Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ 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 [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]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.

The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability.

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) 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 ΔΨm 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 ΔΨm 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.

Cardiac Metabolism and MiRNA Interference.

The aberrant increase in cardio-metabolic diseases over the past couple of decades has drawn researchers' attention to explore and unveil the novel mechanisms implicated in cardiometabolic diseases. Recent evidence disclosed that the derangement of cardiac energy substrate metabolism plays a predominant role in the development and progression of chronic cardiometabolic diseases. Hence, in-depth comprehension of the novel molecular mechanisms behind impaired cardiac metabolism-mediated diseases is crucial to expand treatment strategies. The complex and dynamic pathways of cardiac metabolism are systematically controlled by the novel executor, microRNAs (miRNAs). miRNAs regulate target gene expression by either mRNA degradation or translational repression through base pairing between miRNA and the target transcript, precisely at the 3' seed sequence and conserved heptametrical sequence in the 5' end, respectively. Multiple miRNAs are involved throughout every cardiac energy substrate metabolism and play a differential role based on the variety of target transcripts. Novel theoretical strategies have even entered the clinical phase for treating cardiometabolic diseases, but experimental evidence remains inadequate. In this review, we identify the potent miRNAs, their direct target transcripts, and discuss the remodeling of cardiac metabolism to cast light on further clinical studies and further the expansion of novel therapeutic strategies. This review is categorized into four sections which encompass (i) a review of the fundamental mechanism of cardiac metabolism, (ii) a divulgence of the regulatory role of specific miRNAs on cardiac metabolic pathways, (iii) an understanding of the association between miRNA and impaired cardiac metabolism, and (iv) summary of available miRNA targeting therapeutic approaches.

Chemically synthesized Secoisolariciresinol diglucoside (LGM2605) improves mitochondrial function in cardiac myocytes and alleviates septic cardiomyopathy.

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.

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation.

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 H2 O2 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 H2 O2 -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.

Mitochondrial dysfunction in human immunodeficiency virus-1 transgenic mouse cardiac myocytes.

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 (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m 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 Ψ m 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 human ion channel TRPM2 modulates neuroblastoma cell survival and mitochondrial function through Pyk2, CREB, and MCU activation.

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca2+-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.

Dysregulation of mitochondrial bioenergetics and quality control by HIV-1 Tat in cardiomyocytes.

Cardiovascular disease remains a leading cause of morbidity and mortality in HIV-positive patients, even in those whose viral loads are well controlled with antiretroviral therapy. However, the underlying molecular events responsible for the development of cardiac disease in the setting of HIV remain unknown. The HIV-encoded Tat protein plays a critical role in the activation of HIV gene expression and profoundly impacts homeostasis in both HIV-infected cells and uninfected cells that have taken up released Tat via a bystander effect. Since cardiomyocyte function, including excitation-contraction coupling, greatly depends on energy provided by the mitochondria, in this study, we performed a series of experiments to assess the impact of Tat on mitochondrial function and bioenergetics pathways in a primary cell culture model derived from neonatal rat ventricular cardiomyocytes (NRVCs). Our results show that the presence of Tat in cardiomyocytes is accompanied by a decrease in oxidative phosphorylation, a decline in the levels of ATP, and an accumulation of reactive oxygen species (ROS). Tat impairs the uptake of mitochondrial Ca2+ ([Ca2+ ]m ) and the electrophysiological activity of cardiomyocytes. Tat also affects the protein clearance pathway and autophagy in cardiomyocytes under stress due to hypoxia-reoxygenation conditions. A reduction in the level of ubiquitin along with dysregulated degradation of autophagy proteins including SQSTM1/p62 and a reduction of LC3 II were detected in cardiomyocytes harboring Tat. These results suggest that, by targeting mitochondria and protein quality control, Tat significantly impacts bioenergetics and autophagy resulting in dysregulation of cardiomyocyte health and homeostasis.

Loss of Adult Cardiac Myocyte GSK-3 Leads to Mitotic Catastrophe Resulting in Fatal Dilated Cardiomyopathy.

