The ischemic-injured muscle in CLTI patients is enriched with pro-inflammatory macrophages

To understand the pathological changes in ischemia-injured skeletal muscle, we carried out single-cell RNA-seq analysis of fresh skeletal muscle samples from CLTI patients undergoing limb amputation surgery. The biopsies were taken from both the distal (ischemic) and proximal (non-ischemic) regions of the amputated limb and dissociated into single cell suspensions. It should be noted that amputations occur at a level where the tissue is healthy and normoxic to ensure wound healing. Hence, the proximal tissues are not ischemic. The live cells were FACS sorted and immediately subjected to single-cell transcriptome analysis (Fig 1A). Importantly, obtaining matched proximal and distal tissue from the same individual allowed us to examine the specific pathological changes caused by chronic ischemia while controlling for differences in the genetic background and health conditions of each patient. Using pairs of matched proximal and distal muscle tissues from three representative CLTI patients (Additional file 1: Fig S1A, bottom; Table 1), we recovered a total of 16,201 high-quality cells for downstream bioinformatics analysis. After correcting for batch effect and patient-specific biases (detailed in Materials and Methods), all cells were uniformly dispersed throughout the UMAP space (Additional file 1: Fig S1B). We next analyzed cells based on the anatomic location of the samples from which they were obtained in the amputated limb (distal, ischemic; proximal, non-ischemic) to determine if there was a hypoxic transcriptional signature in cells from the distal tissue. Indeed, the cells from the distal limb displayed increased expression of HIF1A compared to those from the proximal limb (Additional file 1: Fig S1C). This finding, in concert with our computed tomography (CT) scans for each patient (Additional file 1: Fig S1A, bottom), demonstrates that the distal tissue specimens are ischemic compared to the proximal specimens.

Fig. 1

Single-cell transcriptional profiling of human CLTI patients’ limb muscle in non-ischemic versus ischemic conditions. A Schematic diagram illustrating the generation of scRNA-seq datasets using proximal and distal tissue from human CLTI skeletal muscle. B Uniform manifold approximation projection (UMAP) visualization showing cell populations (n = 16,201) from non-ischemic and ischemic tissues of CLTI patients (n = 3 donors, paired proximal and distal tissues were analyzed). C Dot plot displaying the expression of marker genes for each cell population. Dot size represents the percentage of cells that positively detect the transcripts, and the color scale indicates average expression levels. D, E UMAP visualization of macrophages in proximal (blue, non-ischemic) and distal (pink, ischemic) skeletal muscle (D) and sub-clusters (E, C0-C8). F Top five Gene Ontology (GO) terms enriched by differentially expressed genes (P-value < 0.05 & |log2FoldChange| > 0.25) between distal (pink, ischemic) clusters (1 and 2) and proximal (blue, non-ischemic) cluster (0). Pink and blue bars represent the GO terms enriched in distal and proximal conditions, respectively. G Feature plots showing the expression of pro-inflammatory genes in macrophages. H Quantification of representative pro-inflammatory gene expression in proximal versus distal macrophages. Adjusted p-values (adj.P) and log fold changes of average expression (avg.log2FC) were calculated by Wilcoxon rank-sum test. I Quantification of CD11b+/CD206+ and CD11b+/CD206- macrophages in ischemic and non-ischemic CLTI patient muscle specimens. P values were calculated by paired Wilcoxon rank-sum test

Table 1 Human CLTI patient demographics (N = 10)

The 16,201 cells were annotated into ten major cell types, including fibro-adipogenic progenitor cells (FAPs), muscle stem cells (MuSCs), muscle progenitor cells (MPCs), endothelial cells, macrophages, neutrophils, T cells, pericytes, NK cells, and smooth muscle cells (SMCs) based on the expression of well-defined cell type-specific marker genes (Fig. 1B, C; Additional file 1: Fig S1D, S1E). We further analyzed the proportional distribution of each cell type in both proximal and distal conditions from the three CLTI patients. This analysis indicates a decrease in the proportion of endothelial cells, pericytes, T cells, MuSCs, and MPCs, and an increase in the presence of FAPs and neutrophils in the distal tissues compared to the proximal non-ischemic tissues (Additional file 1: Fig S1F). Our single-cell atlas of CLTI patient samples, encompassing matched proximal and distal tissue samples, suggests that chronic ischemic damage alters the cellular landscape of skeletal muscle in CLTI patients.

