Adaptations in mitochondrial quality control and interactions with innate immune signaling within skeletal muscle: A narrative review

Highlights

• •Changes in skeletal muscle activity are associated with adaptations in skeletal muscle mitochondria via mitochondrial quality control processes.
• •Chronic disuse is associated with a reduced and fragmented mitochondrial network that is abnormally functioning. On the other hand, exercise training often induces increases in mitochondrial biogenesis and networking, which promotes efficiency.
• •Mitochondrial dysfunction has been associated with stimulation of innate immune responses through the release of mitochondrial DAMPs which can negatively impact skeletal muscle function.
• •Modulating skeletal muscle activity can potentially serve as a promising therapeutic for managing excess inflammatory responses in skeletal muscle.

Abstract

Skeletal muscle health and function are essential determinants of metabolic health, physical performance, and overall quality of life. The quality of skeletal muscle is heavily dependent on the complex mitochondrial reticulum that contributes toward its unique adaptability. It is now recognized that mitochondrial perturbations can activate various innate immune pathways, such as the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome complex by propagating inflammatory signaling in response to damage-associated molecular patterns (DAMPs). The NLRP3 inflammasome is a multimeric protein complex and is a prominent regulator of innate immunity and cell death by mediating the activation of caspase-1, pro-inflammatory cytokines interleukin-1β and interleukin-18 and pro-pyroptotic protein gasdermin-D. While several studies have begun to demonstrate the relationship between various mitochondrial DAMPs (mtDAMPs) and NLRP3 inflammasome activation, the influence of various metabolic states on the production of these DAMPs and subsequent inflammatory profile remains poorly understood. This narrative review aimed to address this by highlighting the effects of skeletal muscle use and disuse on mitochondrial quality mechanisms including mitochondrial biogenesis, fusion, fission and mitophagy. Secondly, this review summarized the impact of alterations in mitochondrial quality control mechanisms following muscle denervation, aging, and exercise training in relation to NLRP3 inflammasome activation. By consolidating the current body of literature, this work aimed to further the understanding of innate immune signaling within skeletal muscle, which can highlight areas for future research and therapeutic strategies to regulate NLRP3 inflammasome activation during divergent metabolic conditions.

Graphical abstract

1. Search strategy

To investigate the relationship between mitochondria, skeletal muscle activity, and the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome complex, we implemented a specific search strategy for this review. We utilized academic databases including PubMed, Google Scholar, and the York University Omni library with the following keywords: “NLRP3 inflammasome complex”, “skeletal muscle”, “mtDAMPs”, “exercise training”, “skeletal muscle disuse”, “aging”, “mitochondrial quality control”, and “innate immune signaling”. To increase specificity of the search results, these keywords were utilized together (i.e., “skeletal muscle disuse” and “innate immune signaling”). All studies were done on animal models or human models using in vitro or in vivo models. While we focused our results primarily on studies done in skeletal muscle, due to a lack of studies in this field, we included some fundamental work done in other cell types to highlight relevant areas that could be expanded upon in the context of skeletal muscle. These studies are always clearly indicated in the text. If a comparable study was done in skeletal muscle or if an article was not relevant to the scope of our paper, the study was excluded.

2. Skeletal muscle structure and function

Skeletal muscle is a dynamic organ that comprises approximately 40% of total body mass and plays an essential role in many functions, including locomotion, whole-body metabolism, and fine motor skills.¹ Skeletal muscle fibers can be classified as either slow-twitch or fast-twitch, depending on their functional properties. Slow-twitch, or Type I fibers, are classified by high levels of the myosin heavy chain I (MHC I) isoform and increased mitochondrial volume, which contribute toward their increased dependence on aerobic respiration and fatigue resistance. In contrast, Type II fibers are characterized by reduced mitochondrial content and a larger cross-sectional area and motor unit size, which facilitates high force production and rapid contractility at the expense of increased glycolytic reliance and fatigability. In human muscle, fast-twitch muscle fibers are further classified as Type IIa and Type IIx. As a continuum, Type IIa fibers express intermediate characteristics that fall between the Type I and Type IIx extremes.² The relative proportion of these myofibers determine the aerobic and force capacity of skeletal muscle and influence adaptations to chronic use and disuse. In general, chronic endurance training induces a shift in the muscle fiber phenotype toward a more oxidative composition (i.e., dependent on fiber type recruitment during exercise bouts).³ On the other hand, disuse stimuli such as immobilization, long-term bed rest, or microgravity have been shown to particularly affect Type II fibers and lead to reductions in muscle oxidative capacity, force production, myofiber size, and endurance.³

3. Skeletal muscle mitochondria

3.1. Mitochondrial structure and function

Mitochondrial content within skeletal muscle is relatively low compared to other tissues, ranging from ∼2%–10% of total cell volume depending on the fiber type and degree of cellular adaptation. In addition, mitochondria can present with a variety of divergent morphologies depending on their subcellular location. Skeletal muscle mitochondria often exist in a networked reticulum, which is further classified into 2 subpopulations.^4,5 Subsarcolemmal (SS) mitochondria are closely localized beneath the sarcolemmal cell membrane and exhibit a circular structure with reduced networking. These mitochondria are implicated in supplying energy to membrane-related processes such as ion transport and nuclear gene expression.⁶ In contrast, intermyofibrillar (IMF) mitochondria exhibit an elongated reticular phenotype and are located between the myofibrils, which enables them to provide ATP to sustain skeletal muscle contractile activity.⁶

