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Bcl2‐associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle, and many cancers. Deletions and truncations in BAG3 that result in haplo‐insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo‐insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3fl/+). Mice were crossed with α‐MHC‐Cre mice in order to generate mice with cardiac‐specific haplo‐insufficiency (cBAG3+/−) and underwent bi–weekly echocardiography to assess their cardiac phenotype. By 10 weeks of age, cBAG3+/− mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non‐transgenic littermates (Cre−/−BAG3fl/+). Contractility in adult myocytes isolated from cBAG3+/− mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca2+]i) transient amplitudes in myocytes isolated from cBAG3+/− mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo‐insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo‐insufficiency. [ABSTRACT FROM AUTHOR]

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents. [ABSTRACT FROM AUTHOR]

The emerging evidences suggest that posttranslational modification of target protein by ubiquitin (Ub) not only regulate its turnover through ubiquitin proteasome system (UPS) but is a critical regulator of various signaling pathways. During ubiquitination, E3 ligase recognizes the target protein and determines the topology of ubiquitin chains. In current study, we studied the role of TRIM4, a member of the TRIM/RBCC protein family of RING E3 ligase, in regulation of hydrogen peroxide (H 2 O 2 ) induced cell death. TRIM4 is expressed differentially in human tissues and expressed in most of the analyzed human cancer cell lines. The subcellular localization studies showed that TRIM4 forms distinct cytoplasmic speckle like structures which transiently interacts with mitochondria. The expression of TRIM4 induces mitochondrial aggregation and increased level of mitochondrial ROS in the presence of H 2 O 2 . It sensitizes the cells to H 2 O 2 induced death whereas knockdown reversed the effect. TRIM4 potentiates the loss of mitochondrial transmembrane potential and cytochrome c release in the presence of H 2 O 2 . The analysis of TRIM4 interacting proteins showed its interaction with peroxiredoxin 1 (PRX1), including other proteins involved in regulation of mitochondrial and redox homeostasis. TRIM4 interaction with PRX1 is critical for the regulation of H 2 O 2 induced cell death. Collectively, the evidences in the current study suggest the role of TRIM4 in regulation of oxidative stress induced cell death. [ABSTRACT FROM AUTHOR]

The nuclear factor κB (NF-κB) transcription factor family plays a key role in regulation of the inflammatory pathway in response to different physiological stimuli starting from development to ageing. The dysregulation of NF-κB has been associated with many pathological conditions like inflammatory diseases, neurodegeneration, metabolic diseases and various kinds of malignancies. The NF-κB pathway is regulated by number of post-translational modifications, including phosphorylation and ubiquitination. Ubiquitin (Ub) E3 ligases are key regulators of the process of ubiquitination and provide specificity to the pathway as they recognise the substrate and determine the topology of ubiquitination. TRIMs, members of RING family of Ub E3 ligases, are characterised by the presence of three conserved domains, RING, B-Box and coiled- coil (RBCC). Emerging evidence suggests that TRIMs regulate innate immune signalling during infection and different pathological conditions. The studies have demonstrated the role of TRIMs in regulation of inflammatory pathways including NF-κB. Recent reports suggest that TRIMs play a critical role in regulation of the NF-κB pathway by ubiquitinating proteins at different steps. In the current review, we discuss the role of TRIMs as novel NF-κB regulators and their role in different pathophysiological conditions. [ABSTRACT FROM AUTHOR]

