A Perspective on “Hypoxia Resistance is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle”

; Hypoxias; r esistance; inher ent; phenotype; mouse Skeletal muscle is reliant on a constant oxygen supply for mov ement, cellular r espiration, and thermogenesis. Heter oge-neous fibre types exist in skeletal muscle as a continuum from slow- to fast-twitch to facilitate specialized function. Type I (oxidati v e fibr es) pr esent a slow-twitc h phenotype , c har acterized by high oxygen capacity and increased fatigue resistance. In contrast, type IIa (fast oxidati v e gl ycol ytic phenotype) and type IIx (fast gl ycol ytic) pr esent faster twitc h speeds and contr

Skeletal muscle is reliant on a constant oxygen supply for mov ement, cellular r espiration, and thermogenesis. Heter ogeneous fibre types exist in skeletal muscle as a continuum from slow-to fast-twitch to facilitate specialized function. Type I (oxidati v e fibr es) pr esent a slow-twitc h phenotype , c har acterized by high oxygen capacity and increased fatigue resistance. In contrast, type IIa (fast oxidati v e gl ycol ytic phenotype) and type IIx (fast gl ycol ytic) pr esent faster twitc h speeds and contr actile force a faster time to fatigue and a recued reliance on oxidati v e energy metabolism. 1 This reliance on oxygen for functional capacity means that a sustained lack of oxygen supply (hypoxia and/or anoxia) to muscle has been linked to n umer ous pathological conditions such as chronic inflammation, slow tissue r e pair, micr ov ascular complications, and irr ev ersib le dama ge (ie, m uscle necrosis). 2 It has been proposed that the location of the m uscle (pr oximal or distal) to the site of hypoxic insult might dictate the functional and necrosis response to reduced oxygen availability. To test this hypothesis, a recent study published by Amorese et al. in Function 3 used femoral artery transection of the lower limb in (10-12 wk old, C57BL/6NJ) mice to induce hypoxia in vi v o and anoxia ex vi v o to challenge differ ent m uscles [eg, extensor digitorum longus (EDL), soleus (SOL), and flexor digitorium brevis (FDB)] to hypoxia and anoxia insults. Following this process, the authors tested the functional consequence of oxygen av aila bility by measuring muscle force production and metabolic changes during anoxia exposure (3 h). After a hypoxic insult, the authors observed higher fibr otic dama ge in EDL and SOL compared with FDB skeletal muscle, suggesting that muscle location might alter functional capacity in the absence of oxygen.

FDB Skeletal Muscle Shows Unique Phenotypical Responses to Altered Oxygen Availability
Unlike other skeletal muscles located in the distal region, the FDB skeletal muscle presented preserved contractile function during anoxic conditions compared with EDL and SOL suggesting that this unique phenotype might be attributed at least in part by oxygen tension or energetic supply (glycolysis). To test this dir ectl y, Amor ese et al. 3 showed that during anoxia insult in the presence of potassium cyanide (KCN), an inhibitor of mitoc hondrial respir ation, the FDB skeletal muscle maintained for ce 1 pr oduction compar ed to the EDL, despite low er mitoc hondrial content, respiration and membrane potential. Thus, the authors ele gantly demonstr ated that the FDB response to oxygen availa bility differ ed to other m uscles in the low er le g, a response that did not seem to r el y on altered mitochondrial function.

