Skeletal Muscle Regeneration in Advanced Diabetic Peripheral Neuropathy

Abstract

Background:

Decreased lean muscle mass in the lower extremity in diabetic peripheral neuropathy (DPN) is thought to contribute to altered joint loading, immobility, and disability. However, the mechanism behind this loss is unknown and could derive from a reduction in size of myofibers (atrophy), destruction of myofibers (degeneration), or both. Degenerative changes require participation of muscle stem (satellite) cells to regenerate lost myofibers and restore lean mass. Determining the degenerative state and residual regenerative capacity of DPN muscle will inform the utility of regeneration-targeted therapeutic strategies.

Methods:

Biopsies were acquired from 2 muscles in 12 individuals with and without diabetic neuropathy undergoing below-knee amputation surgery. Biopsies were subdivided for histological analysis, transcriptional profiling, and satellite cell isolation and culture.

Results:

Histological analysis revealed evidence of ongoing degeneration and regeneration in DPN muscles. Transcriptional profiling supports these findings, indicating significant upregulation of regeneration-related pathways. However, regeneration appeared to be limited in samples exhibiting the most severe structural pathology as only extremely small, immature regenerated myofibers were found. Immunostaining for satellite cells revealed a significant decrease in their relative frequency only in the subset with severe pathology. Similarly, a reduction in fusion in cultured satellite cells in this group suggests impairment in regenerative capacity in severe DPN pathology.

Conclusion:

DPN muscle exhibited features of degeneration with attempted regeneration. In the most severely pathological muscle samples, regeneration appeared to be stymied and our data suggest that this may be partly due to intrinsic dysfunction of the satellite cell pool in addition to extrinsic structural pathology (eg, nerve damage).

Clinical Relevance:

Restoration of DPN muscle function for improved mobility and physical activity is a goal of surgical and rehabilitation clinicians. Identifying myofiber degeneration and compromised regeneration as contributors to dysfunction suggests that adjuvant cell-based therapies may improve clinical outcomes.

Introduction

Diabetic peripheral neuropathy (DPN) induces substantial muscle weakness that limits mobility and quality of life.^1,4,6 Treating neuropathy-associated muscle dysfunction has the potential to significantly impact the course of DPN by improving gait, normalizing joint loading, and increasing physical activity. However, traditional strengthening interventions have struggled to improve strength and performance in the context of DPN,³⁶ suggesting a compromised ability of DPN muscle to respond to loading cues at the cellular level.

Very little data exist regarding the nature of cellular pathology in DPN muscle. Noninvasive imaging studies have noted a reduction in “lean” muscle volume, finding instead extensive infiltration of fibrous and adipose tissue within the muscle boundary.^3,6,17 However, these modalities are unable to determine whether reductions in lean volume represent atrophic or degenerative processes or a combination thereof. This is a critical distinction when considering potential modes for functional recovery in DPN. Muscle atrophy is a process of intracellular protein breakdown that reduces the size of myofibers.⁴³ This process is theoretically reversible when appropriate hypertrophic stimuli (eg, loading) are reapplied. Conversely, degeneration is the process of selective myofiber destruction by apoptosis and necrosis.² Once the myofiber is lost, a new myofiber must be generated to recover muscle mass. The process of generating new myofibers (regeneration) is critically dependent on a population of muscle-resident stem cells (satellite cells [SCs]),²⁹ while the process of hypertrophy is not.³³

While it has not been investigated in the context of DPN, there is notable evidence for ongoing muscle degeneration with chronic denervation.^21,48 Myofiber necrosis, phagocytosis, splitting, and hypercontraction have been noted in individuals with spinal cord injury,²⁶ Charcot-Marie-Tooth disease,²² Welander distal myopathy,⁷ and prior poliomyelitis.⁸ These findings are echoed by studies in animal models with chronic denervation.^9,11,38 Intriguingly, attempts to experimentally reverse denervation-induced muscle pathology are unable to fully recover function after an extended period of denervation despite full restoration of innervation.¹⁵ A number of studies have noted decline or dysfunction in the SC pool with chronic denervation that could contribute to this persistent functional deficit.^18,25,42

In this work, we evaluated whether degeneration and SC dysfunction are also features of DPN. We investigated the cellular composition, morphology, transcriptional profile, and myogenic capacity in muscle biopsies from individuals with DPN to characterize its degenerative state and regenerative potential. The ultimate goal would be to guide therapeutic interventions to target the limiting factor(s) in functional recovery—specifically, whether SC supplementation could complement other cell-based strategies promoting nerve regeneration^32,44,46 and physical interventions to improve muscle strength.

