Instituto Tecnológico y de Estudios Superiores de Monterrey Campus Monterrey
School of Medicine and Health Sciences
Effect of moderate exercise on skeletal muscle mitochondrial function and dynamics in obese Zucker rats
Dissertation presented by Bianca Daniela Nieblas León
Submitted to the
School of Medicine and Health Sciences
As a partial requirement to obtain the academic degree of Master
in
Biomedical Sciences
Monterrey Nuevo León, June, 2020
5 Dedication
To my teachers of life. My mother, Teresa, your strength and your big heart are my greatest motivation. My father, José Luis, for your dedication to provide me with the tools that have allowed me to fulfill my professional goals.
To my best friends, my sisters Mayela and Belén. As on every path, you have been present on this one.
To my brothers at heart, Marcelo and Claudio.
All my effort and all my work are for you.
6 Acknowledgments
I would like to thank my advisor, Noemí García, MsC. Thank you for your patience and for giving me the responsibility of this project from which I learned a lot. To her team, Perla Pérez, PhD., Fernanda Nájera and Jorge Vela, MD., for their support in the experiments presented in this project.
To the thesis commitee, Fabiola Castorena, PhD., Julio Altamirano, PhD., Ana Laura González, PhD., Carmen Brenes, PhD., and Dr. Gerardo García, PhD. Thank you for sharing your knowledge and support in carrying out this project.
To my family for their constant encouragement.
To my friends, my second family. My admiration and gratitude are eternal.
To my new colleagues, Estefanía, Felipe, Itzel, Mariana and Nikita, may your path be as great as you are. Thank you for making this an unforgettable experience.
To all those who are part of the Cardiology laboratory, it has been an honor to work alongside you.
To Tecnológico de Monterrey for the tuition scholarship and to CONACyT (CVU 450250) for the maintenance scholarship.
7 Effect of moderate exercise on skeletal muscle mitochondrial function
and dynamics in obese Zucker rats Abstract
Obesity and metabolic syndrome are related to a significant reduction in mitochondrial quality.
Current evidence suggests that both conditions promote an unbalance between fusion and fission, the main events of mitochondrial dynamics and this is associated with alterations in mitochondrial function, mainly in highly energetic tissues such as skeletal muscle. In the treatment of obesity and metabolic disorders, the various adaptations resulting from moderate-intensity physical exercise stand out, which are considered to be regulated by AMPK. This project aimed to evaluate modifications in gene and protein expression of the main regulators of mitochondrial dynamics and, using confocal microscopy, determine changes in mitochondrial function through the evaluation of membrane potential and distribution changes in the main mitochondrial subpopulations in a murine model of obesity after chronic moderate exercise. 12 weeks old male Zucker obese rats were randomly divided into a sedentary obese group and an exercise obese group (n=4 per group). The exercise consisted of 4 weeks of swimming training for 60min/5days a week.
After 48 hours of the last exercise bout, animals were euthanized and both gastrocnemius muscles were isolated. A significant increase in fission was evidenced by changes in phosphorylation of Drp1 and AMPK. Subsarcolemmal mitochondria showed a more organized network in comparison with the sedentary group while there was no change in the intermyofibrillar region. An increase in fission regulated by AMPK might be segregating damaged mitochondria and enhancing its removal while activating mitochondrial biogenesis to ensure restoration of mitochondrial mass by generating a healthier population in the subsarcolemmal region.
8 Table of Contents
Chapter 1 – Problem statement ... 11
Background ... 11
Problem statement ... 14
Research objectives ... 15
1. General objective ... 15
2. Specific objectives ... 15
Rationale ... 15
Scope of the study ... 17
Chapter 2 – Theoretical framework ... 18
Etiology and prevalence of Metabolic Syndrome ... 18
Mitochondrial Dysfunction as a mechanism underlying insulin resistance in skeletal muscle ... 19
Skeletal muscle and myofibers ... 20
Mitochondria from skeletal muscle ... 21
Mitochondrial Quality Control ... 23
Mitochondrial Dynamics ... 24
1. Mitochondrial Fusion ... 25
2. Mitochondrial Fission ... 26
Aerobic Exercise ... 27
AMPK ... 28
Mitochondrial dysfunction and oxidative stress ... 30
Chapter 3 – Methodology ... 32
Ethical animal handling ... 32
Experimental Model ... 32
Swimming Training Protocol ... 34
Gastrocnemius muscle isolation ... 35
Gene and protein expression analysis ... 36
RNA Extraction and real-time quantitative PCR (RT-qPCR) ... 36
Western immunoblot analysis ... 37
Confocal Microscopy experiments ... 38
Membrane potential ... 39
Mitochondrial Distribution ... 39
Mitochondrial Density ... 40
9
1. MitoTracker green fluorescence using confocal microscopy. ... 40
2. Citrate synthase activity. ... 41
3. Quantification of Mitochondrial DNA Copy Number. ... 41
1. Catalase Activity. ... 42
2. Lipid Peroxidation. ... 42
3. Protein Carbonylation. ... 43
Chapter 4 – Results ... 44
Body weight evaluation before and after four weeks of swimming training and gastrocnemius muscle weight ... 44
Evaluation of gene expression and levels of mitochondrial dynamics proteins and AMPK on gastrocnemius muscle after four weeks of moderate exercise training ... 44
1. Effect of four-weeks of exercise training on mRNA expression in gastrocnemius muscle ... 44
2. Effect of four-weeks of exercise training on mitochondrial dynamics protein levels in gastrocnemius muscle ... 45
Mitochondrial Fusion proteins ... 45
Mitochondrial Fission proteins ... 46
3. Effect of four weeks of swimming training on oxidative stress ... 47
4. Effect of four-weeks swimming training on AMPK total protein level and activity ... 47
Evaluation of membrane potential changes and mitochondrial distribution on gastrocnemius muscle fibers after four weeks of swimming training ... 48
1. Membrane potential changes after four weeks of swimming training ... 48
2. Mitochondrial Distribution ... 49
Subsarcolemmal Mitochondria ... 49
Intermyofibrillar Mitochondria ... 50
Evaluation of mitochondrial biogenesis on gastrocnemius muscle after four weeks of swimming training ... 51
1. Mitochondrial density ... 51
2. PGC1a mRNA expression and protein level. ... 52
3. Gastrocnemius myofibers area ... 53
Chapter 5 – Analysis and results discussion ... 54
Chapter 6 – Conclusion and Perspectives ... 57
References ... 58
CURRICULUM VITAE ... 69
10 Abbreviations
Δψm: mitochondrial membrane potential
AMPK: adenosine monophosphate-activated protein kinase ATP: adenosine triphosphate
Drp1: dynamin related protein 1 Fis1: mitochondrial fission protein IMM: inner membrane
IMFM: intermyofibrillar mitochondria MFF: mitochondrial fission factor Mfn1: mitofusin 1
Mfn2: mitofusin 2
OMA: overlapping activity with m-AAA protease 1 OMM: outer membrane
Opa1: optic atrophy protein 1
OXPHOS: oxidative phosphorylation
PGC1a: proliferator-activated receptor-g co-activator 1a ROS: reactive oxygen species
RT-qPCR: real-time quantitative polymerase chain reaction SSM: subsarcolemmal mitochondria
TFAM: mitochondrial transcription factor A
11 Chapter 1 – Problem statement
Background
Mitochondrial morphology is determined by the frequency of fusion and fission, the main events of mitochondrial dynamics (T. Yu, Robotham, & Yoon, 2006). The balance of both processes, as well as the maintenance of a functional mitochondrial population, have a fundamental role in the cellular physiology of skeletal muscle, given that biochemical and morphological analyzes have shown the presence of alterations in mitochondria in this tissue in conditions of obesity and insulin resistance (Huei-Fen. Jheng et al., 2012; Luo et al., 2013; Whitley, Engelhart, & Hoppins, 2019).
