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Recent discoveries of the molecular identity of mitochondrial Ca(2+) influx/efflux mechanisms have placed mitochondrial Ca(2+) 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 Ca(2+). 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 Ca(2+) handling in cardiomyocytes and its contribution to excitation-contraction/metabolism coupling in the heart. Though mitochondrial Ca(2+) 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 Ca(2+) 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 Ca(2+) signaling that remain in the field to provide a platform for future discussions and experiments to help close this gap.

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.

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.

Mitochondrial Ca2+homeostasis, the Ca2+influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca2+signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca2+uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca2+influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca2+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 Ca2+uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca2+handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca2+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.

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 at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type 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 the one of the GqPCR 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, βI, βII, 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, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (

Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879.

Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.

Increased production of reactive oxygen species (ROS) from mitochondria is the main cause of hyperglycemic complications. We previously showed that hyperglycemic conditions induce mitochondrial fragmentation that is causal for ROS overproduction. This study was to identify signaling components that induce mitochondrial fragmentation in high-glucose stimulation. We found that exposing cells to the high-glucose concentration evokes increases in cytosolic Ca2+. Chelating Ca2+ in the high-glucose medium prevented not only the Ca2+ transient but also mitochondrial fragmentation and the ROS increase, indicating that the Ca2+ influx across the plasma membrane is an upstream event governing mitochondrial fission and the ROS generation in high-glucose stimulation. We found that the high-glucose-induced Ca2+ increase activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2). The Ca2+ chelation prevented the ERK1/2 activation, and inhibition of the ERK1/2 phosphorylation decreased mitochondrial fragmentation as well as ROS levels in high-glucose stimulation. In addition, the level of the mitochondrial fission protein dynamin-like protein 1 in mitochondria increased in high-glucose incubation in a Ca2+-dependent manner. In vitro kinase assays showed that ERK1/2 is capable of phosphorylating dynamin-like protein 1. These results demonstrate that high-glucose stimulation induces the activation of mitochondrial fission via signals mediated by intracellular Ca2+ and ERK1/2. Antioxid. Redox Signal. 14, 425–437.

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.

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiomyocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed both in human heart failure (HF) and in animal HF models. However, it is still unclear which cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Recent reports suggest that Gq-protein coupled receptor (GqPCR) signaling pathways are critical for the development and progression of HF. Therefore, we hypothesize that GqPCR stimulation induces mitochondrial fragmentation and dysfunction, which initiates cardiomyocyte death. We found that protein kinase D (PKD) activated by GqPCR signaling was translocated to outer mitochondrial membrane (OMM) observed by Western blot analysis of cytosolic and mitochondria-enriched fractionated proteins and by live cell imaging of fluorescence resonance energy transfer (FRET). We also found that GqPCR-mediated PKD activation induced mitochondrial fragmentation, leading to increased reactive oxygen species (ROS) generation as well as increased mitochondrial permeability transition pore (mPTP) opening, which initiates apoptotic signaling activation and cardiomyocyte death. These morphological and functional changes in cardiac mitochondria were mediated via PKD-dependent phosphorylation of mitochondrial fission protein, Dynamin-Like Protein 1 (DLP1) at S637. Moreover, pretreatment with a novel potent PKD inhibitor CRT0066101 effectively inhibited GqPCR-mediated PKD translocation to OMM, DLP1 phosphorylation at S637, mitochondrial fragmentation, ROS generation and mPTP activation. In conclusion, we demonstrate that GqPCR stimulation induces mitochondrial fragmentation and dysfunction through PKD-dependent phosphorylation of DLP1 at S637, which likely contributes to cardiomyocyte injury. Thus, small-molecule PKD inhibitor may become a novel and potent therapeutic for preventing cardiac cell injury and death during HF.

We investigated regulation of various signal transduction pathways during oxidative stresses in the kidney of young and aged rats. Menadione-induced regulation of molecules in PI 3-kinase, MAPK, and AMPK pathways was determined in the young (2 months) and old (24 months) groups. PI 3-kinase activity and Akt phosphorylation were significantly reduced in the old compared with the young. PTEN tumor suppressor was also lower in its expression and phosphorylation levels in the old. Response of the molecules in PI 3-kinase pathway to menadione was minimized. In contrast, over 5-fold induction of ERK1/2 phosphorylation by menadione was observed in both groups. On the other hand, basal activities as well as menadione-induced activities of JNK1 and AMPK were higher in the old than in the young. While p27Kip1, p53, and p21Waf1 were slightly increased by menadione in both groups, the basal induction level in the old was considerably higher. In conclusion, the results suggest that the age-related down-regulation of PI 3-kinase/Akt pathway and up-regulation of JNK1, AMPK, and p53 pathways may be responsible for the increased susceptibility to oxidative stress.