3.2. Reactive oxygen species (ROS)

ROS are unstable oxygen molecules characterized by the presence of highly reactive unpaired electrons. These molecules can be categorized as either radicals including superoxide anions and hydroxyl radicals, or non-radical oxygen derivatives, such as hydrogen peroxide.⁷ While ROS can be considered a cytotoxic stressor, they can also act as potentially beneficial signaling molecules that can influence skeletal muscle adaptations.⁸ The mitochondrial electron transport chain (ETC) has been implicated as a large contributor of ROS, which can occur in response to a sustained protonmotive force and the resulting backpressure on the ETC during respiration. This leads to electron leakage from various respiratory complexes, primarily Complexes I and III, and subsequent ROS production.⁹ Within muscle, ROS can act as important second messengers through the regulation of cellular kinases such as the p38 mitogen-activated protein kinase (p38 MAPKs), which are involved in mitochondrial biogenesis and apoptotic signaling, as well as inflammatory signaling through the regulation of the transcription factor nuclear factor kappa-B (NF-κB).^10,11 Due to their high reactivity, ROS can also oxidize various biomolecules, including lipids, proteins, and nucleic acids.^12,13 For instance, ROS have been implicated in the oxidation of mitochondrial DNA, which is especially susceptible to oxidative damage due to its close proximity to the ETC and lack of protective histones. This can cause mitochondrial DNA (mtDNA) mutations and the propagation of several pro-inflammatory pathways through the release of oxidized mtDNA.^14,15

3.3. The mitochondrial genome

In addition to its complex structure, mitochondria also uniquely contain their own genome through mtDNA, which is independent of the nuclear genome. However, mtDNA replication and transcription are still closely regulated by nuclear gene products such as mitochondrial transcription factor A (TFAM), which is involved in several aspects of mtDNA processing. TFAM has been shown to play an essential role in the compacting of mtDNA into protein–DNA complexes termed mitochondrial nucleoids, which allows for improved stability and increased control of mitochondrial gene expression and replication.^16,17 In addition, during mtDNA replication, the binding of TFAM to mtDNA aids in the unwinding of mtDNA to allow other key proteins to initiate DNA replication and control mtDNA copy number.18, 19, 20 TFAM also plays an essential role in the initiation of mtDNA transcription, which is mediated by its binding and subsequent recruitment of transcription machinery, as well as interactions with specific transcription factors such as p53, to increase mtDNA transcripts.^21,22

3.4. Mitochondrial quality control

Due to the imperative role of mitochondria in skeletal muscle health and functioning, the maintenance of the mitochondrial reticulum is critical. As a result, the mitochondrial network undergoes constant remodelling through a series of processes collectively referred to as “mitochondrial quality control” (Fig. 1). These processes include biogenesis, fission, fusion, and mitophagy, which enable the mitochondrial network to adapt and respond to various metabolic demands.

Fig. 1.

Mitochondrial maintenance and quality control processes. The health and functioning of the mitochondrial pool are highly reliant on a series of quality control processes, including mitochondrial biogenesis, fusion, fission, and mitophagy. While these dynamic processes happen simultaneously, the drive toward a particular process can be mediated by several stimuli, including exercise. Following muscle contraction and an altered metabolic demand, several cellular perturbations often occur, including increases in the AMP:ATP ratio, increased cytosolic calcium release from the sarcoplasmic reticulum, and the transient production of ROS. These molecules act as important regulators of mitochondrial maintenance by activating signaling kinases AMPK, CAMK, and p38 MAPK, respectively. Activation of these kinases ultimately converges on the phosphorylation and subsequent nuclear translocation of the master regulator of mitochondrial biogenesis, PGC-1α, which co-activates various transcription factors, including NRF-1/2, to promote the transcription of a variety of NuGEMPs. Following transcription and translation, these proteins can be imported via mitochondrial import machinery to support the production of newly functional mitochondria through biogenesis. To elongate the mitochondrial reticulum and enhance cellular respiration, mitochondria can undergo the process of fusion, which is mediated by MFN1/2 and OPA1 for fusion of the outer and inner membranes, respectively. While the expansion of the reticulum is critical, mitochondrial health is equally reliant on processes that aid in the removal of dysfunctional mitochondria to conserve the remaining pool. Dysfunctional mitochondria can be characterized by a loss of membrane potential (ΔΨ), impaired import and respiration, and an excessive production of ROS. To segregate these mitochondria, the process of mitochondrial fission is induced through the recruitment of DRP1 via the mitophagy receptor, FIS1, which allows the removal of the dysfunctional organelle from the remainder of the reticulum. Lastly, these abnormally functioning mitochondria become destined for cellular degradation through the lysosome in the process of mitophagy, which allows for the breakdown of mitochondrial proteins via hydrolytic proteases to allow for the recycling of monomers to support other cellular processes. AMPK = adenosine 5’-monophosphate (AMP)-activated protein kinase; CAMK = Ca²⁺/calmodulin-dependent protein kinase; DRP1 = dynamin-related protein 1; ETC = electron transport chain; FIS1 = fission protein 1; MFN1/2 = mitofusin-1 and 2; NRF-1/2 = nuclear respiratory factor 1/2; NuGEMPs = nuclear genes encoding mitochondrial proteins; mtDNA = mitochondrial DNA; OPA1 = optic atrophy 1; p38 MAPK = p38 mitogen-activated protein kinase; PGC-1α = peroxisome proliferator-activated receptor-gamma coactivator-1alpha; PINK = phosphatase and tensin homolog induced putative kinase; ROS = reactive oxygen species; TIM = translocase of the inner mitochondrial membrane; TOM = translocase of the outer mitochondrial membrane.

