Publications by authors named "Jon Sin"

22 Publications

  • Page 1 of 1

Hypothermia promotes mitochondrial elongation In cardiac cells via inhibition of Drp1.

Cryobiology 2021 Jul 29. Epub 2021 Jul 29.

Cedars-Sinai Smidt Heart Institute, Barbra Streisand Women's Heart Center, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32 °C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32 °C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cryobiol.2021.07.013DOI Listing
July 2021

At the heart of mitochondrial quality control: many roads to the top.

Cell Mol Life Sci 2021 Apr 5;78(8):3791-3801. Epub 2021 Feb 5.

Smidt Heart Institute, Cedars-Sinai Medical Center, AHSP9313, 127 S. San Vicente Blvd., Los Angeles, CA, 90048, USA.

Mitochondrial quality control depends upon selective elimination of damaged mitochondria, replacement by mitochondrial biogenesis, redistribution of mitochondrial components across the network by fusion, and segregation of damaged mitochondria by fission prior to mitophagy. In this review, we focus on mitochondrial dynamics (fusion/fission), mitophagy, and other mechanisms supporting mitochondrial quality control including maintenance of mtDNA and the mitochondrial unfolded protein response, particularly in the context of the heart.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-021-03772-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106602PMC
April 2021

Ultraviolet A light effectively reduces bacteria and viruses including coronavirus.

PLoS One 2020 16;15(7):e0236199. Epub 2020 Jul 16.

Medically Associated Science and Technology (MAST) Program, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

Antimicrobial-resistant and novel pathogens continue to emerge, outpacing efforts to contain and treat them. Therefore, there is a crucial need for safe and effective therapies. Ultraviolet-A (UVA) phototherapy is FDA-approved for several dermatological diseases but not for internal applications. We investigated UVA effects on human cells in vitro, mouse colonic tissue in vivo, and UVA efficacy against bacteria, yeast, coxsackievirus group B and coronavirus-229E. Several pathogens and virally transfected human cells were exposed to a series of specific UVA exposure regimens. HeLa, alveolar and primary human tracheal epithelial cell viability was assessed after UVA exposure, and 8-Oxo-2'-deoxyguanosine was measured as an oxidative DNA damage marker. Furthermore, wild-type mice were exposed to intracolonic UVA as an in vivo model to assess safety of internal UVA exposure. Controlled UVA exposure yielded significant reductions in Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Clostridioides difficile, Streptococcus pyogenes, Staphylococcus epidermidis, Proteus mirabilis and Candida albicans. UVA-treated coxsackievirus-transfected HeLa cells exhibited significantly increased cell survival compared to controls. UVA-treated coronavirus-229E-transfected tracheal cells exhibited significant coronavirus spike protein reduction, increased mitochondrial antiviral-signaling protein and decreased coronavirus-229E-induced cell death. Specific controlled UVA exposure had no significant effect on growth or 8-Oxo-2'-deoxyguanosine levels in three types of human cells. Single or repeated in vivo intraluminal UVA exposure produced no discernible endoscopic, histologic or dysplastic changes in mice. These findings suggest that, under specific conditions, UVA reduces various pathogens including coronavirus-229E, and may provide a safe and effective treatment for infectious diseases of internal viscera. Clinical studies are warranted to further elucidate the safety and efficacy of UVA in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236199PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365468PMC
September 2020

Intermittent Use of a Short-Course Glucagon-like Peptide-1 Receptor Agonist Therapy Limits Adverse Cardiac Remodeling via Parkin-dependent Mitochondrial Turnover.

Sci Rep 2020 05 19;10(1):8284. Epub 2020 May 19.

Cedars Sinai Medical Center, Smidt Heart Institute, Los Angeles, USA.

Given that adverse remodeling is the leading cause of heart failure and death in the USA, there is an urgent unmet need to develop new methods in dealing with this devastating disease. Here we evaluated the efficacy of a short-course glucagon-like peptide-1 receptor agonist therapy-specifically 2-quinoxalinamine, 6,7-dichloro-N-(1,1-dimethylethyl)-3-(methylsulfonyl)-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB; aka Compound 2) - in attenuating adverse LV remodeling. We also examined the role, if any, of mitochondrial turnover in this process. Wild-type, Parkin knockout and MitoTimer-expressing mice were subjected to permanent coronary artery ligation, then treated briefly with DMB. LV remodeling and cardiac function were assessed by histology and echocardiography. Autophagy and mitophagy markers were examined by western blot and mitochondrial biogenesis was inferred from MitoTimer protein fluorescence and qPCR. We found that DMB given post-infarction significantly reduced adverse LV remodeling and the decline of cardiac function. This paralleled an increase in autophagy, mitophagy and mitochondrial biogenesis. The salutary effects of the drug were lost in Parkin knockout mice, implicating Parkin-mediated mitophagy as part of its mechanism of action. Our findings suggest that enhancing Parkin-associated mitophagy and mitochondrial biogenesis after infarction is a viable target for therapeutic mitigation of adverse remodeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-64924-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237417PMC
May 2020