Mitochondrial dynamics evaluation, in vivo, by transfection of mitochondrial matrix- targeted photoactivatable green fluorescent protein (mtPAGP) with electroporation of the gastrocnemius muscle of mice, showed organelle communicates, revealing the mitochondria dynamics nature that allows sharing its content. In contrast, dynamic behavior was altered in the skeletal muscle of high-fat diet-induced obesity mice, evidenced by a decrease in fluorescence and the distance at which fluorescence spread in a longitudinal direction (R. Liu et al., 2014).
Through electron microscopy, considered the gold standard for the evaluation of mitochondrial content (Bishop, Granata, & Eynon, 2014), it has been shown that patients with obesity have smaller mitochondria, evidenced by a 35% reduction in the mitochondrial area of the vastus lateralis muscle in comparison with lean subjects, who display a more defined internal mitochondrial structure (Kelley, He, Menshikova, & Ritov, 2002). In experimental models of diet-induced obesity, a decrease in the area has also been seen, where obesity is related to the diminished mitochondrial area and maximum
12 gastrocnemius muscle length in comparison to wild type mice. This same group demonstrated that in differentiated C2C12 cells (skeletal muscle cell line) treated with palmitate changed mitochondrial morphology towards a fragmented and discontinuous network, evidenced by a higher proportion of smaller mitochondria (Huei-Fen. Jheng et al., 2012). Smaller mitochondrial size in these studies was associated with decreased mitochondrial function, specifically due to reduced NADH:O2 oxidoreductase enzyme activity decreased membrane potential, and lower ATP content (H.-F. Jheng et al., 2012;
Kelley et al., 2002).
These morphological alterations observed in the obesity have been associated with changes in the expression profiles of the main proteins involved in mitochondrial dynamics, particularly by upregulation fission proteins and downregulation fusion proteins (Ciaran E.
Fealy, Mulya, Lai, & Kirwan, 2014; R. Liu et al., 2014; T. Yu et al., 2006). In skeletal muscle mitochondria of ob/ob mice and those treated with a high-fat diet for more than 10 weeks, as well as in mitochondria isolated from C2C12 cells of skeletal muscle, the levels of Drp1 and Fis1 are significantly elevated. On the other hand, a high-fat diet can also decrease the expression of Mfn1 and Mfn2 (H.-F. Jheng et al., 2012; R. Liu et al., 2014).
Lifestyle changes that include physical activity and exercise are considered key in the management of obesity (Strasser, 2013). Moderate-intensity aerobic exercise is recognized for its positive effects on weight control, adiposity levels and other serum markers such as fasting glucose and total cholesterol levels, as well as improvements in insulin sensitivity (Axelrod, Fealy, Mulya, & Kirwan, 2019; G. da Rocha et al., 2016). However, mitochondrial function and dynamics changes by chronic exercise, particularly moderate- intensity exercise are unclear.
13 Short term studies in non-obesity conditions that include aerobic exercise have shown a positive regulation of Drp1 at the Ser637 residue, related to the enhancement of fusion in the gastrocnemius muscle and improvements in oxidative metabolism (Moore et al., 2019a).
In skeletal muscle biopsies of patients with obesity and insulin resistance, 7 weeks of moderate aerobic exercise shown a decrease in Drp1 and an increase in Mfn2 (Pattanakuhar et al., 2019). Longer period of exercise (12 weeks) has shown a decrease in Drp1 activity by Ser616 residue phosphorylation (pro-fission) as wells as upregulation of OPA1 (Ciaran E. Fealy et al., 2014). In the same time, lower Fis1 levels and an increase in the fusion:fission ratio has also been seen (Axelrod et al., 2019). Short term evaluation of mitochondrial dynamics protein profile in conditions of obesity has not been studied.
Furthermore, exercise training can have an impact on mitochondrial homeostasis through changes in mitochondrial biogenesis, a process mainly regulated by PGC1a; aerobic exercise training can enhance PGC1a expression in a high-fat diet induced obesity model (Greene et al., 2014)
AMPK activity has been related to the regulatory mechanisms for the adaptive responses of exercise. In non-obese patients, resistance exercise for 8 weeks significantly increases total AMPK levels, and their activity assessed by phosphorylation at the Thr172 residue.
However, in patients with obesity and metabolic syndrome, only a significant change in its total content and not in its activity has been described after 8 weeks of resistance exercise (Layne et al., 2011). However, in experimental models of diabetes, a significant increase has been observed after the same period using aerobic exercise training (H. W. Liu &
Chang, 2018)
14 Finally, in our research group, the effect of 60 min of moderate exercise through a swimming session in the obese Zucker rat was evaluated, resulting in a significant increase in Mfn2 and a decrease in total Drp1 when compared to a sedentary obese control.
Importantly, after evaluation of mitochondrial distribution by confocal microscopy, the reestablishment of the organization in the intermyofibrillar region was shown after this single session (Rivera-Álvarez et al., 2020). Given that the evidence for the effect of moderate physical exercise is not entirely clear and that a single session revealed significant modifications, this study seeks to study the initial effects of chronic moderate-intensity training in this obesity model on proteins that regulate dynamics and mitochondrial function.
Problem statement
The mitochondrion is a dynamic organelle involved in important cellular activities and a disruption of its function is associated with the development of obesity-induced insulin resistance (Heo et al., 2017). However, the role of exercise in obesity-associated mitochondrial abnormalities has not been fully elucidated. More importantly, short-term evidence showing initial adaptations has not been described under conditions of obesity.
Therefore, the purpose of this study was to answer the following research hypothesis:
Moderate physical exercise will regulate mitochondrial dynamics through AMPK activity, leading to an improvement in mitochondrial function in an obese condition.
15 Research objectives
1. General objective
To evaluate the effect of exercise training on protein and gene expression of the main regulators of mitochondrial dynamics, AMPK activity, and mitochondrial function in skeletal muscle from obese Zucker rats.
2. Specific objectives
1. To evaluate the gene expression and levels of mitochondrial dynamics proteins and AMPK in gastrocnemius muscle after four weeks of moderate exercise training.
2. To evaluate mitochondrial function by membrane potential determination and mitochondrial distribution in gastrocnemius muscle fibers after four weeks of moderate exercise training.