The NF-κB family of transcription factors is activated in response to various intracellular or extracellular stimuli and its dysregulation leads to pathological conditions like infection, cancer, neurodegenerative disorders. The post-translational modification by ubiquitination regulates various steps of NF-κB pathway. In the current study, we have described the role of TRIM13, an endoplasmic reticulum (ER) membrane anchored E3 ligase in regulation of NF-κB. The expression of TRIM13 represses TNF induced NF-κB while the knockdown has the opposite effect. The E3 ligase activity and ER localization is essential for NF-κB suppression whereas TRIM13 regulated autophagy is not essential. TRIM13 interacts with NEMO and modulates its ubiquitination and turnover, hence may regulate IKK complex activity. TRIM13 mediated NF-κB repression is essential for negative regulation of clonogenic ability of the cells. This study for the first time demonstrated the role of TRIM13, ER resident RING E3 ligase as a novel regulator of NEMO ubiquitination, negative regulator of NF-κB signaling and its role as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Ischemia-induced metabolic remodeling plays a critical role in the pathogenesis of adverse cardiac remodeling and heart failure however, the underlying molecular mechanism is largely unknown. Here, we assess the potential roles of nicotinamide riboside kinase-2 (NRK-2), a muscle-specific protein, in ischemia-induced metabolic switch and heart failure through employing transcriptomic and metabolomic approaches in ischemic NRK-2 knockout mice. The investigations revealed NRK-2 as a novel regulator of several metabolic processes in the ischemic heart. Cardiac metabolism and mitochondrial function and fibrosis were identified as top dysregulated cellular processes in the KO hearts post-MI. Several genes linked to mitochondrial function, metabolism, and cardiomyocyte structural proteins were severely downregulated in the ischemic NRK-2 KO hearts. Analysis revealed significantly upregulated ECM-related pathways which was accompanied by the upregulation of several key cell signaling pathways including SMAD, MAPK, cGMP, integrin, and Akt in the KO heart post-MI. Metabolomic studies identified profound upregulation of metabolites mevalonic acid, 3,4-dihydroxyphenylglycol, 2-penylbutyric acid, and uridine. However, other metabolites stearic acid, 8,11,14-eicosatrienoic acid, and 2-pyrrolidinone were significantly downregulated in the ischemic KO hearts. Taken together, these findings suggest that NRK-2 promotes metabolic adaptation in the ischemic heart. The aberrant metabolism in the ischemic NRK-2 KO heart is largely driven by dysregulated cGMP and Akt and mitochondrial pathways. Key messages: Post-myocardial infarction metabolic switch critically regulates the pathogenesis of adverse cardiac remodeling and heart failure. Here, we report NRK-2 as a novel regulator of several cellular processes including metabolism and mitochondrial function post-MI. NRK-2 deficiency leads to downregulation of genes important for mitochondrial pathway, metabolism, and cardiomyocyte structural proteins in the ischemic heart. It was accompanied by upregulation of several key cell signaling pathways including SMAD, MAPK, cGMP, integrin, and Akt and dysregulation of numerous metabolites essential for cardiac bioenergetics. Taken together, these findings suggest that NRK-2 is critical for metabolic adaptation of the ischemic heart. [ABSTRACT FROM AUTHOR]

The role of store‐operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV‐dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV‐induced cholesterol biosynthesis was critical for UV‐induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV‐dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis‐dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis. Synopsis: The role of STIM/Orai‐mediated store‐operated Ca2+ entry (SOCE) in melanoma is debated. This work reports a novel signaling axis linking UV exposure to cholesterol biosynthesis and suppression of SOCE, promoting aberrant cancer metabolism and invasive phenotypes.UV radiation suppresses SOCE and enhances invasiveness of melanoma cell lines in vivo.STIM‐Orai inhibition is sufficient to drive metastasis in syngeneic mice.Increased cholesterol biosynthesis is required for UV‐induced SOCE suppression and melanoma metastasis.UV‐induced SOCE reduction enhances glucose uptake and anabolic O‐GlcNAcylation, shifting melanoma cells to a more invasive phenotype.Cholesterol‐mediated SOCE suppression and increased O‐GlcNAcylation are conserved in melanoma patient explants. [ABSTRACT FROM AUTHOR]

The discovery of MICU1 as gatekeeper of mitochondrial calcium (m Ca2+) entry has transformed our understanding of m Ca2+ flux. Recent studies revealed an additional role of MICU1 as a Ca2+ sensor at MICOS (mitochondrial contact site and cristae organizing system). MICU1's presence at MICOS suggests its involvement in coordinating Ca2+ signaling and mitochondrial ultrastructure. Besides its role in Ca2+ regulation, MICU1 influences cellular signaling pathways including transcription, epigenetic regulation, metabolism, and cell death, thereby affecting human health. Here, we summarize recent findings on MICU1's canonical and noncanonical functions, and its relevance to human health and diseases. • MICU1, a mitochondrial Ca2+ sensor, plays a key role in decoding cytoplasmic Ca2+ signals. • Through the mtCU, MICU1 modulates mitochondrial Ca2+ influx. • MICU1 functions extend beyond mtCU, impacting mitochondrial ultrastructure via MICOS. • Loss of MICU1 leads to bioenergetics failure and increased susceptibility to various cell death pathways. • MICU1 mutations are linked to neuromuscular, cognitive, and cardiac impairments. [ABSTRACT FROM AUTHOR]