Methods

Study Design

This study was approved by the Human Research Protection Office. The study was conducted from 2016 to 2019. Eligible patients scheduled for elective below-knee amputations signed an informed consent on the day of surgery prior to any medications. There were 12 people who participated in the study: 7 people (4 female; 57 [SD, 6] years; 30.4 [SD, 6.3] body mass index [BMI]) who had diagnosed diabetes and peripheral neuropathy (DPN) and 5 people (3 female; 46 [SD, 10] years; 29.2 [SD, 2.4] BMI) who were metabolically normal controls (MNCs) without diabetes or neuropathy. All patients undergoing below-knee amputation were eligible for the study. Exclusion criteria were people with diagnosed peripheral vascular disease, infections that extended into the biopsy area, or pregnancy; vulnerable populations as defined by the National Institutes of Health (NIH); and those infected with human immunodeficiency virus or tuberculosis.

Muscle biopsies were taken from 2 locations distal to the amputation site: the medial gastrocnemius (MG) muscle of the calf and the abductor hallucis (AH) muscle of the foot. This design was selected to isolate the effects of peripheral neuropathy from the systemic effects of diabetes. Peripheral nerve degeneration is more advanced in the foot than the calf,²⁷ while both sites experience the same systemic features of diabetes. Due to the advanced state of foot deterioration in our participants, in 2 cases there was insufficient AH muscle to biopsy, and in an additional 5 there was insufficient AH muscle in the biopsy to complete all of the assays performed in this study. This limitation precluded a within-patient analysis, and thus MG and AH muscles are compared by group (see statistical analyses for more details). Numbers per group are indicated for each assay in the text or figure legend.

Muscle Collection and Processing

Surgeons from the Foot & Ankle and Trauma Services performed the amputation surgeries and obtained biopsies for the study. A ~100-mg portion of each tissue was placed on ice for immediate transport back to the lab. Biopsies were cleaned of visible connective tissue and fat under a dissecting microscope and subdivided into 3 parts. One part was embedded in tragacanth gum and flash-frozen in liquid nitrogen–cooled isopentane for histological analyses, 1 part was flash-frozen in liquid nitrogen for RNA sequencing, and 1 part was placed in a digestive solution for cell isolation as described below.

Histological Analyses

Histological analyses were used to quantify the number and types of cells present in the biopsy. Specifically, outcome measures were (1) percent of biopsy containing myofibers (indicating loss of contractile tissue), (2) percent of myofibers with centralized nuclei (indicating past regeneration), (3) relative percentages of 4 fiber types (indicating fiber-type shifts and recent regeneration), and (4) fraction of SCs (indicating the size of the SC pool).

Specimens allocated to histological analyses were cut in the axial plane in 10-μm sections on a cryostat (Leica Biosystems, Wetzlar, Germany). Sections were either stained with hematoxylin and eosin (H&E) to visualize tissue morphology or immunostained to characterize cell populations. Immunostaining was performed on unfixed sections against 4 isoforms of myosin heavy chain (MHC)—type I, type IIa, type IIx, and embryonic (Developmental Studies Hybridoma Bank, Iowa City, IA: BA-F8, SC-71, 6H1, and BF-45, respectively)—to identify the 4 fiber types, and they were counterstained with laminin (ab11575; Abcam, Cambridge, UK) to visualize fiber boundaries. Immunostaining against Pax7 (Developmental Studies Hybridoma Bank: PAX7) was performed following 10 minutes of acetone fixation and counterstained with embryonic MHC, laminin, and 4′,6-diamidino-2-phenylindole (DAPI).

