Posttranslational modifications of mitochondrial fission and fusion proteins in cardiac physiology and pathophysiology.

Mitochondrial fragmentation frequently occurs in chronic pathological conditions as seen in various human diseases. In fact, abnormal mitochondrial morphology and mitochondrial dysfunction are hallmarks of heart failure (HF) in both human patients and HF animal models. A link between mitochondrial fragmentation and cardiac pathologies has been widely proposed, but the physiological relevance of mitochondrial fission and fusion in the heart is still unclear. Recent studies have increasingly shown that posttranslational modifications (PTMs) of fission and fusion proteins are capable of directly modulating the stability, localization, and/or activity of these proteins. These PTMs include phosphorylation, acetylation, ubiquitination, conjugation of small ubiquitin-like modifier proteins, O-linked-N-acetyl-glucosamine glycosylation, and proteolysis. Thus, understanding the PTMs of fission and fusion proteins may allow us to understand the complexities that determine the balance of mitochondrial fission and fusion as well as mitochondrial function in various cell types and organs including cardiomyocytes and the heart. In this review, we summarize present knowledge regarding the function and regulation of mitochondrial fission and fusion in cardiomyocytes, specifically focusing on the PTMs of each mitochondrial fission/fusion protein. We also discuss the molecular mechanisms underlying abnormal mitochondrial morphology in HF and their contributions to the development of cardiac diseases, highlighting the crucial roles of PTMs of mitochondrial fission and fusion proteins. Finally, we discuss the future potential of manipulating PTMs of fission and fusion proteins as a therapeutic strategy for preventing and/or treating HF.

Role of mitochondrial Ca2+ homeostasis in cardiac muscles.

Recent discoveries of the molecular identity of mitochondrial Ca2+ influx/efflux mechanisms have placed mitochondrial Ca2+ transport at center stage in views of cellular regulation in various cell-types/tissues. Indeed, mitochondria in cardiac muscles also possess the molecular components for efficient uptake and extraction of Ca2+. Over the last several years, multiple groups have taken advantage of newly available molecular information about these proteins and applied genetic tools to delineate the precise mechanisms for mitochondrial Ca2+ handling in cardiomyocytes and its contribution to excitation-contraction/metabolism coupling in the heart. Though mitochondrial Ca2+ has been proposed as one of the most crucial secondary messengers in controlling a cardiomyocyte's life and death, the detailed mechanisms of how mitochondrial Ca2+ regulates physiological mitochondrial and cellular functions in cardiac muscles, and how disorders of this mechanism lead to cardiac diseases remain unclear. In this review, we summarize the current controversies and discrepancies regarding cardiac mitochondrial Ca2+ signaling that remain in the field to provide a platform for future discussions and experiments to help close this gap.

Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis.

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

Protein kinase D activation induces mitochondrial fragmentation and dysfunction in cardiomyocytes.

KEY POINTS: Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. ABSTRACT: Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.

The Mitochondrial Ca2+ Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca2+ uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca2+ uptake and our current understanding of mitochondrial Ca2+ homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca2+ uniporter complex.

The mitochondrial Ca2+ uniporter: regulation by auxiliary subunits and signal transduction pathways.

Mitochondrial Ca(2+) homeostasis, the Ca(2+) influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca(2+) signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca(2+) uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca(2+) influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca(2+) uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca(2+) uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca(2+) handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca(2+) uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.

Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells.

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, ßI, ßII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCßI and PKCßII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, ß and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.

Adrenergic signaling controls RGK-dependent trafficking of cardiac voltage-gated L-type Ca2+ channels through PKD1.

RATIONALE: The Rad-Gem/Kir-related family (RGKs) consists of small GTP-binding proteins that strongly inhibit the activity of voltage-gated calcium channels. Among RGKs, Rem1 is strongly and specifically expressed in cardiac tissue. However, the physiological role and regulation of RGKs, and Rem1 in particular, are largely unknown. OBJECTIVE: To determine if Rem1 function is physiologically regulated by adrenergic signaling and thus impacts voltage-gated L-type calcium channel (VLCC) activity in the heart. METHODS AND RESULTS: We found that activation of protein kinase D1, a protein kinase downstream of α(1)-adrenergic signaling, leads to direct phosphorylation of Rem1 at Ser18. This results in an increase of the channel activity and plasma membrane expression observed by using a combination of electrophysiology, live cell confocal microscopy, and immunohistochemistry in heterologous expression system and neonatal cardiomyocytes. In addition, we show that stimulation of α(1)-adrenergic receptor-protein kinase D1-Rem1 signaling increases transverse-tubule VLCC expression that results in increased L-type Ca(2+) current density in adult ventricular myocytes. CONCLUSION: The α(1)-adrenergic stimulation releases Rem1 inhibition of VLCCs through direct phosphorylation of Rem1 at Ser18 by protein kinase D1, resulting in an increase of the channel activity and transverse-tubule expression. Our results uncover a novel molecular regulatory mechanism of VLCC trafficking and function in the heart and provide the first demonstration of physiological regulation of RGK function.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

Alpha1-adrenenoceptor stimulation inhibits cardiac excitation-contraction coupling through tyrosine phosphorylation of beta1-adrenoceptor.

