Mitochondrial Morphology Regulates Organellar Ca2+ Uptake and Changes Cellular Ca2+ Homeostasis

AbstractChanges in mitochondrial size and shape have been implicated in several physiological processes, but their role in mitochondrial Ca2+ uptake regulation and overall cellular Ca2+ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics towards increased fusion through expression of a dominant negative form of the fission protein DRP1 (DRP1‐DN) markedly increased both mitochondrial Ca2+ retention capacity and Ca2+ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion‐promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin 2 (MFN2) strongly reduced mitochondrial Ca2+ uptake speed and capacity in these cells. These changes were not dependent on modifications in inner membrane potentials or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as measured by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium‐replenishing process of store‐operated calcium entry (SOCE) was impaired in MFN2 knockdown cells, while DRP1‐DN‐promoted fusion resulted in faster cytosolic Ca2+ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca2+ uptake, which impacts on cellular Ca2+ homeostasis.

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Cell Death and Disease ◽

10.1038/s41419-021-03502-4 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Qing-Rui Wu ◽

Dan-Lin Zheng ◽

Pei-Ming Liu ◽

Hui Yang ◽

Lu-An Li ◽

...

Keyword(s):

Calcium Channel ◽

Mitochondrial Dysfunction ◽

High Glucose ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Calcium Handling ◽

Cardiomyocyte Hypertrophy ◽

Mitochondrial Morphology ◽

Calmodulin Binding ◽

Downstream Target

AbstractMitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.

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Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

10.1101/2021.10.21.465262 ◽

2021 ◽

Author(s):

Erminia Donnarumma ◽

Michael Kohlhaas ◽

Elodie Vimont ◽

Etienne Kornobis ◽

Thibaut Chaze ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Cell Death ◽

Knockout Mice ◽

Mitochondrial Permeability Transition ◽

Mitochondrial Fission ◽

Sterile Inflammation ◽

Mitochondrial Morphology ◽

Fission Process ◽

General Decline

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

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Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients

Biomolecules ◽

10.3390/biom10030450 ◽

2020 ◽

Vol 10 (3) ◽

pp. 450 ◽

Cited By ~ 1

Author(s):

Takeshi Tokuyama ◽

Asei Hirai ◽

Isshin Shiiba ◽

Naoki Ito ◽

Keigo Matsuno ◽

...

Keyword(s):

Mitochondrial Disease ◽

Mitochondrial Myopathy ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Critical Role ◽

Leigh Syndrome ◽

Mitochondrial Morphology ◽

Mitochondrial Fragmentation ◽

Cellular Toxicity ◽

Morphologic Changes

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

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Mitochondrial Deficits With Neural and Social Damage in Early-Stage Alzheimer’s Disease Model Mice

Frontiers in Aging Neuroscience ◽

10.3389/fnagi.2021.748388 ◽

2021 ◽

Vol 13 ◽

Author(s):

Afzal Misrani ◽

Sidra Tabassum ◽

Qingwei Huo ◽

Sumaiya Tabassum ◽

Jinxiang Jiang ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Neurodegenerative Disorder ◽

Early Stage ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Neuronal Morphology ◽

Therapeutic Interventions ◽

Early Event ◽

Mitochondrial Morphology

Alzheimer’s disease (AD) is the most common neurodegenerative disorder worldwide. Mitochondrial dysfunction is thought to be an early event in the onset and progression of AD; however, the precise underlying mechanisms remain unclear. In this study, we investigated mitochondrial proteins involved in organelle dynamics, morphology and energy production in the medial prefrontal cortex (mPFC) and hippocampus (HIPP) of young (1∼2 months), adult (4∼5 months) and aged (9∼10, 12∼18 months) APP/PS1 mice. We observed increased levels of mitochondrial fission protein, Drp1, and decreased levels of ATP synthase subunit, ATP5A, leading to abnormal mitochondrial morphology, increased oxidative stress, glial activation, apoptosis, and altered neuronal morphology as early as 4∼5 months of age in APP/PS1 mice. Electrophysiological recordings revealed abnormal miniature excitatory postsynaptic current in the mPFC together with a minor connectivity change between the mPFC and HIPP, correlating with social deficits. These results suggest that abnormal mitochondrial dynamics, which worsen with disease progression, could be a biomarker of early-stage AD. Therapeutic interventions that improve mitochondrial function thus represent a promising approach for slowing the progression or delaying the onset of AD.