For the myofiber percentage calculation, an IsoData threshold was applied to the H&E-stained image and eosin-positive pixels were divided by the total pixels in the image. Also, on H&E stained images, myofibers with a cross-sectional area greater than 1000 μm² with centrally localized nuclei were counted and normalized by the total myofiber number. In smaller-diameter fibers, it was impossible to determine whether a nucleus was central or peripheral. Fiber-type distributions were determined by MHC fluorescent signals. SCs were identified by co-localization of Pax7 and DAPI beneath the laminin-positive myofiber basal lamina and normalized to the image area or fiber number. All histological outcome measures were averaged from 2 non-overlapping 10× images per biopsy.

RNA Sequencing and Pathway Analysis

RNA sequencing was performed to generate a comprehensive picture of the transcriptional state of the muscle. This analysis identifies changes in expression of specific genes and gene networks (pathways) that guide changes in cell and tissue physiology (eg, atrophy and regeneration).

RNA was isolated from muscle samples by homogenization in TRIzol reagent (Invitrogen, Carlsbad, CA), followed by chloroform extraction and purification by RNEasy columns (Qiagen, Hilden, Germany). Briefly, libraries were prepared using 500 ng to 1 μg of total RNA validated for size and purity. mRNA was fragmented and reverse transcribed using SuperScript III RT enzyme (Life Technologies, Carlsbad, CA), with a second strand reaction to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were sequenced on an Illumina HiSeq-3000, using single reads extending 50 base pairs (Illumina, Inc., San Diego, CA). mRNA-seq reads were aligned to the human genome hg38 with STAR version 2.5.4b.²⁰ Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:feature version 1.4.6,³⁰ with gene-body annotation of human GENCODE version 27.²⁴ The RNA sequencing data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE143979. Differential expression of transcripts with counts per million >1.0 in at least half samples was determined using R package edgeR⁴¹ with the exactTest function applied. The false discovery rate (FDR) was determined using the Benjamini and Hochberg correction with an FDR <0.05 considered significant. Principal component analysis was performed using plotPC³¹ and plotted using the ggplot2 package.

Ingenuity Pathway Analysis (IPA; Qiagen) was applied to significant genes to identify up- or downregulated transcriptional networks in DPN AH versus MG. These networks were grouped by diseases and functions. Significance was determined at P < .05. Network maps were based on those of Smith et al⁴⁷ with the addition of a new regeneration map based on published networks.^13,50

SC Isolation and Culture

Isolation and culture of SCs from biopsies provides an indication of their ability to initiate, and participate in, the formation of new myofibers (regeneration).

SC isolation was performed by fluorescence-activated cell sorting (FACS) as previously described.³⁵ Briefly, cells were dissociated from biopsies in a collagenase- and dispase-based digestive solution with mechanical disruption. Cell suspensions were filtered and washed, expanded in culture to 50% confluence, trypisinized, and frozen for long-term storage. Thawed cell suspensions were incubated with the following fluorophore-conjugated primary antibodies: anti-NCAM*PE (BD Biosciences, San Jose, CA, catalog #: 555516), anti-CD31*FITC (Invitrogen, catalog #: 11031942), and anti-CD45*Pacific Blue (Biolegend, San Diego, CA, catalog #: 304022). Labeled suspensions were passed through a FACS AriaII (BD) and CD31–/CD45–/NCAM+ cells were isolated and expanded in culture.

All culture experiments were performed in triplicate at passage 3. SCs were grown to 90% confluence in a myogenic growth media (20% fetal bovine serum, 0.4 ng/mL basic fibroblast growth factor, 1% penicillin/streptomycin, and a 50/50 mix of low-glucose Dulbecco’s modified Eagle’s medium [DMEM] and Hamm’s F-10) and then switched to a myogenic differentiation media (5% horse serum, 10 μg/mL insulin, and 1% penicillin/streptomycin in low-glucose DMEM) for 3 days. Following differentiation, cells were fixed for 10 minutes in ice-cold methanol, washed, and immunostained for MHC (Developmental Studies Hybridoma Bank: MF-20). Myogenic differentiation was quantified as the fraction of MHC-positive (MHC+) pixels identified by IsoData thresholding.

