Publications by authors named "Bruno B Queliconi"

13 Publications

  • Page 1 of 1

Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy.

J Mol Cell Cardiol 2019 05 11;130:160-169. Epub 2019 Apr 11.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart.

Methods: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction.

Results: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality.

Conclusions: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.
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http://dx.doi.org/10.1016/j.yjmcc.2019.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948926PMC
May 2019

A selective inhibitor of mitofusin 1-βIIPKC association improves heart failure outcome in rats.

Nat Commun 2019 01 18;10(1):329. Epub 2019 Jan 18.

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, 94305-5174, CA, USA.

We previously demonstrated that beta II protein kinase C (βIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that βIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. βIIPKC siRNA or a βIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-βIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMβA, a rationally-designed peptide that selectively antagonizes Mfn1-βIIPKC association. SAMβA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMβA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMβA may be a potential treatment for patients with heart failure.
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http://dx.doi.org/10.1038/s41467-018-08276-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338754PMC
January 2019

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure.

Autophagy 2017 Aug;13(8):1304-1317

a Department of Anatomy , Institute of Biomedical Sciences, University of Sao Paulo , Sao Paulo , Brazil.

We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O consumption, elevated HO release and increased Ca-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.
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http://dx.doi.org/10.1080/15548627.2017.1325062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584854PMC
August 2017

Bicarbonate Increases Ischemia-Reperfusion Damage by Inhibiting Mitophagy.

PLoS One 2016 14;11(12):e0167678. Epub 2016 Dec 14.

Cedars-Sinai Heart Institute, Los Angeles, California, United States of America.

During an ischemic event, bicarbonate and CO2 concentration increase as a consequence of O2 consumption and lack of blood flow. This event is important as bicarbonate/CO2 is determinant for several redox and enzymatic reactions, in addition to pH regulation. Until now, most work done on the role of bicarbonate in ischemia-reperfusion injury focused on pH changes; although reperfusion solutions have a fixed pH, cardiac resuscitation protocols commonly employ bicarbonate to correct the profound acidosis associated with respiratory arrest. However, we previously showed that bicarbonate can increase tissue damage and protein oxidative damage independent of pH. Here we show the molecular basis of bicarbonate-induced reperfusion damage: the presence of bicarbonate selectively impairs mitophagy, with no detectable effect on autophagy, proteasome activity, reactive oxygen species production or protein oxidation. We also show that inhibition of autophagy reproduces the effects of bicarbonate in reperfusion injury, providing additional evidence in support of this mechanism. This phenomenon is especially important because bicarbonate is widely used in resuscitation protocols after cardiac arrest, and while effective as a buffer, may also contribute to myocardial injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167678PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156406PMC
July 2017

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

J Biol Chem 2015 Oct 10;290(42):25199-211. Epub 2015 Aug 10.

From the Ubiquitin Project and

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.
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http://dx.doi.org/10.1074/jbc.M115.671446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646171PMC
October 2015

A time to reap, a time to sow: mitophagy and biogenesis in cardiac pathophysiology.

J Mol Cell Cardiol 2015 Jan 16;78:62-72. Epub 2014 Oct 16.

Cedars-Sinai Heart Institute and Barbra Streisand Women's Heart Center. Electronic address:

Balancing mitophagy and mitochondrial biogenesis is essential for maintaining a healthy population of mitochondria and cellular homeostasis. Coordinated interplay between these two forces that govern mitochondrial turnover plays an important role as an adaptive response against various cellular stresses that can compromise cell survival. Failure to maintain the critical balance between mitophagy and mitochondrial biogenesis or homeostatic turnover of mitochondria results in a population of dysfunctional mitochondria that contribute to various disease processes. In this review we outline the mechanics and relationships between mitophagy and mitochondrial biogenesis, and discuss the implications of a disrupted balance between these two forces, with an emphasis on cardiac physiology. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
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http://dx.doi.org/10.1016/j.yjmcc.2014.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268279PMC
January 2015

An anoxia-starvation model for ischemia/reperfusion in C. elegans.