Antiviral Effects of Menthol on Coxsackievirus B.

Viruses 2020 03 28;12(4). Epub 2020 Mar 28.

The Smidt Heart Institute and the Barbra Streisand Women's Heart Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

Coxsackievirus B (CVB) is a common human enterovirus that causes systemic infection but specifically replicates to high titers in the pancreas. It was reported that certain viruses induce mitochondrial fission to support infection. We documented that CVB triggers mitochondrial fission and blocking mitochondrial fission limits infection. The transient receptor potential channels have been implicated in regulating mitochondrial dynamics; namely, the heat and capsaicin receptor transient receptor potential cation channel subfamily V member 1 (TRPV1) contributes to mitochondrial depolarization and fission. When we transiently warmed HeLa cells to 39 °C prior to CVB exposure, infection was heightened, whereas cooling cells to 25 °C reduced infection. Inducing "cold" by stimulating transient receptor potential cation channel subfamily M member 8 (TRPM8) with menthol led to reduced infection and also resulted in lower levels of mitochondrial fission during infection. Additionally, menthol stabilized levels of mitochondrial antiviral signaling (MAVS) which is known to be tied to mitochondrial dynamics. Taken together, this highlights a novel pathway wherein CVB relies on TRPV1 to initiate proviral mitochondrial fission, which may contribute to the disruption of antiviral immunity. TRPM8 has been shown to antagonize TRPV1, and thus we hypothesize that stimulating TRPM8 blocks TRPV1-mediated mitochondrial fragmentation following CVB exposure and attenuates infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v12040373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232514PMC
March 2020

Sex differences in inflammation, redox biology, mitochondria and autoimmunity.

Redox Biol 2020 04 4;31:101482. Epub 2020 Mar 4.

Center for Clinical and Translational Science, Mayo Clinic, Jacksonville, FL, USA; Department of Cardiovascular Medicine, Mayo Clinic, Jacksonville, FL, USA; Department of Immunology, Mayo Clinic, Jacksonville, FL, USA; Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA. Electronic address:

Autoimmune diseases are characterized by circulating antibodies and immune complexes directed against self-tissues that result in both systemic and organ-specific inflammation and pathology. Most autoimmune diseases occur more often in women than men. One exception is myocarditis, which is an inflammation of the myocardium that is typically caused by viral infections. Sex differences in the immune response and the role of the sex hormones estrogen and testosterone are well established based on animal models of autoimmune viral myocarditis as well as in mitochondrial function leading to reactive oxygen species production. RNA viruses like coxsackievirus B3, the primary cause of myocarditis in the US, activate the inflammasome through mitochondrial antiviral signaling protein located on the mitochondrial outer membrane. Toll-like receptor 4 and the inflammasome are the primary signaling pathways that increase inflammation during myocarditis, which is increased by testosterone. This review describes what is known about sex differences in inflammation, redox biology and mitochondrial function in the male-dominant autoimmune disease myocarditis and highlights gaps in the literature and future directions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212489PMC
April 2020

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis.

Biochim Biophys Acta Mol Basis Dis 2019 11 6;1865(11):165530. Epub 2019 Aug 6.

Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Electronic address:

Background: During pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.

Methods: We examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.

Results: Pancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.

Conclusion: Our findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2019.08.006DOI Listing
November 2019

Mitophagy protects against statin-mediated skeletal muscle toxicity.

FASEB J 2019 11 23;33(11):11857-11869. Epub 2019 Aug 23.

Cedars-Sinai Medical Center, Los Angeles, California, USA.