3. To evaluate mitochondrial biogenesis in gastrocnemius muscle after four weeks of moderate exercise training.
Rationale
Mitochondrial dysfunction in skeletal muscle is considered a hallmark of obesity and insulin resistance, which is characterized by impaired bioenergetics, altered morphology, and high production of reactive oxygen species (ROS) (Greene et al., 2015). Evidence indicates that mitochondrial morphology and function are closely regulated (Diaz-Vegas et al., 2020) and that the maintenance of both are necessary for the treatment of patients with metabolic syndrome. However, the molecular mechanisms between the metabolic
16 syndrome and the structural and functional changes of the mitochondria are not entirely clear (Bhatti, Bhatti, & Reddy, 2017; P Hardwick, 2014). Conditions such as obesity have been described to promote fragmentation of the mitochondrial network, an event that is involved in the development of metabolic diseases (Axelrod et al., 2019), alteration that can be improved by physical exercise (Greene et al., 2015). Although the effect of physical exercise on mitochondrial volume, number, and density is widely known, its effect on mitochondrial dynamics is unclear (Axelrod et al., 2019). On the other hand, existing data is limited to the expression of mRNA and fusion and fission regulatory proteins, however, the development of dynamic methods that allow visualizing both processes in skeletal muscle in conjunction with molecular markers that are specific to mitochondrial health is of great importance (Drake, Wilson, & Yan, 2016). Since dynamic regulation is essential in the response of cells to various physiological challenges, it is of utmost importance to understand the molecular basis of the modulation of mitochondrial dynamics (R. Yu, Jin, Lendahl, Nistér, & Zhao, 2019). Skeletal muscle has specialized features that raise important questions about the role of mitochondrial fusion and fission that include fiber type heterogeneity and diverse mitochondrial subpopulations; for these reasons, is important to consider selective remodeling of the network (Heo et al., 2017; Mishra &
Chan, 2016; Moore et al., 2019b). In summary, maintaining skeletal muscle homeostasis through balancing mitochondrial morphology and restoring its function may offer a novel therapeutic strategy for the treatment of metabolic syndrome (Chan, 2020; Huei-Fen. Jheng et al., 2012; H. W. Liu & Chang, 2018).
17 Scope of the study
The results of this project will provide evidence of the initial chronic adaptations of moderate physical exercise on the morphology and mitochondrial function in skeletal muscle of an experimental model of obesity, as well as modifications in the distribution of the main mitochondrial subpopulations, the subsarcolemmal and intermyofibrillar mitochondrial regions.
On the other hand, this study has various limitations, since changes in metabolic pathways such as insulin signaling or fatty acid oxidation that would allow us to have a more complete picture of the effects of exercise will not be explored; in addition, in this study we will only obesity conditions being assessed, a lean group will not be included.
18 Chapter 2 – Theoretical framework
Etiology and prevalence of Metabolic Syndrome
Obesity, defined as the abnormal or excessive accumulation of adipose tissue in the body (World Health Organization, 2020), is a chronic disease of multifactorial origin, dependent on the complex interaction between genetic, environmental, behavioral and epigenetic factors that produce a state of positive energy balance leading to an increase in body weight (G. da Rocha et al., 2016; Kadouh & Acosta, 2017). In this regard, a sedentary lifestyle due to physical inactivity is considered an important contributing factor for escalating the prevalence of obesity and other metabolic abnormalities (Bhatti et al., 2017).
Obesity conditions is associated with metabolic syndrome, first identified by Raven et al in 1988 as “X Syndrome”, which is characterized by the presence of hypertension, dyslipidemia, insulin resistance and a state of prediabetes, manifested with an impaired fasting glucose or glucose intolerance. Consequently, metabolic syndrome carries an increased risk of cardiovascular disease, type 2 diabetes and certain types of cancer, resulting in a reduction in life quality (Bagry, Raghavendran, Carli, & Phil, 2010; Baskin, Winders, & Olson, 2015; G. da Rocha et al., 2016). A systematic meta-analysis reported that metabolic syndrome has a prevalence of 41% in Mexican adults (Gutiérrez-Solis, Datta Banik, & Méndez-González, 2018).
Clinical and experimental trials have provided evidence that obesity-induced insulin resistance in high energy consumption tissues, such as skeletal muscle, constitutes a characteristic feature of metabolic dysfunction due to a strong association with most
19 components of the syndrome (Gutiérrez-Rodelo, Roura-Guiberna, & Olivares-Reyes, 2017).
Mitochondrial Dysfunction as a mechanism underlying insulin resistance in skeletal muscle
Insulin resistance is a condition where higher circulating insulin levels are necessary to achieve its glucose-lowering response (Petersen & Shulman, 2018). The underlying mechanisms involve an increase in fatty acid (FA) uptake that contributes to increased lipid accumulation in skeletal muscle, leading to lipotoxicity, which is known to impair muscle insulin sensitivity. Increased FA uptake contributes to accumulation of proinflammatory lipid metabolites (fatty acyl-CoAs, diacylglycerols, and/or ceramides); these intracellular lipid metabolites have been shown to activate serine/threonine protein kinases and suppress insulin actions (Huei-Fen. Jheng et al., 2012).
The manifestations of insulin-resistant glucose metabolism include reduced glucose transport due to impaired GLUT4 translocation and reduced insulin-receptor tyrosine phosphorylation, IRS-1 tyrosine phosphorylation and reduced rates of glycogen synthesis.
In skeletal muscle, various factors may contribute to disturbances in glucose and FA metabolism, including an alteration in the functional capacity of mitochondria. Skeletal muscle is particularly susceptible to mitochondrial dysfunction due to its high content of mitochondria and its strong dependence on oxidative phosphorylation to produce energy (Kelley et al., 2002).
20 Skeletal muscle and myofibers
Skeletal muscle comprises 40-60% of human body weight and contributes to multiple functions. From a metabolic point of view, it is a major determinant of whole-body aerobic capacity due to its crucial role in the regulation of fatty acid uptake, fatty acid oxidation, basal energy metabolism and is the primary tissue responsible for glucose disposal (Frontera & Ochala, 2015; Greene et al., 2014; Guadalupe-Grau et al., 2018).
Skeletal muscle is characterized by an arrangement of multinucleated muscle fibers;
important cellular elements constitute myofibers, including a transverse tubular system, the sarcoplasmic reticulum (SR) and a mitochondrial network (Frontera & Ochala, 2015).
Every muscle contains a mixture of distinct fiber types that are specialized according to the work they perform (Mishra & Chan, 2016). Muscle contractile properties are underpinned by a contractile protein called myosin, composed of two heavy chains and four light-chain subunits. Four isoforms of myosin heavy chain (MyHC) expressed in adult skeletal muscle tissues have been classified as MyHCI, -IIA, -IIX, and -IIB (IIB is not found on human tissues) (Komiya et al., 2015; Murgia et al., 2017). Type I fibers are characterized by slow contraction rates, high mitochondrial content and increased reliance on oxidative phosphorylation (OXPHOS). On the other hand, type II fibers have fast contractions, lower mitochondrial content, and a decrease reliance on OXPHOS (Mishra & Chan, 2016).
In skeletal muscle, mitochondria form a highly dynamic network that adapts to changing energetic stimuli by modifying their reticular nature and their content (Arribat et al., 2019).
Bioenergetic fluctuations within a cell result in mitochondrial adaptations concerning the stimuli, particularly in the volume of mitochondria per volume of muscle fiber, known as mitochondrial volume density (Meinild Lundby et al., 2018).