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Mitochondrial dynamics and mitophagy are necessary for proper invasive growth in Rice Blast

10.1101/592915 ◽

2019 ◽

Author(s):

Yanjun Kou ◽

Yunlong He ◽

Jiehua Qiu ◽

Shu Yazhou ◽

Fan Yang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Morphological Changes ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Blast Disease ◽

Cereal Crops ◽

Mitochondrial Morphology ◽

Invasive Growth ◽

In Planta ◽

Antioxidant Treatment

SUMMARYMagnaporthe oryzaecauses Blast disease, which is one of the most devastating infections in rice and several important cereal crops.M. oryzaeneeds to coordinate gene regulation, morphological changes, nutrient acquisition, and host evasion, in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growthin plantahave remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology duringMagnaporthe-Rice interaction. Interestingly, loss of either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction inM. oryzaepathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growthin planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy, and are required for proper induction and establishment of the blast disease in rice.

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Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e16-04-0208 ◽

2017 ◽

Vol 28 (3) ◽

pp. 396-410 ◽

Cited By ~ 37

Author(s):

Edward Cherok ◽

Shan Xu ◽

Sunan Li ◽

Shweta Das ◽

W. Alex Meltzer ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Critical Role ◽

E3 Ubiquitin Ligase ◽

Mitochondrial Morphology ◽

Ubiquitinated Proteins ◽

Terminal Domain ◽

Degradation Rates ◽

And Control

MARCH5, an OMM-associated E3 ubiquitin ligase, controls mitochondrial function. Despite its importance, the mechanism and factors controlling MARCH5 activity are largely unknown. Here we report that the MARCH5 C-terminal domain plays a critical role in degradation of MARCH5 substrates, likely by facilitating release of ubiquitinated proteins from the OMM. We also found that the mitochondrial fission proteins Drp1 and Mff negatively regulate MARCH5’s activity toward MiD49 and Mcl1. Knockouts of either Drp1 or Mff led to reduced expression, shorter half-lives, and increased ubiquitination of MiD49 and Mcl1. Effects of Mff and Drp1 depletion on degradation rates and ubiquitination of Mcl1 and MiD49 were eliminated in Drp1−/−/MARCH5−/− and Mff−/−/MARCH5−/− cells. Our data show that it is not mitochondrial morphology per se but rather Mff and Drp1 that directly control MARCH5. Consistently, we find that Mff is an integral component of the MARCH5/p97/Npl4 complex, which is also controlled by MARCH5’s C-terminal domain. Furthermore, not only mitochondrial fission but also fusion is regulated through Mff and Drp1 protein activities. Thus, in addition to their canonical roles in mitochondrial fission, Mff and Drp1 also act as regulatory factors that control mitochondrial fission and fusion.

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Abstract 393: MCL-1 Promotes Survival and Influences Mitochondrial Dynamics in Cardiac Myocytes

Circulation Research ◽

10.1161/res.117.suppl_1.393 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Alexandra G Moyzis ◽

Robert L Thomas ◽

Jennifer Kuo ◽

Åsa B Gustafsson

Keyword(s):

Cell Death ◽

Cardiac Myocytes ◽

Apoptotic Cell ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Apoptotic Cell Death ◽

Mitochondrial Fusion ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Rapid Degradation