Why Does the FDB Display Altered Adaptation to Reduced Oxygen Availability?
Gi v en the KCN results, the authors speculated that the attenuated force and energy production of the FDB may be attributed to an upregulation of glycolytic metabolism. To test this hypothesis, the authors incubated FDB muscle with the gl ycol ysis inhibitor br omopyruv ate (BrP) for 3 h. All thr ee skeletal m uscles (FDB, EDL, and SOL) treated with BrP displayed a force reduction at a similar timepoint, as well as similar glycogen depletion in ano xia e xposure, confirming that FDB does not de pend on gl ycol ysis mor e than other muscles. To further delineate the unique phenotype of the FDB skeletal muscle, Amorese and colleagues compar ed the pr oteome of the thr ee skeletal m uscles (FDB, EDL, and SOL) using tandem mass tag (TMT)-labeled mass spectrometr y and anal yzed differ ential expr ession patterns of ov er 2000 pr oteins acr oss skeletal m uscles. This appr oach identified that the number of glycolytic proteins normalized to the total number of identified proteins was significantly lower in the FDB compared to EDL but not SOL.
Considering the deleterious effect of the gl ycol ytic inhibitor on the force production of the FDB skeletal muscle, the authors examined the abundance of the tr ansmembr ane glucose transporter protein type 1 (GLUT1), a rate limiting step in glucose metabolism. The results showed that GLUT1 protein content in FDB skeletal muscle was 1.9-fold higher than EDL and 2.1-fold higher than SOL. Additionally, the authors investigated whether GLUT1 plays a decisi v e r ole in fatigue r esistance in FDB utilizing skeletal muscle-specific GLUT1 knockout mice (GLUT1 KO). The deletion of GLUT1 in the FDB skeletal m uscle r esulted in a marked loss of force production following anoxia exposure wher eas this r esponse w as pr eserv ed in wild-type mice. Furthermore , for ce production in FDB skeletal muscle was significantly different between genotypes after 100 min of anoxia exposure whereas no genotype effect was found under oxygenated conditions.

What can the FDB Teach us About Muscle Adaptation to Environmental Stress?
The work from Amorese and colleagues is a wonderful example of how skeletal muscle has evolved to deal with specific envir onmental str esses to meet energetic demands. This r emarka b le plasticity to adapt to environmental, physiological, and pathological conditions exhibited by skeletal muscle is conferred by changes in muscle contractile phenotype and metabolic shift. 4 Protecting skeletal muscle from hypoxia is a critical need to impr ov e tissue survi v al during states of acute hypoxia or during m uscle w asting. Thus, understanding the potential mechanisms by which FDB can maintain function in the absence of oxygen may provide important new information as to how and wh y h ypo xia and ano xia ar e detrimental to m uscle function in clinical scenarios. 5 Disuse atrophy, c har acterized as a loss of muscle mass, r educed m uscle str ength, and meta bolic dysfunction, 6 ar e common features of sedentary behavior resulting from hospitalization, limb immobilization, or bedrest. Systemic hypoxia, has been identified as contributing to poor musculoskeletal states in multiple clinical populations, including chronic obstructive pulmonary disease (COPD) 7 and heart failure. 8 Amorese and collea gues hav e identified a unique phenotype of the FDB muscle and its ability to maintain its function in the complete absence of oxygen. Thus, studies such as this, where differ ent m uscles ar e examined in r esponse to fundamental meta bolic/envir onmental str essors may identify novel biology to explain why muscles can or cannot adapt to alterations in demand. Certainly, these results support the inclusion of numerous tissue analysis in rodent metabolic studies factoring in the r elev ance of anatomical properties of muscle in specific disease pathologies. To support this notion, recent work from Naruse et al. (2023) hav e r e ported that muscle-specific atrophy is varied in human skeletal muscle in response to aging. 9 Furthermore, the trajector y of m uscle loss w as v aria b le when comparing across m uscle gr oups (quadrice ps, 0.66%/yr; hamstrings 1.18%/yr), pr oviding useful information for development of str ate gies to prev ent m uscle loss. Indeed, measuring mechanistic responses in both atr ophy-pr one and atr ophy-r esistant m uscles, has r ecentl y been proposed as a valuable approach to identifying novel pathways in disuse atrophy models, which may provide novel targets for therapeutic intervention. 10 Thus, rather than considering di v ergent r esponses as a biological anomal y, perhaps studying muscle such as the FDB can provide valuable information into the unique adapti v e potential of skeletal muscle in health and disease.

Funding
Dr Philp gratefully aknowledges financial support from the Graham Painton Foundation.