21 Mitochondria from skeletal muscle
Mitochondria have five compartments that carry out specialized functions: the outer mitochondrial membrane (OMM), the inter-membrane space, the inner mitochondrial membrane (IMM), the cristae and the matrix. Mitochondria have a fundamental role in providing the energy required for everyday activities. In skeletal muscle, mitochondria are organized in a reticular network and their main role is the production of adenosine triphosphate (ATP). The production of ATP takes place during the reactions of the tricarboxylic acid (TCA) cycle, located within the matrix, and via the electron transport chain (ETC), located along the IMM. The ETC comprises five multi-polypeptide complexes (complexes I-V) embedded in the IMM that receive electrons from the reduced forms of nicotinamide adenine dinucleotide (NADH + H+) and flavin adenine dinucleotide (FADH2), generated mainly in the TCA cycle. During the initial step, electrons are transferred through complexes I and complex II to IV of the ETC, to its final acceptor, O2. Through this process, protons are pumped out of the matrix into the inter-membrane space generating an electrochemical gradient (Δψm) that is the driving force enabling Complex V to generate ATP by phosphorylation of adenosine diphosphate (ADP) (Figure 1). The combination of these last two processes is described as oxidative phosphorylation (OXPHOS) (Bishop et al., 2014).
22
Figure 1. Mitochondrial Bioenergetics. Adapted from (Marroqui et al., 2018)
In addition, skeletal muscle cells display distinct mitochondrial subpopulations that differ in morphology and function. Subsarcolemmal mitochondria (SS) reside beneath the sarcolemma and are generally globular with a few branches, whereas intermyofibrillar mitochondria (IMFM) are located between myofibrils (Figure 2A) arranged in pairs (Figure 2B) at the z-band of each sarcomere and have elongated tubular shapes (Vendelin et al., 2005; Vincent et al., 2019; Wahwah, Kras, Roust, & Katsanos, 2020)
Figure 2. A) Mitochondrial subpopulations localization. B) Pair arrangement of mitochondria network in gastrocnemius muscle. Adapted from (Vendelin et al., 2005; Wahwah et al., 2020)
Each mitochondrial subpopulation has distinctive lipid and protein content, as well as different capacities for mitochondrial respiration and endogenous protein synthesis. IMF mitochondrial subpopulation is suggested to specialize in energy production for contractile
A B
23 activity, whereas SS mitochondrial has a role in providing energy for metabolic processes that are carried out at the sarcolemmal level (Wahwah et al., 2020).
In agreement with these, the analysis of the autofluorescence signal of flavoproteins in subsarcolemmal mitochondria presents a different organization, characterized by the formation of mitochondrial groups in this region. A higher oxidized state in subsarcolemmal mitochondria has been demonstrated by an increase in flavoprotein signal using confocal microscopy, a measurement that reflects ETC activity, which could be related to metabolic status and activity (Kuznetsov et al., 2006). This heterogeneous response may reflect not only a different structural composition but also the existence of various regulatory mechanisms for mitochondrial homeostasis within different regions of the muscle cell, which will have an impact on mitochondrial quality control, which is ensured through the coordination of mitochondrial biogenesis, dynamics, and mitophagy.
(Diaz-Vegas et al., 2020; Ferreira et al., 2010; Wahwah et al., 2020).
Mitochondrial Quality Control
Mitochondrial biogenesis, defined as the synthesis of new mitochondria from pre-existing ones, is mainly coordinated by the proliferator-activated receptor-g co-activator 1a (PGC1a) (Diaz-Vegas et al., 2020). Upregulation of PGC1a and other transcription factors start the transcription of nuclear genes encoding mitochondrial proteins such as mitochondrial transcription factor A (TFAM). TFAM is then imported into mitochondria by the protein import machinery to mitochondrial DNA (mtDNA) bind, in order to upregulate the expression of genes encoding ETC subunits, resulting in increased oxygen consumption, ATP synthesis, and mitochondrial content. Mitochondrial fusion and fission,
24 the main events of mitochondrial dynamics regulate mitochondrial turnover by facilitating the dilution and clearance of damaged organelles through a specialized autophagic pathway, known as mitophagy (Picca et al., 2018). The dynamic nature of the mitochondrial quality control helps maintain a constant mitochondrial mass by creating balance between biogenesis and degradation (Diaz-Vegas et al., 2020).
Mitochondrial dynamics shape the mitochondrial network and contribute to mitochondrial function and quality control and both fusion and fission are integrated into diverse cellular functions that respond to changes in cell physiology (Whitley et al., 2019)
Mitochondrial Dynamics
Recent advances in molecular biology have redefined to mitochondria as a plastic network sensitive to the bioenergetic demand of the cell (Axelrod et al., 2019).
The overall morphology of mitochondria is maintained through the balance between mitochondrial fusion and fission, the main events of mitochondrial dynamics. The role of mitochondrial dynamics is enabling content mixing of both mitochondrial matrix and membranous components among the mitochondrial population (R. Liu et al., 2014) allowing metabolic adaptations and its quality control (Diaz-Vegas et al., 2020).
The frequencies of fusion and fission events are balanced to maintain the overall morphology of the mitochondrial population (Huei-Fen. Jheng et al., 2012). This balance is required for maintaining the integrity of the mitochondrial genome (mtDNA), which encodes essential components of the ETC (Chen et al., 2010; Mishra & Chan, 2016;
Whitley et al., 2019) and to support mitochondrial function with the objective to prevent some disease development (Tilokani, Nagashima, Paupe, & Prudent, 2018).
25 The main proteins composing the core machinery of mitochondrial dynamics are large GTPase proteins belonging to the dynamin family. These mechanoenzymes can oligomerize and change the mitochondrial conformation to drive membrane remodeling, constriction, fission and/or fusion. Even within the same cell, mitochondrial can exist in a variety of ever-changing shapes (Diaz-Vegas et al., 2020). Under most physiological conditions, mitochondrial shape and size remain relatively constant; the deregulation of these spatio-temporal events favoring either fusion or fission results in either a fragmented network characterized by a large number of small round-shape mitochondria or a hyperfused network with elongated and highly connected mitochondria (Tilokani et al., 2018).
1. Mitochondrial Fusion
Fusion of mitochondrial network depends on the activity of large, membrane localized GTPases and involves mixing of matrix contents. Fusion plays a key role particularly after nutrient deprivation, since elongation of mitochondria prevents degradation by mitophagy(Chan, 2006b; Diaz-Vegas et al., 2020).
In mammalian cells, the fusion of mitochondria is regulated by two mitofusins, MFN1 and MFN2 situated in the outer mitochondrial membrane (OMM), and OPA1, optic atrophy protein, located in the inner mitochondrial membrane (IMM). MFN1 and MFN2 display a very high degree of homology, with 80% of sequence similarity in humans, and similar structural organization but display different functions: MFN1 is a core component of the fusion reaction together with OPA1, whereas the exact role of MFN2 in fusion remains elusive and it has been shown to participate in mitochondrion-mitochondrion interaction,
26 as well as in juxtaposition of mitochondria with other organelles (in particular with the ER) (Chan, 2006a; Giacomello, Pyakurel, Glytsou, & Scorrano, 2020).
2. Mitochondrial Fission
Mitochondrial fission requires the coordinated action of other factors. Actin-mediated division at mitochondria-SR contact sites activates downstream signals in the fission pathway (Whitley et al., 2019).