RATIONALE: Cardiac myocyte-specific deletion of either glycogen synthase kinase (GSK)-3α and GSK-3ß leads to cardiac protection after myocardial infarction, suggesting that deletion of both isoforms may provide synergistic protection. This is an important consideration because of the fact that all GSK-3-targeted drugs, including the drugs already in clinical trial target both isoforms of GSK-3, and none are isoform specific. OBJECTIVE: To identify the consequences of combined deletion of cardiac myocyte GSK-3α and GSK-3ß in heart function. METHODS AND RESULTS: We generated tamoxifen-inducible cardiac myocyte-specific mice lacking both GSK-3 isoforms (double knockout). We unexpectedly found that cardiac myocyte GSK-3 is essential for cardiac homeostasis and overall survival. Serial echocardiographic analysis reveals that within 2 weeks of tamoxifen treatment, double-knockout hearts leads to excessive dilatative remodeling and ventricular dysfunction. Further experimentation with isolated adult cardiac myocytes and fibroblasts from double-knockout implicated cardiac myocytes intrinsic factors responsible for observed phenotype. Mechanistically, loss of GSK-3 in adult cardiac myocytes resulted in induction of mitotic catastrophe, a previously unreported event in cardiac myocytes. Double-knockout cardiac myocytes showed cell cycle progression resulting in increased DNA content and multinucleation. However, increased cell cycle activity was rivaled by marked activation of DNA damage, cell cycle checkpoint activation, and mitotic catastrophe-induced apoptotic cell death. Importantly, mitotic catastrophe was also confirmed in isolated adult cardiac myocytes. CONCLUSIONS: Together, our findings suggest that cardiac myocyte GSK-3 is required to maintain normal cardiac homeostasis, and its loss is incompatible with life because of cell cycle dysregulation that ultimately results in a severe fatal dilated cardiomyopathy.

Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress.

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca(2+)]i) response in vascular endothelial cells (ECs). [Ca(2+)]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca(2+)]i response is, in part, due to Ca(2+) release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca(2+) store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca(2+)]i in sheared ECs. Cultured ECs, loaded with a Ca(2+)-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca(2+)]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca(2+)]i transients/oscillations were present when experiments were conducted in Ca(2+)-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca(2+)]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca(2+) uniporter also inhibited the shear-induced [Ca(2+)]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca(2+) uptake/release, regulate the temporal profile of shear-induced ER Ca(2+) release. [Ca(2+)]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca(2+)]i response.

TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria.

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

A splice variant of the human ion channel TRPM2 modulates neuroblastoma tumor growth through hypoxia-inducible factor (HIF)-1/2α.

The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy.

Ca²âº entry via Trpm2 is essential for cardiac myocyte bioenergetics maintenance.

Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca(2+) concentration increase after Trpm2 activation was due to direct Ca(2+) influx and not indirectly via reverse Na(+)/Ca(2+) exchange. To elucidate whether Ca(2+) entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2(.-)) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2(.-) levels in gKO myocytes and only in the presence of extracellular Ca(2+), indicating sustained Ca(2+) entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/dt) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/dt. We conclude 1) cardiac Trpm2-mediated Ca(2+) influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca(2+) influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.

LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation.

Dysregulation of mitochondrial Ca(2+)-dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca(2+) transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca(2+) transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca(2+) transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca(2+) influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, (D676A D688K)LETM1 mutant-overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS-derived fibroblasts, the mitochondrial Ca(2+) uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch-clamp current (IMCU) was largely unaffected in LETM1-knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca(2+) transport and bioenergetics. These findings reveal the role of LETM1-dependent mitochondrial Ca(2+) flux in shaping cellular bioenergetics.

Synergistic effect of amphotericin B and tyrosol on biofilm formed by Candida krusei and Candida tropicalis from intrauterine device users.

The presence of intrauterine contraceptive devices (IUDs) provides a solid surface for attachment of microorganisms and an ideal niche for the biofilm to form and flourish. Vaginal candidiasis is often associated with the use of IUDs. Treatment of vaginal candidiasis that develops in connection with IUD use requires their immediate removal. Here, we present in vitro evidence to support the use of combination therapy to inhibit Candida biofilm. Twenty-three clinical Candida isolates (10 C. krusei and 13 C. tropicalis) recovered from endocervical swabs obtained from IUD and non-IUD users were assessed for biofilm-formation ability. The rate of isolation of Candida did not differ significantly among IUD and non-IUD users (P = 0.183), but the biofilm-formation ability of isolates differed significantly (P = 0.02). An in vitro biofilm model with the obtained isolates was subjected to treatment with amphotericin B, tyrosol, and a combination of amphotericin B and tyrosol. Inhibition of biofilm by amphotericin B or tyrosol was found to be concentration dependent, with 50% reduction (P < 0.05) at 4 mg/l and 80 µM, respectively. Hence, a combination effect of tyrosol and amphotericin B was studied. Interestingly, approximately 90% reduction in biofilm was observed with use of 80 µM tyrosol combined with 4 mg/l amphotericin B (P < 0.001). This represents a first step in establishing an appropriate antibiofilm therapy when yeasts are present.