The synthesis of new mitochondria, a process referred to as mitochondrial biogenesis, is reliant on the complex interplay of numerous signaling pathways. A variety of signals, such as cytosolic Ca²⁺ levels, an increased adenosine 5’-monophosphate (AMP)/ATP ratio, and ROS, can lead to the subsequent activation of Ca²⁺/calmodulin-dependent protein kinase (CAMK), AMP-activated protein kinase (AMPK), and p38 MAPK, respectively, which all converge on the transcriptional activation of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α).²³ PGC-1α is a coactivator that is commonly known as the “master regulator of mitochondrial biogenesis” due to its ability to associate with numerous transcription factors, such as nuclear respiratory factor 1 (NRF-1) and peroxisome proliferators-activated receptor gamma (PPARγ), to promote the expression of nuclear genes encoding mitochondrial proteins (NuGEMPs).²⁴ This process supports the generation of new mitochondria through the production of essential proteins, including import machinery components, ETC subunits, and proteins involved in the transcription of the mitochondrial genome such as TFAM.²⁴ Mitochondrial fusion refers to the elongation of the reticulum and is highly regulated through the GTP hydrolases (GTPases), mitofusin-1 and 2 (MFN1/2) and optic atrophy 1 (OPA1), which are responsible for the fusion of the outer and inner mitochondrial membrane, respectively. The fusion of multiple mitochondria promotes increased aerobic respiration efficiency due to the sharing of metabolites and resources.^25,26 However, in certain cases of metabolic stress, segments of the mitochondrial network can become poorly functioning, which is generally characterized by reductions in membrane potential, inefficient aerobic respiration, and accelerated ROS emission. Thus, to retain the integrity of the mitochondrial network, these mitochondria need to be degraded through the processes of mitochondrial fission and mitophagy. Fission promotes the segregation of abnormally functioning mitochondria from the reticulum and is coordinated by the GTPase dynamin-related protein 1 (DRP1) and its associated mitochondrial receptor, mitochondrial fission protein 1 (FIS1).^25,26 If mitochondrial dysfunction cannot be resolved, mitochondria are tagged for degradation through the process of mitophagy to avoid the accumulation of damage-associated molecular patterns such as mitochondrial ROS. Mitophagy is a complex process that can be initiated by several signaling cascades; however, the most widely studied mitophagy system is mediated by the phosphatase and tensin homolog induced putative kinase 1 (PINK1) and Parkin.²⁷ In this process, the recruitment of Parkin by PINK1 leads to the ubiquitination of outer mitochondrial membrane proteins, marking them for interaction with and engulfment by an autophagosomal membrane. Following this, receptors on the autophagosome can bind to lysosomal receptors, facilitating fusion and subsequent mitochondrial degradation and cellular recycling.²⁸

4. Plasticity of skeletal muscle mitochondria

One of the most notable features of skeletal muscle is its ability to adapt to various metabolic demands, which can be partially attributed to the highly plastic nature of the mitochondrial reticulum and the coordination of various mitochondrial quality control processes. However, depending on the stimulus and metabolic perturbations present, the mitochondrial pool can undergo both beneficial and maladaptive changes that ultimately impact the skeletal muscle phenotype (Fig. 2).

Fig. 2.

Adaptive plasticity of skeletal muscle mitochondria. In response to different metabolic states, the mitochondrial reticulum within skeletal muscle undergoes various adaptations that impact the overall skeletal muscle phenotype. During prolonged periods of muscle disuse, the mitochondrial reticulum demonstrates apparent reduction in both mitochondrial content and function in both the SS and IMF subpopulations. For instance, the ability to both generate new mitochondria and degrade dysfunctional organelles results in an accumulation of fragmented and dysfunctional mitochondria within the pool. These mitochondria become characterized by reductions in mitochondrial membrane potential, inefficient aerobic respiration, and an excessive production of mtROS. Overall, this contributes to a less oxidative muscle phenotype and a greater reliance on glycolytic metabolism. In contrast, following chronic endurance training, biogenesis and fusion pathways are upregulated leading to increases in mitochondrial volume and a more networked morphology that improves metabolic efficiency. In addition, mitochondrial turnover is improved and promotes the fission and subsequent degradation of dysfunctional mitochondria. This results in the remaining pool exhibiting improved quality characterized by increases in aerobic capacity, improved ATP production, and reductions in mtROS. IMF = intermyofibrillar; mtROS = mitochondrial reactive oxygen species; SS = subsarcolemmal.

4.1. Chronic endurance exercise

Early pioneering work by Holloszy and Booth,²⁹ Gollnick and King,³⁰ and Kirkwood et al.,³¹ established that endurance exercise could lead to adaptive changes in mitochondrial reticulum content in both rodent models and humans. This highly correlated with improvements in endurance performance, largely due to reduced rates of lactic acidosis, glycogen sparing, and enhanced lipid oxidation.³² These foundational studies sparked interest surrounding the molecular mechanisms that can modulate the phenotype of SS and IMF mitochondria. As a result, various animal models have been employed to study exercise adaptations, including treadmill training, electrical stimulation-induced chronic contractile activity (CCA), and voluntary wheel running. While treadmill training and CCA provide a consistent exercise stimulus with highly controlled parameters, voluntary wheel running is more variable and relies on the behavioral patterns of the subject.³³ Thus, these exercise models lead to varying degrees of adaptation,^11,34,35 depending on exercise duration, intensity, and modality.

Acute exercise has been shown to induce transient metabolic disturbances, promoting the activation of various upstream kinases such as p38 MAPK, CAMK, and AMPK, which positively regulate the nuclear translocation of key mitochondrial proteins such as PGC-1α.^21,36, 37, 38, 39 However, while acute exercise can promote transient changes in cellular signaling, repeated exercise bouts are required to induce long-term adaptations in the skeletal muscle phenotype. Changes in mitochondrial content can be partially attributed to an exercise-induced drive toward mitochondrial biogenesis through increases in PGC-1α transcription and protein content.⁴⁰ This ultimately leads to an increased expression of NuGEMPs, along with TFAM-mediated increases in mtDNA replication and transcription.⁴¹ This increase in protein expression works in conjunction with increases in mitochondrial protein import to promote the generation of new mitochondria.^42,43

While the biogenesis of new mitochondria is beneficial to increasing mitochondrial volume, mitochondrial health is also dependent on the regular turnover of the mitochondrial reticulum. Through increases in the fusion:fission ratio, exercise training can promote an elongated mitochondrial phenotype by increasing the expression of pro-fusion proteins MFN1/2 and OPA1 while downregulating the expression of proteins involved in mitochondrial fission, such as DRP1 and FIS1.44, 45, 46 Furthermore, the turnover of dysfunctional mitochondria relies on the complex interplay between mitophagy initiation and the quality of the terminal lysosomes. Chronic endurance exercise stimulates a larger capacity for mitophagy in conjunction with an increased drive toward lysosomal biogenesis, which is evident through increases in the expression of Parkin and the transcription factor EB (TFEB), which is closely linked to lysosomal biogenesis.^34,47 Consequently, improvements in mitochondrial density and function have repeatedly demonstrated a positive correlation with oxidative efficiency and functional parameters in skeletal muscle, such as maximal oxygen consumption (VO_2max) and lactate production.^48,49