Mitochondria serve as a hub for many cellular processes, including bioenergetics, metabolism, cellular signaling, redox balance, calcium homeostasis, and cell death. The mitochondrial proteome includes over a thousand proteins, encoded by both the mitochondrial and nuclear genomes. The majority (~99%) of proteins are nuclear encoded that are synthesized in the cytosol and subsequently imported into the mitochondria. Within the mitochondria, polypeptides fold and assemble into their native functional form. Mitochondria health and integrity depend on correct protein import, folding, and regulated turnover termed as mitochondrial protein quality control (MPQC). Failure to maintain these processes can cause mitochondrial dysfunction that leads to various pathophysiological outcomes and the commencement of diseases. Here, we summarize the current knowledge about the role of different MPQC regulatory systems such as mitochondrial chaperones, proteases, the ubiquitin-proteasome system, mitochondrial unfolded protein response, mitophagy, and mitochondria-derived vesicles in the maintenance of mitochondrial proteome and health. The proper understanding of mitochondrial protein quality control mechanisms will provide relevant insights to treat multiple human diseases. [ABSTRACT FROM AUTHOR]

Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin binding protein, predominantly expresses in skeletal muscle with a trace amount expressed in healthy cardiac tissue. NRK-2 expression dramatically increases in mouse and human ischemic heart however, the specific role of NRK-2 in the pathophysiology of ischemic cardiac diseases is unknown. We employed NRK2 knockout (KO) mice to identify the role of NRK-2 in ischemia-induced cardiac remodeling and dysfunction. Following myocardial infarction (MI), or sham surgeries, serial echocardiography was performed in the KO and littermate control mice. Cardiac contractile function rapidly declined and left ventricular interior dimension (LVID) was significantly increased in the ischemic KO vs. control mice at 2 weeks post-MI. An increase in mortality was observed in the KO vs. control group. The KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric analysis. Consistently, histological assessment revealed an extensive and thin scar and dilated LV chamber accompanied with elevated fibrosis in the KOs post-MI. Mechanistically, we observed that loss of NRK-2 enhanced p38α activation following ischemic injury. Consistently, ex vivo studies demonstrated that the gain of NRK-2 function suppresses the p38α as well as fibroblast activation (α-SMA expression) upon TGF-β stimulation, and limits cardiomyocytes death upon hypoxia/re‑oxygenation. Collectively our findings show, for the first time, that NRK-2 plays a critical role in heart failure progression following ischemic injury. NRK-2 deficiency promotes post-MI scar expansion, rapid LV chamber dilatation, cardiac dysfunction and fibrosis possibly due to increased p38α activation. • NRK-2 expression increases several fold in both mouse and human ischemic hearts. • NRK-2 protects heart from dilatative cardiac remodeling post-ischemia. • Loss of NRK-2 promotes post-MI scar expansion and thinning, fibrosis, LV dilatation, and heart failure. • Gain of NRK-2 function limits hypoxia re-oxygenation -induced cardiomyopathy. • NRK-2 specifically inhibits p38α activation to protect heart from ischemic insult. [ABSTRACT FROM AUTHOR]