Cardiac fibroblasts (CFs) are one o the most abundant cell types in the heart and play key roles in regulating myocardial physiological function and pathophysiological remodeling especially for the cardiac fibrosis. The levels of Angiotensin II (AT-II) are increased in the remodeling heart and Angiotensin signaling participates in pathological CF proliferation. It has been shown that CF proliferation may occur via the increased levels of cellular reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiac myofibroblast cell line H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhances mitochondrial Ca2+-induced mtSO generation in primary CFs, which can activate ROS-dependent proliferation signaling in primary CFs. First, we confirmed that AT-II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake assess by mitochondria-targeted Ca2+ biosensor in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT-II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. We also confirmed that AT-II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria-targeted antioxidant, Mito-tempo significantly inhibited AT-II-mediated activation of the mtSO production as well as proliferative pathway without changing the AT-II-induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation via MCU serves as an important regulator for the Ang II-induced proliferation in CFs.

Mitochondrial Ca(2+) controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca(2+) signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca(2+) accumulation is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU), but recent reports also indicate that mitochondrial Ca(2+)-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca(2+)-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca(2+) uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca(2+)-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca(2+) through this channel in response to cytosolic Ca(2+) elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca(2+) uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca(2+) uptake mechanism in response to the elevation of [Ca(2+)]c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca(2+)-mediated functions such as energy metabolism.

Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006.

Ca+ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca2+ 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 Ca2+ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-induced ATP production in cardiac H9c2 myoblasts.

Introduction: Mutations in skeletal muscle type ryanodine receptor (RyR1) exhibit life-treating human disorders including malignant hyperthermia (MH) and central core disease, which features skeletal muscle rigidity triggered by anesthesia and body temperature elevation. However, cardiac arrhythmias and/or sudden cardiac death in these patients are also frequently reported during anesthesia or even under the conscious condition. Hypothesis: Chronic mitochondrial Ca 2+ overload through leaky mutant RyR1 expressed at cardiac mitochondria leads to mitochondria injury and cardiac arrhythmia. Methods: We determined the cardiac phenotype of knock-in mice carrying a leaky RyR1 MH mutation Y522S (YS). Results: WT- and YS - RyR1 were expressed at mitochondria but not at SR in heart. Ultra-structure of heterozygous YS hearts exhibited severe abnormal/disrupted mitochondrial morphology as well as myofibril contractures (Fig. A-E). YS cardiac mitochondria showed high susceptibility to Ca 2+ -induced opening of mitochondrial permeability transition pores and depolarized mitochondrial membrane potential compared to WT, suggesting compromised mitochondrial functions. Langendorff perfusion developed similar LV pressure in response to high dose bolus of Isoproterenol in WT and YS, but only YS heart frequently developed multiple ventricular extrasystoles after Isoproterenol (Fig. F). Conclusion: Chronic mitochondrial damage by Ca 2+ overload through leaky mutant RyR1 induces mitochondrial structural and functional disruption, which facilitates arrhythmogenic outbursts under acute catecholaminergic stress.

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.

Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism–secretion coupling in pancreatic β-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications. Antioxid. Redox Signal. 14, 439–457.

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.

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.

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 Cγ/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.

Background Plasminogen activator inhibitor (PAI)–1 is a key regulator of the fibrinolytic system. PAI-1 levels are markedly elevated in the asthmatic airways. The 4G/5G polymorphism of the PAI-1 gene is associated with allergic asthma. Objective To characterize the mechanisms of the 4G/5G-dependent PAI-1 expression in mast cells (MCs), a major source of PAI-1 and key effector cells in asthma. Methods Transcription of PAI-1 was assessed by transiently transfecting human MC line (HMC-1) cells with the luciferase-tagged PAI-1 promoters containing the 4G or 5G allele (4G–PAI-1 or 5G–PAI-1 promoter). Upstream stimulatory factor (USF)–1 and the E-box interactions were studied by electrophoretic mobility shift assays and supershift assays. Expression of USF-1 was determined by Western blot analysis. Results The 4G–PAI-1 promoter has higher promoter activity than the 5G–PAI-1 promoter in stimulated HMC-1 cells, and the E-box adjacent to the 4G/5G site (E-4G/5G) regulates the genotype-specific PAI-1 transcription. USF-1 binds to the E-4G with greater affinity than to the E-5G. USF-1 level is increased in HMC-1 cells after stimulation, and elevated USF-1 enhances PAI-1 transcription. Overexpression of wild-type USF-1 or dominant-negative USF remedies the 4G/5G-dependent PAI-1 transcription. Conclusion Binding of USF-1 to the E-4G/5G regulates the 4G/5G polymorphism–dependent PAI-1 expression in MCs.