4.2. Muscle disuse

In contrast to the adaptive increases in mitochondrial content brought about by regular exercise, chronic muscle disuse can have notable deleterious effects on the mitochondrial pool and skeletal muscle health. In human studies, muscle disuse can be induced through models like bed rest or unilateral limb immobilization. On the other hand, several animal models can be used to partially or completely limit muscle activity in relatively short time periods, including sciatic nerve transection, hindlimb immobilization, and hindlimb suspension.⁵⁰

4.2.1. Skeletal muscle denervation

Skeletal muscle denervation is a widely used experimental model for studying muscle disuse. However, it is important to recognize that the complete removal of neural input adds an additional layer of complexity compared to immobilization models in which the nerve remains intact. As a result, denervation closely resembles the disuse induced by conditions of neural trauma or peripheral neuropathies. As expected, the affected muscles are characterized by rapid decreases in whole muscle size and myofiber cross-sectional area. Prior work has demonstrated that 7 days of unilateral hindlimb denervation is sufficient to reduce the nuclear expression of PGC-1α alongside reductions in the mtDNA regulator TFAM. These changes impede the generation of new mitochondria through reductions in biogenesis pathways and contribute toward the observed declines in organelle content.51, 52, 53, 54, 55 Within the remaining mitochondrial pool, mitochondria also demonstrate increased signs of dysfunction, abnormal morphology, and a fragmented reticulum. Consequently, these mitochondria exhibit the reductions in oxygen consumption and increased ROS emission that precede any evident muscle atrophy (Fig. 2).56, 57, 58 In addition, to compensate for abnormally functioning mitochondria, the expression of various mitophagy and lysosomal markers such as TFEB, PINK1, Parkin, and p62 are upregulated.^54,59,60 However, while mitophagy machinery is upregulated, the active degradation of dysfunctional mitochondria, measured by mitophagy flux, shows an initial transient increase that diminishes over time as denervation progresses.^51,53 Altogether, these findings demonstrate that while signaling toward mitophagy is activated following periods of disuse, due to impaired degradation capacity, dysfunctional mitochondria accumulate within denervated skeletal muscle, which further propagates catabolic signaling and muscle atrophy.

4.2.2. The aging phenotype

The natural progress of aging provides a multifaceted stimulus that involves whole-body adaptations and inter-tissue crosstalk that can impact skeletal muscle health and quality of life.⁶¹ Several studies have shown that beginning as early as the age of 50, aging populations lose ∼1%–2% of muscle mass per year.62, 63, 64 As a result, this progressive loss of muscle mass with aging, termed sarcopenia, has become a prominent area of research to better understand the cellular changes associated with its development. Similar to the changes seen following muscle denervation, aged muscle exhibits a decrease in mitochondrial content that promotes higher glycolytic reliance.⁶⁵ The mitochondrial reticulum demonstrates a pro-fission phenotype with increased fragmentation, which stimulates a larger drive toward mitophagy and mitochondrial-mediated apoptosis.^46,66,67 In comparison to young counterparts, aged muscle also demonstrates an impaired ability to initiate autophagy in response to metabolic stressors.⁶⁸ This balance in mitochondrial quality is further altered through a reduced drive toward mitochondrial biogenesis, evident through decreases in PGC-1α at both the transcriptional and protein level, which can be improved with both acute and chronic bouts of exercise.^69,70 Chronic exercise has been universally highlighted as an effective strategy to preserve skeletal muscle mass and functional performance with age, in part by improving the health of the mitochondrial pool.71, 72, 73 Since aging is a whole-body phenomenon, it is also important to recognize the wide variety of cellular changes in alternative tissue types that can also directly impact skeletal muscle health and function. For instance, an emerging role of immune cells is being increasingly explored in the context of aging due to the observed systemic low-grade chronic inflammation and senescence-associated secretory phenotype, which has been coined “inflammaging”.⁷⁴

5. Innate immune signaling

5.1. The first line of defense

All cellular organisms require the ability to defend themselves against cellular perturbations, including external pathogens such as bacteria and viruses as well as endogenous damage brought about by injury or internal disease. The immune system, comprised of both the innate immune system and adaptive immune system, works to recognize abnormal entities and swiftly launch effector responses to eliminate the source and maintain homeostatic conditions. Specifically, the innate immune system is often termed “the first line of defense” because it provides non-specific and rapid responses against foreign substances or damaged molecules. In contrast, the adaptive immune system is responsible for the specific recognition and targeting of foreign antigens, as well as for immunological memory of pathogens.⁷⁵ Despite being distinct responses, the innate immune system acts as a critical initiator of inflammation that provides sufficient time for an adaptive response to be mounted.⁷⁶ Additionally, in contrast to the original notion, several studies have begun to demonstrate that the innate immune system is capable of “trained immunity” to prevent reinfection within invertebrates, which lack an adaptive immune system.⁷⁷ This could implicate the innate immune system as a potential contributor to immunological memory alongside the adaptive immune system.

5.2. Pattern recognition receptors (PRRs)

To better understand the role of the innate immune system in responding to foreign stimuli, it is critical to investigate the precise mechanism through which recognition occurs. PRRs are able to recognize pathogen-derived compounds called pathogen-associated molecular patterns (PAMPs) that are not expressed through the host organism to launch a corresponding immune response.⁷⁸ In addition, PRRs can be activated by danger signals produced from damaged host cells termed damage-associated molecular patterns (DAMPs).⁷⁹ To ensure that signals are not going undetected, PRRs are located strategically within various compartments, including cell membranes and cytosol. In brief, PRRs bind to structures through their ligand recognition domains, which recruit various adaptor proteins to launch downstream signaling cascades to activate effector molecules.⁸⁰ To respond effectively to different stimuli, there are various classes of PRRs that possess unique structural characteristics and cellular localization, including Toll-like receptors (TLRs) and NOD-like receptors (NLRs).⁸⁰

5.2.1. TLRs and NLRs

TLRs are a group of transmembrane receptors that share common pathways of activation to initiate an immune response. The stimulation of TLRs by their respective ligands prompts the activation of intracellular signaling pathways mediated by adaptor proteins such as myeloid differentiation primary response gene 88 (MyD88), which converges upon the activation of transcription factors such as NF-κB and interferon regulatory factor 7 (IRF7). This transcriptionally upregulates the production of pro-inflammatory proteins, such as cytokines and inflammasome components, to facilitate immune signaling.