Intriguingly, when macrophages were segregated into non-overlapping populations on a new UMAP space with increased resolution, significant differences were seen between cells derived from proximal versus distal tissue (Fig. 1D). The macrophages were separated into nine sub-clusters (Fig. 1E). Of these, cluster 0 was composed primarily of cells from non-ischemic tissue, while clusters 1 and 2 were predominantly composed of macrophages from ischemic-injured distal tissue (Fig. 1E). We further identified genes differentially expressed in clusters 1 and 2 versus cluster 0 (Wilcoxon test, p-value < 0.05, log2 fold change > 0.25, Additional file 3: Table S2) and found that the genes highly expressed in clusters 1 and 2 were enriched for Gene Ontology (GO) terms related to pro-inflammatory pathways (Fig. 1F). Several well characterized pro-inflammatory genes, such as TNF, IL1B, CCL3, and CCL4 [50], as well as the key hypoxia-responsive gene HIF1A [51], were expressed at significantly higher levels in macrophages from distal tissue versus those from proximal muscle (Fig. 1G, H; Additional file 1: Fig S1G). These results demonstrate that macrophages in the distal tissues of CLTI patients are impacted by chronic ischemic-injury and display a pro-inflammatory phenotype.

To experimentally validate these findings, we collected distal and proximal skeletal muscle samples from another seven CLTI patients (Table 1) and immunostained the tissue sections with antibodies against the pan-macrophage marker CD11b and the anti-inflammatory macrophage marker CD206 (Additional file 1: Fig S1H, S1I). According to previously published criteria [52, 53], we designated the CD11b+/CD206+ cells as anti-inflammatory macrophages and the CD11b+/CD206- cells as inflammatory macrophages. In the ischemic-injured distal muscle, we found on average 1.97-fold more pro-inflammatory macrophages compared to the non-ischemic condition (Fig. 1I, p-value = 0.02, paired samples Wilcoxon test). In contrast, there were on average 2.07-fold more anti-inflammatory macrophages in the non-ischemic proximal muscles (Fig. 1I, I-value = 0.02, paired samples Wilcoxon test). Considering all these results, we conclude that pro-inflammatory macrophages are indeed enriched in the ischemic-damaged limb muscle of CLTI patients.

Single-cell transcriptome analysis of regenerative versus CLTI-like mouse limb muscle following hind-limb ischemia surgery

Next, we sought to determine whether the change of the inflammatory response of ischemic limb muscle is associated with the tissue loss in CLTI. Since it is not feasible to obtain clinical samples from CLTI patients through disease progression over time, we employed a murine model of CLTI in which hind limb ischemia (HLI) surgery is used to ligate the femoral artery in BALB/c and C57BL/6 mice to assess the temporal dynamics of limb tissue loss in CLTI (Fig. 2A, B). As noted, following HLI surgery, BALB/c mice develop a CLTI-like, profound tissue loss phenotype and paw necrosis, and a significant reduction of Cd31+ endothelial cells as assessed by capillary density analysis compared to C57BL/6 mice (Fig. 2B, right; Additional file 1: Fig S2A) [13, 19, 54]. In contrast, C57BL/6 mice display very minor, if any, tissue loss, despite experiencing a similar 80–90% reduction in limb blood flow after HLI (Fig. 2B). Consistent with previous results [13], the ischemic tibialis anterior (TA) muscle of C57BL/6 mice exhibited a potent muscle regenerative response following HLI, indicated by 6.2-fold greater expression of embryonic myosin heavy chain (eMHC) and 3.1-fold more Pax7+ satellite cells compared to BALB/c mice at 7 days post-injury (dpi) (Fig. 2C, D; Additional file 1: Fig S2B). Therefore, BALB/c mice represent a murine model of CLTI with permanent tissue loss, whereas C57BL/6 mice are resistant to ischemia-induced muscle damage and display a potent skeletal muscle regenerative program following HLI.