Statistical Analyses

Comparisons between groups (DPN:MNC) and muscles (AH:MG) were performed by 2-way ANOVA with a Sidak posttest to detect significance between individual groups. Comparisons between groups within a muscle were performed by t test or 1-way ANOVA where appropriate. Significance was set at P < .05. Results are presented as mean ± standard error.

Results

DPN Muscle Exhibited Histological Evidence of Degeneration and Regeneration

Compared with muscle biopsies from MNCs, biopsies from individuals with DPN exhibited extensive evidence of pathology on H&E-stained sections (Figure 1A). DPN muscle exhibited dramatic and localized myofiber atrophy, fibrous replacement (section sign), and fatty infiltration (asterisk). These pathological features were more prevalent in AH muscles than MG muscles. To assess this difference, the percentage of each H&E image attributed to myofibers was quantified (Figure 1B). Two-way ANOVA revealed a significant main effect of group (DPN:MNC, P < .001) and muscle (AH:MG, P = .004) with a significant interaction (P = .006), indicating that the difference between AH and MG biopsies was unique to the DPN group. Indeed, posttest analysis revealed a significant difference between groups within MG and AH muscles, but only a significant difference between muscles in the DPN group. There was a large variance in the distribution of myofiber percentage in the DPN AH group, with some biopsies exhibiting localized regions of pathology (Figure 1A, bottom center) and some exhibiting extensive atrophy and fibro/fatty infiltration (Figure 1A, bottom right). For select subsequent analyses, these are subdivided into moderate (mod) and severe (sev) groups by >30% or <30% myofiber percentage, respectively.

Figure 1.

Histological evidence for degeneration and regeneration in diabetic peripheral neuropathy (DPN) muscle. (A) Representative hematoxylin and eosin–stained sections from metabolically normal control (MNC) and DPN groups. DPN samples exhibit infiltration of fibrous (section sign) and adipose (asterisk) tissues with evidence of fiber degeneration including myofiber necrosis (arrowhead), myofiber splitting (white/black arrow), and hypercontracted myofibers (double arrow). Regenerated fibers, marked with a centrally localized nucleus (arrows), are also abundant. (B) Quantification of (eosin-positive) myofibers in biopsies demonstrates significantly reduced myofiber percentages in DPN compared with MNC, and in DPN abductor hallucis (AH) compared with DPN medial gastrocnemius (MG). DPN AH samples were stratified into moderate (mod) and severe (sev) pathology by whether the percentage was above or below 30. (C) Quantification of centrally nucleated fibers demonstrates increased prevalence in both DPN muscles. MNC MG (n = 5), DPN MG (n = 6), MNC AH (n = 4), DPN AH (mod) (n = 3), DPN AH (sev) (n = 3). *P < .05, **P < .01, ***P < .005, ****P < .001.

Interestingly, many of the biopsies in the DPN group also exhibited evidence of degeneration and past regeneration. These include myofiber necrosis (arrowhead), myofiber splitting (white/black arrow), hypercontracted myofibers (double arrow), and myofibers with centrally localized nuclei (arrow). Centrally placed nuclei frequently indicate a past regenerative event.³⁴ The percentage of fibers with centrally placed nuclei was significantly higher in DPN MG compared with MNC MG and in DPN AH (sev) compared with MNC AH (Figure 1C). However, these were not different between DPN MG and AH groups. Taken together, these data indicate that degeneration and regeneration are associated with DPN pathology.

