RESUMO
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.
Assuntos
Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Masculino , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Sarcolema/metabolismo , Canais de Cátion TRPC/química , Canais de Cátion TRPC/genéticaRESUMO
Light pruning and deficit irrigation regimes are practices which are widely used in high yielding commercial vineyards in the warm climate regions of Australia. Little information is available on their impacts on carbohydrate dynamics in vegetative organs within and between seasons, and on the resulting plant capacity to maintain productivity and ripen fruits. This study was conducted to address this gap in knowledge over five vintages on Vitis vinifera L. cv. Cabernet Franc, Shiraz, and Cabernet Sauvignon in the Sunraysia region of Victoria, Australia. Lighter pruning did not change the total carbohydrates concentration and composition in wood and roots within seasons in Cabernet Franc and Shiraz. However, the total carbohydrate pool (starch and soluble sugars) at the end of dormancy increased under lighter pruning, due to higher vine size, associated with retention and growth of old-wood (trunk and cordons). Water deficit negatively impacted trunk and leaf starch concentrations, over the day and within seasons in Cabernet Sauvignon. Soluble sugars concentrations in these tissues tended to be higher under limited water supply, possibly due to higher sugar mobilization as photosynthesis decreased. Trunk carbohydrate concentrations markedly varied within and between seasons, highlighting the importance of interactive factors such as crop load and climate on carbon status. The period between fruit-set and véraison was shown to be critical for its impact on the balance between carbon accretion and depletion, especially under water deficit. The lower leaf and trunk starch concentration under water deficit resulted in a decrease of yield components at harvest, while similar yields were reached for all pruning systems. The sugar allocated to berries at harvest remained remarkably stable for all practices and seasons, irrespective of vine yield and carbohydrate status in vegetative organs in Shiraz and Cabernet Sauvignon.
RESUMO
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.
Assuntos
Autofagia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Sítios de Ligação , Compostos de Boro/farmacologia , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Ventrículos do Coração/citologia , Proteínas de Choque Térmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fagossomos/metabolismo , Fosfatidiletanolaminas/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Ratos , Ratos Sprague-Dawley , Proteína Sequestossoma-1 , Transdução de SinaisRESUMO
BACKGROUND: We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20-like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. METHODS AND RESULTS: To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)-/-) had been deleted (DCM-2Tg×IP3-R(2)-/-) and transverse aortic constriction was performed on IP3-R(2)-/- mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)-/- were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. CONCLUSIONS: We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miocárdio/metabolismo , Animais , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Eletrocardiografia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genótipo , Bloqueio Cardíaco/metabolismo , Bloqueio Cardíaco/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/patologia , Fenótipo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Tempo , Função Ventricular Esquerda , Pressão Ventricular , Remodelação VentricularRESUMO
Chronic atrial fibrillation (AF) is a complication associated with the dilated atria of patients with valvular heart disease and contributes to worsened pathology. We examined microRNA (miRNA) expression profiles in right and left atrial appendage tissue from valvular heart disease (VHD) patients. Right atrial (RA) appendage from patients undergoing coronary artery bypass grafting and left atrial (LA) appendage from healthy hearts, not used for transplant, were used as controls. There was no detectable effect of chronic AF on miRNA expression in LA tissue, but miRNA expression in RA was strongly influenced by AF, with 47 miRNAs (15 higher, 32 lower) showing differential expression between the AF and control sinus rhythm groups. VHD induced different changes in miRNA expression in LA compared with RA. Fifty-three (12 higher, 41 lower) miRNAs were altered by VHD in LA, compared with 5 (4 higher, 1 lower) in RA tissue. miRNA profiles also differed between VHD-LA and VHD-RA (13 higher, 26 lower). We conclude that VHD and AF influence miRNA expression patterns in LA and RA, but these are affected differently by disease progression and by the development of AF. These findings provide new insights into the progression of VHD.
Assuntos
Apêndice Atrial/metabolismo , Fibrilação Atrial/etiologia , Fibrilação Atrial/metabolismo , Regulação da Expressão Gênica/fisiologia , Doenças das Valvas Cardíacas/complicações , MicroRNAs/metabolismo , Idoso , Análise de Variância , Feminino , Perfilação da Expressão Gênica , Doenças das Valvas Cardíacas/fisiopatologia , Humanos , Modelos Lineares , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VitóriaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento Alternativo/fisiologia , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Fosfolipase C beta/metabolismo , Sarcolema/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cardiomegalia/metabolismo , Cardiotônicos/farmacologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Microdomínios da Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas do Tecido Nervoso , Fenilefrina/farmacologia , Fosfolipase C beta/química , Fosfolipase C beta/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Domínios de Homologia de src/fisiologiaRESUMO
BACKGROUND: The generation of large amounts of microarray data presents challenges for data collection, annotation, exchange and analysis. Although there are now widely accepted formats, minimum standards for data content and ontologies for microarray data, only a few groups are using them together to build and populate large-scale databases. Structured environments for data management are crucial for making full use of these data. DESCRIPTION: The MiMiR database provides a comprehensive infrastructure for microarray data annotation, storage and exchange and is based on the MAGE format. MiMiR is MIAME-supportive, customised for use with data generated on the Affymetrix platform and includes a tool for data annotation using ontologies. Detailed information on the experiment, methods, reagents and signal intensity data can be captured in a systematic format. Reports screens permit the user to query the database, to view annotation on individual experiments and provide summary statistics. MiMiR has tools for automatic upload of the data from the microarray scanner and export to databases using MAGE-ML. CONCLUSION: MiMiR facilitates microarray data management, annotation and exchange, in line with international guidelines. The database is valuable for underpinning research activities and promotes a systematic approach to data handling. Copies of MiMiR are freely available to academic groups under licence.