Permeabilized myocardial fibers as model to detect mitochondrial dysfunction during sepsis and melatonin effects without disruption of mitochondrial network

doi:10.1016/j.mito.2015.12.010

Mitochondrion

Volume 27, March 2016, Pages 56-63

https://doi.org/10.1016/j.mito.2015.12.010 Get rights and content

Highlights

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Permeabilized fibers of mouse myocardial yield accurate mitochondrial bioenergetic information.
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Key defects in OXPHOS and ETS capacities were identified in septic mice myocardial.
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ETC supercomplex assembly may underlie mitochondrial respiration impairment.
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Melatonin maintained mitochondrial function and supercomplex assembly during sepsis.

Abstract

Analysis of mitochondrial function is crucial to understand their involvement in a given disease. High-resolution respirometry of permeabilized myocardial fibers in septic mice allows the evaluation of the bioenergetic system, maintaining mitochondrial ultrastructure and intracellular interactions, which are critical for an adequate functionality. OXPHOS and electron transport system (ETS) capacities were assessed using different substrate combinations. Our findings show a severe septic-dependent impairment in OXPHOS and ETS capacities with mitochondrial uncoupling at early and late phases of sepsis. Moreover, sepsis triggers complex III (CIII)-linked alterations in supercomplexes structure, and loss of mitochondrial density. In these conditions, melatonin administration to septic mice prevented sepsis-dependent mitochondrial injury in mitochondrial respiration. Likewise, melatonin improved cytochrome b content and ameliorated the assembly of CIII in supercomplexes. These results support the use of permeabilized fibers to identify properly the respiratory deficits and specific melatonin effects in sepsis.

Introduction

Sepsis, defined as the systemic inflammatory response to infection, has a significant health and economic impact associated with high mortality and morbidity (Mayr et al., 2006, Mayr et al., 2014). There is currently evidences supporting mitochondrial dysfunction in sepsis (Brealey and Singer, 2003, Crouser, 2004, d'Avila et al., 2008, Li et al., 2013, Garrabou et al., 2012). During sepsis, an induction of inducible nitric oxide synthase (iNOS) occurs, providing a significant increase of nitric oxide (NO•) levels (Álvarez and Boveris, 2004, Escames et al., 2006, Ortiz et al., 2014). In parallel, sepsis courses high reactive oxygen species (ROS) production, mainly represented by superoxide anion (O₂•−). These conditions favor the reaction between NO• and O₂•− generating the highly toxic peroxynitrite anions (ONOO −) (Escames et al., 2003), which irreversibly inhibit the respiratory complexes. Hence, sepsis results in oxidative-nitrosative stress that might impair the mitochondrial electron transfer system (ETS) components, damaging proteins, DNA and membrane lipids, and causing severe mitochondrial dysfunction (Álvarez and Boveris, 2004, Cassina and Radi, 1996, Escames et al., 2003). Therefore, the analysis of mitochondrial function is critical to understand the pathophysiology of this disease. A number of studies demonstrate several advantages of working with permeabilized fibers instead of isolated mitochondria. Permeabilized fibers allow normal mitochondrial interactions and organization within cells compared to standard procedures. Moreover, permeabilized fibers preserve mitochondria properties, yield an appropriate pool of all mitochondrial subpopulations (mitochondrial content ≥ 95%), and harvest a representative mixture of functional and damaged mitochondria (Gnaiger, 2009, Kuznetsov et al., 2008, Picard et al., 2011, Saks et al., 1998). Previous reports demonstrated the beneficial effects of melatonin against diseases accompained by oxidative-nitrosative stress. Melatonin prevents septic shock and multiple organ failure reducing the expression of iNOS (and other proinflammatory molecules), blunting the elevated levels of NO• and ROS, and restoring ETS activity and ATP production (Escames et al., 2007, Escames et al., 2006, Wu et al., 2008, Lowes et al., 2013). Here, we measured mitochondrial respiration in permeabilized mouse myocardial fibers at early and latter septic process by high-resolution respirometry. Our objective was the analysis of their bioenergetic profile without their disruption due to mitochondrial isolation procedure, looking for specific ETS impairments and to identify melatonin targets during sepsis.

Section snippets

Animals

Wild-type C57BL/6 male mice (n = 80), provided by Harland Laboratories (Barcelona, Spain), were housed in clear plastic cages (four mice per cage) and maintained in the University of Granada's facility in a specific pathogen-free barrier zone with a controlled 12 h light/dark cycle with lights on at 08:00 h, and a constant room temperature (22 °C ± 1 °C). Mice were fed ad libitum and with free access to water. We used 3 month old mice for all experiments. All experiments were conducted in accordance

Results and discussion

It is known that sepsis induces myocardial dysfunction and mitochondrial impairment (Álvarez and Boveris, 2004, Li et al., 2013, Ortiz et al., 2014). However, specific mitochondrial ETS defects cannot be properly identified due to, among other issues, the loss of mitochondrial integrity and/or mitochondrial content during the isolation process, which mainly affect damaged mitochondria. Furthermore, although there is relevant evidence regarding the properties of melatonin against sepsis,

Conflict of interest

Authors declare that there are no conflicts of interest in relation to the work described.

Acknowledgments

The authors thank to Erich Gnaiger for providing knowledge and assistant, and Iryna Rusanova for her technical support. CD and JAG are supported by the Instituto de Salud Carlos III, Spain; HV is a PhD student supported by a FPU fellowship from the Ministerio de Educación, Spain; JAG is a PhD student from the Fundación General Universidad de Granada Empresa; MED-C and ML-S are PhD students from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain, and LCL is supported by

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¹: Present address: Oroboros Instruments, High-Resolution Respirometry, Innsbruck, Austria.

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MitoMattersPermeabilized myocardial fibers as model to detect mitochondrial dysfunction during sepsis and melatonin effects without disruption of mitochondrial network

Highlights

Abstract

Introduction

Section snippets

Animals

Results and discussion

Conflict of interest

Acknowledgments

Mol. Cell

Free Radic. Biol. Med.

Anal. Biochem.

Cell Metab.

Arch. Biochem. Biophys.

Mitochondrion

Life Sci.

Int. J. Biochem. Cell Biol.

Arch. Biochem. Biophys.

Br. J. Anaesth.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Surg. Res.

J. Biol. Chem.

Melatonin-mitochondria interplay in health and disease

Curr. Top. Med. Chem.

Mitochondrial dysfunction in sepsis

Curr. Infect. dis. Rep.

Diaphragm and cardiac mitochondrial creatine kinases are impaired in sepsis

J. Appl. Physiol.

Sepsis induces brain mitochondrial dysfunction

Crit. Care Med.

Melatonin counteracts lipopolysaccharide-induced expression and activity of mitochondrial nitric oxide synthase in rats

FASEB J.

Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice

J. Pineal Res.

MitoMatters
Permeabilized myocardial fibers as model to detect mitochondrial dysfunction during sepsis and melatonin effects without disruption of mitochondrial network