Pulmonary emphysema is characterized by alveolar type II (ATII) cell death, destruction of alveolar wall septa, and irreversible airflow limitation. Cigarette smoke induces oxidative stress and is the main risk factor for this disease development. ATII cells isolated from nonsmokers, smokers, and patients with emphysema were used for this study. ATII cell apoptosis in individuals with this disease was detected. DJ-1 and S100A8 have cytoprotective functions against oxidative stress-induced cell injury. Reduced DJ-1 and S100A8 interaction was found in ATII cells in patients with emphysema. The molecular function of S100A8 was determined by an analysis of the oxidation status of its cysteine residues using chemoselective probes. Decreased S100A8 sulfination was observed in emphysema patients. In addition, its lower levels correlated with higher cell apoptosis induced by cigarette smoke extract in vitro. Cysteine at position 106 within DJ-1 is a central redox-sensitive residue. DJ-1 C106A mutant construct abolished the cytoprotective activity of DJ-1 against cell injury induced by cigarette smoke extract. Furthermore, a molecular and complementary relationship between DJ-1 and S100A8 was detected using gain- and loss-of-function studies. DJ-1 knockdown sensitized cells to apoptosis induced by cigarette smoke extract, and S100A8 overexpression provided cytoprotection in the absence of DJ-1. DJ-1 knockout mice were more susceptible to ATII cell apoptosis induced by cigarette smoke compared with wild-type mice. Our results indicate that the impairment of DJ-1 and S100A8 function may contribute to cigarette smoke-induced ATII cell injury and emphysema pathogenesis [ABSTRACT FROM AUTHOR]

Background: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology.Methods: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex.Results: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa2+ uptake impair mitochondrial energetics and contractile function.Conclusions: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology. [ABSTRACT FROM AUTHOR]

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure. [ABSTRACT FROM AUTHOR]

JC virus encodes an important regulatory protein, known as Agnoprotein (Agno). We have recently reported Agno's first protein-interactome with its cellular partners revealing that it targets various cellular networks and organelles, including mitochondria. Here, we report further characterization of the functional consequences of its mitochondrial targeting and demonstrated its co-localization with the mitochondrial networks and with the mitochondrial outer membrane. The mitochondrial targeting sequence (MTS) of Agno and its dimerization domain together play major roles in this targeting. Data also showed alterations in various mitochondrial functions in Agno-positive cells; including a significant reduction in mitochondrial membrane potential, respiration rates and ATP production. In contrast, a substantial increase in ROS production and Ca2+ uptake by the mitochondria were also observed. Finally, findings also revealed a significant decrease in viral replication when Agno MTS was deleted, highlighting a role for MTS in the function of Agno during the viral life cycle. • Agno is one of the critical regulatory proteins of JC virus and plays important roles during the JC virus life cycle. • Agno targets mitochondria through its MTS and dimerization domain; and negatively affects mitochondrial functions. • Replication efficiency of JCV is significantly reduced when the Agno MTS is deleted. [ABSTRACT FROM AUTHOR]

The alveolus participates in gas exchange, which can be impaired by environmental factors and toxins. There is an increase in using electronic cigarettes (e-cigarettes); however, their effect on human primary alveolar epithelial cells is unknown. Human lungs were obtained from nonsmoker organ donors to isolate alveolar type II (ATII) cells. ATII cells produce and secrete pulmonary surfactant and restore the epithelium after damage, and mitochondrial function is important for their metabolism. Our data indicate that human ATII cell exposure to e-cigarette aerosol increased IL-8 levels and induced DNA damage and apoptosis. We also studied the cytoprotective effect of DJ-1 against ATII cell injury. DJ-1 knockdown in human primary ATII cells sensitized cells to mitochondrial dysfunction as detected by high mitochondrial superoxide production, decreased mitochondrial membrane potential, and calcium elevation. DJ-1 knockout (KO) mice were more susceptible to ATII cell apoptosis and lung injury induced by e-cigarette aerosol compared with wild-type mice. Regulation of the oxidative phosphorylation (OXPHOS) is important for mitochondrial function and protection against oxidative stress. Major subunits of the OXPHOS system are encoded by both nuclear and mitochondrial DNA. We found dysregulation of OXPHOS complexes in DJ-1 KO mice after exposure to e-cigarette aerosol, which could disrupt the nuclear/mitochondrial stoichiometry, resulting in mitochondrial dysfunction. Together, our results indicate that DJ-1 deficiency sensitizes ATII cells to damage induced by e-cigarette aerosol leading to lung injury. [ABSTRACT FROM AUTHOR]

