Introduction:Variants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2 -related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. Methods:We generated transgenic zebrafish expressing wild-type (Tg WT ) or pathogenic variant (Tg R299Q ) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. Results:Tg R299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, Tg R299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca 2+ transient durations. We observed decreased AMPK phosphorylation in the Tg R299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in Tg R299Q . Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPKγ2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPKγ2 localization to the myofilament. Na⁺/Ca²⁺ exchanger (NCX) inhibition increased Ca 2+ duration and diastolic Ca 2+ in Tg WT but not Tg R299Q hearts, indicating reduced free cytosolic Ca 2+ for NCX-mediated extrusion in Tg R299Q . These findings suggest that enhanced AMPKγ2-myosin interaction may promote myofilament Ca²⁺ retention, thereby prolonging Ca²⁺ transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPKγ2-myosin interaction in Tg R299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. Conclusions:The PRKAG2 variant altered cardiac excitability, contractility, and Ca 2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPKγ2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

BACKGROUND:Variants in PRKAG2 cause hypertrophic cardiomyopathy and conduction disturbances. Although prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many hypertrophic cardiomyopathy phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. METHODS:We generated transgenic zebrafish expressing wild-type or pathogenic variant Prkag2 under a myocardium-specific promoter, Tg(cmcl2:Prkag2WT) (TgWT) and Tg(cmcl2: Prkag2R299Q) (TgR299Q), respectively, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. RESULTS:TgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human hypertrophic cardiomyopathy phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6 days post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations, compared to TgWT and wild-type (Tübingen/AB [TuAB]). We observed decreased AMPK (AMP-activated protein kinase) phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and coimmunoprecipitation identified a physical interaction between AMPKγ2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPKγ2 localization to the myofilament. NCX (Na+/Ca2+ exchanger) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPKγ2-myosin interaction may promote myofilament Ca2+ retention, thereby prolonging Ca2+ transient duration and action potential duration in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPKγ2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. CONCLUSIONS:The PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPKγ2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

Introduction: Mutations in the PRKAG2 gene cause a complex myocardial disorder characterized by hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2 -linked hypertrophy with increased glycogen storage, many HCM phenotypes in the disorder remain unexplained by this effect alone. We aimed to uncover the molecular mechanisms by which PRKAG2 mutations induce myocyte hypertrophy and electrical changes during cardiac development. Methods: We created transgenic zebrafish expressing WT (Tg WT ) and pathological mutant (Tg R299Q ) murine Prkag2 cDNA under a myocardium-specific promoter. Using these models, we studied the impact of Prkag2 variant on myocyte electrical activity, metabolism, and cytoarchitecture across cardiogenesis and in mature hearts. Results: Tg R299Q adult fish showed hypertrophic cardiomyocytes, swollen mitochondria, and shortened sarcomere length compared to Tg WT and WT, mirroring human HCM phenotypes. The glycogen content was elevated in adult hearts but not during early cardiogenesis. Despite the absence of glycogen accumulation at 6-day post fertilization, Tg R299Q hearts showed electrical abnormalities, including a reduced conduction velocity, decreased Ca 2+ amplitude, and prolonged action potential durations in both atria and ventricles, compared to Tg WT and WT. Co-immunoprecipitation and proximity ligation assay revealed physical interactions between Prkag2 and myosin heavy chain (MYH), with the R299Q variant enhancing this interaction and altering the subcellular localization of Prkag2 from mitochondria to sarcomeres. Knockdown of the fish homolog of MYH7 ( vmhcl ) restored the early electrical disturbances caused by Tg R299Q . This abnormal MYH-Prkag2 interaction disturbed mitochondria and sarcomere organization contributing to altered myocyte cytoarchitecture observed in Tg R299Q . Conclusions: Mutant PRKAG2 altered cardiac excitability and Ca 2+ handling during embryogenesis, preceding glycogen accumulation. Enhanced binding between Prkag2 and MYHs contributed to these early changes. Our study revealed a novel link between sarcomere proteins and metabolic regulators in cardiac hypertrophy, suggesting a new therapeutic avenue for treating HCM.

AimsThe genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them.Methods and ResultsWe performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands.ConclusionsOf 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.Translational PerspectiveWhole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD.