In addition to TLRs, NLRs are an alternative class of PRRs that also aim to induce innate immune responses. However, while both receptors lead to the upregulation of similar signaling pathways (e.g., NF-κB and IRFs), they possess several distinct features. NLRs are located within the cellular cytoplasm, which influences the variety of PAMPs and DAMPs that are detected. For example, NLRs are primed to recognize various internal danger signals that occur with cellular stress, including extracellular ATP, non-nuclear DNA, and ROS.⁸¹ Several NLRs also possess the ability to assemble into multi-protein complexes termed inflammasomes; these include NLRP1, NLRP2, NLRP3, and NLR family CARD domain containing 4 (NLRC4).⁸² Due to its unique ability to respond to a wide variety of intracellular DAMPs, this review will focus on the NLRP3 protein and its mechanism of action.

5.2.2. Cyclic guanosine monophosphate (GMP) –AMP synthase (cGAS)–stimulator of interferon genes (STING) activation

Beyond TLRs and NLRs, another key component of innate immune pattern recognition is the cGAS–STING DNA sensing pathway. cGAS is an intracellular PRR found within the cytoplasm that specifically recognizes double-stranded DNA (dsDNA). Various studies have demonstrated that mitochondrial DNA, released upon severe mitochondrial dysfunction, can be a potent activator of this immune response and so can mediate inflammasome activation, senescence-associated phenotypes, and chronic inflammation.^83,84 In brief, the allosteric binding of dsDNA to cGAS activates its enzymatic activity and promotes the synthesis of cyclic GMP–AMP (cGAMP). This potent second messenger can translocate to the endoplasmic reticulum and interact with the STING protein, leading to the downstream activation of the tank-binding kinase 1 (TBK1). In addition to activating the Type I interferon response via IRF3, TBK1 can also lead to the release and nuclear translocation of NF-κB from its negative regulator inhibitor of NF-κB (IκB).⁸⁵

5.3. Inflammation and skeletal muscle function

Skeletal muscle displays impressive adaptive capabilities that are influenced by the intricate crosstalk between various systems to support its diverse functions. For example, following acute muscle injury, various circulating immune cells, such as neutrophils and monocytes, as well as tissue-resident macrophages are recruited to the site of injury by the release of damage molecules, including ROS and extracellular matrix components.86, 87, 88 Furthermore, acute exercise is associated with increased oxidative stress and acute muscle damage that is partly mediated by the nuclear translocation of the transcription factor NF-κB.⁸⁹ In contrast, while acute exercise is considered transiently pro-inflammatory, exercise adaptations over time are often associated with an attenuated inflammatory response. For instance, chronic endurance training has been shown to decrease skeletal muscle macrophage content.^90,91 Consequently, this is associated with reductions in circulating pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and increases in classical anti-inflammatory cytokines, such as IL-10.91, 92, 93 However, some studies have shown opposing results suggesting that chronic high-intensity endurance training is associated with increases in skeletal muscle cytokines, including IL-1β, IL-12, and anti-inflammatory interleukin-1 receptor antagonist (IL-1ra), possibly suggesting an impact of exercise intensity and modality on immune adaptations.94, 95, 96

While the adaptive capability of the immune system is highly beneficial in response to cellular perturbations, inflammatory responses that become prolonged or dysregulated can be detrimental. For example, many conditions, such as aging, are associated with chronic inflammation and maladaptive effects on mitochondrial and skeletal muscle health. This emphasizes the need to investigate potential therapeutics and associated mechanisms to alleviate excess inflammatory responses within muscle.⁹⁷

6. The NLRP3 inflammasome complex

6.1. NLRP3 structural characteristics

NLRP3 has emerged as the most well-characterized inflammasome due to its ability to respond to a variety of DAMPs and PAMPs, marking its involvement in both pathogenic and sterile inflammation.^98,99 NLRP3 is a tripartite protein comprised of 3 domains, including the amino (N)-terminal pyrin domain (PYD), the central nucleotide-binding and oligomerization domain (NACHT), and the carboxy (C)-terminal leucine-rich repeat (LRR) domain.¹⁰⁰ (Fig. 3) The PYD domain has been implicated in mediating protein-protein interactions, which is required for inflammasome activation. The assembly of the NLRP3 inflammasome complex is reliant on the interactions between the NLRP3 PYD domain and the apoptosis-associated speck-like protein (ASC) containing a caspase activation and recruitment domain (CARD). ASC is an adaptor protein that contains an N-terminal PYD domain and a C-terminal CARD domain, making it an essential mediator of the interaction between NLRP3 and the recruited procaspase-1.^101,102 Furthermore, the NACHT domain exhibits ATPase activity, which aids in the oligomerization of NLRP3 monomers to promote inflammasome formation.^103,104 Lastly, the LRR domain has been most investigated for its role in ligand detection and as a site for various protein interactions and post-translational modifications to tightly regulate NLRP3 inflammasome activation. However, the importance of this region in inflammasome activation is still up for debate. For instance, while some works have shown that the deletion of the LRR domain inhibits NLRP3 inflammasome activation, others have demonstrated that it is not required for adequate inflammation activity.^105,106

Fig. 3.