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress‐induced cardiac injury remain unclear. Here, the role of proline‐rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H2O2 resulted in 10‐ to 21‐fold increases in Pyk2 phosphorylation in wild‐type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2‐mediated Ca2+ increase with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca2+ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca2+ uptake and enhance adenosine triphosphate generation. Although Trpm2‐mediated Ca2+ influx phosphorylated Ca2+‐calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H2O2‐treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO‐I/R when compared with WT‐I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria‐targeted superoxide scavenger MitoTempo. Our results suggested that Ca2+ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury. [ABSTRACT FROM AUTHOR]

In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (I Ca) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and I Ca amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on I Ca and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism. [ABSTRACT FROM AUTHOR]

Introduction: Recent studies demonstrated that plasma exosomes from diabetic patients and animals lose their ability to maintain angiogenetic properties of endothelial progenitor cells (EPCs)/endothelial cells. However, the underlying mechanisms remain incompletely understood. Hypothesis: Systemic blocking of exosome synthesis/release improves ischemic hindlimb repair in diabetes. Methods: Unilateral hindlimb ischemia (HLI) surgery was conducted by ligation of left femoral artery in 12-week male db/db and db/+ mice which were administrated with either exosome synthesis inhibitor GW4869 (GW, 2 mg/kg body weight, i.p) or vehicle for 4 weeks starting from one week before HLI. Blood perfusion in ischemic hindlimbs was examined by Laser Doppler at pre-HLI, and 0, 3, 7, 14 and 21 days post-HLI. Plasma exosomes were isolated and analyzed by NanoSight. Results: Administration of GW increased blood concentration of CD31+/Sca-1+ cell, decreased cell death in the muscles of ischemic hindlimbs of db/db mice 3-day post-HLI. GW also decreased necrosis and loss of toe/toenail; improved blood flow; enhanced capillary/arterial density determined by CD31 and SMA-α staining and decreased fibrosis in the muscles of ischemic hindlimbs in db/db mice 21-day post-HLI. Plasma exosomes from db/db mice significantly impaired human cardiac microvascular endothelial cells (HCMVECs) tube formation and migration. Mechanistically, diabetic plasma exosomes increased mitochondria proton leak and mitochondrial uncoupling protein-2 (UCP-2) which allows a more rapid electron flux thus reducing membrane potential resulting in reduced reactive oxygen species (ROS) production creating proton leaks across the inner mitochondria membrane to 3.34-fold in HCMVECs. Diabetic plasma exosomes also decreased superoxide dismutase 1 (SOD1), a major antioxidase levels in mitochondria, and mitoROS in HCMVECs. Finally, RNA-seq studies identified highly enriched exosomal microRNA-17 which may directly target SOD1. Conclusions: Systemic inhibition of exosome synthesis/release by GW improved ischemic limb repair in diabetic db/db mice. Mechanistically, although preliminary data, increased mitochondria proton leak/exosome-curried miR-17-induced downregulation of SOD1/mitochondria oxidative stress cascade, at least partially, is involved in plasma exosomes-impaired angiogenic property in diabetes. [ABSTRACT FROM AUTHOR]

