Deficiency of mitochondrial calcium uniporter abrogates iron overload-induced cardiac dysfunction by reducing ferroptosis.

Iron overload associated cardiac dysfunction remains a significant clinical challenge whose underlying mechanism(s) have yet to be defined. We aim to evaluate the involvement of the mitochondrial Ca2+ uniporter (MCU) in cardiac dysfunction and determine its role in the occurrence of ferroptosis. Iron overload was established in control (MCUfl/fl) and conditional MCU knockout (MCUfl/fl-MCM) mice. LV function was reduced by chronic iron loading in MCUfl/fl mice, but not in MCUfl/fl-MCM mice. The level of mitochondrial iron and reactive oxygen species were increased and mitochondrial membrane potential and spare respiratory capacity (SRC) were reduced in MCUfl/fl cardiomyocytes, but not in MCUfl/fl-MCM cardiomyocytes. After iron loading, lipid oxidation levels were increased in MCUfl/fl, but not in MCUfl/fl-MCM hearts. Ferrostatin-1, a selective ferroptosis inhibitor, reduced lipid peroxidation and maintained LV function in vivo after chronic iron treatment in MCUfl/fl hearts. Isolated cardiomyocytes from MCUfl/fl mice demonstrated ferroptosis after acute iron treatment. Moreover, Ca2+ transient amplitude and cell contractility were both significantly reduced in isolated cardiomyocytes from chronically Fe treated MCUfl/fl hearts. However, ferroptosis was not induced in cardiomyocytes from MCUfl/fl-MCM hearts nor was there a reduction in Ca2+ transient amplitude or cardiomyocyte contractility. We conclude that mitochondrial iron uptake is dependent on MCU, which plays an essential role in causing mitochondrial dysfunction and ferroptosis under iron overload conditions in the heart. Cardiac-specific deficiency of MCU prevents the development of ferroptosis and iron overload-induced cardiac dysfunction.

Calcium and Heart Failure: How Did We Get Here and Where Are We Going?

The occurrence and prevalence of heart failure remain high in the United States as well as globally. One person dies every 30 s from heart disease. Recognizing the importance of heart failure, clinicians and scientists have sought better therapeutic strategies and even cures for end-stage heart failure. This exploration has resulted in many failed clinical trials testing novel classes of pharmaceutical drugs and even gene therapy. As a result, along the way, there have been paradigm shifts toward and away from differing therapeutic approaches. The continued prevalence of death from heart failure, however, clearly demonstrates that the heart is not simply a pump and instead forces us to consider the complexity of simplicity in the pathophysiology of heart failure and reinforces the need to discover new therapeutic approaches.

Activation of TRPC (Transient Receptor Potential Canonical) Channel Currents in Iron Overloaded Cardiac Myocytes.

BACKGROUND: Arrhythmias and heart failure are common cardiac complications leading to substantial morbidity and mortality in patients with hemochromatosis, yet mechanistic insights remain incomplete. We investigated the effects of iron (Fe) on electrophysiological properties and intracellular Ca2+ (Ca2+i) handling in mouse left ventricular cardiomyocytes. METHODS: Cardiomyocytes were isolated from the left ventricle of mouse hearts and were superfused with Fe3+/8-hydroxyquinoline complex (5-100 µM). Membrane potential and ionic currents including TRPC (transient receptor potential canonical) were recorded using the patch-clamp technique. Ca2+i was evaluated by using Fluo-4. Cell contraction was measured with a video-based edge detection system. The role of TRPCs in the genesis of arrhythmias was also investigated by using a mathematical model of a mouse ventricular myocyte with the incorporation of the TRPC component. RESULTS: We observed prolongation of the action potential duration and induction of early and delayed afterdepolarizations in myocytes superfused with 15 µmol/L Fe3+/8-hydroxyquinoline complex. Iron treatment decreased the peak amplitude of the L-type Ca2+ current and total K+ current, altered Ca2+i dynamics, and decreased cell contractility. During the final phase of Fe treatment, sustained Ca2+i waves and repolarization failure occurred and ventricular cells became unexcitable. Gadolinium abolished Ca2+i waves and restored the resting membrane potential to the normal range. The involvement of TRPC activation was confirmed by TRPC channel current recordings in the absence or presence of functional TRPC channel antibodies. Computer modeling captured the same action potential and Ca2+i dynamics and provided additional mechanistic insights. CONCLUSIONS: We conclude that iron overload induces cardiac dysfunction that is associated with TRPC channel activation and alterations in membrane potential and Ca2+i dynamics.

Proteomic analysis of mitochondrial biogenesis in cardiomyocytes differentiated from human induced pluripotent stem cells.

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.

Iron Overload, Oxidative Stress and Calcium Mishandling in Cardiomyocytes: Role of the Mitochondrial Permeability Transition Pore.