Transcriptional Analysis Revealed Upregulation of Regeneration-Related Pathways

To further explore the dynamic between atrophic, degenerative, and regenerative signaling in DPN, RNA sequencing was performed on a subset of muscle samples with adequate-quality RNA extraction. Of the 15 658 transcripts detected, only 231 were significantly different comparing MNC AH with MNC MG, indicating that in a metabolically normal, nonneuropathic state AH and MG muscles are transcriptionally very similar. By contrast, 1292 genes were significantly different between DPN AH and DPN MG, of which 1182 were unique to the DPN group (Figure 2A). This indicates that AH and MG muscles were more transcriptionally dissimilar in DPN than in MNC. This conclusion is also supported by a principal component analysis where principal component 1 (PC1) is able to explain 52% of the variance in the data and effectively separate DPN MG and AH from each other and from both MNC muscles (Figure 2B). This suggests a set of correlated genes whose expression is able to describe advancing peripheral neuropathy.

Figure 2.

Transcriptional activation of developmental and regenerative pathways in diabetic peripheral neuropathy (DPN) muscle. (A) Venn diagram of genes with significantly different expression between muscles (abductor hallucis:medial gastrocnemius [AH:MG]) in the 2 groups showing limited overlap with the most differentially expressed transcripts in the DPN group. (B) Principal component analysis of differentially expressed transcripts effectively separates DPN AH and MG samples from each other and from metabolically normal controls (MNCs). (C) Pathway analysis of DPN AH:MG reveals differential expression of categories related to tissue structure (medium gray bars) and tissue development (light gray bars). Relevant components of the pathways are pulled out in horizontal bar graphs. All graphs are plotted as a function of significance level (–log(P value)), and pathway component graphs are color-coded by Z score, indicating whether they are predicted to be promoted (red) or inhibited (blue). (D) Network map of muscle-specific pathways, organized by anatomical localization of proteins and physiological function. Individual circles are genes, color-coded by fold change (DPN AH:MG), with red and blue shades indicating up- and downregulation, respectively. Only genes with significant changes are colored; others are gray. Connecting lines show connectivity between genes in a pathway. Most pathways exhibit predominantly downregulated genes, except for the neuromuscular junction, extracellular matrix, and regeneration pathways. MNC MG (n = 4), DPN MG (n = 4), MNC AH (n = 3), DPN AH (n = 4). Online issue will display colors in Figure 2.

To identify these genes and the pathways through which they are linked, we applied the IPA toolbox to the 1292 genes significantly different between DPN MG and AH. Grouping gene expression by diseases and functions, IPA identified 147 differentially regulated categories. The 20 categories of highest significance include 5 related to tissue structure and 5 related to development (Figure 2C, medium and light gray bars). Probing the functional sub-categories revealed differential regulation of a number of pathways related to muscle contractile function, fiber-type distribution, fibrosis, and development. These pathway changes are suggestive of a myopathic state with possible regenerative features (development-related pathways), but they are not muscle specific. To further probe skeletal muscle–specific pathways, we developed a muscle physiology gene network map (Figure 2D) based on previously published networks.⁴⁷ In the network map, expression of individual significantly differentially regulated genes is color-coded in circle “nodes,” with interactions between nodes in a pathway indicated. Gene nodes are depicted where the resulting protein is typically found in the muscle cell and organized by physiological function.

Interestingly, although genes encoding contractile and cytoskeletal proteins were downregulated in DPN AH compared with MG (expected of an atrophic state), there was not a corresponding upregulation of genes involved in protein breakdown. The so-called “atrogenes” Fbxo32 and Trim63²⁸ were both significantly downregulated, as were Fbxo40, Mettl21, and Vcp—other genes involved in protein degradation.⁴⁵ The 3 genes significantly upregulated in the hypertrophy and atrophy network were Igf1, Slpi, and Fst—all encoding proteins that promote muscle hypertrophy.⁴³ This suggests a state of attempted muscle growth rather than ongoing atrophy. Few other genes were upregulated in any of the muscle-specific networks. They were primarily found in the neuromuscular junction, extracellular matrix, and regeneration networks. The increased expression of genes encoding neuromuscular junction components likely represents attempts by the myofiber to reestablish a connection to a functioning motor neuron as has been described in experimental models of denervation.⁵ Increased expression of genes encoding components of the extracellular matrix likely reflects a more advanced fibrotic environment in DPN AH muscle, which is supported by histological findings (Figure 1).