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and its activation during T cell receptor stimulation has recently been reported. In this study, we examined the role of AMPK in interleukin (IL)-2 production in T cells. Inhibition of AMPK by compound C, a specific inhibitor of AMPK or small interfering RNA of AMPKalpha1 suppressed IL-2 production in Jurkat T cells and peripheral blood lymphocytes stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. We then showed that AMPK inhibition reduced PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. Moreover, inhibition of AMPK suppressed transcriptional activation of NF-AT and AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that compound C inhibited PMA/Io-induced phosphorylation of p38, JNK, and GSK-3beta but not of ERK. These results suggest that AMPK mediates IL-2 production by regulating NF-AT and AP-1activation during T cell stimulation.

We examined the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), the dephosphorylated form of AICA ribotide (also termed “ZMP”), an intermediate of purine biosynthesis, on interleukin (IL)-2 production in T cells. AICAR inhibited IL-2 production in Jurkat T cells and peripheral blood lymphocytes activated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. Pretreatment with 5′-iodotubercidin, an adenosine kinase inhibitor, enhanced AICAR suppression of IL-2 production, suggesting that AICAR, not ZMP, is responsible for IL-2 suppression. We then showed that AICAR inhibited PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. AICAR inhibited DNA binding and transcriptional activation of NF-AT and to a lesser extent AP-1, but not NF-κB, in PMA/Io-activated Jurkat cells. Finally, we found that AICAR inhibited PMA/Io-induced phosphorylation of GSK-3 but not phosphorylation of ERK1/2, p38, and JNK. These results suggest that AICAR exerts its immunosuppressive effect in activated Jurkat cells by inhibiting GSK-3 phosphorylation and NF-AT activation.

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.

Fission and fusion of mitochondrial tubules are the major processes regulating mitochondrial morphology. However, the physiological significance of mitochondrial shape change is poorly understood. Glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells requires mitochondrial ATP production which evokes Ca(2+) influx through plasma membrane depolarization, triggering insulin vesicle exocytosis. Therefore, GSIS reflects mitochondrial function and can be used for evaluating functional changes associated with morphological alterations of mitochondria. Using the insulin-secreting cell line INS-1E, we found that glucose stimulation induced rapid mitochondrial shortening and recovery. Inhibition of mitochondrial fission through expression of the dominant-negative mutant DLP1-K38A eliminated this dynamic mitochondrial shape change and, importantly, blocked GSIS. We found that abolishing mitochondrial morphology change in glucose stimulation increased the mitochondrial inner membrane proton leak, and thus significantly diminished the mitochondrial ATP producing capacity in response to glucose stimulation. These results demonstrate that dynamic change of mitochondrial morphology is a previously unrecognized component for metabolism-secretion coupling of pancreatic β-cells by participating in efficient ATP production in response to elevated glucose levels.

Mitochondria in cells undergo constant morphological changes mainly through fission and fusion. However, functional significance of mitochondrial fission and fusion is not fully understood. To test the importance of mitochondrial morphology in maintaining mitochondrial function, first, we used glucose-stimulated insulin secretion in pancreatic β-cells as an experimental model because insulin secretion upon elevated plasma glucose concentration requires intact mitochondrial function. Increased ATP production in mitochondria from glucose metabolism induces plasma membrane depolarization and subsequent increase of cytosolic Ca2+ triggers insulin exocytosis. We found that glucose stimulation of the β-cell line INS-1E induces transient mitochondrial shortening and recovery. Inhibiting mitochondrial fission by expressing the dominant-negative fission mutant DLP1-K38A abolished the dynamic change of mitochondrial morphology in glucose stimulation. Importantly, we discovered that abolition of the glucose-induced mitochondrial morphology change suppresses glucose-stimulated insulin secretion. Measuring respiration under fission inhibition showed an increase of mitochondrial uncoupling, and thus significantly diminished the mitochondrial ATP production in response to glucose stimulation. Further evaluation of mitochondrial membrane potential in primary hepatocytes revealed that inhibition of mitochondrial fission induces large-scale fluctuations of the potentiometric fluorescence in mitochondria within cells. Frequencies and intervals of the fluorescence oscillation were random and insensitive to inhibitors of anion channels and mitochondrial permeability, and superoxide scavenger. This suggests that the fission inhibition-induced fluctuation of the inner membrane potential is a previously unrecognized unique phenomenon. These observations demonstrate that inhibition of mitochondrial fission induces a large-scale fluctuation of the mitochondrial inner membrane potential, which is functionally reflected in mitochondrial uncoupling. Taken together, our findings indicate that mitochondrial fission plays a role in regulating the coupling efficiency of oxidative phosphorylation.