The deleterious effects of statins on skeletal muscle are well known, but the mechanism associated with these effects remains unresolved. Statins are associated with mitochondrial damage, which may contribute to muscle myopathy. Here we demonstrate that simvastatin induces mitophagy in skeletal muscle cells and hypothesized that attenuating this process by silencing the mitophagy adapter p62/sequestosome-1 (SQSTM1) might mitigate myotoxicity. Surprisingly, silencing p62/SQSTM1 in differentiated C2C12 muscle cells exacerbated rather than attenuated myotoxicity. This inhibition of mitophagy in the face of statin challenge correlated with increased release of cytochrome to the cytosol, activation of caspase-3, and lactate dehydrogenase (LDH) release. Correspondingly, targeted knockdown of Parkin, a canonical E3 ubiquitin ligase important for mitophagy, mirrored the effects of p62/SQSTM1 silencing. To corroborate these findings , we treated Parkin knockout mice with simvastatin for 2 wk. In line with our findings , these mitophagy-compromised mice displayed reduced spontaneous activity, loss of grip strength, and increased circulating levels of muscle damage marker LDH. Our findings demonstrate that mitophagy is an important mechanism to resist statin-induced skeletal muscle damage.-Ramesh, M., Campos, J. C., Lee, P., Song, Y., Hernandez, G., Sin, J., Tucker, K. C., Saadaeijahromi, H., Gurney, M., Ferreira, J. C. B., Andres, A. M. Mitophagy protects against statin-mediated skeletal muscle toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201900807RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902735PMC
November 2019

Myocardial hypothermia increases autophagic flux, mitochondrial mass and myocardial function after ischemia-reperfusion injury.

Sci Rep 2019 07 10;9(1):10001. Epub 2019 Jul 10.

Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.

Animal studies have demonstrated beneficial effects of therapeutic hypothermia on myocardial function, yet exact mechanisms remain unclear. Impaired autophagy leads to heart failure and mitophagy is important for mitigating ischemia/reperfusion injury. This study aims to investigate whether the beneficial effects of therapeutic hypothermia are due to preserved autophagy and mitophagy. Under general anesthesia, the left anterior descending coronary artery of 19 female farm pigs was occluded for 90 minutes with consecutive reperfusion. 30 minutes after reperfusion, we performed pericardial irrigation with warm or cold saline for 60 minutes. Myocardial tissue analysis was performed one and four weeks after infarction. Therapeutic hypothermia induced a significant increase in autophagic flux, mitophagy, mitochondrial mass and function in the myocardium after infarction. Cell stress, apoptosis, inflammation as well as fibrosis were reduced, with significant preservation of systolic and diastolic function four weeks post infarction. We found similar biochemical changes in human samples undergoing open chest surgery under hypothermic conditions when compared to the warm. These results suggest that autophagic flux and mitophagy are important mechanisms implicated in cardiomyocyte recovery after myocardial infarction under hypothermic conditions. New therapeutic strategies targeting these pathways directly could lead to improvements in prevention of heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-46452-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620356PMC
July 2019

Intercepting the Lipid-Induced Integrated Stress Response Reduces Atherosclerosis.

J Am Coll Cardiol 2019 03;73(10):1149-1169

Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey; National Nanotechnology Center, Bilkent University, Ankara, Turkey; Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California; Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California. Electronic address:

Background: Eukaryotic cells can respond to diverse stimuli by converging at serine-51 phosphorylation on eukaryotic initiation factor 2 alpha (eIF2α) and activate the integrated stress response (ISR). This is a key step in translational control and must be tightly regulated; however, persistent eIF2α phosphorylation is observed in mouse and human atheroma.

Objectives: Potent ISR inhibitors that modulate neurodegenerative disorders have been identified. Here, the authors evaluated the potential benefits of intercepting ISR in a chronic metabolic and inflammatory disease, atherosclerosis.

Methods: The authors investigated ISR's role in lipid-induced inflammasome activation and atherogenesis by taking advantage of 3 different small molecules and the ATP-analog sensitive kinase allele technology to intercept ISR at multiple molecular nodes.

Results: The results show lipid-activated eIF2α signaling induces a mitochondrial protease, Lon protease 1 (LONP1), that degrades phosphatase and tensin-induced putative kinase 1 and blocks Parkin-mediated mitophagy, resulting in greater mitochondrial oxidative stress, inflammasome activation, and interleukin-1β secretion in macrophages. Furthermore, ISR inhibitors suppress hyperlipidemia-induced inflammasome activation and inflammation, and reduce atherosclerosis.