The BCL-2 family proteins are important regulators of mitochondrial structure and integrity. MCL-1 is an anti-apoptotic BCL-2 protein that is highly expressed in the myocardium compared to the other anti-apoptotic proteins BCL-2 and BCL-X L. Recently, we reported that MCL-1 is essential for myocardial homeostasis. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Surprisingly, MCL-1 deficient myocytes did not undergo apoptotic cell death. Instead, the cells displayed signs of mitochondrial deterioration and necrotic cell death, suggesting that MCL-1 has an additional role in maintaining mitochondrial function in cardiac myocytes. Similarly, deletion of MCL-1 in fibroblasts caused rapid mitochondrial fragmentation followed by cell death at 72 hours. Interestingly, the MCL-1 deficient fibroblasts retained cytochrome c in the mitochondria , confirming that the cells were not undergoing apoptotic cell death. We have also identified that MCL-1 localizes to the mitochondrial outer membrane (OM) and the matrix in the myocardium and that the two forms respond differently to stress. MCL-1 OM was rapidly degraded after myocardial infarction or fasting, whereas MCL-1 Matrix levels were maintained. Similarly, starvation of MEFs resulted in rapid degradation of MCL-1 OM , whereas MCL-1 Matrix showed delayed degradation. Treatment with the mitochondrial uncoupler FCCP led to rapid degradation of both forms. This suggests that the susceptibility to degradation is dependent on its localization and the nature of the stress. Our data also suggests that these two forms perform distinct functions in regulating mitochondrial morphology and survival. Overexpression of MCL-1 Matrix promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission and clearance by autophagosomes. Thus, our data suggest that MCL-1 exists in two separate locations where it performs different functions. MCL-1 Matrix promotes mitochondrial fusion, which protects cells against excessive mitochondrial clearance during unfavorable conditions.

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Mitochondrial morphology transitions and functions: implications for retrograde signaling?

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00584.2012 ◽

2013 ◽

Vol 304 (6) ◽

pp. R393-R406 ◽

Cited By ~ 125

Author(s):

Martin Picard ◽

Orian S. Shirihai ◽

Benoit J. Gentil ◽

Yan Burelle

Keyword(s):

Cell Function ◽

Mitochondrial Permeability Transition ◽

Mitochondrial Dynamics ◽

Permeability Transition ◽

Retrograde Signaling ◽

Mitochondrial Morphology ◽

Transport Chain ◽

Dynamic Interface ◽

Morphological Transitions ◽

Species Production

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment.

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Loss of the Mitochondrial Fission GTPase Drp1 Contributes to Neurodegeneration in a Drosophila Model of Hereditary Spastic Paraplegia

Brain Sciences ◽

10.3390/brainsci10090646 ◽

2020 ◽

Vol 10 (9) ◽

pp. 646

Author(s):

Philippa C. Fowler ◽

Dwayne J. Byrne ◽

Craig Blackstone ◽

Niamh C. O'Sullivan

Keyword(s):

Hereditary Spastic Paraplegia ◽

Neurodegenerative Disorder ◽

Mitochondrial Fission ◽

Dominant Negative ◽

In Vivo Model ◽

Mitochondrial Morphology ◽

Autophagic Flux ◽

Spastic Paraplegia ◽

Drosophila Model

Mitochondrial morphology, distribution and function are maintained by the opposing forces of mitochondrial fission and fusion, the perturbation of which gives rise to several neurodegenerative disorders. The large guanosine triphosphate (GTP)ase dynamin-related protein 1 (Drp1) is a critical regulator of mitochondrial fission by mediating membrane scission, often at points of mitochondrial constriction at endoplasmic reticulum (ER)-mitochondrial contacts. Hereditary spastic paraplegia (HSP) subtype SPG61 is a rare neurodegenerative disorder caused by mutations in the ER-shaping protein Arl6IP1. We have previously reported defects in both the ER and mitochondrial networks in a Drosophila model of SPG61. In this study, we report that knockdown of Arl6IP1 lowers Drp1 protein levels, resulting in reduced ER–mitochondrial contacts and impaired mitochondrial load at the distal ends of long motor neurons. Increasing mitochondrial fission, by overexpression of wild-type Drp1 but not a dominant negative Drp1, increases ER–mitochondrial contacts, restores mitochondrial load within axons and partially rescues locomotor deficits. Arl6IP1 knockdown Drosophila also demonstrate impaired autophagic flux and an accumulation of ubiquitinated proteins, which occur independent of Drp1-mediated mitochondrial fission defects. Together, these findings provide evidence that impaired mitochondrial fission contributes to neurodegeneration in this in vivo model of HSP.

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