 828±31 µm(2), respectively, P<0.05). Homer proteins were localized near the sarcolemma, associated with Shank3 and phospholipase Cß1b. We conclude that Gq-mediated hypertrophy involves activation of PLCß1b scaffolded onto a Shank3/Homer complex. Signaling downstream of Homer 1c is necessary and sufficient for Gq-initiated hypertrophy.

Phospholipase Cbeta1b 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 Cß1b (PLCß1b), because only this PLCß subtype localizes at the cardiac sarcolemma. In the current study, we investigated the proteins involved in targeting PLCß1b to the sarcolemma in neonatal rat cardiomyocytes. PLCß1b, but not PLCß1a, 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 PLCß1b also associated with Shank3 and α-fodrin, indicating that PLCß1b binds via the C-terminal sequence. Shank3 colocalized with PLCß1b 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 PLCß1b 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.

Selective activation of the "b" splice variant of phospholipase Cbeta1 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.

Gq-initiated cardiomyocyte hypertrophy is mediated by phospholipase Cbeta1b.

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 Cbeta (PLCbeta) subtypes, resulting in generation of the intracellular messengers inositol(1,4,5)tris-phosphate [Ins(1,4,5)P(3)] and sn-1,2-diacylglycerol (DAG), which regulate intracellular Ca(2+) and conventional protein kinase C subtypes, respectively. Gq can also signal independently of PLCbeta, and the involvement of either Ins(1,4,5)P(3) or DAG in cardiomyocyte hypertrophy has not been unequivocally established. Overexpression of one splice variant of PLCbeta1, specifically PLCbeta1b, 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, PLCbeta1a, had no effect. Expression of a 32-aa C-terminal PLCbeta1b peptide, which competes with PLCbeta1b for sarcolemmal association, prevented PLC activation and eliminated hypertrophic responses initiated by Gq or Gq-coupled alpha(1)-adrenergic receptors. In contrast, a PLCbeta1a C-terminal peptide altered neither PLC activity nor cellular hypertrophy. We conclude that hypertrophic responses initiated by Gq are mediated specifically by PLCbeta1b. Preventing PLCbeta1b association with the sarcolemma may provide a useful therapeutic target to limit hypertrophy.

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 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 extreme C-terminal region of phospholipase Cbeta1 determines subcellular localization and function; the "b" splice variant mediates alpha1-adrenergic receptor responses in cardiomyocytes.

Phospholipase Cbeta1 (PLCbeta1) exists as two splice variants, PLCbeta1a (150 kDa) and PLCbeta1b (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 PLCbeta1a comprise a PDZ-interacting domain, whereas the PLCbeta1b sequence has no PDZ-interacting domain but contains unique proline-rich domain 5 residues from the C terminus. PLCbeta1a is localized in the cytoplasm, whereas PLCbeta1b targets to the sarcolemma and is enriched in caveolae. Deletion of 3 amino acids from the C terminus of PLCbeta1b 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 PLCbeta1b selectively dissociated N-terminally GFP-tagged PLCbeta1b from the sarcolemma and inhibited PLC responses to alpha(1)-adrenergic agonists, with a half maximal effective concentration of 12 +/- 1.6 microM (mean+/-SE, n=3). A similar peptide from PLCbeta1a was without effect at concentrations below 100 microM. Thus, the extreme C-terminal sequences of the PLCbeta1 splice variants determine localization and, thus, function. In cardiomyocytes, responses initiated by alpha(1)-adrenergic receptor activation involve only PLCbeta1b, and the selective targeting of this splice variant to the sarcolemma provides a potential therapeutic target to reduce hypertrophy, apoptosis, and arrhythmias.