Iron (Fe) plays an essential role in many physiological processes. Hereditary hemochromatosis or frequent blood transfusions often cause iron overload (IO), which can lead to cardiomyopathy and arrhythmias; however, the underlying mechanism is not well defined. In the present study, we assess the hypothesis that IO promotes arrhythmias via reactive oxygen species (ROS) production, mitochondrial membrane potential (∆Ψm) depolarization, and disruption of cytosolic Ca dynamics. In ventricular myocytes isolated from wild type (WT) mice, both cytosolic and mitochondrial Fe levels were elevated following perfusion with the Fe3+/8-hydroxyquinoline (8-HQ) complex. IO promoted mitochondrial superoxide generation (measured using MitoSOX Red) and induced the depolarization of the ΔΨm (measured using tetramethylrhodamine methyl ester, TMRM) in a dose-dependent manner. IO significantly increased the rate of Ca wave (CaW) formation measured in isolated ventricular myocytes using Fluo-4. Furthermore, in ex-vivo Langendorff-perfused hearts, IO increased arrhythmia scores as evaluated by ECG recordings under programmed S1-S2 stimulation protocols. We also carried out similar experiments in cyclophilin D knockout (CypD KO) mice in which the mitochondrial permeability transition pore (mPTP) opening is impaired. While comparable cytosolic and mitochondrial Fe load, mitochondrial ROS production, and depolarization of the ∆Ψm were observed in ventricular myocytes isolated from both WT and CypD KO mice, the rate of CaW formation in isolated cells and the arrhythmia scores in ex-vivo hearts were significantly lower in CypD KO mice compared to those observed in WT mice under conditions of IO. The mPTP inhibitor cyclosporine A (CsA, 1 µM) also exhibited a protective effect. In conclusion, our results suggest that IO induces mitochondrial ROS generation and ∆Ψm depolarization, thus opening the mPTP, thereby promoting CaWs and cardiac arrhythmias. Conversely, the inhibition of mPTP ameliorates the proarrhythmic effects of IO.

Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy.

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

S-nitrosylation of connexin43 hemichannels elicits cardiac stress-induced arrhythmias in Duchenne muscular dystrophy mice.

Patients with Duchenne muscular dystrophy (DMD) commonly present with severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute ß-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the ß-adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx Cx43+/- mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization, and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels plays a key role in the development of arrhythmias in DMD mice and that these channels may serve as therapeutic targets to prevent fatal arrhythmias in patients with DMD .

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

RATIONALE: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. OBJECTIVE: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). METHODS AND RESULTS: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. CONCLUSIONS: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Cellular Electrophysiology of Iron-Overloaded Cardiomyocytes.

Iron, the most abundant transition metal element in the human body, plays an essential role in many physiological processes. However, without a physiologically active excretory pathway, iron is subject to strict homeostatic processes acting upon its absorption, storage, mobilization, and utilization. These intricate controls are perturbed in primary and secondary hemochromatoses, leading to a deposition of excess iron in multiple vital organs including the heart. Iron overload cardiomyopathy is the leading cause of mortality in patients with iron overload conditions. Apart from mechanical deterioration of the siderotic myocardium, arrhythmias reportedly contribute to a substantial portion of cardiac death associated with iron overload. Despite this significant impact, the cellular mechanisms of electrical disturbances in an iron-overloaded heart are still incompletely characterized. This review article focuses on cellular electrophysiological studies that directly investigate the effects of iron overload on the function of cardiac ion channels, including trans-sarcolemmal and sarcoplasmic reticulum Ca2+ fluxes, as well as cardiac action potential morphology. Our ultimate aim is to provide a comprehensive summary of the currently available information that will encourage and facilitate further mechanistic elucidation of iron-induced pathoelectrophysiological changes in the heart.

Involvement of cytosolic and mitochondrial iron in iron overload cardiomyopathy: an update.

Iron overload cardiomyopathy (IOC) is a major cause of death in patients with diseases associated with chronic anemia such as thalassemia or sickle cell disease after chronic blood transfusions. Associated with iron overload conditions, there is excess free iron that enters cardiomyocytes through both L- and T-type calcium channels thereby resulting in increased reactive oxygen species being generated via Haber-Weiss and Fenton reactions. It is thought that an increase in reactive oxygen species contributes to high morbidity and mortality rates. Recent studies have, however, suggested that it is iron overload in mitochondria that contributes to cellular oxidative stress, mitochondrial damage, cardiac arrhythmias, as well as the development of cardiomyopathy. Iron chelators, antioxidants, and/or calcium channel blockers have been demonstrated to prevent and ameliorate cardiac dysfunction in animal models as well as in patients suffering from cardiac iron overload. Hence, either a mono-therapy or combination therapies with any of the aforementioned agents may serve as a novel treatment in iron-overload patients in the near future. In the present article, we review the mechanisms of cytosolic and/or mitochondrial iron load in the heart which may contribute synergistically or independently to the development of iron-associated cardiomyopathy. We also review available as well as potential future novel treatments.

