Abstract 409: Investigating the Role of the Mitochondrial Calcium Uniporter (mcu) in Calcium Regulation of Cardiac Mitochondria Using Transmural Spectroscopy and an Integrating Sphere

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Anna Kosmach ◽  
Junhui Sun ◽  
Armel Femnou ◽  
Robert S Balaban ◽  
Elizabeth Murphy
Keyword(s):  
White Light ◽  
Mitochondrial Outer Membrane ◽  
Integrating Sphere ◽  
Mitochondrial Calcium ◽  
Mouse Heart ◽  
Light Sources ◽  
Cardiac Mitochondria ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-2-AM at 30°C and then perfused at 37°C to washout uncleaved dye. We determined that rhod-2 localized primarily to the mitochondria under our loading conditions, as shown by loading in a heart expressing a GFP-tagged mitochondrial outer membrane protein and analyzing heart slices under super resolution microscopy. Further, addition of Mn 2+ , which quenches cytosolic rhod-2, has little effect on rhod-2 signal. We insert an optical catheter with both white light and 532nm laser into the left ventricle and interleaved the collection of spectra from both light sources. The perfused heart is center mounted in an integrating sphere for spectra collection. The light passes through the ventricle and reflects off the integrating sphere resulting in a near uniform sampling of the transmitted light. Spectral properties from both light sources are determined using a rapid scanning spectrophotometer. Myoglobin oxygenation, cytochrome redox state, and rhod-2 loading are determined by white light absorbance. Ca 2+ bound rhod-2 emission is determined by removing background tissue effects from laser emission spectra and normalizing to tissue absorbance. Using this method we are able to measure changes in Ca 2+ and cytochromes during treatments such as isoproterenol and ischemia-reperfusion. The use of an integrating sphere transmural spectroscopy provides us an unique method to study mitochondrial Ca 2+ signaling in perfused mouse heart loaded with Rhod-2.

scholarly journals The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter

2013 ◽  
Vol 15 (12) ◽  
pp. 1464-1472 ◽  
Author(s):  
Xin Pan ◽  
Jie Liu ◽  
Tiffany Nguyen ◽  
Chengyu Liu ◽  
Junhui Sun ◽  
...  
Keyword(s):  
Physiological Role ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

scholarly journals Actin enhances ER-mitochondrial calcium transfer and IMM constriction during mitochondrial division

2017 ◽  
Author(s):  
Rajarshi Chakrabarti ◽  
Wei-Ke Ji ◽  
Radu V. Stan ◽  
Jaime de Juan Sanz ◽  
Timothy A. Ryan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Mitochondrial Calcium ◽  
Mitochondrial Membranes ◽  
Independent Manner ◽  
Mitochondrial Division ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Transport Chain

SummaryMitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through the ER-bound formin INF2 stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

scholarly journals MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Mayara S. Bertolini ◽  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Anibal E. Vercesi ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Cell Lines ◽  
Citrate Synthase ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Content Type ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Eukaryotic Supergroup

ABSTRACT The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

scholarly journals Mitochondrial Calcium Uniporter Deficiency in Zebrafish Causes Cardiomyopathy With Arrhythmia

2020 ◽  
Vol 11 ◽  
Author(s):  
Adam D. Langenbacher ◽  
Hirohito Shimizu ◽  
Welkin Hsu ◽  
Yali Zhao ◽  
Alexandria Borges ◽  
...  
Keyword(s):  
Energy Production ◽  
Mitochondrial Calcium ◽  
Sinus Arrest ◽  
Compact Layer ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Trabecular Density ◽  
And Function ◽  
Production Cell

Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.

scholarly journals Exploring the In Vivo Role of the Mitochondrial Calcium Uniporter in Brown Fat Bioenergetics

Cell Reports ◽  
2019 ◽  
Vol 27 (5) ◽  
pp. 1364-1375.e5 ◽  
Author(s):  
Daniel Flicker ◽  
Yasemin Sancak ◽  
Eran Mick ◽  
Olga Goldberger ◽  
Vamsi K. Mootha
Keyword(s):  
Brown Fat ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

scholarly journals Increased mitochondrial calcium uniporter in adipocytes underlies mitochondrial alterations associated with insulin resistance

2017 ◽  
Vol 313 (6) ◽  
pp. E641-E650 ◽  
Author(s):  
Lauren E. Wright ◽  
Denis Vecellio Reane ◽  
Gabriella Milan ◽  
Anna Terrin ◽  
Giorgia Di Bello ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Calcium Uptake ◽  
Genetic Manipulation ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Insulin Resistant ◽  
Calcium Uniporter ◽  
Obesity And Diabetes ◽  
Mitochondrial Calcium Uptake

Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the mitochondrial calcium uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca2+ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analyzed from several models of obese and diabetic rodents and in 72 patients with obesity as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca2+]mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and reactive oxygen species formation. Finally, our data suggest a strong relationship between [Ca2+]mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes and may be associated with the differential metabolic profiles of VAT and SAT.

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