The regeneration network had the highest fraction of differentially regulated genes between DPN AH and MG. These include genes involved in SC activation, differentiation into a myoblast state, fusion to an immature fiber, and embryonic/neonatal isoforms of contractile and cytoskeletal proteins typically found in newly regenerated (immature) fibers (delineated in Figure 3). Activation of the regeneration network is even more dramatic when comparing DPN AH to MNC AH (Figure 3). Numerous genes involved in guiding late-stage myoblast differentiation (eg, Sfrp1–4 and Efna5) and components of immature myofibers (eg, Tnnt2, Actc1, Myl5, Myl4, and Myh3) were more than 5-fold upregulated in both DPN AH (mod) and DPN AH (sev) samples compared with MNC AH. Together, this broad transcriptional analysis suggests ongoing efforts at regeneration that are at least partially successful in generating immature myofibers.

Figure 3.

Both diabetic peripheral neuropathy (DPN) abductor hallucis (AH) (mod) and AH (sev) samples have increased regeneration pathway activation compared with metabolically normal control (MNCs). (A) Delineation of regeneration pathway components, color-coded to depict fold changes between MNC AH and DPN AH (mod). (B) Delineation of regeneration pathway components, color-coded to depict fold changes between MNC AH and DPN AH (sev). Red and blue shades indicate up- and downregulation, respectively. MNC AH (n = 3), DPN AH (mod) (n = 2), DPN AH (sev) (n = 2). Online issue will display colors in Figure 3.

Some Small-Diameter Fibers in DPN Muscle Are Regenerated Rather Than Atrophic

We next turned back to histology to confirm the existence of newly regenerated, immature fibers. Immunostaining of fiber-type–specific MHC isoforms revealed an increase in frequency of small-diameter fibers in DPN muscle identified as primarily fast subtypes MHC IIa and IIx (Figure 4A). However, a small subset of small-diameter fibers stained positively for embryonic MHC, a marker for newly regenerated fibers (Figure 4B). This effect was more dramatic in AH than MG samples (Figure 4C). This supports the transcriptional findings that slow-subtype isoforms are highly downregulated (Figure 2D, contraction [slow fibers]), followed by fast isoforms that are modestly downregulated (Figure 2D, contraction [fast fibers]), followed by immature isoforms that are upregulated (Figure 2D, regeneration; Figure 3). Together, this suggests that DPN muscle is able to produce immature fibers and thus retains some capacity for regeneration.

Figure 4.

Increased frequency of regenerated fibers expressing embryonic myosin heavy chain (MHC) in diabetic peripheral neuropathy (DPN) muscle. (A) Representative images of immunostaining for the 3 mature isoforms of MHC reveal small fibers in DPN abductor hallucis (AH) that are largely fast isoforms IIa and IIx. (B) Representative images of immunostaining for the embryonic MHC isoform reveal a few small fibers to be regenerated. (C) Quantification of fiber-type distributions across muscles and groups finds a slow to fast fiber-type shift in DPN muscle with a significant increase in the percentage of fibers expressing embryonic MHC. Metabolically normal control (MNC) medial gastrocnemius (MG) (n = 5), DPN MG (n = 6), MNC AH (n = 4), DPN AH (mod) (n = 3), DPN AH (sev) (n = 3). *P < .05.

The SC Population Is Reduced in Severe DPN Muscle Pathology

SCs represent the primary cellular source for regeneration in skeletal muscle.²⁹ To explore whether these cells remain intact in DPN muscle, we performed a histological stain for Pax7, a transcription factor marker of SCs (Figure 5A). The density of SCs in histological sections was not significantly different between any groups, although the mean density was approximately 50% lower in the DPN AH (sev) group compared with the DPN MG group (Figure 5B). When varying fiber size was taken into account by normalizing the number of SCs to the number of fibers, significant effects emerged (Figure 5C). DPN MG had significantly more SCs per 100 fibers than MNC MG, perhaps due to an expansion of this population as the muscle attempts regeneration in the early stages of pathology. Conversely, DPN AH (sev) samples had significantly fewer SCs per 100 fibers compared with DPN MG and MNC AH, with a trend toward fewer compared with DPN (mod). The reduced numbers of SCs in DPN AH (sev) muscle suggests that the regenerative cellular pool is eventually compromised as DPN pathology advances.