Conclusions: These results reveal endoplasmic reticulum controls mitochondrial clearance by activating eIF2α-LONP1 signaling, contributing to an amplified oxidative stress response that triggers robust inflammasome activation and interleukin-1β secretion by dietary fats. These findings underscore the intricate exchange of information and coordination of both organelles' responses to lipids is important for metabolic health. Modulation of ISR to alleviate organelle stress can prevent inflammasome activation by dietary fats and may be a strategy to reduce lipid-induced inflammation and atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacc.2018.12.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424590PMC
March 2019

Myocardial fibrosis after adrenergic stimulation as a long-term sequela in a mouse model of Kawasaki disease vasculitis.

JCI Insight 2019 Feb 7;4(3). Epub 2019 Feb 7.

Departments of Biomedical Sciences and Pediatrics, Divisions of Infectious Diseases and Immunology.

Kawasaki disease (KD), the leading cause of acquired cardiac disease among children, is often associated with myocarditis that may lead to long-term myocardial dysfunction and fibrosis. Although those myocardial changes develop during the acute phase, they may persist for decades and closely correlate with long-term myocardial sequelae. Using the Lactobacillus casei cell wall extract-induced (LCWE-induced) KD vasculitis murine model, we investigated long-term cardiovascular sequelae, such as myocardial dysfunction, fibrosis, and coronary microvascular lesions following adrenergic stimuli after established KD vasculitis. We found that adrenergic stimulation with isoproterenol following LCWE-induced KD vasculitis in mice was associated with increased risk of cardiac hypertrophy and myocardial fibrosis, diminished ejection fraction, and increased serum levels of brain natriuretic peptide. Myocardial fibrosis resulting from pharmacologic-induced exercise after KD development was IL-1 signaling dependent and was associated with a significant reduction in myocardial capillary CD31 expression, indicative of a rarefied myocardial capillary bed. These observations suggest that adrenergic stimulation after KD vasculitis may lead to cardiac hypertrophy and bridging fibrosis in the myocardium in the LCWE-induced KD vasculitis mouse model and that this process involves IL-1 signaling and diminished microvascular circulation in the myocardium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.126279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413776PMC
February 2019

Coxsackievirus B infection induces the extracellular release of miR-590-5p, a proviral microRNA.

Virology 2019 03 30;529:169-176. Epub 2019 Jan 30.

The Smidt Heart Institute and the Barbra Streisand Women's Heart Center, Cedars-Sinai Medical Center, Los Angeles, CA, United States. Electronic address:

Coxsackievirus B is a significant human pathogen and is a leading cause of myocarditis. We and others have observed that certain enteroviruses including coxsackievirus B cause infected cells to shed extracellular vesicles containing infectious virus. Recent reports have shown that vesicle-bound virus can infect more efficiently than free virus. Though microRNAs are differentially regulated in cells following infection, few have been associated with the vesicles shed from infected cells. Here we report exclusive trafficking of specific microRNAs into viral vesicles compared to vesicles from non-infected cells. We found that the most highly-expressed unique microRNA in viral vesicles was miR-590-5p, which facilitates prolonged viral replication by blocking apoptotic factors. Cells over-expressing this miR were significantly more susceptible to infection. This may be a mechanism by which coxsackievirus B boosts subsequent rounds of infection by co-packaging virus and a select set of pro-viral microRNAs in extracellular vesicles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2019.01.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382511PMC
March 2019

Coxsackievirus B Escapes the Infected Cell in Ejected Mitophagosomes.

J Virol 2017 12 30;91(24). Epub 2017 Nov 30.

The Cedars-Sinai Heart Institute and the Barbra Streisand Women's Heart Center, Cedars-Sinai Medical Center, Los Angeles, California, USA