Antioxidant defense and protection against cardiac arrhythmias: lessons from a mammalian hibernator (the woodchuck).

Hibernating animals show resistance to hypothermia-induced cardiac arrhythmias. However, it is not clear whether and how mammalian hibernators are resistant to ischemia-induced arrhythmias. The goal of this investigation was to determine the susceptibility of woodchucks ( Marmota monax) to arrhythmias and their mechanisms after coronary artery occlusion at the same room temperature in both winter, the time for hibernation, and summer, when they do not hibernate. By monitoring telemetric electrocardiograms, we found significantly higher arrhythmia scores, calculated as the severity of arrhythmias, with incidence of ventricular tachycardia, ventricular fibrillation, and thus sudden cardiac death (SCD) in woodchucks in summer than they had in winter. The level of catalase expression in woodchuck hearts was significantly higher, whereas the level of oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII) was lower in winter than it was in summer. Ventricular myocytes isolated from woodchucks in winter were more resistant to H2O2-induced early afterdepolarizations (EADs) compared with myocytes isolated from woodchucks in summer. The EADs were eliminated by inhibiting CaMKII (with KN-93), l-type Ca current (with nifedipine), or late Na+ current (with ranolazine). In woodchucks, in the summer, the arrhythmia score was significantly reduced by overexpression of catalase ( via adenoviral vectors) or the inhibition of CaMKII (with KN-93) in the heart. This study suggests that the heart of the mammalian hibernator is more resistant to ischemia-induced arrhythmias and SCD in winter. Increased antioxidative capacity and reduced CaMKII activity may confer resistance in woodchuck hearts against EADs and arrhythmias during winter. The profound protection conferred by catalase overexpression or CaMKII inhibition in this novel natural animal model may provide insights into clinical directions for therapy of arrhythmias.-Zhao, Z., Kudej, R. K., Wen, H., Fefelova, N., Yan, L., Vatner, D. E., Vatner, S. F., Xie, L.-H. Antioxidant defense and protection against cardiac arrhythmias: lessons from a mammalian hibernator (the woodchuck).

Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice.

Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr -/-) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr -/- mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.

Effect of densely ionizing radiation on cardiomyocyte differentiation from human-induced pluripotent stem cells.

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.

The valosin-containing protein is a novel mediator of mitochondrial respiration and cell survival in the heart in vivo.

The valosin-containing protein (VCP) participates in signaling pathways essential for cell homeostasis in multiple tissues, however, its function in the heart in vivo remains unknown. Here we offer the first description of the expression, function and mechanism of action of VCP in the mammalian heart in vivo in both normal and stress conditions. By using a transgenic (TG) mouse with cardiac-specific overexpression (3.5-fold) of VCP, we demonstrate that VCP is a new and powerful mediator of cardiac protection against cell death in vivo, as evidenced by a 50% reduction of infarct size after ischemia/reperfusion versus wild type. We also identify a novel role of VCP in preserving mitochondrial respiration and in preventing the opening of mitochondrial permeability transition pore in cardiac myocytes under stress. In particular, by genetic deletion of inducible isoform of nitric oxide synthase (iNOS) from VCP TG mouse and by pharmacological inhibition of iNOS in isolated cardiac myocytes, we reveal that an increase of expression and activity of iNOS in cardiomyocytes by VCP is an essential mechanistic link of VCP-mediated preservation of mitochondrial function. These data together demonstrate that VCP may represent a novel therapeutic avenue for the prevention of myocardial ischemia.

Transient receptor potential cation 3 channel regulates melanoma proliferation and migration.

Melanoma has an extremely poor prognosis due to its rapidly progressive and highly metastatic nature. Several therapeutic drugs have recently become available, but are effective only against melanoma with specific BRAF gene mutation. Thus, there is a need to identify other target molecules. We show here that Transient receptor potential, canonical 3 (TRPC3) is widely expressed in human melanoma. We found that pharmacological inhibition of TRPC3 with a pyrazole compound, Pyr3, decreased melanoma cell proliferation and migration. Similar inhibition was observed when the TRPC3 gene was silenced with short-hairpin RNA (shRNA). Pyr3 induced dephosphorylation of signal transducer and activator of transcription (STAT) 5 and Akt. Administration of Pyr3 (0.05 mg/kg) to mice implanted with human melanoma cells (C8161) significantly inhibited tumor growth. Our findings indicate that TRPC3 plays an important role in melanoma growth, and may be a novel target for treating melanoma in patients.

Oxygen-glucose deprivation enhancement of cell death/apoptosis in PC12 cells and hippocampal neurons correlates with changes in neuronal excitatory amino acid neurotransmitter signaling and potassium currents.