Figure 5.

Satellite cell (SC) numbers are reduced with severe diabetic peripheral neuropathy (DPN) muscle pathology. (A) Representative immunostaining for the SC marker Pax7 reveals the presence of SCs (arrows) in all muscles and all groups. (B) Quantification of the density of SCs per square millimeter image area suggests a decrease in density in DPN abductor hallucis (AH) (sev) samples. (C) Normalization of SC numbers to the number of fibers per image area finds a significant increase in DPN medial gastrocnemius (MG) compared with metabolically normal controls (MNCs) and a significant decrease in DPN AH (sev) compared with DPN MG and MNC AH. MNC MG (n = 5), DPN MG (n = 6), MNC AH (n = 4), DPN AH (mod) (n = 3), DPN AH (sev) (n = 3). *P < .05, **P < .01.

Isolated SCs in Severe DPN Exhibit a Fusion Deficit in Culture

To determine the functional capacity of the SC population in DPN, SCs were isolated from a subset of samples with sufficient remaining biopsy material. When induced to differentiate in culture and fuse into immature MHC+ myofibers (myotubes), SCs from MNC and DPN moderate groups exhibited a similar 40% to 60 % MHC+ area regardless of muscle type (Figure 6). However, SCs from the 1 DPN AH (sev) sample with sufficient biopsy material had substantially reduced rates of fusion, resulting in just an 8% MHC+ area. While it is difficult to draw firm conclusions from a single sample, in combination with the lower SC population numbers (Figure 5), this fusion deficit is suggestive of a compromised regenerative capacity in severe DPN muscle pathology.

Figure 6.

Satellite cells (SCs) isolated from a diabetic peripheral neuropathy (DPN) abductor hallucis (AH) (sev) sample demonstrate compromised fusion in culture. (A) Representative immunostaining for myosin heavy chain–positive (MHC+) structures (immature myofibers) in cultures of isolated SCs. (B) Quantification of MHC+ area in culture images reveals comparatively little fusion in the DPN (sev) sample compared with other groups. Metabolically normal control (MNC) medial gastrocnemius (MG) (n = 4), DPN MG (n = 4), MNC AH (n = 3), DPN AH (mod) (n = 1), DPN AH (sev) (n = 1).

Discussion

In this study, we present compelling evidence that myofiber degeneration accompanies atrophy in lower extremity muscles in individuals with DPN. Although some studies describe neuropathy-associated muscle pathology as strictly atrophic, a number of detailed histological studies in disorders with chronic neuropathy have noted similar degenerative changes to those described here.^{7,8,21,22,26,48} In fact, comparison of histological images between these studies and those presented here shows striking similarities in fiber morphology and patterning with sporadic degenerative events. However, the extent of degenerative changes observed in a subset of the biopsies (DPN AH [sev]) dramatically exceeded changes reported in other neuropathies. We were not able to find any demographic variable that explained why some AH samples were more degenerated than others. Because the biopsies represent only a fraction of the muscle volume, it is impossible to determine with certainty how much of the muscle contractile material has been lost to degeneration. However, in the DPN AH (sev) subset where contractile material occupies less than 30% of the section, it is likely a significant amount. This is important to note because traditional physical interventions primarily target hypertrophic pathways in existing fibers and alone are unlikely to fully restore mass and function if a significant number of fibers have been lost—even if all remaining fibers were reinnervated.

