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1.
Circ Res ; 127(9): 1159-1178, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32821022

RESUMO

RATIONALE: CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. OBJECTIVE: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. METHODS AND RESULTS: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. CONCLUSIONS: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Aorta , Arritmias Cardíacas/etiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Estimulação Cardíaca Artificial , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Constrição , Citosol/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica , Insuficiência Cardíaca/etiologia , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , Ativação Transcricional
2.
Basic Res Cardiol ; 116(1): 11, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33590335

RESUMO

Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Insuficiência Cardíaca/enzimologia , Histona Desacetilases/metabolismo , Miócitos Cardíacos/enzimologia , Transporte Ativo do Núcleo Celular , Agonistas Adrenérgicos beta/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Histona Desacetilases/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Coelhos , Proteínas Repressoras , Transdução de Sinais , Remodelação Ventricular
3.
J Mol Cell Cardiol ; 125: 18-28, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30321537

RESUMO

Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Imuno-Histoquímica , Fosforilação , Coelhos , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/fisiologia
4.
Circ Res ; 118(2): e19-28, 2016 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-26643875

RESUMO

RATIONALE: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE: To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS: Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.


Assuntos
Cardiomiopatias/metabolismo , Acoplamento Excitação-Contração , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Imagem Multimodal/métodos , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Compostos de Anilina , Animais , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Corantes Fluorescentes , Cinética , Masculino , Mecanotransdução Celular , Camundongos , Ratos Sprague-Dawley , Estresse Mecânico , Xantenos
5.
Circ Res ; 114(9): 1398-409, 2014 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-24643961

RESUMO

RATIONALE: Both ß-adrenergic receptor (ß-AR) and Gq-coupled receptor (GqR) agonist-driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. OBJECTIVE: To assess the spatiotemporal dynamics of PKD activation in response to ß-AR signaling alone and on coactivation with GqR-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. METHODS AND RESULTS: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer-based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that ß-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of ß-AR/cAMP/PKA signaling on GqR-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction-induced hypertrophy. CONCLUSIONS: ß-AR signaling triggers local nuclear signaling and inhibits GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for ß-adrenergic and Gq-coupled stimuli.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Proteína Quinase C/genética , Transporte Proteico , Coelhos , Ratos , Receptores Adrenérgicos beta/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção
6.
Elife ; 102021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34292155

RESUMO

Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. We investigated dispersion of Hedgehog (Hh), Wnt homolog Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc. We discovered that delivery of Hh, Wg, and Dpp to their respective targets is regulated. We found that <5% of Hh and <25% of Wg are taken up by disc cells and activate signaling. The amount of morphogen that is taken up and initiates signaling did not change when the level of morphogen expression was varied between 50 and 200% (Hh) or 50 and 350% (Wg). Similar properties were observed for Dpp. We analyzed an area of 150 µm×150 µm that includes Hh-responding cells of the disc as well as overlying tracheal cells and myoblasts that are also activated by disc-produced Hh. We found that the extent of signaling in the disc was unaffected by the presence or absence of the tracheal and myoblast cells, suggesting that the mechanism that disperses Hh specifies its destinations to particular cells, and that target cells do not take up Hh from a common pool.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/metabolismo , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Morfogênese , Asas de Animais/embriologia , Proteína Wnt1/genética
7.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33734293

RESUMO

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Mioblastos/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Asas de Animais/metabolismo
8.
Front Pharmacol ; 8: 9, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28174535

RESUMO

Protein Kinase D isoforms (PKD 1-3) are key mediators of neurohormonal, oxidative, and metabolic stress signals. PKDs impact a wide variety of signaling pathways and cellular functions including actin dynamics, vesicle trafficking, cell motility, survival, contractility, energy substrate utilization, and gene transcription. PKD activity is also increasingly linked to cancer, immune regulation, pain modulation, memory, angiogenesis, and cardiovascular disease. This increasing complexity and diversity of PKD function, highlights the importance of tight spatiotemporal control of the kinase via protein-protein interactions, post-translational modifications or targeting via scaffolding proteins. In this review, we focus on the spatiotemporal regulation and effects of PKD signaling in response to neurohormonal, oxidant and metabolic signals that have implications for myocardial disease. Precise targeting of these mechanisms will be crucial in the design of PKD-based therapeutic strategies.

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