Fig. 2

Single-cell RNA-seq analysis of hind limb ischemia (HLI) surgery induced limb muscle regeneration and damage responses in C57BL/6 and BALB/c mice. A Perfusion imaging of C57BL/6 (top) and BALB/c (bottom) mouse strains before and after HLI surgery (Pre-operatively and Post-operatively, respectively) and on post-op days 1, 3, and 7. B (Left) Quantification of limb perfusion as determined by perfusion imaging at indicated timepoints. Left hindlimb (HLI surgery) perfusion normalized to right hindlimb perfusion for each mouse. n = 3 per mouse strain, per timepoint. *p < 0.01. (Right) Representative images of mice on post-op days 1, 3, and 7 following HLI surgery. Red arrow indicates ischemic changes apparent on post-op days 3 and 7 in BALB/c mice. C Representative immunofluorescence staining images of mice on post-op day 7 following HLI surgery. eMHC (red) indicates regenerated muscle fibers; Pax7 (green) indicates satellite cells. D Quantification of eMHC+ area (left panel) and Pax7+ cell counts (right panel) shown in C. n = 6 mice per group. Data expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. E Experimental design of mice scRNA-seq analysis of mouse models of HLI (n = 2 per mouse strain, per timepoint). F UMAP visualization of the scRNA-seq atlas assembled from all samples and time points. G The expression of cell type marker genes used for each cell type/cluster annotated in F

Next, we performed scRNA-seq analysis utilizing cells collected from hindlimb muscles, including tibialis anterior (TA), gastrocnemius, and soleus, from BALB/c and C57BL/6 mice, following HLI surgery, at intervals ranging from 1 to 7 days post-injury (dpi). Cells obtained from the unligated limb, following sham surgery, served as the control group. We incorporated two biological replicates for each experimental condition (Fig. 2E). In total, we recovered 84,362 high-quality single cells from the two mouse strains at four-times (Fig. 2F, Additional file 1: Fig S2C). We identified 17 major cell types, including MuSC/MPCs, immune cells, and FAPs (Fig. 2F). These annotated cell types express high levels of expected marker genes that were defined in previous studies of scRNA-seq analysis of mouse skeletal muscle regeneration (Fig. 2G, Additional file 1: Fig S2D). Notably, a marked increase in HIF1A expression was observed in cells harvested from post-HLI limbs in both mouse strains, relative to cells from non-ischemic limbs, thereby demonstrating the ischemic impact on cells following HLI (Additional file 1: Fig S2E). These scRNA-seq datasets thus provide the first reference atlas to examine the temporal dynamics of cell populations and their gene expression patterns in mouse strains that display either effective or failed skeletal muscle regeneration following limb ischemia.

Pro-inflammatory macrophages are enriched in the ischemic-damaged limb muscle of mice subjected to HLI

We next explored whether differences exist in the macrophage populations in the ischemic hindlimbs of C57BL/6 and BALB/c mice. Fine resolution sub-clustering analysis of a total of 26,991 macrophages revealed 12 sub-clusters (Additional file 1: Fig S3A, clusters 0-11), which display temporal-, strain-, and cluster-specific gene expression patterns (Fig. 3A, Additional file 1: Fig S3B, Additional file 4: Table S3). Notably, clusters 4 and 5 were dominated by BALB/c cells, while clusters 1, 2, 3, 7, 8, and 9 were made up primarily of C57BL/6 macrophages (Fig. 3A, right). At 3 dpi, the macrophages from BALB/c and C57BL/6 mice were segregated into two non-overlapping populations on the UMAP (Fig. 3A, right, day 3), indicating drastically different macrophage gene expression programs at day 3 in the two strains. Significantly, the BALB/c-specific cluster 5 displayed a strong pro-inflammatory gene expression signature, as demonstrated by a high inflammatory response score, which was computed based on the expression levels of genes associated with the GO term “inflammatory response” (Fig. 3B, Additional file 3: Table S2, detailed in material and methods). Macrophages can be classified largely into the pro-inflammatory M1 and anti-inflammatory/pro-regenerative M2 states based on their in vitro phenotype in response to inflammation stimulation [55]. Using this convention, we found that anti-inflammatory M2 genes were highly expressed in the C57BL/6 macrophages, while pro-inflammatory M1 genes were highly expressed in macrophages from BALB/c mice (Fig. 3C, D). These results suggest that following HLI, the non-regenerative BALB/c limb muscles are enriched with macrophages exhibiting a pro-inflammatory phenotype compared to those in the regenerative C57BL/6 muscle.