The mechanism of full recovery from a degenerated state lies in myofiber regeneration. It is clear from the evidence that we present here that DPN muscle is attempting to combat degeneration with regeneration. Regeneration-related genes and pathways were upregulated both between DPN and MNC and within the DPN group between the more affected AH and the less affected MG. Histological findings of newly generated fibers were also increased in DPN samples. These findings echo those of animal models, indicating that ongoing attempted regeneration is a general feature of chronic denervation.^10,16,18,49 What is perhaps more surprising is the absence of increased atrophic signaling in the transcriptional data. Genes commonly upregulated across multiple atrophic states were significantly downregulated in our samples—both between DPN and MNC and in DPN AH compared with MG. Instead, major hypertrophic mediators, Igf1 and Fst, were significantly upregulated. These signaling patterns are in clear contrast to the evidence for extensive myofiber atrophy in histological images. This incongruity could arise from a stabilization of the atrophic state, protein-level activation of existing atrophic mediators, protein breakdown through alternative pathways, or atrophy driven by decreased protein synthesis. However, even assuming ongoing atrophy and degeneration, it is clear that the muscle is concurrently attempting regeneration and growth—a promising sign for recovery capacity. Of particular interest is the dramatic increase in Wnt antagonists Sfrp1 and Sfrp2, which were more than 50-fold upregulated in DPN AH (sev) samples compared with MNC AH. These genes are involved in modulating Wnt signaling during regeneration, and an overabundance of sFRPs has been shown to negatively impact terminal myogenesis.¹⁹ Furthermore, altered Wnt signaling has been implicated in SC dysfunction with aging,¹² demonstrating the importance of this signaling pathway in the maintenance of regenerative capacity. Thus, we plan to explore further the role of dysregulated Wnt signaling in the impaired differentiation capacity of DPN AH (sev) myoblasts.

The question remains, however, whether regeneration is functional or impaired. Without a motor neuron to innervate a regenerated fiber, maturation and function will be impeded.³⁷ Our data suggest that in moderate DPN pathology, where myofibers still comprise greater than 30% of the muscle, the SC pool is still present and functional. This is an encouraging result, suggesting that if innervation could be restored, muscle mass could be rebuilt through regeneration. However, in severe DPN pathology, depletion of the SC pool and intrinsic dysfunction in cells themselves might combine with other systemic factors (eg, lack of innervation) to limit muscle regenerative capacity. This hypothesis has been proposed in animal models of denervation as well, where depletion of the SC pool has been noted.^18,25,42 A handful of studies have had moderate success promoting regeneration in denervated animal models by supplementing the SC pool with injected myoblasts.^14,23,40 Myoblast supplementation has been evaluated extensively in the context of muscular dystrophy in both mice and humans,³⁹ so the concept of supplementing physical interventions with injected myoblast is realistic. The concept of combining cell-based therapies to promote both myofiber regeneration and reinnervation is realistic as a number of groups are working to develop cell injections to promote nerve regrowth in animal models of denervation.^32,44,46

To our knowledge, this is the first study to describe degeneration and regeneration in DPN. The strengths of this study include a diverse team comprising surgeons, clinicians, and basic scientists that enabled cell and molecular measures on human tissue and the acquisition of biopsy material that was sufficient in many cases to assay regeneration at the level of the gene, cell, and tissue. There are several limitations to our study and conclusions that should also be noted. First, though we found significant relationships, our sample sizes were small. Our main limitation was the relatively small number of individuals seeking amputation without vascular compromise. Second, most individuals enrolled in this study had structural compromise of the foot (surgical hardware failure, bony collapse, ulceration) combined with a period of unloading on the affected leg (casting, boot, wheelchair), which likely contributed to the pathology within the AH muscle. Many of these were common between MNC and DPN groups but could still uniquely interact with diabetes. Third, many of the study participants also had comorbidities that could contribute to muscle pathology. We attempted to control for systemic effects by comparing a distal to a proximal muscle and excluded those with diagnosed vascular disease (which also advances distal to proximal), but we could not control for a number of other factors.

Conclusion

Taken together, the data presented here demonstrate that varied levels of degeneration and attempted regeneration accompany atrophy as features of DPN muscle pathology. The significance of this finding lies in the ability or inability of the intrinsic regenerative capacity to rebuild muscle to improve strength and function. Evidence for deficits in the SC pool in advanced degeneration suggests that adjuvant cell replacement or cell-targeted therapies may be beneficial in combination with standard or emerging therapies.