Mitochondrial division is executed by the cytosolic GTPase dynamin-related protein 1 (Drp1). The lack of a membrane-localizing pleckstrin homology (PH) domain, a transmembrane (TM), or any other membrane-anchoring domain makes it necessary for Drp1 to be actively translocated to mitochondria in order to execute its function by MOM- anchored receptors, mitochondrial fission protein 1 (Fis1) and mitochondrial fission factor (MFF) membrane that are translocated to the mitochondrial surface during mitochondrial division, where it assembles into higher-order complexes at the endoplasmic reticulum (ER)–mitochondrial contact sites to wrap around the mitochondria inducing mitochondrial fission via its GTPase activity (R. Yu et al., 2019). Additionally, post-translational modifications regulate Drp1 recruitment or detachment from mitochondria. During mitosis, phosphorylation of Drp1 at Ser616 by cyclin dependent kinase (CDK) 1/Cyclin B or CDK5 promotes mitochondrial fission. Drp1 phosphorylation at Ser637 by protein kinase A (PKA) induces its separation from mitochondria, inhibiting fission; in contrast, calcineurin mediated dephosphorylation at S637 enables its translocation to mitochondria (Ko, Hyun, Min, & Kim, 2016). Mitochondrial fission predominates during elevated stress levels and cell death; however it is also observed in the phase G2/M of the cell cycle and
27 is needed for mitochondrial motility, quality control and mtDNA inheritance (Tilokani et al., 2018).
The complex regulation of mitochondrial dynamics is essential to facilitate diverse cellular functions and as described before, an abnormal mitochondria structure is associated with its dysfunction and contribution to disease pathology, either because they are attributed to altered expression of the mitochondrial fusion and fission proteins or because abnormal signaling pathways are predicted to modify mitochondrial dynamics (Whitley et al., 2019).
Therefore, therapeutics that address mitochondrial dysfunction have a potentially broad clinical impact. Especially, those that re-establish the balance between fusion and fission since they might improve cellular function, leading to improved patient outcomes (Whitley et al., 2019). Among non-drug treatment strategies, continuous aerobic exercise performed at low/moderate intensities result in several adaptations on muscular systems (G. da Rocha et al., 2016).
Aerobic Exercise
Endurance exercise has long been recognized to bring about beneficial health outcomes and enhance physical performance. Repetitive muscular contractions generate specific mechanic stimuli that promote significant changes in the structure and function of the cellular elements in skeletal muscle, which are highly dependent on the parameters of the physical exercise protocols such as intensity, duration and frequency (Frontera & Ochala, 2015; Hackney, 2019). These adaptations include increases in mitochondrial mass, higher oxidative phosphorylation, and cristae remodeling in the IMM (Diaz-Vegas et al., 2020).
28 Accumulating evidence indicates that the enzyme adenosine monophosphate-activated protein kinase (AMPK), which is stimulated upon increases in AMP/ATP, plays an important role in mediating several cellular and metabolic processes during exercise (Sriwijitkamol et al., 2007).
AMPK
AMPK is a ubiquitously expressed serine/threonine protein kinase with a heterotrimeric structure, consisting of an a (a1, a2) catalytic subunit, and two regulatory subunits, b (b1, b2) and g (g1, g2, g3) (Day, Ford, & Steinberg, 2017).
Liver kinase B1 (LKB1), a tumor suppressor, is the major upstream kinase activating AMPK by phosphorylation at Thr172. Also, increases in AMP:ATP ratio and a consequent binding of AMP to the g subunit causes conformational changes in AMPK that promote its activation. Ca2+/calmodulin-dependent protein kinase CAMKKB can also phosphorylate AMPK at Thr172, providing a Ca2+ activated pathway to switch on AMPK (Hardie, 2011).
AMPK is considered a metabolic master switch, which turns off several anabolic processes at the same time that turns on catabolic processes with the purpose of increasing the energy level of the cell, as shown in figure 3 (Morales-Alamo & Calbet, 2016).
Figure 3. Structure and regulation of AMPK. Adapted from Long et al (Long & Zierath, 2006).
a b
g
29 Activation of AMPK leads to the phosphorylation of key metabolic substrates and transcriptional regulators that are linked to nearly all branches of cellular metabolism, including glucose uptake and fatty acid oxidation (Day et al., 2017).
In relation with mitochondrial quality control, AMPK coordinately modulate mitochondrial biogenesis and mitophagy (Diaz-Vegas et al., 2020).The stimulation of AMPK by exercise leads to activation of PGC1a by direct phosphorylation of threonine and serine residues. This phosphorylation event may ultimately promote mitochondrial biogenesis. AMPK can mediate fission and mitophagy by phosphorylation of MFF and ULK1, respectively (Figure 4) (Bhatti et al., 2017).
Figure 4. AMPK regulation on mitochondrial quality control. Adapted from (Herzig & Shaw, 2018; Long
& Zierath, 2006)
Collectively, AMPK activation contributes to the beneficial effects of exercise on glucose and lipid metabolism by acutely increasing muscle glucose disposal and fatty acid oxidation and, chronically, by enhancing mitochondrial number and function (Sriwijitkamol et al., 2007).
30 During exercise performance, reactive oxygen species (ROS) are generated and are also dependent on the intensity, duration and training status and are emerging as an essential signal in mitochondrial adaptation to exercise in skeletal muscle (Morales-Alamo &
Calbet, 2016)
Mitochondrial dysfunction and oxidative stress
Mitochondria generate ROS in a physiological range as a consequence of normal OXPHOS reactions Obesity generates mitochondrial alterations leading to a dysfunctional organelle that impacts in all cellular functions in which mitochondria is involved, including regulation of ROS. Parallel changes in ROS levels and mitochondrial morphology have been reported (R. Liu et al., 2014; Willems, Rossignol, Dieteren, Murphy, & Koopman, 2015) (R. Liu et al., 2014).
In insulin resistance and hence, metabolic syndrome, mitochondrial fission seems to be triggered, an increase in oxidative stress, defined as an imbalance between ROS generation and antioxidant cellular defenses (P Hardwick, 2014; M. Rocha, Apostolova, Diaz-Rua, Muntane, & Victor, 2020). Upon cellular stress, Drp1 is translocated to mitochondria and activation follows. Meanwhile, Mfn1 is inhibited by Bak, a protein involved in apoptosis, resulting in an increase in the ratio fission:fusion and mitochondrial fragmentation, which is sensitized by Bax and triggering cell death (Zhan, Brooks, Liu, Sun, & Dong, 2013).
In summary, mitochondrial dysfunction is defined as the failure of mitochondria to adapt appropriately or sufficiently to its environment with a consequent state of mismatch between mitochondrial activities and cellular demands and it can manifest as altered
31 mitochondrial biogenesis, changes in membrane potential, and the decrease in mitochondrial number and altered activities of oxidative proteins (Bhatti et al., 2017; Diaz- Vegas et al., 2020; Tilokani et al., 2018). As mentioned before, many factors involved in metabolic alterations can damage mitochondria or interfere with mitochondrial repair.
However, the detailed mechanisms and causal relationship between mitochondrial dysfunction and insulin resistance in obesity are not clear (R. Liu et al., 2014). A proposed model of this relationship developed by Fealy et al is presented. Lipotoxicity and the consequent imbalance between supply and demand of fatty acids lead to hyperpolarization of mitochondria, generating ROS. ROS can activate Drp1 as described before leading to leaking of intermediate metabolites, inhibiting insulin signaling. Loss of membrane potential as a result of mitochondrial fission, results in dissipation of the nutrient excess, and a reduced AMP:ATP ratio downregulates AMPK, PGC1a and reduction of fusion, leading to increases in fission:fusion (Figure 5).