Fig. 3

scRNA-seq analyses reveals that the pro-inflammatory macrophages are enriched in the BALB/c limb following HLI surgery. A UMAP plots illustrating the distribution of macrophages (depicted in blue) and other cells (depicted in grey) from C57BL/6 and BALB/c mice at specified time intervals. The left UMAP plot aggregates all cells from the two strains across all time points. In the right-hand UMAP plots, macrophages for the distinct time points and individual strains are highlighted in blue, while all remaining cells are represented in grey. B The inflammatory gene module score is high in cluster 5 cells, which are specific to BALB/c mice. Red color indicates high gene module score. C GSEA enrichment analysis reveals that M1 macrophage markers are highly expressed in BALB/c macrophages, while M2 macrophage markers are highly expressed in C57BL/6 macrophages. Macrophage gene expression patterns are assessed using scRNA-seq data. D Dot plot showing differentially expressed genes in macrophages between C57BL/6 and BALB/c mice on day 3 post-ischemia. Dot size represents -log10 FDR; color scale indicates log2-fold change in gene expression. Pink and blue colors indicate genes upregulated in C57BL/6 and BALB/c, respectively. E Volcano plot displaying differentially expressed genes from bulk RNA-seq analysis of macrophages purified from BALB/c and C57BL/6 mice at 3 days post-HLI. Red and blue dots represent upregulated genes in C57BL/6 and BALB/c mice, respectively. F: Genome browser snapshots showcasing bulk RNA-seq data at specified gene loci in macrophages derived from both strains on day 3 post-injury. Two biological replicates (rep1, rep2) are included for each condition. G Top five GO terms enriched by differentially expressed genes (FDR < 0.05, log2FoldChange>1) from panel E. Red and blue bars correspond to GO terms enriched in C57BL/6 and BALB/c mice, respectively

To experimentally validate the findings from scRNA-seq analyses, we purified CD11b+/F4/80+ macrophages by FACS from the hindlimb muscle of C57BL/6 and BALB/c mice at 3 dpi following HLI for bulk RNA-seq analysis (Additional file 1: Fig S3C). We identified 289 down-regulated and 320 up-regulated genes in C57BL/6 versus BALB/c macrophages (Fig 3E, Additional file 5: Table S4, DEseq2, fold change > 2, FDR < 0.05). Many pro-inflammatory genes, such as Arg1, Cxcl3, and Cxcl1 [30], were highly expressed in the BALB/c macrophages (Fig. 3E, F). In contrast, anti-inflammatory and pro-regenerative genes, such as Chil3, Igf1, Gdf15, and Gdf3 [56, 57], were highly expressed in C57BL/6 3 dpi macrophages (Fig. 3E, F; Additional file 1: Fig S3D). The genes highly expressed in 3 dpi BALB/c macrophages were enriched for GO terms associated with inflammatory response, chemotaxis, and immune response (Fig. 3G, Additional file 5: Table S4). These results collectively highlight the clear transcriptional differences in macrophages present in the regenerative (C57BL/6) and non-regenerative (BALB/c) limbs following HLI and demonstrate that a strong pro-inflammatory transcriptional signature of macrophages, particularly at 3 dpi, is associated with the limb tissue loss phenotype of BALB/c mice.

Impaired muscle regeneration in BALB/c ischemic limbs characterized by premature myogenic differentiation and proliferative deficit of MuSCs

MuSCs directly contribute to muscle regeneration through their activation from the initial quiescent state and subsequent proliferation, differentiation, and fusion [58]. To delineate strain-specific responses of MuSCs and MuSC-derived muscle precursor cells (MPCs) following HLI, we performed an in-depth analysis of all the scRNA-seq data on Pax7+ MuSCs and Myod+ MPCs. First, the MuSCs/MPCs were classified into quiescent (Pax7 high), activated/proliferating (MyoD, Ki67 high), early-differentiating (MyoG+), and late-differentiating (Ckm+) states (Fig. 4A, Additional file 1: Fig S4A). Next, we conducted pseudotime trajectory analysis to rank the MuSCs/MPCs based on their transcriptome similarities (Additional file 1: Fig S4B). This approach showed that MuSCs/MPCs ranked in the early part of the pseudotime trajectory expressed high levels of quiescence marker genes (Hes1, Calcr, Cd34, Pax7, Myf5, Notch1/3), while cells ranked later in the pseudotime trajectory expressed high levels of the activation marker gene MyoD and cell cycle-related genes (Cdnb1/2, Cdc20, Cdk1) or high levels of marker genes associated with myogenic differentiation (MyoG, Ckm, Myh1). Therefore, the MuSCs/MPCs in our dataset, when ranked along this pseudotime trajectory, exhibit the expected cellular states, ranging from quiescence to activation/proliferation and differentiation (Fig. 4B). Significantly, along the pseudotime trajectory, we noted that MuSCs/MPCs from both BALB/c and C57BL/6 mice begin at the initial quiescent state (Fig. 4C, top) and transition into the activation/proliferation and differentiation states (Fig. 4C, middle and bottom), demonstrating that MuSCs/MPCs from the BALB/c strain do not intrinsically lack regenerative capacity.