Coxsackievirus B (CVB) is a common enterovirus that can cause various systemic inflammatory diseases. Because CVB lacks an envelope, it has been thought to be inherently cytolytic, wherein CVB can escape from the infected host cell only by causing it to rupture. In recent years, however, we and others have observed that various naked viruses, such as CVB, can trigger the release of infectious extracellular microvesicles (EMVs) that contain viral material. This mode of cellular escape has been suggested to allow the virus to be masked from the adaptive immune system. Additionally, we have previously reported that these viral EMVs have LC3, suggesting that they originated from autophagosomes. We now report that CVB-infected cells trigger DRP1-mediated fragmentation of mitochondria, which is a precursor to autophagic mitochondrial elimination (mitophagy). However, rather than being degraded by lysosomes, mitochondrion-containing autophagosomes are released from the cell. We believe that CVB localizes to mitochondria, induces mitophagy, and subsequently disseminates from the cell in an autophagosome-bound mitochondrion-virus complex. Suppressing the mitophagy pathway in HL-1 cardiomyocytes with either small interfering RNA (siRNA) or Mdivi-1 caused marked reduction in virus production. The findings in this study suggest that CVB subverts mitophagy machinery to support viral dissemination in released EMVs. Coxsackievirus B (CVB) can cause a number of life-threatening inflammatory diseases. Though CVB is well known to disseminate via cytolysis, recent reports have revealed a second pathway in which CVB can become encapsulated in host membrane components to escape the cell in an exosome-like particle. Here we report that these membrane-bound structures derive from mitophagosomes. Blocking various steps in the mitophagy pathway reduced levels of intracellular and extracellular virus. Not only does this study reveal a novel mechanism of picornaviral dissemination, but also it sheds light on new therapeutic targets to treat CVB and potentially other picornaviral infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01347-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709598PMC
December 2017

Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

Autophagy 2016 ;12(2):369-80

a The Cedars-Sinai Heart Institute and the Barbra Streisand Women's Heart Center Cedars-Sinai Medical Center , Los Angeles , CA , USA.

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2015.1115172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836019PMC
December 2016

Recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis.

Virology 2015 Oct 1;484:288-304. Epub 2015 Jul 1.

The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, CA 92182-4614, USA. Electronic address:

Coxsackieviruses (CVs) are relatively common viruses associated with a number of serious human diseases, including myocarditis and meningo-encephalitis. These viruses are considered cytolytic yet can persist for extended periods of time within certain host tissues requiring evasion from the host immune response and a greatly reduced rate of replication. A member of Picornaviridae family, CVs have been historically considered non-enveloped viruses - although recent evidence suggest that CV and other picornaviruses hijack host membranes and acquire an envelope. Acquisition of an envelope might provide distinct benefits to CV virions, such as resistance to neutralizing antibodies and efficient nonlytic viral spread. CV exhibits a unique tropism for progenitor cells in the host which may help to explain the susceptibility of the young host to infection and the establishment of chronic disease in adults. CVs have also been shown to exploit autophagy to maximize viral replication and assist in unconventional release from target cells. In this article, we review recent progress in clarifying virus replication and dissemination within the host cell, identifying determinants of tropism, and defining strategies utilized by the virus to evade the host immune response. Also, we will highlight unanswered questions and provide future perspectives regarding the potential mechanisms of CV pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2015.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567421PMC
October 2015

Untangling autophagy measurements: all fluxed up.

Circ Res 2015 Jan;116(3):504-14

From the Cedars-Sinai Heart Institute and the Barbra Streisand Women's Heart Center Cedars-Sinai Medical Center, Los Angeles, CA.

Autophagy is an important physiological process in the heart, and alterations in autophagic activity can exacerbate or mitigate injury during various pathological processes. Methods to assess autophagy have changed rapidly because the field of research has expanded. As with any new field, methods and standards for data analysis and interpretation evolve as investigators acquire experience and insight. The purpose of this review is to summarize current methods to measure autophagy, selective mitochondrial autophagy (mitophagy), and autophagic flux. We will examine several published studies where confusion arose in data interpretation, to illustrate the challenges. Finally, we will discuss methods to assess autophagy in vivo and in patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.116.303787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313387PMC
January 2015

Measuring cardiac autophagic flux in vitro and in vivo.

Methods Mol Biol 2015 ;1219:187-97

Donald P. Shiley BioScience Center, San Diego State University, 5500 Campanile Dr., San Diego, CA, 92182-4650, USA.

Autophagy is a lysosomal-dependent catabolic pathway that recycles various cytoplasmic-borne components, such as organelles and proteins, through the lysosomes. This process creates energy and biomolecules that are used to maintain homeostasis and to serve as an energy source under conditions of acute stress. Autophagic flux is a measure of efficiency or throughput of the pathway. Here, we describe a method for determining autophagic flux in vitro and in vivo using the autophagosomal/lysosomal fusion inhibitors chloroquine or bafilomycin A1 and then probing for the autophagosomal marker LC3-II via Western Blot.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-1661-0_14DOI Listing
June 2015

The impact of juvenile coxsackievirus infection on cardiac progenitor cells and postnatal heart development.