Mrs2-mediated mitochondrial magnesium uptake is essential for the regulation of MCU-mediated mitochondrial Ca2+ uptake and viability.

Mitochondrial Ca2+ uptake is essential in regulating bioenergetics, cell death, and cytosolic Ca2+ transients. Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here, we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed that decreased matrix [Mg2+] is associated with increased MCU activity and significantly prompted mitochondrial permeability transition pore opening. Our findings support the critical role of mMg2+ in regulating MCU activity.

BRCA1 and BRCA2 mutations in the ovarian cancer population across race and ethnicity: special reference to Asia.

OBJECTIVE: To evaluate the prevalence and spectrum of BRCA mutations among ovarian carcinoma patients of different races and ethnicity with special reference to Asia. METHODS: A systematic review of the literature was undertaken to evaluate the prevalence of BRCA mutations among people belonging to different races. The electronic search strategy was developed specifically for the different databases concerned and via cross-referencing. RESULTS: The frequency of BRCA1 and BRCA2 mutations ranged from 1.1 to 39.7 and from 0 to 13.9, respectively. BRCA1 mutations are more common among ovarian cancer cases than BRCA2 mutations, although the ratio of BRCA1 to BRCA2 varies between populations. The Swedish and Indian populations showed 12 and 7 times as many BRCA1 as BRCA2 mutations, respectively, whilst in a study from Iceland the ratio was 0.5:1. These wide-ranging estimates of the mutation prevalence suggest genetic heterogeneity between different populations. CONCLUSION: The ability to identify BRCA1/2 mutations was found to be successful in the clinical management of ovarian cancer. Given the implications for clinical care and for advances in cancer prevention, identifying racial difference in genetic or lifestyle factors, which may modify the cancer risk due to BRCA1/2 mutations, is a high priority for future research.

Polymorphism and overexpression of HER2/neu among ovarian carcinoma women from Tiruchirapalli, Tamil Nadu, India.

PURPOSE: Alteration and overexpression of HER2 proto-oncogene have been implicated in the carcinogenesis and prognosis of ovarian cancer. We evaluated this hypothesis among women with ovarian carcinoma patients from Tiruchirapalli, Tamil Nadu, India. METHODS: Genomic DNA was extracted from 72 case patients and 288 control subjects and was examined for I655V polymorphism by PCR-RFLP based assay. Immunohistochemistry analysis was carried out in order to study the overexpression of HER2 protein. The observed number of each genotype was compared with that expected for a population in the Hardy-Weinberg equilibrium. In analysing the relation between genotype and overexpression of HER2 protein, Cochran-Mantel-Haenszel statistics was used. RESULTS: We found that 20.8% of the case patients and 16.3% of the control subjects were heterozygous for the Val allele and 10 case patients (13.8%) and 3 control subjects (1.1%) were homozygous for this allele (P < 0.001). Compared with women with Ile/Ile genotype, women with Val/Val genotype had an elevated risk of ovarian cancer. The genotype distributions were consistent with the Hardy-Weinberg equilibrium. The risk increased with the number of Val allele and women homozygous for the Val allele had 15-fold (OR = 15.3; 95%CI = 4.09-57.31) increased risk of cancer. The patients homozygous for the Valine allele showed strong HER2 protein expression. CONCLUSION: The results showed that the valine allele may be an indicator of genetic susceptibility to ovarian carcinoma in the study population.

Mitochondrial Magnesium is the cationic rheostat for MCU-mediated mitochondrial Ca2+ uptake.

Calcium (Ca2+) uptake by mitochondria is essential in regulating bioenergetics, cell death, and cytosolic Ca2+ transients. Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. MCU is a heterooligomeric complex with a pore-forming component and accessory proteins required for channel activity. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed Mrs2 as the authentic mammalian mitochondrial Mg2+ channel using the planar lipid bilayer recordings. Using a liver-specific Mrs2 KO mouse model, we showed that decreased matrix [Mg2+] is associated with increased MCU activity and matrix Ca2+ overload. The disruption of Mg2+dependent MCU regulation significantly prompted mitochondrial permeability transition pore opening-mediated cell death during tissue IR injury. Our findings support a critical role for mMg2+ in regulating MCU activity and attenuating mCa2+ overload.