Fig. 4

MuSCs/MPCs in BALB/c mice undergo precocious differentiation after HLI surgery. A UMAP representation displaying quiescent (depicted in yellow), activated/proliferative (in blue), and differentiating (in pink) muscle stem cells (MuSCs) and muscle precursor cells (MPCs). Total MuSC/MPC count stands at 9,217. B MuSCs/MPCs from part A are arranged in a pseudotime sequence, initiating from quiescent cells (left) and advancing to activated/proliferative (middle) and subsequently to differentiating cells (right). The heatmap's color gradient represents the expression intensity of the specified genes in MuSCs/MPCs in accordance with the pseudotime progression. C (Top) Violin plot showing the distribution of quiescent (depicted in yellow), activated/proliferative (in blue), and differentiating (in pink) MuSCs/MPCs in the two strains along the pseudotime trajectory shown in B. (Bottom) Curve plot showing the distribution of MuSC/MPCs at indicated time points, before (sham) and 1, 3, and 7 dpi post-HLI, in the two mouse strains along the pseudotime trajectory shown in B. D Gene set enrichment analysis (GSEA) highlighting the genes related to “skeletal muscle cell proliferation” are significantly enriched in the up-regulated genes in MuSCs/MPCs in C57BL/6 mice compared to those in BALB/c mice. E, F At 7 dpi, TA muscles were collected from both C57BL/6 and BALB/c for immunostaining using antibodies against Pax7 and cell proliferation marker Mki67. E n = 5 mice per strain. Data expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01. F Representative Pax7 (red) and Mki67 (green) immunofluorescence staining images along with DAPI (blue). G Representative RNAscope data showing that Adgre1+ macrophages (F4/80, green) and Myod1+ MuSC/MPCs (red) are spatially proximal to each other in the limb muscle of BALB/c and C57BL/6 mice at day 3 after HLI. Three mice per strain were used for RNAscope analysis. H Inferred ligand-receptor interactions between macrophages and MuSCs in BALB/c and C57BL/6 following HLI at 3 days post HLI surgery. I IGF1 promotes proliferation of primary MPCs purified from BALB/c and C57BL/6 strains. (Left) Representative images of EdU incorporation by C57BL/6 and BALB/c primary MPCs cultured with or without recombinant IGF1 for 72 h. EdU was added to the culture medium 6 h prior to cell fixation and imaging. Nuclei stained with Hoechst. Arrows indicate EdU+ cells. (Right) Quantification of the percentage of EdU+ cells for the indicated strains and treatment conditions. *P < 0.05, **P < 0.005

It is well established that the MuSCs/MPCs proliferation phase is critical for efficient muscle regeneration as it is required to produce sufficient MPCs for myogenic differentiation and fusion and that the timing of the transition from the proliferative state to the differentiation state is also important [59]. We found that before HLI (sham) and at 7 dpi, the MuSCs/MPCs collected from the two mouse strains were well-aligned on the pseudotime trajectory and mirrored each other closely in both the early (quiescent) and late (differentiation) stages of pseudotime (Fig. 4D, sham and day 7). In contrast, the MuSCs/MPCs exhibited drastic strain-specific differences at 1 dpi and 3 dpi (Fig. 4D, day 1 and day 3). At both timepoints, a larger fraction of C57BL/6 MuSCs/MPCs (blue lines) were in the activation/proliferation phase, whereas the vast majority of the BALB/c cells (red lines) occupied the late, differentiation stage of the pseudotime trajectory (Fig. 4D, day 1 and day 3). This pattern suggests that the BALB/c MPCs committed to differentiation prematurely without sufficient proliferation. Indeed, the genes highly expressed in C57BL/6 compared to BALB/c MPCs at 3 dpi were significantly enriched for GO terms “skeletal muscle cell proliferation” (Fig. 4F). Finally, through immunofluorescence analysis of post-HLI limb muscle, we found that in BALB/c mice the proportion of proliferative Ki67/Pax7-double positive MuSCs was substantially less than in C57BL/6 mice (Fig. 4E). Collectively, these results suggest that the failure of skeletal muscle regeneration in the BALB/c model of CLTI is at least in part due to inadequate proliferation and premature differentiation of MuSCs/MPCs.