Neuronal death is a pathophysiological process that is often caused by hypoxia/ischemia. However, the causes of hypoxia/ischemia-induced neuronal death are debated, and additional experimental data are needed to resolve this debate. In the present study, we applied oxygen-glucose deprivation (OGD) to PC12 cells and hippocampal neurons to establish a hypoxia/ischemia model. We evaluated the effects of OGD on cell death/apoptosis and on the levels of two excitatory amino acid neurotransmitters, aspartic acid and glutamic acid, in both hippocampal neurons and the medium used to culture the hippocampal neurons. We also evaluated GluR2 expression in hippocampal neurons as well as the effects of OGD on whole-cell potassium currents in PC12 cells and hippocampal neurons. Our experimental results showed that OGD significantly decreased cell viability and markedly enhanced apoptosis in PC12 cells and hippocampal neurons. OGD treatment for 3 h increased the levels of Asp and Glu in the medium used to culture hippocampal neurons, but decreased both the levels of Asp and Glu and GluR2 expression in hippocampal neurons. Furthermore, OGD altered the electrophysiological properties of voltage-dependent potassium channels in PC12 cells and hippocampal neurons in different ways; OGD decreased the voltage-dependent potassium current in PC12 cells, but increased this current in hippocampal neurons. On the basis of these results, we concluded that OGD enhanced neuronal cell death/apoptosis in addition to altering neuronal excitatory amino acid neurotransmitter signaling and whole-cell voltage-dependent potassium currents.

Selective Connexin43 Inhibition Prevents Isoproterenol-Induced Arrhythmias and Lethality in Muscular Dystrophy Mice.

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. We targeted the gap junction protein connexin43 (Cx43) responsible for maintaining cardiac conduction. In mild mdx and severe mdx:utr mouse models of DMD, and human DMD tissues, Cx43 was found to be pathologically mislocalized to lateral sides of cardiomyocytes. In addition, overall Cx43 protein levels were markedly increased in mouse and human DMD heart tissues examined. Electrocardiography on isoproterenol challenged mice showed that both models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetics to inhibit lateralized Cx43 function prior to challenge protected mdx mice from arrhythmogenesis and death, while mdx:utr mice displayed markedly improved ECG scores. These findings suggest that Cx43 lateralization contributes significantly to DMD arrhythmogenesis and that selective inhibition may provide substantial benefit.

The Zinc Ion Chelating Agent TPEN Attenuates Neuronal Death/apoptosis Caused by Hypoxia/ischemia Via Mediating the Pathophysiological Cascade Including Excitotoxicity, Oxidative Stress, and Inflammation.

AIMS: We aim to determine the significant effect of TPEN, a Zn(2+) chelator, in mediating the pathophysiological cascade in neuron death/apoptosis induced by hypoxia/ischemia. METHODS: We conducted both in vivo and in vitro experiments in this study. PC12 cells were used to establish hypoxia/ischemia model by applying oxygen-glucose deprivation (OGD). SHR-SP rats were used to establish an acute ischemic model by electrocoagulating middle cerebral artery occlusion. The effect of TPEN on neuron death/apoptosis was evaluated. In addition, the relative biomarks of excitotoxicity, oxidative stress, and inflammation reactions in hypoxia/ischemia PC12 cell model as well as in SHR-SP rat hypoxia/ischemia model were also assessed. RESULTS: TPEN significantly attenuates the neurological deficit, reduced the cerebral infarction area and the ratio of apoptotic neurons, and increased the expression of GluR2 in the rat hypoxia/ischemia brain. TPEN also increased blood SOD activity, decreased blood NOS activity and blood MDA and IL-6 contents in rats under hypoxia/ischemia. In addition, TPEN significantly inhibited the death and apoptosis of cells and attenuated the alteration of GluR2 and NR2 expression caused by OGD or OGD plus high Zn(2+) treatments. CONCLUSIONS: Zn(2+) is involved in neural cell apoptosis and/or death caused by hypoxia/ischemia via mediating excitotoxicity, oxidative stress, and inflammation.

Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen-Glucose Deprivation (OGD).

To elucidate the role of Zn(2+)-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia-ischemia, PC12 cells were exposed to Oxygen-Glucose Deprivation (OGD) solution mimicking the hypoxic-ischemic condition in neuron, and the effect of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn(2+) chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3h, 6h and 24h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1h, but decrease at 3h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3h and 6h, P < 0.01 at 24h respectively compared to control) and GluR2 expression (P < 0.05 at 3h, 6h and 24h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic-ischemic condition.

Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration.

Store-operated Ca(2+) entry (SOCE) is a major mechanism of Ca(2) (+) import from extracellular to intracellular space, involving detection of Ca(2+) store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca(2+) channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.