Introduction: Mutations in the lamin A/C ( LMNA ) gene have been causally linked to atrial arrhythmias and cardiac conduction disease (CCD) in young adults. However, the mechanism by which the laminopathy leads to perturbed cardiac electrophysiology has not been fully elucidated. Hypothesis: We hypothesize that protein-truncating variants in LMNA will impair the mechano-protection force in the nuclear envelope and will lead to early-onset cardiomyocyte degeneration and CCD. Methods: In a multi-regional registry of early-onset CCD and atrial fibrillation in Japan, we performed whole-exome DNA sequencing of 23 probands. Using CRISPR/Cas9, we generated indels of candidate gene homologues in zebrafish and characterized cardiac physiology using optical mapping technology and immunohistochemistry. Results: Among all probands, our study revealed four rare nonsense variants in the nuclear protein-coding genes. We focused on a LMNA protein-truncating variant, c.339dupT (p.K114X fsX1), and created a variant in the zebrafish lmna that produced a similar truncation. Lmna -/- zebrafish larvae showed shortened atrial action potential duration (APD) (msec.) compared to wild-type controls (162 ± 19 vs 227 ± 79, p<0.05), while conduction velocities (CV) did not vary between the genotypes. In contrast, the atrioventricular canal of lmna -/- embryos exhibited prolonged APD (399 ± 51 vs 322 ± 47, p<0.05) and slower CV (mm/sec.) (0.37 ± 0.12 vs 0.57 ± 0.12, p<0.05) than controls. Immunohistochemistry demonstrated that atrial cardiomyocytes of embryonic lmna -/- zebrafish displayed significantly decreased cell numbers and smaller cell size compared to those of controls, which in turn developed into abnormal nuclear structures in adult. Conclusions: These findings suggest that lamin A is a prerequisite for proper atrial cardiomyocyte morphology in embryonic zebrafish, and is indispensable for correct cardiac electrophysiology and -conduction.

Mutations in the lamin A/C ( LMNA ) gene have been causally linked to dilated cardiomyopathy with conduction disease. However, the mechanism by which the laminopathy leads to electrophysiological disorders has not been fully elucidated. In a multi-regional registry of early-onset cardiac conduction

Objective: Phosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDPethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. Methods: To determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. Results: PSD knockdown disrupted muscle phospholipid homeostasis leading to an similar to 25% reduction in PtdEtn and an similar to 45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. Conclusions: The PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass. (C) 2019 The Authors. Published by Elsevier GmbH.

Growth factors such as thrombin and transforming growth factor (TGF)-beta facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-beta signals via canonical carboxy terminal phosphorylation of R-Smads and also non -canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-beta receptor leading to both canonical and non -canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattem phosphorylation was maximal at 15 mm (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes -XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved.

Introduction: Next-generation sequencing has generated many gene variants associated with sudden cardiac death in diseases like cardiac conduction disease (CCD). Determining which of these are caus...

Rationale: Cardiac metabolism is thought to be altered in insulin resistance and type 2 diabetes (T2D). Our understanding of the regulation of cardiac substrate metabolism and insulin sensitivity has largely been derived from ex vivo preparations which are not subject to the same metabolic regulation as in the intact heart in vivo. Studies are therefore required to examine in vivo cardiac glucose metabolism under physiologically relevant conditions.Objective: To determine the temporal pattern of the development of cardiac insulin resistance and to compare with dynamic approaches to interrogate cardiac glucose and intermediary metabolism in vivo.Methods and results: Studies were conducted to determine the evolution of cardiac insulin resistance in C57Bl/6 mice fed a high-fat diet (HFD) for between 1 and 16 weeks. Dynamic in vivo cardiac glucose metabolism was determined following oral administration of [U-C-13] glucose. Hearts were collected after 15 and 60 min and flux profiling was determined by measuring C-13 mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates. Cardiac insulin resistance, determined by euglycemic hyperinsulinemic clamp, was evident after 3 weeks of HFD. Despite the presence of insulin resistance, in vivo cardiac glucose metabolism following oral glucose administration was not compromised in HFD mice. This contrasts our recent findings in skeletal muscle, where TCA cycle activity was reduced in mice fed a HFD. Similar to our report in muscle, glucose derived pyruvate entry into the TCA cycle in the heart was almost exclusively via pyruvate dehydrogenase, with pyruvate carboxylase mediated anaplerosis being negligible after oral glucose administration.Conclusions: Under experimental conditions which closely mimic the postprandial state, the insulin resistant mouse heart retains the ability to stimulate glucose metabolism. (C) 2015 Elsevier Inc. All rights reserved.