The pro-inflammatory niche is associated with premature differentiation of MuSCs in BALB/c mice following HLI

MuSC regenerative events are substantively coordinated and supported by macrophage-derived ligands [28, 60, 61]. To address whether the inflammatory macrophages in BALB/c muscle are associated with premature differentiation of MuSCs following HLI, we sought to identify candidate ligand-receptor pairs to account for the disparate, strain-specific macrophage-MuSC cross-talk. We assessed the probability of intercellular communication between macrophages and MuSCs using a computational method called CellPhoneDB [37]. We found that at 3 dpi, C57BL/6 MuSCs are likely responsive to well-characterized pro-regenerative cytokines secreted by macrophages, such as TGFB1 and IGF1 (Fig. 4G), which play critical roles in promoting MPC proliferation and preventing their premature differentiation [56, 62, 63]. In contrast, TGFB1 and IGF1 pathways were not detected in BALB/c mice in macrophage-MuSC intercellular communication (Fig. 4G). Notably, at 3 dpi, the IGF1 receptor (Igf1r) is specifically expressed in C57BL/6 MuSCs/MPCs (Additional file 1: Fig S4E) and the expression of Igf1 in C57BL/6 macrophages is elevated compared to BALB/c macrophages at the same timepoint (Additional file 1: Fig S3D). This data suggests that signaling communication, specifically within the IGF1 pathway, between macrophages and MuSC/MPCs plays a pivotal role in fostering MuSC/MPC proliferation and averting premature differentiation in C57BL/6 mice.

To test this idea, we purified primary MuSCs from both mouse strains for in vitro cell proliferation assays using EdU incorporation. Upon treatment with recombinant IGF1, new DNA synthesis in MuSCs/MPCs, isolated from both mouse strains, significantly increased by 2–3-fold (Fig. 4H). Consequently, we reasoned that the observed failure of muscle regeneration in BALB/c mice does not stem from an inherent deficiency in the proliferative ability of MuSCs/MPCs, for example in response to stimulation by IGF1. Rather, it appears to result from a deficiency in the secretion of pro-regenerative cytokines like IGF1 by macrophages, facilitating at least in part, the premature differentiation of MuSCs/MPCs and ultimately the failure of muscle regeneration in BALB/c mice following hindlimb ischemia. Indeed, recent research corroborates this perspective, illustrating that the administration of IGF1, via AAV9, significantly improved muscle mass and function post-HLI in BALB/c mice [64]. These findings highlight the potential of cytokines such as IGF1 to foster muscle regeneration in ischemic limbs, by promoting MuSCs/MPCs proliferation. Notably the beneficial pro-regenerative effects of cytokines such as IGF1 are conspicuously absent in BALB/c mice given the predominance of pro-inflammatory macrophages observed in this strain following HLI.

Disruption of MuSCs fate switch is associated with aberrant macrophage-MuSC signaling crosstalk in human CLTI

Finally, we sought to translate our findings from murine models of HLI to human CLTI patients. We found that the distal muscle samples of three CLTI patients analyzed by scRNA-seq contained fewer MuSCs/MPCs compared to matched proximal samples (Additional file 1: Fig S5A). To further explore these findings, we conducted Pax7 immunostaining on cross-sections of limb muscles collected from an additional seven CLTI patients (Additional file 1: Fig S5B, Table 1). In four out of the seven patients, the number of Pax7+ MuSCs in the ischemic distal muscle were ~10–60% less than those in the matched non-ischemic proximal muscle (Additional file 1: Fig S5C). These results indicate that in at least a subset of CLTI patients (approximately 57–67%), ischemic-damaged limb muscle contains fewer MuSCs/MPCs compared to non-ischemic muscle.