PLoS Pathog 2014 Jul 31;10(7):e1004249. Epub 2014 Jul 31.

The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.

Coxsackievirus B (CVB) is an enterovirus that most commonly causes a self-limited febrile illness in infants, but cases of severe infection can manifest in acute myocarditis. Chronic consequences of mild CVB infection are unknown, though there is an epidemiologic association between early subclinical infections and late heart failure, raising the possibility of subtle damage leading to late-onset dysfunction, or chronic ongoing injury due to inflammatory reactions during latent infection. Here we describe a mouse model of juvenile infection with a subclinical dose of coxsackievirus B3 (CVB3) which showed no evident symptoms, either immediately following infection or in adult mice. However following physiological or pharmacologically-induced cardiac stress, juvenile-infected adult mice underwent cardiac hypertrophy and dilation indicative of progression to heart failure. Evaluation of the vasculature in the hearts of adult mice subjected to cardiac stress showed a compensatory increase in CD31+ blood vessel formation, although this effect was suppressed in juvenile-infected mice. Moreover, CVB3 efficiently infected juvenile c-kit+ cells, and cardiac progenitor cell numbers were reduced in the hearts of juvenile-infected adult mice. These results suggest that the exhausted cardiac progenitor cell pool following juvenile CVB3 infection may impair the heart's ability to increase capillary density to adapt to increased load.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117602PMC
July 2014

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

PLoS Pathog 2014 Apr 10;10(4):e1004045. Epub 2014 Apr 10.

The Integrated Regenerative Research Institute (IRRI) at San Diego State University, Cell & Molecular Biology Joint Doctoral Program, Department of Biology, San Diego State University, San Diego, California, United States of America.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983045PMC
April 2014

MitoTimer: a novel tool for monitoring mitochondrial turnover.

Autophagy 2013 Nov 11;9(11):1852-61. Epub 2013 Oct 11.

San Diego State University; San Diego, CA USA.

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial-targeting sequence (coined "MitoTimer") and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and production was induced with doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Dox addition for as little as 1 h was sufficient to induce MitoTimer expression within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent transition from green to red over 48 h and was amenable to analysis by fluorescence microscopy and flow cytometry of whole cells or isolated mitochondria. A second Dox administration 48 h after the initial induction resulted in a second round of expression of green MitoTimer. The extent of new protein incorporation during a second pulse was increased by administration of a mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/auto.26501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028337PMC
November 2013

Mitophagy is required for acute cardioprotection by simvastatin.

Antioxid Redox Signal 2014 Nov 20;21(14):1960-73. Epub 2013 Sep 20.

Donald P. Shiley BioScience Center, San Diego State University , San Diego, California.

Aims: We have shown that autophagy and mitophagy are required for preconditioning. While statin's cardioprotective effects are well known, the role of autophagy/mitophagy in statin-mediated cardioprotection is not. In this study, we used HL-1 cardiomyocytes and mice subjected to ischemia/reperfusion to elucidate the mechanism of statin-mediated cardioprotection.

Results: HL-1 cardiomyocytes exposed to simvastatin for 24 h exhibited diminished protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling, increased activation of unc-51-like kinase 1, and upregulation of autophagy and mitophagy. Similar findings were obtained in hearts of mice given simvastatin. Mevalonate abolished simvastatin's effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin-treated HL-1 cells exhibited mitochondrial translocation of Parkin and p62/SQSTM1, fission, and mitophagy. Because Parkin is required for mitophagy and is expressed in heart, we investigated the effect of simvastatin on infarct size in Parkin knockout mice. Simvastatin reduced infarct size in wild-type mice but showed no benefit in Parkin knockout mice. Inhibition of HMG-CoA reductase limits mevalonate availability for both cholesterol and coenzyme Q10 (CoQ) biosynthesis. CoQ supplementation had no effect on statin-induced Akt/mTOR dephosphorylation or macroautophagy in HL-1 cells, but it potently blocked mitophagy. Importantly, CoQ supplementation abolished statin-mediated cardioprotection in vivo.

Innovation And Conclusion: Acute simvastatin treatment suppresses mTOR signaling and triggers Parkin-dependent mitophagy, the latter which is required for cardioprotection. Coadministration of CoQ with simvastatin impairs mitophagy and cardioprotection. These results raise the concern that CoQ may interfere with anti-ischemic benefits of statins mediated through stimulation of mitophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ars.2013.5416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4208607PMC
November 2014
-->