Structure of the NLRP3 inflammasome complex. The NLRP3 inflammasome is a multiprotein complex comprised of 3 proteins to facilitate inflammasome assembly, including NLRP3, ASC, and procaspase-1. The sensor protein NLRP3 is comprised of 1033 amino acids (mus musculus) and can be further divided into 3 domains. The carboxy-terminal (C-terminal) LRR domain has been primarily implicated in ligand detection and stabilizing the inflammasome structure. The central NACHT domain is critical in the hydrolysis of ATP that promotes inflammasome oligomerization and regulation to promote the intricate multi-protein assembly. Lastly, the PYD domain promotes the recruitment and mediates homotypic protein-protein interactions with the PYD domain located on the 193-amino acid adaptor protein, ASC. As NLRP3 itself lacks a CARD, this interaction is critical in promoting procaspase-1 recruitment through the formation of ASC filaments and subsequent CARD–CARD protein interaction. The effector protein of the inflammasome complex, procaspase-1, is comprised of an N-terminal CARD domain, as well as 2 primary catalytic subunits that act as cleavage sites for its activation, resulting in a 402 amino-acid protein. Through the CARD–CARD interactions, the procaspase-1 proteins form a similar filament structure that allows for the proximity-induced autocatalytic cleavage between the p20 and p10 subunits to obtain the enzymatically active caspase-1 protein. ASC = apoptosis-associated speck-like protein; CARD = caspase recruitment and activation domain; LRR = leucine-rich repeat; NACHT = nucleotide-binding and oligomerization domain; NLRP3 = nucleotide oligomerization domain-like receptor protein 3; PYD = animo-terminal (N-terminal) pyrin.

6.2. NLPR3 inflammasome: Canonical mechanism of action

Due to its role in innate immune activation, the NLRP3 inflammasome must be closely regulated to avoid overactivation and detrimental health effects. Canonical activation of the NLPR3 inflammasome generally requires a 2-step activation pattern that consists of the priming stage followed by subsequent assembly and activation (Fig. 4). The priming stage is initiated at the level of TLRs, which upon interacting with various PAMPs and DAMPs, promote the nuclear translocation of NF-κB and binding to NF-κB response elements within the promotor regions of NLRP3, pro-interleukin-1β (pro-IL-1β), and pro-IL-18.⁸² As under basal conditions these genes are not abundantly expressed, the priming stage allows for sufficient expression prior to inflammasome assembly.⁸² The second step of activation requires an additional stress signal, which includes various PAMPs and DAMPs such as extracellular ATP, ROS, and viral RNA.^82,107 Following this signal, activated NLRP3 monomers can oligomerize into the classic inflammasome structure through its interactions with ASC and subsequent recruitment of procaspase-1. Upon assembly, procaspase-1 is autocatalytically cleaved into its active caspase-1 protein, which can mediate the activation of pro-inflammatory cytokines pro-IL-1β (Asp116) and pro-IL-18 (Asp36). IL-1β is recognized as a critical initiator of the immune response through the stimulation of other cytokines and the recruitment of additional immune cells through vasodilation.¹⁰⁸ While this canonical pathway is most recognized, additional signaling pathways such as TNF-α-mediated inflammation can also converge on inflammasome activation in the absence of PAMP priming.^109,110

Fig. 4.

Canonical mechanism of NLRP3 inflammasome activation. To maintain tight regulation of its activity, the NLRP3 inflammasome complex is activated by a 2-step mechanism of priming and subsequent assembly. The priming stage is mediated by the recognition of various extracellular and intracellular PAMPs/DAMPs, which promote the nuclear translocation of NF-κB, where it can bind to NF-κB binding sites located within the promoter regions of various genes involved in the NLRP3 inflammasome pathway. This ultimately leads to the transcriptional upregulation and subsequent translation of NLRP3, pro-IL-1β, and pro-IL-18. Following the priming stage, inflammasome assembly requires an additional signal through various DAMPs, including mtDNA and mtROS, which can be released from poorly functioning mitochondria through mitochondrial-mediated permeabilization pores. This promotes the assembly of NLRP3, ASC, and procaspase-1 into the active NLRP3 inflammasome complex, promoting the cleavage and maturation of procaspase-1. The enzymatically active caspase-1 promotes the cleavage of GSDMD and of pro-inflammatory cytokines, pro-IL-1β and pro-IL-18, releasing GSDMD-N and mature IL-1β and IL-18. GSDMD-N can embed itself within the plasma membrane, disrupting osmotic pressure and promoting pyroptotic cell death and the release of pro-inflammatory proteins to induce local inflammation. AMP = adenosine 5’-monophosphate; ASC = apoptosis-associated speck-like protein; cGAS = cyclic GMP–AMP synthase; DAMPs = damage-associated molecular patterns; GMP = guanosine monophosphate; GSDMD-C = the C-terminal of gasdermin-D; GSDMD-N = the N-terminal of gasdermin-D; IL = interleukin; mtDNA = mitochondrial DNA; mtROS = mitochondrial reactive oxygen species; NF-κB = nuclear factor kappa-B; NLRP3 = nucleotide oligomerization domain -like receptor protein 3; PAMPs = pathogen-associated molecular patterns; STING = stimulator of interferon genes; TLR = toll-like receptor.

6.3. Mitochondria and NLRP3 inflammasome activation

Mitochondria are prominent contributors to the innate immune response due to their endosymbiotic origins and ability to produce various endogenous damage molecules. For instance, several mitochondrial components have been implicated as mitochondrial DAMPs (mtDAMPs) that can be released under stressful conditions, such as cardiolipin,¹¹¹ ROS,¹¹² and mtDNA.¹¹³ Specifically, many of these mtDAMPs have been linked to immune signaling, immune cell infiltration, and the direct activation of the NLRP3 inflammasome complex. In addition, several mitochondrial proteins can facilitate physical interactions with NLRP3 components, including the mitochondrial fusion protein MFN2, and mitochondrial antiviral proteins.^112,114,115

6.3.1. MtDNA as an mtDAMP

Early work has demonstrated that mtDNA can elicit inflammatory responses due to its susceptibility to oxidative damage and its bacterial origins.¹¹⁶ Moreover, oxidized mtDNA (identified by a marker of DNA oxidation, 8-hydroxy-2 deoxyguanosine (8-OHdG)) has been shown to directly bind to the NRLP3 protein and promote inflammasome activation.¹¹⁷ This is further supported by studies showing mtDNA release can occur in response to mitochondrial outer membrane permeabilization, which can be mediated through B-cell lymphoma-2 (BCL-2)-associated X protein (BAX) and BCL2 antagonist/killer 1B (BAK) pore formation and voltage-dependent anion channel (VDAC) oligomerization.^15,118,119 Following its release, mtDNA can interact with various PRRs, including NLRs, cGAS, and TLRs, to initiate inflammatory responses through primarily NF-κB signaling and the Type I interferon response.