Furthermore, to assess changes in gene expression and signaling activity of MuSCs/MPCs in human CLTI, we performed an integrative analysis of our human CLTI scRNA-seq data in conjunction with published human muscle scRNA-seq data generated from ten healthy individuals [65] (Fig. 5A, Additional file 1: Fig S5D). This analysis revealed Pax7+ MuSCs (cluster 2), MyoG+ MPCs (cluster 13) populations in human muscle, and a C3AR1+ macrophage population (cluster 7) (Fig. 5A, 5B; Additional file 1: Fig S5E). Incorporating data from healthy human skeletal muscles allows us to benchmark gene expression patterns of MuSCs/MPCs observed in CLTI patients against those in healthy individuals. To further elucidate the alterations in gene expression in MuSCs/MPCs in human CLTI, we segregated all the MuSC/MPCs from healthy and CLTI individuals onto a distinct UMAP space with increased resolution (Fig. 5C, Additional file 1: Fig S5F, S5G). Within this new UMPA space, we identified two major populations: Pax7+ MuSCs and MPCs committed to myogenic differentiation, which express MyoG or CKM (Fig. 5C, D). Importantly, the Pax7+ MuSCs from distal tissues exhibited substantially diminished levels of the quiescence/self-renewal marker SPRY1, as well as the cell cycle marker CDK6, when compared to their proximal counterparts and healthy MuSCs (Fig. 5E). This altered gene expression pattern hints at a decline in the self-renewal and proliferative capacity of MuSCs in the CLTI limbs affected by chronic ischemic damage. Additionally, the committed MPCs in distal tissues demonstrated significantly elevated levels of the early differentiation marker MYOG and terminal differentiation marker MYH3, compared to those in the proximal tissues of CLTI and in healthy muscles (Fig. 5E). These findings support the model that MuSCs/MPCs in the distal ischemic muscle of human CLTI patients are undergoing a loss of stem cell quiescence, a decline in cell proliferative capacity, and premature differentiation. These trends mirror the phenomena we observed in the murine CLTI model of HLI in BALB/c mice, thereby reinforcing the validity of our findings that premature differentiation of MuSCs/MPCs is associated with muscle regeneration failure in CLTI.

Fig. 5

Increased pro-inflammatory signaling pathways between macrophages and MuSC/MPCs is associated with premature differentiation phenotype of MuSC/MPCs in the ischemia-damaged human limb of CLTI patients. A, B UMAP visualization presenting single-cell data gathered from human skeletal muscle tissue samples encompassing 3 CLTI patients under both proximal and distal conditions, along with data from 10 healthy individuals. A A total of 34,950 cells are represented in the UMAP graph. B The cells in clusters 2 and 13, illustrated in purple, exhibit Pax7 (left) and MyoG (right) expression respectively. C Cells from Pax7+ and MyoG+ clusters delineated in B are extracted from total cells and projected with an increased resolution onto a separate UMAP space that contain 5 sub-clusters (cluster 0–4), depicted in different colors. D Within this refined UMAP representation in D, cells in clusters 2 and 4 are denoted as “MPC”, demonstrating the expression of myogenic differentiation markers like MyoG and CKM. Conversely, the cells congregated in clusters 0 and 1 are identified as MuSCs, characterized by the expression of quiescent MuSCs markers Pax7 and Myf5. E The MPC and MuSCs defined in D are separated into healthy (yellow), distal (red), and proximal (blue) conditions for gene expression analyses. Violin plots show the expression of MyoG, MYH3, SPRY1, and CKD6 expression in MPCs (left) and in MuSCs (right), respectively. *P-value < 0.05; **P-value < 0.01. P-values were calculated by the Wilcoxon rank-sum test. F The numbers (left) and interaction strength (right) of inferred signaling interactions calculated by CellChat between macrophages and MuSCs in distal (pink, ischemic) and proximal (blue, non-ischemic) muscles of CLTI patients. G The significant signaling pathways between macrophages and MuSCs/MPCs are ranked based on their inferred strength differences between distal (ischemic) and proximal (non-ischemic) skeletal muscles. Signaling pathways colored in red are enriched in distal muscle, while those colored in blue are enriched in proximal tissues between macrophages and MuSCs/MPCs. H Ligand-receptor interactions inferred by CellPhoneDB between macrophages and MPCs or MuSCs in distal (ischemic) and proximal (non-ischemic) conditions. In distal conditions, we observed stronger pro-inflammatory signaling pathways, such as IL6, CCL4, and SPP1, compared to proximal conditions

To further elucidate the underlying mechanisms associated with permanent tissue loss in CLTI patients, we investigated the potential association between the transcriptome signatures of MuSCs/MPCs and macrophages in our human CLTI scRNA-seq data. Leveraging intercellular communications inferred from scRNA-seq data [39], we found that the inter-cellular communication signaling pathways between macrophages and MuSCs/MPCs were markedly more pronounced in the ischemic muscle relative to the non-ischemic proximal muscle, both in terms of number and strength of signaling interactions (Fig.