Figure 5. Adapted from (CiarÁn E. Fealy, Mulya, Axelrod, & Kirwan, 2018)
32 Chapter 3 – Methodology
Ethical animal handling
Animals were handled in accordance with the regulations of the Mexican Official Norm (NOM-062-ZOO-1999) regarding technical specifications for production, care, and use of laboratory animals. All procedures were approved by the animal use and care committee of the School of Medicine, Tecnologico de Monterrey (Protocol #2019–007).
Experimental Model
Zucker obese rats show apparent obesity from ~4 week. Obesity is due a congenital mutation in the leptin receptor leading to hyperphagia (King & Austin, 2016). Leptin is a hormone mainly produced by adipocytes in white adipose tissue; when in circulation, it is taken up into the brain where it regulates food intake, appetite control behavior and energy expenditure after binding to its receptor and activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) in the hypothalamus (Wang, Chandrasekera, &
Pippin, 2014). There is evidence that an increase in fatty acids uptake, related to a higher expression of muscle plasma membrane fatty acid–binding protein (FABPM) and alteration in the fatty acids disposal to storage may underlie the development of insulin resistance in these rats (Turcotte, Swenberger, Tucker, & Yee, 2001).
Table 1 shows biochemical differences between Lean and Obese Zucker rat in a study developed in our group by Romero-Nava et al. Except for systolic blood pressure (SBP), significant differences are found between lean and Zucker rat.
33
Table 1. Biochemical characteristics between lean and obese Zucker rat
Variable Lean Obese
Weight (g) 367 ± 37 469 ± 10
SBP (mmHg) 140 ± 7.6 151 ± 11.4
Glucose (mg/dL) 113.7 ± 2.6 166.7 ± 10.3 Total cholesterol (mg/dL) 45.6 ± 1.7 93.3 ± 4.06 HDL cholesterol (mg/dL) 19.4 ± 0.46 31.8 ± 1.3 LDL cholesterol (mg/dL 26.27 ± 2.08 61.75 ± 3.3 Triglycerides (mg/dL) 24.3 ± 2.6 142 ± 10
Adapted from (Romero-Nava et al., 2017)
Ten-week-old male Zucker obese (fa/fa) rat (n=8) were maintained on light/dark cycle of 12 h and under controlled temperature environment during this study. Two animals were housed per cage with water and food ad libitum. Body weight was measured weekly.
Figure 6. Obese Zucker rat.
Animals were randomly divided into two groups: sedentary obese group (n=4) and exercise obese group (n=4). The animals from the exercise obese group performed their respective exercise for a period of four weeks (as described below), while animals from the control group (obese sedentary) were kept on their cage for the same period.
34 Swimming Training Protocol
In genetic and diet-induced obesity experimental models, swimming training has been used to show the adaptations to acute and chronic exercise and is considered a modulator of cellular metabolism via changes in muscle bioenergetics and oxidative stress (Flis et al., 2019). When swimming pattern is continuous (avoiding floating) and without addition of weight to the tail or body it is documented to be performed at 45-65% of maximal oxygen uptake (VO2 max) and is considered a moderate intensity exercise (J. H. Jones, 2007).
Swimming training protocol was divided in three phases, pre-adaptation, adaptation and training. In pre-adaptation, rats were exposed for 10 and 15 min/two days to shallow water at 36 ± 1ºC. Adaptation phase consisted in increasing the time of exposition to water. The purpose of the water and time adaption was to reduce stress. The time was gradually increased up to 60 min (G. da Rocha et al., 2016; Lima et al., 2013).
Swimming training sessions were performed in a polymer plastic container (59.7 cm long x 42.9 cm width x 31.1 cm height) with water temperature controlled at 36 ± 1ºC. The swimming training was conducted during 4 weeks with a weekly frequency of 5 days for 60 min and 2 days of rest as shown in table 2.
Table 2. Swimming training protocol
Day 1 2
two day rest
1 2 3 4 5
two day rest
1 2 3 4 5
two day rest
Time 10 15 20 20 30 40 60 60 60 60 60 60
Pre- Adaptation
Week 1: Adaptation Week 2-4: Training
After every exercise session, animals were towel-dried and returned to the bioterium standard conditions.
35 At the end of the four weeks, animals were euthanized 48 hours after the last exercise session and 8 hours without food. After induction and maintenance of anesthesia at 3-3.5%
sevoflurane inhalation respectively, euthanasia was performed by removal of the heart.
Blood samples were collected, and plasma was obtained for biochemical analysis.
Additionally, samples of white adipose tissue, liver, kidney, and soleus muscle were removed, snap frozen in liquid nitrogen and stored at -80ºC.
Figure 7. Obese Zucker rat swimming
Gastrocnemius muscle isolation
As mentioned before, skeletal muscle contains type I and type II fibers. Gastrocnemius muscle has a lateral and a medial head and is mainly composed of type IID/X fibers.
Type II fibers are predominant in MS patients and are associated to higher mitochondrial dysfunction (Cornachione, Benedini-Elias, Polizello, Carvalho, & Mattiello-Sverzut, 2011; Layne et al., 2011; Stuart et al., 2013).
Left and right gastrocnemius muscle were dissected, connective tissue and fat were removed, and the medial head was separated from the whole muscle. Left gastrocnemius muscle medial head was immediately frozen in liquid nitrogen and stored at -80ºC until analysis.
36 Gene and protein expression analysis
Understanding molecular mechanisms provides insight to the physiological capacity.
Genomic and proteomic approaches that include gene and protein expression and functional control of enzymes/proteins have been key drivers of advancement in the field of biological sciences (Hackney, 2019)
RNA Extraction and real-time quantitative PCR (RT-qPCR)
RT-qPCR involves three steps: the reverse transcriptase (RT)-dependent conversion of RNA into complementary DNA (cDNA), amplification of the cDNA using the PCR and detection and quantification of amplification products in real time (Nolan, Hands, &
Bustin, 2006). About 20 mg of tissue samples was homogenized for extraction of total RNA using TRIzol™ reagent (Invitrogen) and quantified by spectrophotometry (260/280nm). cDNA was prepared by reverse transcription of 2.5 𝜇g total RNA using First- Strand cDNA synthesis kit (Promega) using random primer. The PCR reaction included the following component: each primer at a concentration of 300 nM, cDNA template (10 ng), and SYBR Green Master Mix (Bioline). The thermocycling conditions for PCR were as follows: 95∘C for 1 min, followed by 40 cycles of 95∘C for 15 s, 61∘C for 30 s, and 72∘C for 45 s. Each sample was run in triplicate and relative changes in gene expression were calculated according to 2^DDCt method. β-actin primers were included as an internal control to normalize mRNA levels. Primer sequences are shown:
37
Table 3. List of RT-qPCR primers.