The pathophysiology of human immunodeficiency virus (HIV)‐associated cardiomyopathy remains uncertain. We used HIV‐1 transgenic (Tg26) mice to explore mechanisms by which HIV‐related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨm) under normoxic conditions but lower Δ Ψm after hypoxia/reoxygenation (H/R). In addition, Δ Ψm in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post‐H/R, mitochondrial superoxide (O 2•–) levels were higher in Tg26 compared to WT myocytes. Overexpression of B‐cell lymphoma 2‐associated athanogene 3 (BAG3) reduced O 2•– levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post‐H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post‐H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post‐H/R, Δ Ψm was lower, mitochondrial O 2•– levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2•– levels and restored contraction amplitudes to normal in Tg26 myocytes post‐H/R in the presence of isoproterenol. The hemizygous NL4‐3 Δgag/pol transgenic (Tg26) mouse with replication‐deficient, noninfectious human immunodeficiency virus type 1 (HIV‐1) but with HIV‐1 proteins present in tissues simulates contemporary HIV‐1‐infected patients treated with combination antiretroviral therapy and is an excellent model to study chronic complications of HIV‐1 infection such as HIV‐associated cardiomyopathy. Cardiac myocytes isolated from Tg26 mice demonstrate mitochondrial dysfunction and elevated reactive oxygen species (ROS) that are exacerbated by hypoxia/reoxygenation and adrenergic stress. B‐cell lymphoma 2‐associated athanogene 3 (BAG3) regulates autophagy, apoptosis, excitation‐contraction coupling, mitochondrial quality control, and sarcomere stability in cardiac myocytes and can associate with the HIV‐1 protein Tat. Overexpression of BAG3 in Tg26 myocytes restores the elevated ROS levels to normal and improves contractile dysfunction. BAG3 may be a useful therapeutic modality in the treatment of HIV‐associated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Abstract Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular metabolic health, we tested if the synthetic anti-oxidant lignan secoisolariciresinol diglucoside (SDG; LGM2605) would alleviate septic cardiac dysfunction and investigated the underlying mechanism. Using the cecal ligation and puncture (CLP) mouse model of peritonitis-induced sepsis, we observed impairment of cardiac function beginning at 4 h post-CLP surgery. Treatment of mice with LGM2605 (100 mg/kg body weight, i.p.) 6 h post-CLP surgery reduced cardiac ROS accumulation and restored cardiac function. Assessment of mitochondrial respiration (Seahorse XF) in primary cardiomyocytes obtained from adult C57BL/6 mice that had undergone CLP and treatment with LGM2605 showed restored basal and maximal respiration, as well as preserved oxygen consumption rate (OCR) associated with spare capacity. Further analyses aiming to identify the cellular mechanisms that may account for improved cardiac function showed that LGM2605 restored mitochondria abundance , increased mitochondrial calcium uptake and preserved mitochondrial membrane potential. In addition to protecting against cardiac dysfunction, daily treatment with LGM2605 and antibiotic ertapenem (70 mg/kg) protected against CLP-associated mortality and reversed hypothermia when compared against mice receiving ertapenem and saline. Therefore, treatment of septic mice with LGM2605 emerges as a novel pharmacological approach that reduces cardiac ROS accumulation, protects cardiac mitochondrial function, alleviates cardiac dysfunction, and improves survival. Highlights • Sepsis results in progressive cardiac dysfunction associated with oxidative stress. • ROS formation underlies mitochondrial dysfunction in septic cardiomyopathy. • Antioxidant LGM2605 scavenges ROS and alleviates mitochondrial dysfunction. • LGM2605 treats septic cardiac dysfunction and improves survival. [ABSTRACT FROM AUTHOR]

The physiological cardiac hypertrophy is an adaptive condition without myocyte cell death, while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. This study explores the miRNome of α-2M-induced physiologically hypertrophied cardiomyocytes and the role of miRNA-99 family during cardiac hypertrophy. Physiological and pathological cardiac hypertrophy was induced in H9c2 cardiomyoblast cell lines using α-2M and isoproterenol respectively. Total RNA isolation and small RNA sequencing were executed for physiological hypertrophy model. The differentially expressed miRNAs and its target mRNAs were validated in animal models. Transcription factor binding sites were predicted in the promoter of specific miRNAs and validated by ChIP-PCR. Subsequently, the selected miRNA was functionally characterized by overexpression and silencing. The effects of silencing of upstream regulator and downstream target gene were studied. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy, of which miR-99 family was highly downregulated upon α-2M treatment. However, this miR-99 family expression was upregulated during pathological hypertrophy and confirmed in animal models. ChIP-PCR confirms the binding of Egr-1 transcription factor to the miR-99 promoter. Further, silencing of Egr-1 decreased the expression of miR-99. The overexpression or silencing of miR-99 diverges the physiological hypertrophy to pathological hypertrophy and vice versa by regulating Akt-1 pathway. Silencing of Akt-1 replicates the effect of overexpression of miR-99. Conclusion The results proved Egr-1 mediated regulation of miR-99 family that plays a key role in determining the fate of cardiac hypertrophy by regulating Akt-1 signaling. [ABSTRACT FROM AUTHOR]