Adrenoceptor stimulation is a key determinant of cardiac excitation-contraction coupling mainly through the activation of serine/threonine kinases. However, little is known about the role of protein tyrosine kinases (PTKs) activated by adrenergic signaling on cardiac excitation-contraction coupling. A cytoplasmic tyrosine residue in ß1-adrenoceptor is estimated to regulate Gs-protein binding affinity from crystal structure studies, but the signaling pathway leading to the phosphorylation of these residues is unknown. Here we show α1-adrenergic signaling inhibits ß-adrenergically activated Ca(2+) current, Ca(2+) transients and contractile force through phosphorylation of tyrosine residues in ß1-adrenoceptor by PTK. Our results indicate that inhibition of ß-adrenoceptor-mediated Ca(2+) elevation by α1-adrenoceptor-PTK signaling serves as an important regulatory feedback mechanism when the catecholamine level increases to protect cardiomyocytes from cytosolic Ca(2+) overload.

PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy.

Dynamic nucleocytoplasmic shuttling of class IIa histone deacetylases (HDACs) is a fundamental mechanism regulating gene transcription. Recent studies have identified several protein kinases that phosphorylate HDAC5, leading to its exportation from the nucleus. However, the negative regulatory mechanisms for HDAC5 nuclear exclusion remain largely unknown. Here we show that cAMP-activated protein kinase A (PKA) specifically phosphorylates HDAC5 and prevents its export from the nucleus, leading to suppression of gene transcription. PKA interacts directly with HDAC5 and phosphorylates HDAC5 at serine 280, an evolutionarily conserved site. Phosphorylation of HDAC5 by PKA interrupts the association of HDAC5 with protein chaperone 14-3-3 and hence inhibits stress signal-induced nuclear export of HDAC5. An HDAC5 mutant that mimics PKA-dependent phosphorylation localizes in the nucleus and acts as a dominant inhibitor for myocyte enhancer factor 2 transcriptional activity. Molecular manipulations of HDAC5 show that PKA-phosphorylated HDAC5 inhibits cardiac fetal gene expression and cardiomyocyte hypertrophy. Our findings identify HDAC5 as a substrate of PKA and reveal a cAMP/PKA-dependent pathway that controls HDAC5 nucleocytoplasmic shuttling and represses gene transcription. This pathway may represent a mechanism by which cAMP/PKA signaling modulates a wide range of biological functions and human diseases such as cardiomyopathy.

Intermittent Fasting Activates AMP-Kinase to Restructure Right Ventricular Lipid Metabolism and Microtubules.

Intermittent fasting (IF) extends life span via pleotropic mechanisms, but one important molecular mediator is adenosine monophosphate-activated protein kinase (AMPK). AMPK enhances lipid metabolism and modulates microtubule dynamics. Dysregulation of these molecular pathways causes right ventricular (RV) failure in patients with pulmonary arterial hypertension. In rodent pulmonary arterial hypertension, IF activates RV AMPK, which restores mitochondrial and peroxisomal morphology and restructures mitochondrial and peroxisomal lipid metabolism protein regulation. In addition, IF increases electron transport chain protein abundance and activity in the right ventricle. Echocardiographic and hemodynamic measures of RV function are positively associated with fatty acid oxidation and electron transport chain protein levels. IF also combats heightened microtubule density, which normalizes transverse tubule structure.

Mitochondrial Ca 2+ uniporter (MCU) variants form plasma-membrane channels.

MCU is widely recognized as a responsible gene for encoding a pore-forming subunit of highly mitochondrial-specific and Ca 2+ -selective channel, mitochondrial Ca 2+ uniporter complex (mtCUC). Here, we report a novel short variant derived from the MCU gene (termed MCU-S) which lacks mitochondria-targeted sequence and forms a Ca 2+ - permeable channel outside of mitochondria. MCU-S was ubiquitously expressed in all cell-types/tissues, with particularly high expression in human platelets. MCU-S formed Ca 2+ channels at the plasma membrane, which exhibited similar channel properties to those observed in mtCUC. MCU-S channels at the plasma membrane served as an additional Ca 2+ influx pathway for platelet activation. Our finding is completely distinct from the originally reported MCU gene function and provides novel insights into the molecular basis of MCU variant-dependent cellular Ca 2+ handling.

Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS.