Gen Forward (5’-3’) Reverse (5’-3’)
AMPK 5’-CCAGAGAACGTGTTGCTGGA-3’ 5’-CGCATACAGCCTTCCTGAGAT-3’
PGC1a 5’-CAACCAGTACAACAATGAGC-3’ 5’-ACCTACTTCTGCCTAACG-3’
b-Actin 5’TCTGTGTGGATTGGTGGCTC-3’ 5’-ACGCAGCTCAGTAACCGTCC-3’
Drp1 5’-GGTGGAATTGGAGATGGTGGTCGA -3’ 5’-TTCGTGCAACTGGAACTGGCACA-3’
MFN2 5’-TGAAGGATGACCTCGTGCTG-3’ 5’-AACTGCTTCTCCGTCTGCATC-3’
OPA1 5’-AAGGCATCCACCACAGGAAG-3’ 5’-CTCCAACCACAACAACCCGT-3’
OMA 5’-AGTGCGCGCTCACGATAA-3’ 5’-TTACTGCTGGGAGCCTCGAT-3’
Fis1 5´-GACGACATCCGTAGAGGCAT-3’ 5’-CATATTCCTTGAGCCGGTAGT-3’
Western immunoblot analysis
Western blot is a useful technique often used to separate and identify proteins in a mixture after separation by molecular weight through gel electrophoresis. Transfer to a membrane and incubation with label antibodies specific to the protein of interest (Mahmood & Yang, 2012). Approximately 30-40 mg of frozen muscle was homogenized in RIPA buffer containing phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged at for at 13000 rpm at 4ºC. Protein content of the supernatant was determined using the Lowry method with BSA as standard. Aliquots of supernatant were mixed with Laemmli buffer and subsequently boiled at 95ºC for 5 min. 20-40 µg of proteins were loaded onto 12% gels with running buffer at 80-100 V for 2 hr., transferred to polyvinylidene fluoride (PVDF) membranes with transfer buffer at 0.30 A for 1-2 hr. Membranes were blocked with 5%
skimmed milk or 2% BSA in PBS containing 0.1% Tween-20 at room temperature for 2 hr. The membranes were incubated over-night at 4°C with the following primary antibodies: Santa Cruz Biotechnology and Cell Signaling Technology; MFN2 (1:1000,
#9482) OPA1 (1:1000, sc-393296), OMA (1:1000, sc-515788), Drp1 (1:1000, #8570), pDRP616(1:1000, #3455), Fis1(1:1000, sc98900), PGC1a (1:1000, sc-517380), TFAM (1:1000, sc-166965) AMPK(1:1000, #2532) Phospho-AMPKa(Thr172) (1:1000, 40H9)
38 GAPDH (1:1000, sc-25778). The membranes that reacted with the primary antibody were incubated at room temperature for 2 hr. with horseradish peroxidase- conjugated anti- mouse or anti-rabbit secondary antibodies. The bands were detected by using the enhanced chemiluminescence detection re-agent Clarity TM Western ECL (Bio-Rad). Densitometry of band was analyzed using ImageJ software (http://rsb.info.nih.gov/ij/).
Confocal Microscopy experiments
For confocal microscopy experiments, right gastrocnemius muscle was isolated. After dissection, whole gastrocnemius muscle (Figure 8, panel A) was placed on a dish containing relaxation buffer containing potassium aspartate 100 mM, KCl 20 mM, HEPES 20 mM, L-glutamate 3 mM, malic acid 3 mM, EGTA 10 mM, MgCl2 200 mM, CaCl2 22.5 mM, ATP 5 mM, phosphocreatine and creatine kinase. Medial head was separated from the whole muscle (Figure 8, panel B) and as a final step, fiber bundles were separated and used for confocal imaging (Figure 8, panel C)
Figure 8. Gastrocnemius muscle isolation. A)Whole gastrocnemius muscle B) medial head of gastrocnemius muscle C) gastrocnemius muscle fiber bundles
B
A C
39 Membrane potential
Mitochondrial membrane potential (Δψm) is considered an important physiological mitochondrial parameter and a key indicator of cell health, since is related to the capacity of the cell to generate ATP by OXPHOS. Use of fluorescent dyes has become commonly used tools for monitoring Δψm changes. As a class, these dyes are typically lipophilic cationic compounds that accumulate into the mitochondrial membrane matrix space in inverse proportion to Δψm (Perry, Norman, Barbieri, Brown, & Harris, 2011).
Gastrocnemius myofibers were placed in 2 ml Relax Buffer and loaded with 250 nM tetramethylrhodamine ethyl ester (TMRE) for 25 min at room temperature. Confocal imaging was performed with a Leica TCS SP5 microscope (Leica Microsystems, Wetzlar, Germany). Excitation and emission wavelengths were 552 nm and 574 nm, respectively.
Images were acquired, saved and analysis was performed using ImageJ software. A total number of 37 images for the sedentary obese group and a total of 46 images for the exercise obese group were analyzed.
Mitochondrial Distribution
Pasqualin et al, developed an ImageJ plugin to quantify the organization of t-tubules (TT), using fast Fourier transformation (FFT). The regularity of striations and TT within the cell results in the emergence of a main peak in the FFT spectrum of the image. The position of the peak is used to determine the period. Its amplitude is considered an indicator of the organization index (Power). TT power is the best indicator of the transversal organization level in the transverse-axial tubular system (Pasqualin, Gannier, Malécot, Bredeloux, &
40 Maupoil, 2015). Due to the proximity of the mitochondrial network with t-tubules, we used this plugin to quantify the organization in the subsarcolemmal and the intermyofibrillar regions.
Figure 9. Gastrocnemius muscle sarcomere. Adapted from (Rivera-Álvarez et al., 2020).
Mitochondrial Density
The generation of new mitochondria components requires the coordination of the nuclear and mitochondrial genome for the complete compliment of mitochondrial proteins that control aerobic energy production. Approaches based on measurements of mitochondrial and signaling are important biomarkers of mitochondrial biogenesis (Miller & Hamilton, 2012).
1. MitoTracker green fluorescence using confocal microscopy.
Gastrocnemius myofibers were placed in 2 ml Relaxing Buffer and loaded with 50 nM MitoTracker Green for 15 min at room temperature. Confocal imaging was performed with a Leica TCS SP5 microscope (Leica Microsystems, Wetzlar, Germany). Excitation and emission wavelengths were 490 nm and 515 nm, respectively. Images were acquired, stored and analysis of TMRE fluorescence over mitochondrial regions of interest was
41 performed using ImageJ software.
2. Citrate synthase activity.
Citrate synthase activity was assessed in a medium containing (in mM) 100 KH2PO4, 0.2 oxaloacetate, 0.1 acetyl-CoA, 0.03 DTNB (5,5′-Dithio-bis-[2- nitrobenzoic acid]), and 0.05% Triton X-100 (pH 7.4), and 100 μg of mitochondrial protein.
Measurement of CoA was performed by assessing thiol (CoA-SH) formation in the presence of DTNB. The latter reacts with thiol forming TNB- SH-CoA (5-thio-2- nitrobenzoic acid), and this reaction can be monitored at 412 nm. Units of activity were estimated with the extinction coefficient of TNB (13.3 mM−1 × cm−1).
3. Quantification of Mitochondrial DNA Copy Number.
Mitochondrial DNA relative content was measured by assessing mtDNA copy number to nDNA copy number ratio (mtDNA/nDNA) by RT-qPCR as described by Vela-Guajardo et al. (Vela-Guajardo et al., 2017). A mitochondrial gene (D-loop) was amplified for detection of mtDNA and was compared to a nuclear gene (actin). The assay was performed in a total volume of 20 µL containing 20 ng DNA template, 10 µL of 2x and SYBR Green Master Mix (Bioline) and primers at a 200 nM concentration. Amplification was carried out with a single denaturation step for 2 minutes at 95ºC, 30 seconds of annealing/elongation at 60ºC with one additional step of 10 minutes for final elongation at 72ºC.
42 Oxidative stress
There is overwhelming evidence leading to the conclusion that obesity is a state of increased oxidative stress and as described before, upon cellular stress, ROS can promote a state of mitochondrial fragmentation. Protein carbonylation and lipid peroxidation are measurements of damage induced by oxidative stress, while catalase, an enzyme that hydrolyzes hydrogen peroxide to water was measured to evaluate the antioxidant capacity after exercise training.