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 wasa major species in this transfer.PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition byPMPs.Conversely, Par4-deleted mice, whichhad reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability. [ABSTRACT FROM AUTHOR]

Summary Ca 2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca 2+ ] m uptake rate, elevated mROS, and enhanced [Ca 2+ ] m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity. [ABSTRACT FROM AUTHOR]

The modulation of mitochondrial functions is important for maintaining cellular homeostasis. Mitochondria essentially depend on the import of RNAs and proteins encoded by the nuclear genome. MicroRNAs encoded in the nucleus can translocate to mitochondria and target the genome, affecting mitochondrial function. Here, we analyzed the role of miR-4485 in the regulation of mitochondrial functions. We showed that miR-4485 translocated to mitochondria where its levels varied in response to different stress conditions. A direct binding of miR-4485 to mitochondrial 16S rRNA was demonstrated. MiR-4485 regulated the processing of pre-rRNA at the 16S rRNA-ND1 junction and the translation of downstream transcripts. MiR-4485 modulated mitochondrial complex I activity, the production of ATP, ROS levels, caspase-3/7 activation, and apoptosis. Transfection of a miR-4485 mimic downregulated the expression of regulatory glycolytic pathway genes and reduced the clonogenic ability of breast cancer cells. Ectopic expression of miR-4485 in MDA-MB-231 breast carcinoma cells decreased the tumorigenicity in a nude mouse xenograft model. Furthermore, levels of both precursor and mature miR-4485 are decreased in tumor tissue of breast cancer patients. We conclude that the mitochondria-targeted miR-4485 may act as a tumor suppressor in breast carcinoma cells by negatively regulating mitochondrial RNA processing and mitochondrial functions. [ABSTRACT FROM AUTHOR]

Summary Calcium (Ca 2+ ) flux into the matrix is tightly controlled by the mitochondrial Ca 2+ uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca 2+ or Mg 2+ destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg 2+ loading or blockade of mitochondrial Ca 2+ extrusion suppresses MCU Ca 2+ -uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg 2+ and Ca 2+ binding. [ABSTRACT FROM AUTHOR]

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na + –K + –ATPase and L-type Ca 2 + channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca 2 + channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca 2 + ] i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca 2 + current (I Ca ) and sarcoplasmic reticulum (SR) Ca 2 + content but not Na + /Ca 2 + exchange current (I NaCa ) or SR Ca 2 + uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored I Ca amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD 50 and APD 90 were prolonged in shBAG3 myocytes. Protein levels of Ca 2 + entry molecules and other important excitation–contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca 2 + channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. [ABSTRACT FROM AUTHOR]

Summary Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨ m ) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca 2+ - and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca 2+ retention, similar to cyclophilin D (CypD, PPIF ) knockdown with sustained ΔΨ m during both Ca 2+ and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca 2+ - and ROS-induced ΔΨ m depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site. [ABSTRACT FROM AUTHOR]

Chronic inflammation in tumor microenvironment plays an important role at different stages of tumor development. The specific mechanisms of the association and its role in providing a survival advantage to the tumor cells are not well understood. Mitochondria are emerging as a central platform for the assembly of signaling complexes regulating inflammatory pathways, including the activation of type-I IFN and NF-κB. These complexes in turn may affect metabolic functions of mitochondria and promote tumorigenesis. NLRX1, a mitochondrial NOD-like receptor protein, regulate inflammatory pathways, however its role in regulation of cross talk of cell death and metabolism and its implication in tumorigenesis is not well understood. Here we demonstrate that NLRX1 sensitizes cells to TNF-α induced cell death by activating Caspase-8. In the presence of TNF-α, NLRX1 and active subunits of Caspase-8 are preferentially localized to mitochondria and regulate the mitochondrial ROS generation. NLRX1 regulates mitochondrial Complex I and Complex III activities to maintain ATP levels in the presence of TNF-α. The expression of NLRX1 compromises clonogenicity, anchorage-independent growth, migration of cancer cells in vitro and suppresses tumorigenicity in vivo in nude mice. We conclude that NLRX1 acts as a potential tumor suppressor by regulating the TNF-α induced cell death and metabolism. [ABSTRACT FROM AUTHOR]