Background: Recent break-through discovery in the molecular identity of mitochondrial Ca2+ uniporter (MCU) opens the new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported in human sample mass spectroscopy, but the post translational modifications of MCU are completely unknown.Hypothesis: Tyrosine phosphorylation of MCU can modulate the mitochondrial Ca2+-uptake rate in cardiac cells.Methods: MCU was transiently or stably overexpressed in cardiac H9C2 cells. Tyrosine phosphorylation of MCU was detected by a general anti-phospho-tyrosine antibody. Mitochondrial Ca2+ concentration ([Ca2+]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam).Results: α1-adrenergic stimulation by phenylephrine enhanced the translocation of a Ca2+-dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) from cytosol to mitochondria followed by the increase in mitochondrial Pyk2 activity. Overexpressed MCU was exclusively localized at mitochondria and tyrosine residues in MCU were phosphorylated after Pyk2 activation. In addition, Pyk2 was bound to MCU at the basal condition and this interaction was enhanced by phenylephrine treatment. Moreover, Pyk2-dependent phosphorylation of MCU enhances MCU olgomerization observed by a conventional native PAGE. These effects were abolished by the co-transfection of kinase-dead Pyk2. In MCU-overexpressed cells, [Ca2+]m increased rapidly and reached to higher levels in response to cytosolic Ca2+ transients evoked by thapsigargin compared to non-transfected cells. Moreover, peak [Ca2+]m in MCU-overexpressed cells reached to much higher levels by phenylephrine pretreatment compared to non-treated cells.Conclusion: α1-adrenergic stimulation accelerates mitochondrial Ca2+ uptake through Pyk2-dependent direct phospholylation of MCU, which promotes the formation of tetrametric MCU channel pore. Our findings open up an exciting opportunity for investigating the first candidate cell signaling pathway for the MCU post translational modifications in cardiac cells.

Background:Cytosolic Ca2+ elevation is an important trigger for both mitochondrial dynamics and ATP generation in various cell types including cardiac myocytes. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Recent studies have identified additional Ca2+ uptake pathways, which exhibit different biophysical properties from MCU including skeletal-muscle type ryanodine receptor isoform1 (RyR1). However it is still unclear that which mitochondrial Ca2+ influx mechanism mainly participates in the regulation of mitochondrial morphology/dynamics and energetic functions in cardiac myocytes. Aim:To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology and function in cardiac cells. Methods:GFP- or non-tagged RyR1 was transiently or stably overexpressed in cardiac H9C2 cells. MCU and RyR2 are also expressed for comparisons. Mitochondrial localization of over-expressed RyR1 was observed by co-expression of mitochondrial matrix-targeted RFP (mtRFP) using confocal microscope and also quantified by Western blotting using mitochondria fractionation. Mitochondrial morphology was evaluated by the calculation of aspect ratio and form factor from mtRFP pictures. Mitochondrial Ca2+ concentration ([Ca2+]m) and ATP ([ATP]m) was measured by mitochondrial matrix-targeted Ca2+-sensitive inverse pericam (Mitycam) and FRET-based indicators for ATP (ATeam), respectively.Results:Overexpressed RyR1 was partially localized in mitochondria, but the another RyR isoform RyR2 did not show any co-localization with mitochondria. Over-expression of RyR1 but not MCU and RyR2 showed the mitochondrial fragmentation. Fragmented mitochondria showed higher [Ca2+]m at peak and sustained Ca2+ transients compared to elongated mitochondria. RyR1 overexpressed cells had higher [ATP]m at the basal condition and showed more ATP production in response to [Ca2+]c elevation compared to non-transfected cells.Conclusion:Mitochondrial RyR1 strongly modulates mitochondrial morphology, Ca2+ influx and Ca2+-induced ATP production in cardiac cells.

Mitochondrial Ca2+ has a critical role for balancing cell survival and death. Ca2+ influx into mitochondrial matrix is mediated primarily by the mitochondrial calcium uniporter (MCU). However, the signaling pathways that regulate MCU channel functions via post-translational modifications of MCU are completely unknown. Here we show that adrenergic signaling induces MCU tyrosine phosphorylation and accelerates mitochondrial Ca2+ uptake in cardiac cells. Adrenergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine residues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane domains and 6 in the C-terminus), which are conserved across all eukaryotic species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphorylation candidate sites for protein tyrosine kinases using phosphorylation prediction programs. We mutated these tyrosine residues to phenylalanine and generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We confirmed that only two tyrosine sites were phosphorylated in response to adrenergic stimulation in situ using cell lines stably expressing MCU-YFs. In addition, overexpression of these MCU-YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation by thapsigargin, whereas wild-type MCU transfection dramatically accelerates mitochondrial Ca2+ uptake compared to non-transfected cells. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU can modulate its channel functions and regulate mitochondrial Ca2+ uptake.