1. Catalase Activity.
Catalase was indirectly measured through oximetry quantification. Before this assay the oximeter was calibrated using distilled H2O to stablish the O2 concentration in water as a 100% for analysis purposes. The protocol includes 5 mM H2O2, 100 µg of protein, and 50 mM KH2PO4 pH 7 in a total volume of 1 ml. The protocol has a duration of 1 min at 25ºC.
Catalase produces H2O and O2 with a directly proportional relationship after adding H2O2
to the system. H2O2 should be added last after a stable O2 % is obtained from the buffer and sample mix for 10 sec. The range of percentage change was used for analysis.
2. Lipid Peroxidation.
Lipid peroxidation was measured using Lipid Peroxidation (MDA) Assay kit (Colorimetric/Fluorometric) (Abcam, ab118970) in 20 ml of plasma. In this assay, free MDA present in the sample reacts with Thiobarbituric Acid (TBA) and generate an MDA- TBA adduct, which can be quantified colorimetrically at 532 nm.
43 3. Protein Carbonylation.
Protein carbonyl groups are an important and immediate biomarker of oxidative stress.
This marker was measured using the Protein Carbonyl Content Assay Kit (Abcam, ab126287) in 20 mg of muscle. DNP hydrazones formed from the reaction are quantified at 375 nm.
Statistical Analysis
Data are expressed as mean ± standard error of the mean or median and interquartile range, as specified. Unpaired t-test was performed to compare the effects of four weeks of swimming training between the sedentary obese group and the exercise obese group.
Significance level was set at 0.05. Statistical analysis including graphs were produced using GraphPad Prism version 8.0.0 para Mac (GraphPad Software, San Diego, California USA).
Chapter 4 – Results
Body weight evaluation before and after four weeks of swimming training and gastrocnemius muscle weight
As mentioned before, body weight was measured on a weekly basis. As shown in table 4, significant differences in both groups between their initial and final weight were found.
After four weeks of swimming training, there was not significant weight changes between groups (p=0.597).
Table 4. Body weight changes.
Sedentary obese Exercise Obese
Before After p-value Before After p-value
Body weight (g) 430.2± 20.07 652.8± 27.02 0.0006 402.6±7.76 636.8±4.43 <0.0001 Data are presented as mean ± SEM. Unpaired two-tail t-test.
Evaluation of gene expression and levels of mitochondrial dynamics proteins and AMPK on gastrocnemius muscle after four weeks of moderate exercise training
1. Effect of four-weeks of exercise training on mRNA expression in gastrocnemius muscle
Expression levels of genes associated with fusion and fission were measured in the sedentary obese group and the exercise obese group. No difference in the expression levels of genes involved in mitochondrial fusion Mfn2, OPA1 and OMA1 (p>0.05) or mitochondrial fission, Drp1 and Fis1 (p>0.05) were detected in gastrocnemius muscle after four weeks of swimming training (figure 10).
45
Figure 10. Relative expression of genes involved in mitochondrial dynamics in gastrocnemius muscle.
Results are presented as median and interquartile range; n=4 per group. Unpaired one-tail t-test.
2. Effect of four-weeks of exercise training on mitochondrial dynamics protein levels in gastrocnemius muscle.
Mitochondrial Fusion proteins
Protein levels of the mail mitochondrial dynamics regulators were evaluated in the gastrocnemius muscle of the obese sedentary group and the obese exercise group. For the fusion proteins, the Mfn2 protein content is significantly lower (p=0.002) in the obese exercise group in comparison with the control group. As described before OMA1 protein is related to the proteolytic processing of OPA1 in two main isoforms, L-OPA1 and S- OPA1, a lower ratio, is related to mitochondrial fission. Even though OMA1 protein level had a tendency to be higher (p=0.06); total OPA1 content, as well as the ratio of L-OPA1/S- OPA1showed no significant differences (p=0.10 and p=0.33 respectively, figure 11).
MFN2OPA1
OMADRP1 FIS
MFN2OPA1
OMADRP1 FIS 0.0
0.5 1.0 1.5 2.0
Relative mRNA level
Sedentary group Exercise group
46
Figure 11. Mitochondrial fusion protein content in gastrocnemius muscle. A) Representative immunoblots for Mfn2, Opa1 and Oma and corresponding GAPDH. B) Quantification of Mfn2, Opa1 and Oma protein
content. C) L-Opa1/S-Opa1 ratio; n=4 per group. Data are presented as mean ± SEM, *p<0.05
Mitochondrial Fission proteins
Changes in mitochondrial fission proteins were evaluated after four weeks of swimming training. In the obese exercise group, no changes in total Drp1 protein content were found;
however, pDrp1 at Ser616 was significantly increased (p=0.008), while Fis1 was significantly lower (p=0.04), figure 12.
These results indicate that, four weeks of swimming training upregulated mitochondrial fission, evidenced by a decreased level of Mnf2 and an increase in pDrp1.
Figure 12. Mitochondrial fission protein content in gastrocnemius muscle. A) Quantification of Drp1, B) pDrp1Ser616 and C) Fis1 protein content with its respective representative immunoblot; n= 4 per group.
Data are presented as mean ± SEM. Unpaired one-tail t-test, *p<0.05
MFN2 OPA1 OMA 0.0
0.5 1.0 1.5 2.0
Normalized to GAPDH
Sedentary group Exercise group
*
Sedentary obese Exercise obese 0.0
0.5 1.0 1.5
Fis/GAPDH
*
Sedentary obese Exercise obese 0.0
0.5 1.0 1.5 2.0
DRP1/GAPDH
Sedentary obese Exercise obese 0.0
0.5 1.0 1.5 2.0
pDRP1Ser616/DRP1
*
A B C
A B C
Mfn2 GAPDH Opa1
GAPDH OMA GAPDH
Fis GAPDH
Drp1 GAPDH
pDrp1 Drp1
2.8 3.0 3.2 3.4 3.6 3.8
L-OPA1/S-OPA1
Sedentary obese Exercise obese
47 3. Effect of four weeks of swimming training on oxidative stress
Obesity is associated with higher ROS emission and damaged to macromolecules such as lipids and proteins. On the other hand, oxidative stress is suggested as a mechanism underlying mitochondrial fission. After four weeks of swimming training, catalase activity was not modified. No damage assessed by lipid peroxidation on plasma and protein carbonylation in tissue extract after four weeks of swimming training were evident in the obese exercise group.
Table 5. Oxidative stress markers
Sedentary group Exercise group p-value
Catalase (U/mg) 215.4 ± 102 128.4 ± 11.53 0.228
Protein carbonylation (nmol/mg) 17.33 ± 2.5 30.23 ± 9.1 0.113
MDA (pmol/100µl) 76.28 ± 24.3 85.74 ± 8.2 0.363
Data are expressed as mean ± SEM. Unpaired one-tail t-test.
4. Effect of four-weeks swimming training on AMPK total protein level and activity
To identify a possible mechanism by which exercise could be modifying mitochondrial dynamics gene and protein expression, AMPK relative mRNA expression and protein level was evaluated by RT-qPCR and western blot, respectively. Four weeks of swimming training showed a trend for mRNA expression to be higher (p=0.06), as shown in figure 13.
