Santhanam Shanmughapriya

The Mitochondrial Calcium Uniporter Matches Energetic Supply with Cardiac Workload during Stress and Modulates Permeability Transition

Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca(2+)), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu(-/-) mice display no overt baseline phenotype and are protected against mCa(2+) overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu(-/-) mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca(2+)-dependent metabolism during acute stress.

Antimicrobial activity of seaweeds extracts against multiresistant pathogens

Fourteen seaweeds collected from the intertidal zone of Southwest coast of India were tested against ten human pathogen bacteria and one human pathogen fungus using the well diffusion test in the casitone agar medium. The species used in the present study include five Chlorophyta (Bryopsis plumosa, Ulva fasciata, Acrosiphonia orientalis, Chaetomorpha antennina, Grateloupia filicina), five Rhodophyta (Hypnea pannosa, Gracilaria corticata, Centroceras clavulatum, Portieria hornemannii, Cheilosporum spectabile) and four Phaeophyta (Padina tetrastromatica, Sargassum wightii, Stocheospermum marginatum, Chnoospora bicanaliculata). Of these, seven species were determined to be highly bioactive and screened on the multiresistant pathogens. We found that drying process has eliminated the active principles in the seaweeds. In the present study, methanol:toluene (3∶1) was found to be the best solvent for extracting the antimicrobial principles from fresh algae. However, the ethanolic extract showed no antibacterial activity.Acrosiphonia orientalis showed activity against 70% of the tested organisms.Stocheospermum marginatum was the only seaweed that showed activity againstKlebsiella pneumoniae. The extract fromGracilaria corticata was highly active againstProteus mirabilis, a Gram negative pathogenic bacterium. The present findings revealed that the tested seaweeds were highly active against Gram negative bacteria than Gram positive bacteria. The antimicrobial principle from seaweed was found to be a lipophilic compound. The compound was stable over a wide range of temperature (30–60 °C). The active principles of highly active seaweedsAcrosiphonia orientalis andStocheospermum marginatum were bactericidal.

Production and characterization of lipopeptide biosurfactant by a sponge-associated marine actinomycetes Nocardiopsis alba MSA10.

A sponge-associated marine actinomycetes Nocardiopsis alba MSA10 was screened and evaluated for the production of biosurfactant. Biosurfactant production was confirmed by conventional screening methods including hemolytic activity, drop collapsing test, oil displacement method, lipase production and emulsification index. The active compound was extracted with three solvents including ethyl acetate, diethyl ether and dichloromethane. The diethyl ether extract was fractionated by TLC and semi-preparative HPLC to isolate the pure compound. In TLC, a single discrete spot was obtained with the Rf 0.60 and it was extrapolated as valine. Based on the chemical characterization, the active compound was partially confirmed as lipopeptide. The optimum production was attained at pH 7, temperature 30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. Considering the biosurfactant production potential of N. alba, the strain could be developed for large-scale production of lipopeptide biosurfactant.

SLC25A23 augments mitochondrial Ca2+ uptake, interacts with MCU, and induces oxidative stress–mediated cell death

Knockdown of SLC25A23 decreases mitochondrial Ca2+ uptake, and SLC25A23 interacts with MCU and MICU1, components of mitochondrial Ca2+ uniporter. Expression of SLC25A23 EF-hand-domain mutants has a dominant-negative phenotype of reduced mitochondrial Ca2+ uptake. It also attenuates basal ROS and oxidant-induced ATP decline and cell death.

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury ROLE OF MITOCHONDRIA

Background: TRPM2 channels are present in the heart, but their function is unknown. Results: Genetic ablation of TRPM2 results in cardiac mitochondrial dysfunction, enhanced ROS production, and exacerbated cardiac ischemic injury. Conclusion: TRPM2 channels preserve cardiac mitochondrial bioenergetics and protect cardiac myocytes from ischemic injury. Significance: TRPM2 is a rational target for treatment of ischemic heart disease. 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 Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ 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.

Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU

Calcium signaling stimulates the accumulation of the mitochondrial calcium uniporter to regulate mitochondrial metabolism. Maintaining mitochondrial calcium uptake The calcium uniporter complex, which includes the protein MCU, mediates mitochondrial calcium uptake, a process that buffers excess cytosolic calcium and regulates mitochondrial metabolism. Shanmughapriya et al. examined mitochondrial calcium uptake and function in a B lymphocyte cell line deficient in one or more proteins necessary for mediating two types of calcium signals—IICR, calcium released from the endoplasmic reticulum through the calcium-permeable IP3 receptors, and SOCE, calcium influx through store-operated calcium channels. Without IICR or SOCE, the activity of the transcription factor CREB, which bound to the MCU promoter, and the expression and abundance of MCU were reduced, mitochondrial calcium uptake was compromised, and mitochondrial metabolism was altered. Cells deficient in IICR or SOCE lacked an oscillating basal calcium signal. Thus, IICR and SOCE control the capacity of mitochondria to uptake calcium and therefore regulate mitochondrial metabolism. Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB (cyclic adenosine monophosphate response element–binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

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

Dysregulation of mitochondrial Ca2+‐dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca2+ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1‐mediated Ca2+ transport remains unresolved. This study examines LETM1‐mediated mitochondrial Ca2+ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf‐Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca2+ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, D676A D688KLETM1 mutant‐overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS‐derived fibroblasts, the mitochondrial Ca2+ 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 Ca2+ transport and bioenergetics. These findings reveal the role of LETM1‐dependent mitochondrial Ca2+ flux in shaping cellular bioenergetics.—Doonan, P J., Chandramoorthy, H. C., Hoffman, N. E., Zhang, X., Cárdenas, C., Shanmughapriya, S., Rajan, S., Vallem, S., Chen, X., Foskett, J. K., Cheung, J. Y., Houser, S. R., Madesh, M., LETM1‐dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation. FASEB J. 28, 4936–4949 (2014). www.fasebj.org

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.

Prevalence and risk factors of HPV infection among women from various provinces of the world

ObjectiveWe set to estimate the genotype-specific prevalence of human papilloma virus (HPV) and its associated risk factors responsible among women with normal and abnormal cytology by systematic literature survey.MethodsReports on HPV prevalence published between 2000 and 2011 were retrieved. To be included, studies required information on cervical cytology, plus detailed descriptions of study populations, methods used to collect cervical samples, and assays used for HPV DNA detection and typing. Final analyses included 280 studies of which 120 were included in the final analysis. The OR, 95% CI and P value were calculated using SPSS 16.0.ResultsOverall HPV prevalence in 576,281 women was estimated to be 32.1% (95% CI 32.098, 32.102). Corresponding estimates by region showed Eastern Asia (China) with the highest prevalence of about 57.7% of the HPV infection followed by South Central Asia (Indian subcontinent). The HPV prevalence was higher in less developed countries (42.2%) than in more developed regions (22.6%). The type-specific HPV prevalence study showed HPV 16 (9.5%) and 18 (6.2%) to be the prevalent type irrespective of the region of study. First coitus at a younger (≤15) age, increased number of pregnancies, increased number of sexual partners, use of contraceptives, smoking and chewing habit and early age at marriage were recognized as the significant risk factors for HPV infection. The phylogenetic analysis of HPV-16 showed the clustering of Indian sequence with the European and American sequences suggesting a similarity between HPV types. Even though the oncogenic proteins of HPV-16 detected in more developed and less developed regions clustered, the prevalence and the severity of the diseases among the less developed regions could be well explained as the exposure of the population to the possible associated risk factors concerning to the living conditions and nature of the life style adopted by the population.