J Vis Exp 2014 Mar 11(85). Epub 2014 Mar 11.

Department of Medicine, Nephrology Division, University of Rochester Medical Center, School of Medicine and Dentistry.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr (simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.
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http://dx.doi.org/10.3791/51231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4089474PMC
March 2014

Exercise training restores cardiac protein quality control in heart failure.

PLoS One 2012 27;7(12):e52764. Epub 2012 Dec 27.

Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H₂O₂ release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H₂O₂ resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca²⁺-induced permeability transition and reduced H₂O₂ release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052764PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531365PMC
June 2013

Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion.

Free Radic Biol Med 2013 Feb 27;55:46-53. Epub 2012 Nov 27.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil.

The carbon dioxide/bicarbonate (CO(2)/HCO(3)(-)) pair is the main biological pH buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO(2)/HCO(3)(-) on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart, and Caenorhabditis elegans). We found that, although various concentrations of CO(2)/HCO(3)(-) do not affect function under basal conditions, ischemia-reperfusion or similar insults in the presence of higher CO(2)/HCO(3)(-) resulted in greater functional loss associated with higher oxidative damage in all models. Because the effect of CO(2)/HCO(3)(-) was observed in all models tested, we believe this buffer is an important determinant of oxidative damage after ischemia-reperfusion.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995138PMC
February 2013

Mitochondrial compartmentalization of redox processes.

Free Radic Biol Med 2012 Jun 1-15;52(11-12):2201-8. Epub 2012 Apr 26.

Departamento de Bioquímica, Instituto de Química, Brazil.

Knowledge of location and intracellular subcompartmentalization is essential for the understanding of redox processes, because oxidants, owing to their reactive nature, must be generated close to the molecules modified in both signaling and damaging processes. Here we discuss known redox characteristics of various mitochondrial microenvironments. Points covered are the locations of mitochondrial oxidant generation, characteristics of antioxidant systems in various mitochondrial compartments, and diffusion characteristics of oxidants in mitochondria. We also review techniques used to measure redox state in mitochondrial subcompartments, antioxidants targeted to mitochondrial subcompartments, and methodological concerns that must be addressed when using these tools.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.03.008DOI Listing
October 2012

Redox regulation of the mitochondrial K(ATP) channel in cardioprotection.

Biochim Biophys Acta 2011 Jul 20;1813(7):1309-15. Epub 2010 Nov 20.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.
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http://dx.doi.org/10.1016/j.bbamcr.2010.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109179PMC
July 2011

Mitochondrial ion transport pathways: role in metabolic diseases.

Biochim Biophys Acta 2010 Jun-Jul;1797(6-7):832-8. Epub 2010 Jan 5.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H+ transport (promoted by uncoupling proteins, UCPs), Ca2+ and K+ uniporters which may be determinant in metabolic disorders.
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http://dx.doi.org/10.1016/j.bbabio.2009.12.017DOI Listing
January 2011

Pharmacological and physiological stimuli do not promote Ca(2+)-sensitive K+ channel activity in isolated heart mitochondria.

Cardiovasc Res 2007 Mar 30;73(4):720-8. Epub 2006 Nov 30.

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Objective: Mitochondrial calcium-activated K(+) (mitoK(Ca)) channels have been described as channels that are activated by Ca(2+), inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK(Ca) channels. Here, we show several weaknesses in this hypothesis.

Methods: Isolated mitochondria from rat hearts were tested for evidence of mitoK(Ca) activity by analyzing functional parameters in K(+)-rich and K(+)-free media.

Results: NS-1619 promoted mitochondrial depolarization both in K(+)-rich and K(+)-free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK(Ca) channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K(+) in the media was substituted with tetraethylammonium (TEA(+)), which is not transported by any known K(+) carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K(+). In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK(Ca) activity by altering Ca(2+) concentrations in the medium and inducing mitochondrial uncoupling.

Conclusion: Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K(+) transport in isolated heart mitochondria compatible with mitoK(Ca) opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK(Ca) and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.
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http://dx.doi.org/10.1016/j.cardiores.2006.11.035DOI Listing
March 2007