Several studies have investigated the role of mitochondrial quality control mechanisms in mitigating mitochondrial dysfunction and subsequent mtDNA release to elucidate the importance of mitochondrial health in mediating innate inflammation. For example, excessive mitochondrial fragmentation induced by Mfn1 knockdown led to increased VDAC-mediated mtDNA release and the upregulation of endosomal TLR9 signaling and NLRP3 inflammasome components. Furthermore, autophagy plays an essential role in the degradation of potential DAMPs, thereby reducing their potential to evoke inflammatory responses. Several groups have demonstrated that impairments in autophagy processes stimulated by the absence of the mitophagy receptor BCL2 interacting protein 3 (BNIP3), as well as upstream regulator beclin-1, have been linked to an accumulation of mtDNA and ROS, which drives TLR9 inflammatory signaling.^113,120 The protective role of mitophagy can further be demonstrated through the use of mitophagy inducers, such as the nutraceutical Urolithin A, which has been shown to blunt the release of mtDNA and, therefore, downstream inflammatory responses.⁸³

6.3.2. Mitochondrial ROS (mtROS)

In addition to mtDNA, mtROS are another well-established mtDAMP that can be excessively produced under metabolic stress. Zhou and colleagues¹¹² demonstrated that the inhibition of the mitochondrial ETC led to an upregulation of mtROS and NLRP3 inflammasome activation. While the specific interaction site between mtROS and NLRP3 has not been fully identified, the crystal structure of NLRP3 demonstrates disulfide bonds between cysteine residues that could potentially be regulated through oxidative stress.¹²¹ Furthermore, several papers have implicated a role for thioredoxin-interacting protein (TXNIP) in mediating ROS-mediated inflammasome activation through direct interaction with the NLRP3 protein.¹²²

However, while it has been made clear that there is a strong relationship between mitochondria and the NLRP3 inflammasome, there is some conflicting evidence as to whether mitochondrial dysfunction is a cause or consequence of NLRP3 inflammasome activation, contributing to its complexity. For instance, Park and colleagues¹²³ found that the knockdown of the mitochondrial fission GTPase, DRP1, was associated with increased activation of the NLRP3 inflammasome, mtROS, and mtDNA release in response to ATP treatment. Similar to findings from Nakahira et al.,¹¹³ the inhibition of caspase-1 prevented mitochondrial fragmentation and the release of mtDNA, which suggests that mitochondrial damage can be a consequence of inflammasome activation. In contrast to this, other studies have demonstrated that mtROS production and the binding of released mtDNA to NLRP3 occurs independent of caspase-1 activity, suggesting upstream involvement of mitochondrial damage prior to inflammasome activation.^113,117

Overall, these studies highlight the important role of the mitochondria in mediating NLRP3 inflammasome activation. By further elucidating the mechanisms surrounding mitochondrial-mediated DAMP release, various sites of intervention can be targeted to regulate the activation of the NLRP3 inflammasome complex and maintain cellular homeostasis.

6.4. NLRP3 adaptations to skeletal muscle disuse

6.4.1. Muscle denervation

As outlined above, muscle denervation is a severe model of muscle disuse that is characterized by reductions in muscle strength and increased atrophy, alongside impairments in mitochondrial health.⁵¹ Due to the adaptable nature of the mitochondrial reticulum, periods of prolonged muscle disuse can contribute to increased fragmentation, impaired energy production, and an accumulation of mtDAMPs that can propagate immune responses. Following muscle disuse by peripheral nerve injury, skeletal muscle transitions toward a pro-inflammatory environment that involves immune cell infiltration and the production of cytokines such as IL-6 and TNF-α, which can further propagate signaling toward muscle atrophy.¹²⁴ In addition, other studies have demonstrated an active role for NLRP3 inflammasome activation in response to various inducers of muscle atrophy, including IL-1β and angiotensin-II treatment, which has been linked to mitochondrial dysfunction and increased inflammasome activation. This atrophy can be attenuated by NLRP3 knockout (KO) or the upregulation of anti-inflammatory signaling.^125,126 However, while the role of the NLRP3 inflammasome has been explored in the context of skeletal muscle atrophy, there is still a lack of information surrounding its involvement in skeletal muscle denervation. A study by You et al.¹²⁷ demonstrated that 14–28 days of skeletal muscle denervation was sufficient to induce NLRP3 inflammasome activation and downstream components, including caspase-1, N-terminal of gasdermin-D (GSDMD-N), IL-1β, and IL-18. Furthermore, this was associated with an upregulation of atrogenes, muscle RING-finger protein-1 (Murf-1), and Atrogin-1, which can facilitate protein degradation through the ubiquitin-proteosome system to stimulate muscle atrophy. In the absence of NLRP3 via a whole-body KO, these responses were blunted, which suggests that NLRP3 inflammasome activation may be a contributor to denervation-induced skeletal muscle atrophy.¹²⁷ While this study implicates NLRP3 inflammasome activation in denervation-induced atrophy, more work needs to be done to determine the relationship between mitochondrial health and this pro-inflammatory environment.