Fluid shear stress generated by steady laminar blood flow protects vessels from atherosclerosis. Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) are fluid shear stress-responsive genes and key mediators in flow anti-inflammatory and antiatherosclerotic actions. However, the molecular mechanisms underlying flow induction of KLF2 and eNOS remain largely unknown. Here, we show a novel role of histone deacetylase 5 (HDAC5) in flow-mediated KLF2 and eNOS expression. We found for the first time that fluid shear stress stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a calcium/calmodulin-dependent pathway. Consequently, flow induced the dissociation of HDAC5 and myocyte enhancer factor-2 (MEF2) and enhanced MEF2 transcriptional activity, which leads to expression of KLF2 and eNOS. Adenoviral overexpression of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 were replaced by Ala259/Ala498, HDAC5-S/A), which shows resistance to flow-induced nuclear export, suppressed flow-mediated MEF2 transcriptional activity and expression of KLF2 and eNOS. Importantly, HDAC5-S/A attenuated the flow-inhibitory effect on monocyte adhesion to endothelial cells. Taken together, our results reveal that phosphorylation-dependent derepression of HDAC5 mediates flow-induced KLF2 and eNOS expression as well as flow anti-inflammation, and suggest that HDAC5 could be a potential therapeutic target for the prevention of atherosclerosis.

α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

VEGF stimulates HDAC7 phosphorylation and cytoplasmic accumulation modulating matrix metalloproteinase expression and angiogenesis.

OBJECTIVE: Histone acetylation/deacetylation plays an important role in the control of gene expression, tissue growth, and development. In particular, histone deacetylases 7 (HDAC7), a member of class IIa HDACs, is crucial in maintaining vascular integrity. However, whether HDAC7 is involved in the processes of vascular endothelial signaling and angiogenesis remains unclear. Here, we investigated the role of HDAC7 in vascular endothelial growth factor (VEGF) signaling and angiogenesis. METHODS AND RESULTS: We show for the first time that VEGF stimulated phosphorylation of HDAC7 at the sites of Ser178, Ser344, and Ser479 in a dose- and time-dependent manner, which leads to the cytoplasmic accumulation of HDAC7. Using pharmacological inhibitors, siRNA, and adenoviruses carrying dominant-negative mutants, we found that phospholipase Cgamma/protein kinase C/protein kinase D1 (PKD1)-dependent signal pathway mediated HDAC7 phosphorylation and cytoplasmic accumulation by VEGF. Infection of ECs with adenoviruses encoding a mutant of HDAC7 specifically deficient in PKD1-dependent phosphorylation inhibited VEGF-induced angiogenic gene expression, including matrix metalloproteinases MT1-matrix metalloproteinase (MMP) and MMP10. Moreover, HDAC7 and its targeting genes were involved in VEGF-stimulated endothelial cell migration, tube formation, and microvessel sprouting. CONCLUSIONS: Our results demonstrate that VEGF stimulates PKD1-dependent HDAC7 phosphorylation and cytoplasmic accumulation in endothelial cells modulating gene expression and angiogenesis.

Molecular mechanisms of ghrelin-mediated endothelial nitric oxide synthase activation.

Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.

Adrenergic Regulation of Drp1-Driven Mitochondrial Fission in Cardiac Physio-Pathology.

Abnormal mitochondrial morphology, especially fragmented mitochondria, and mitochondrial dysfunction are hallmarks of a variety of human diseases including heart failure (HF). Although emerging evidence suggests a link between mitochondrial fragmentation and cardiac dysfunction, it is still not well described which cardiac signaling pathway regulates mitochondrial morphology and function under pathophysiological conditions such as HF. Mitochondria change their shape and location via the activity of mitochondrial fission and fusion proteins. This mechanism is suggested as an important modulator for mitochondrial and cellular functions including bioenergetics, reactive oxygen species (ROS) generation, spatiotemporal dynamics of Ca2+ signaling, cell growth, and death in the mammalian cell- and tissue-specific manners. Recent reports show that a mitochondrial fission protein, dynamin-like/related protein 1 (DLP1/Drp1), is post-translationally modified via cell signaling pathways, which control its subcellular localization, stability, and activity in cardiomyocytes/heart. In this review, we summarize the possible molecular mechanisms for causing post-translational modifications (PTMs) of DLP1/Drp1 in cardiomyocytes, and further discuss how these PTMs of DLP1/Drp1 mediate abnormal mitochondrial morphology and mitochondrial dysfunction under adrenergic signaling activation that contributes to the development and progression of HF.

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of PI 3-kinase/Akt and NF-kappaB activation in murine BV2 microglial cells.

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A3 receptor ligands on LPS-induced tumor necrosis factor (TNF-alpha) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A3 receptor agonist, suppressed LPS-induced TNF-alpha protein and mRNA levels. Moreover, MRS1523, a selective A3 receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-alpha. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-kappaB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-kappaB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A3 receptor activation suppresses TNF-alpha production by inhibiting PI 3-kinase/Akt and NF-kappaB activation in LPS-treated BV2 microglial cells.