G protein-coupled receptors (GPCR) are one of the most important targets for therapeutics due to their abundance and diversity. The G protein-coupled receptor for thrombin can transactivate protein tyrosine kinase receptors (PTKR) and we have recently established that it can also transactivate serine/threonine kinase receptors (S/TKR). A comprehensive knowledge of the signalling pathways that GPCR transactivation elicits is necessary to fully understand the implications of both GPCR activation and the impact of target drugs. Here, we demonstrate that thrombin elicits dual transactivation-dependent signalling pathways to stimulate mRNA expression of glycosaminoglycan synthesizing enzymes chondroitin 4-O-sulfotransferase 1 and chondroitin sulfate synthase 1. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of the MAP kinase Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminal serine residues and does not involve matrix metalloproteinases. This work shows that all of the thrombin mediated signalling to glycosaminoglycan synthesizing enzyme gene expression occurs via transactivation-dependent pathways and does not involve transactivation-independent signalling. These findings highlight the complexity of thrombin-mediated transactivation signalling and the broader implications of GPCR targeted therapeutics.

Rationale: Defects in muscle glucose metabolism are linked to type 2 diabetes. Mechanistic studies examining these defects rely on the use of high fat-fed rodent models and typically involve the determination of muscle glucose uptake under insulin-stimulated conditions. While insightful, they do not necessarily reflect the physiology of the postprandial state. In addition, most studies do not examine aspects of glucose metabolism beyond the uptake process. Here we present an approach to study rodent muscle glucose and intermediary metabolism under the dynamic and physiologically relevant setting of the oral glucose tolerance test (OGTT).Methods and results: In vivo muscle glucose and intermediary metabolism was investigated following oral administration of [U-C-13] glucose. Quadriceps muscles were collected 15 and 60 min after glucose administration and metabolite flux profiling was determined by measuring C-13 mass isotopomers in glycolytic and tricarboxylic acid (TCA) cycle intermediates via gas chromatography-mass spectrometry. While no dietary effects were noted in the glycolytic pathway, muscle from mice fed a high fat diet (HFD) exhibited a reduction in labelling in TCA intermediates. Interestingly, this appeared to be independent of alterations in flux through pyruvate dehydrogenase. In addition, our findings suggest that TCA cycle anaplerosis is negligible in muscle during an OGTT.Conclusions: Under the dynamic physiologically relevant conditions of the OGTT, skeletal muscle from HFD fed mice exhibits alterations in glucose metabolism at the level of the TCA cycle. (C) 2015 Elsevier Inc. All rights reserved.

Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP: phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis.

Introduction: Mutations in the cardiac myosin-binding protein C ( MYBPC3 ) gene have been implicated in over one third of hypertrophic cardiomyopathy (HCM) cases. These mutations have been reported to be associated with delayed expression of hypertrophy and a relatively good prognosis. However, we previously reported that a patients with a S593Pfs*9 mutation (a single base deletion of a thymidine residue at nucleotide 11645 in exon 18) in this gene deteriorated into the“end-stage” HCM, characterized by left ventricular systolic dysfunction, cavity dilation, and heart failure. This mutation causes a truncated MYBPC3. The precise functional and pathological consequences of this mutation are not fully characterized. The aim of this study was to recapitulate the S593Pfs*9 mutation in the zebrafish to define the pathologic mechanisms. Methods: We used the CRISPR/Cas9 system to generate a genocopy of the S593Pfs*9 with a specific guide RNA injected with Cas9 into one-cell stage embryos. Results: Gross morphological examination of the mutants showed a smaller ventricle and pericardial edema compared with controls. There were significant reductions in heart rate (125 +/- 31 vs 148 +/- 9 /min), end-diastolic volume (EDV) (0.41 +/- 0.16 vs 0.72 +/- 0.18 nl), end-systolic volume (ESV) (0.24 +/- 0.08 vs 0.36 +/- 0.13 nl), stroke volume (SV) (0.16 +/- 0.12 vs 0.36 +/- 0.09 nl) in mutants compared with control embryos at 48 hours post fertilization (hpf). In addition, the calcium transient amplitude (0.17 +/- 0.02 vs 0.23 +/- 0.02 ratio units) was also significantly reduced in the mutant hearts compared with the control hearts at 48 hpf. Conclusions: Our data demonstrate that the S593Pfs*9 mutants exhibit reduced EDV, ESV, and SV and define early abnormalities of Ca 2+ handling that may drive the development of heart failure at later stages. This model will allow the mechanistic dissection of MYBPC-associated heart failure and enable chemical screens for novel drugs.

Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions.

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, beta-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-beta receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.

Transforming growth factor-β (TGF-β) is a secreted homodimeric protein that plays an important role in regulating various cellular responses including cell proliferation and differentiation, extracellular matrix production, embryonic development and apoptosis. Disruption of the TGF-β signalling pathway is associated with diverse disease states including cancer, renal and cardiac fibrosis and atherosclerosis. At the cell surface TGF-β complex consists of two type I and two type II transmembrane receptors (TβRI and TβRII respectively) which have serine/threonine kinase activity. Upon TGF-β engagement TβRII phosphorylates TβRI which in turn phosphorylates Smad2/3 on two serine residues at their C-terminus which enables binding to Smad4 to form heteromeric Smad complexes that enter the nucleus to initiate gene transcription including for extracellular matrix proteins. TGF-β signalling is also known to activate other serine/threonine kinase signalling that results in the phosphorylation of the linker region of Smad2. The Smad linker region is defined as the domain which lies between the MH1 and MH2 domains of a Smad protein. Serine/threonine kinases that are known to phosphorylate the Smad linker region include mitogen-activated protein kinases, extracellular-signal regulated kinase, Jun N-terminal kinase and p38 kinase, the tyrosine kinase Src, phosphatidylinositol 3′-kinase, cyclin-dependent kinases, rho-associated protein kinase, calcium calmodulin-dependent kinase and glycogen synthase kinase-3. This review will cover the role of Smad linker region phosphorylation downstream of TGF-β signalling in vascular cells. Key factors including the identification of the kinases that phosphorylate individual Smad residues, the upstream agents that activate these kinases, the cellular location of the phosphorylation event and the importance of the linker region in regulation and expression of genes induced by TGF-β are covered.

OBJECTIVES:This review discusses the latest developments in G protein coupled receptor (GPCR) signalling related to the transactivation of cell surface protein kinase receptors and the therapeutic implications.KEY FINDINGS:Multiple GPCRs have been known to transactivate protein tyrosine kinase receptors for almost two decades. More recently it has been discovered that GPCRs can also transactivate protein serine/threonine kinase receptors such as that for transforming growth factor (TGF)-β. Using the model of proteoglycan synthesis and glycosaminoglycan elongation in human vascular smooth muscle cells which is a component of an in vitro model of atherosclerosis, the dual tyrosine and serine/threonine kinase receptor transactivation pathways appear to account for all of the response to the agonists, endothelin and thrombin.SUMMARY:The broadening of the paradigm of GPCR receptor transactivation explains the broad range of activities of these receptors and also the efficacy of GPCR antagonists in cardiovascular therapeutics. Deciphering the mechanisms of transactivation with the aim of identifying a common therapeutic target remains the next challenge.

The current paradigm of G protein coupled receptor signaling involves a classical pathway being the activation of phospholipase C and the generation of 1,4,5-inositol trisphosphate, signaling through β-arrestin scaffold molecules and the transactivation of tyrosine kinase growth factor receptors. Transactivation greatly expands the range of signaling pathways and responses attributable to the receptor. Recently it has been revealed that G protein coupled receptor agonists can also transactivate the serine/threonine kinase cell surface receptor for transforming growth factor-β (Alk5). This leads to the generation of carboxyl terminal phosphorylated Smad2 which is the immediate downstream product of the activated Alk5. Thus, the current paradigm of G protein coupled signaling can be expanded to include the transactivation of the serine kinase receptor Alk5. These insights expand the possibilities for outcomes of therapeutically targeting GPCRs where more substantive and prolonged actions such as the synthesis of extracellular matrix may be affected.

The canonical TGF-β signalling pathway involves Smad transcription factors through direct serine phosphorylation of the carboxy termini, nuclear translocation and regulation of transcription by receptor-regulated (R)-Smad complexes. Smads can also be phosphorylated in the linker region most prominently by the action of mitogen-activated protein (MAP) kinases, which in turn have been activated by TGF-β or a multitude of other growth factors and hormones. Linker region phosphorylation can prevent nuclear translocation of Smads and inhibit TGF-β signalling, potentially leading to oncogenesis. However, some evidence has revealed that linker region phosphorylated Smads can be translocated to the nucleus where they regulate transcription particularly of the synthesis of extracellular matrix molecules. Matrix molecules such as collagen and proteoglycans are involved in diseases such a fibrosis and atherosclerosis, respectively, and the involvement of linker region phosphorylation may represent a new therapeutic target.