6.4.2. NLRP3 inflammasome in the aging phenotype

In 2000, Franceschi and colleagues⁷⁴ coined the term “inflammaging” which describes the pro-inflammatory aging phenotype, which is characterized by a senescence-associated secretory phenotype that encompasses increases in senescent markers, increased processing of pro-inflammatory receptors and cytokines, as well as reductions in anti-inflammatory signaling.^74,128 Basally with aging, several immune factors are upregulated, including the increased expression of various TLRs, associated adaptors, MyD88, and NF-κB components, all of which suggest an increased drive toward pro-inflammatory signaling in skeletal muscle.¹²⁹ Furthermore, as mentioned above, aged muscle often suffers from age-related increases in protein catabolism and deficits in mitochondrial function, which contribute toward the development of sarcopenia and associated reductions in skeletal muscle mass. Due to its involvement in activating caspases and pro-inflammatory cell death, several studies have aimed to characterize the potential role of NLRP3 inflammasome activation in sarcopenia. Works from Mcbride et al.¹³⁰ and Antuña et al.¹³¹ have shown basal increases in several proteins associated with NLRP3 inflammasome signaling, including caspase-1 and IL-1β, which were associated with reductions in myofiber size, increased muscle damage, and the increased circulation of mtDAMPs, including extracellular ATP. However, in the absence of NLRP3, 24-month-old mice were protected from age-related losses in muscle mass and, specifically, in the preservation of Type II fibers, which was also associated with improvements in skeletal muscle functional parameters.¹³⁰ Furthermore, several deficits in mitochondrial quality control have been implicated in the pro-inflammatory phenotype driven by age. For instance, a reduced drive toward mitophagy with age is associated with an accumulation of dysfunctional mitochondria and subsequent activation of pro-inflammatory pathways, including TLR9–NLRP3-mediated signaling, as well as with increased activation of alternative DNA sensing pathways such as cGAS–STING. Interestingly, this can be attenuated through an increased drive toward mitophagy induced by external agents or by the overexpression of mitophagy receptors like BNIP3.^83,120 Overall, the literature provides some evidence of age-related increases in NLRP3 signaling within skeletal muscle; however, the connection between inflammasome activation and mtDAMPs, as well as the potential mitigation through exercise, has yet to be investigated.

6.5. NLRP3 inflammasome adaptations to endurance training

With regard to skeletal muscle health, exercise is considered a highly effective intervention to improve the quality of muscle and the mitochondrial pool. Chronic endurance training has been associated with enhanced mitochondrial quality control mechanisms and reduced oxidative stress, ultimately contributing to an improved muscle oxidative capacity.^48,131,132 While TLR signaling is most commonly associated with the inflammatory response, several studies have demonstrated that the absence of TLR4 attenuates the upregulation of mitochondrial content, biogenesis, and cytokine production that is usually observed following long term-endurance training.¹³³ In addition, in relation to mtDAMPs, chronic training has been associated with reductions in mtROS production due to increased mitochondrial efficiency and an improved clearance of dysfunctional organelles, which could inhibit mtDNA release; however, the data on this are scarce in the context of innate immune activation. Furthermore, there is currently very little evidence surrounding the impact of endurance training on mtDNA release in skeletal muscle. With regard to the immune system, chronic endurance training has also been praised for its role in shifting the inflammatory environment toward an anti-inflammatory phenotype through the upregulation of anti-inflammatory cytokines and suppression of pro-inflammatory cytokines.¹³⁴ In addition, chronic endurance exercise has been shown to induce PGC-1α expression, which has been linked to the repression of pro-inflammatory NF-κB signaling in skeletal muscle as well as the inhibition of NLRP3 inflammasome signaling by promoting improved mitochondrial health in renal tubular epithelial cells.^135,136 While there is little to no evidence surrounding the effects of endurance training on NLRP3 inflammasome activation within skeletal muscle, a few studies have demonstrated the possible role of endurance training in regulating this pathway. For instance, in cardiomyocytes, 8 weeks of moderate endurance training has been associated with reductions in mitochondrial stress responses as well as NLRP3 activation and subsequent IL-1β release in a model of cardiac overload hypertrophy.¹³⁷ Furthermore, in high-fat-diet-induced obesity, 14 weeks of voluntary wheel running is capable of attenuating NLRP3 inflammasome signaling and oxidative stress originating from nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases.¹³⁸ A similar reduction in inflammasome signaling can be observed in neuronal cells derived from a mouse model of Parkinson’s disease and in adipose tissue from obese mouse models, following 10 weeks and 6 weeks of treadmill training, respectively.^139,140 Considering the highly adaptive nature of skeletal muscle mitochondria, along with the effects of exercise training on the NLRP3 inflammasome in alternative tissue types, there is a strong foundation for further exploring the potential of this relationship in future studies.

7. Conclusion

Inflammatory signaling plays an essential role in cellular homeostasis and skeletal muscle health. While it is now recognized that mitochondrial dysfunction can propagate innate immune pathways, the relationship between mitochondrial turnover in various metabolic conditions and innate immune signaling is not completely elucidated. This narrative review offers a novel perspective on several pro-inflammatory conditions in the context of mitochondria, and specifically skeletal muscle, which is often underexplored. By highlighting the importance of mitochondrial signaling in relation to innate immune activation, this review presents alternative pathways to alleviate excessive inflammation by improving skeletal muscle health and mitochondrial function. The current body of literature has demonstrated that chronic muscle use and disuse can alter the inflammatory response by modulating skeletal muscle health and mitochondrial quality control mechanisms such as biogenesis, fission, fusion, and mitophagy. Evaluating this relationship more completely could elucidate potential mechanisms and therapeutics to regulate inflammatory pathway activation under abnormal metabolic conditions in an effort to retain cellular homeostasis and muscle health.

8. Limitations and future directions

There are several limitations related to this literature review that should be considered. When investigating the impact of skeletal muscle adaptations on innate immune signaling, the differences in disuse and exercise training protocols made direct comparisons across various studies difficult. Furthermore, while this study focused primarily on the impact of the NLRP3 inflammasome complex and its activation in the context of skeletal muscle mitochondria, there are various other redundant signaling pathways that should be further explored in future work. Similarly, while the scope of this review is focused on investigating mitochondrial function in relation to innate immune signaling, there are many areas that can be expanded upon with future work. While this review focuses on the interactions between innate immune signaling and skeletal muscle activity, it is important to acknowledge that both of these conditions involve a systemic whole-body response that involves alternative tissue types. Thus, future studies should work toward investigating effects of tissue-crosstalk on NLRP3 inflammasome activation in skeletal muscle and the effects of existing clinical therapeutics in alleviating this inflammatory pressure. Additionally, because this review only includes endurance training adaptations, it would be interesting to compare the effectiveness of other exercise modalities, such as interval or resistance training. Furthermore, there is a lack of literature elucidating the specific mitochondrial stimuli that are activating the NLRP3 inflammasome complex and eliciting these downstream effects in skeletal muscle. As a result, more work needs to be done to evaluate various mtDAMPs that could possibly be involved in these adaptations in order to better identify targets for intervention.