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. 2024 Feb 11:5:100194.
doi: 10.1016/j.jvssci.2024.100194. eCollection 2024.

Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia

Ali Navi  1 , Hemanshu Patel  1 , Xu Shiwen  2 , Daryll Baker  1 , David Abraham  2 , Janice Tsui  1
Affiliations

Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia

Ali Navi et al. JVS Vasc Sci. .

Abstract

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI.

Methods: The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model.

Results: TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb.

Conclusions: TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application.

Keywords: Critical limb-threatening ischemia (CLTI); Inflammation; Peripheral arterial disease (PAD); Skeletal muscle; Toll-like receptor (TLR).

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Conflict of interest statement

None.

Figures

Fig 1
Fig 1
(A) Representative immunoblots from biopsied muscles for Toll-like receptor 4 (TLR4) and tubulin loading control and densitometric analysis of the TLR4 immunoblots (n = 6; P < .05; Mann-Whitney U test). (B) Representative immunoblots for phosphorylated nuclear factor κB (P-NFκB) and Jun N-terminal kinase (P-JNK) and densitometric analysis of the P-NFκB and P-JNK immunoblots (n = 4; P < .05; Mann-Whitney U test, between control and chronic limb-threatening ischemia [CLTI] samples). (C) Representative immunoblots from muscle biopsies for cleaved caspase 3 and tubulin loading control and densitometric analysis of the cleaved caspase 3 immunoblots (n = 4; P < .05; Mann-Whitney U test). (D) Representative immunoblots from muscle biopsies for HSP70, HSP60, and tubulin loading control and densitometric analysis of the HSP70 and HSP60 immunoblots (n = 4; P < .05; Mann-Whitney U test; between control and CLTI samples).
Fig 2
Fig 2
(A) Representative immunoblots from cultured myotubes from patients with no peripheral arterial disease (PAD) for TL4, cleaved caspase 3, and tubulin loading control and densitometric analysis of the TLR4 immunoblots (n = 4; P < .05; Mann-Whitney U test) (B) Representative immunoblots from cultured myotubes from patients with chronic limb-threatening ischemia (CLTI) for TL4, cleaved caspase-3, and tubulin loading control and densitometric analysis of the TLR4 immunoblots (n = 6; P < .05; Mann-Whitney U test). (C) Representative Western blots for phosphorylated nuclear factor κB (P-NFκB) with TLR4 agonist (synthetic LPS) or TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides [RS-LPS]) pretreatment before simulated ischemia in myotubes from patients with CLTI (B) Densitometric analyses of Western blots of P-NFκB with TLR4 agonist (synthetic LPS) or TLR4 antagonist (RS-LPS) pretreatment before simulated ischemia in myotubes from patients with CLTI (n = 6; P < .05; Mann-Whitney U test) (D) Representative Western blot of cleaved caspase-3 from patients with CLTI, which were pretreated with TLR4 antagonist (RS-LPS) or TLR4 neutralizing antibody before simulated ischemia (n = 6; P < .05; Mann-Whitney U test). (E) Effect of ischemia on myotubes from patients with CLTI. Representative Western blots of cleaved caspase-3 after inhibition of adaptor proteins and densitometric analyses of Western blots showing downregulation of cleaved caspase-3 after inhibition of MyD88 (n = 6; P < .05; Mann-Whitney U test).
Fig 3
Fig 3
Effect of Toll-like receptor 4 (TLR4) antagonism and ischemia on myotubes from patients with chronic limb-threatening ischemia (CLTI). (A) TLR4 antagonist pretreatment attenuates ischemia-induced IL6 release (n = 6; P < .05; Mann-Whitney U test). (B) TLR4 antagonist pretreatment attenuates ischemia-induced tumor necrosis factor α (TNF-α) release (n = 6; P < .05; Mann-Whitney U test). Enzyme-linked immunosorbent assay analyses of (C) interleukin 6 (IL-6) and (D) TNF-α from cultured myotubes from patients with CLTI, with or without pretreatment with MyD88 and TRIF inhibitor (n = 6). ∗P < .05; Mann-Whitney U test.
Fig 4
Fig 4
(A) Representative laser Doppler perfusion images obtained with mice positioned supine under the scan head on a low-temperature heating pad. Serial color-coded perfusion images (A-C) of 12-week-old C57BL/6 mice on postoperative days 3, 7, and 21, respectively. (D-F) Twelve-week-old C57BL/6 mice with genetically deleted Toll-like receptor 4 (TLR4) on postoperative days 3, 7, and 21, respectively. (G-I) Twelve-week-old C57BL/6 treated with exogenous TLR4 inhibition on postoperative days 3, 7, and 21, respectively. (B) Perfusion recovery after surgery. The ischemic/nonischemic lower limb perfusion ratio showed a significant difference on days 3, 7, and 21 between the TLR4−/− group and control and TLR4 antagonist (LPS-RS) groups (∗P < .001 at days 3 and 7; ∗∗P = .01 at day 21; Kruskal-Wallis test). The difference between each group was also significant (P < .005; Mann-Whitney U test) on days 3, 7, and 21. RS-LPS, lipopolysaccharide from Rhodobacter sphaeroides.
Fig 5
Fig 5
Functional and ischemic scoring of the mice in all groups showed faster recovery in Toll-like receptor (TLR4)−/− and lipopolysaccharide (LPS-RS) groups compared with the vehicle group (∗P < .05 between groups; Kruskal-Wallis test; n = 6).
Fig 6
Fig 6
(A) Fluorescent immunohistochemical staining for cleaved caspase-3 (red color) in C57BL/6 mouse gastrocnemius muscle, post hindlimb ischemia day 21 (paraffin embedded, DAPI [blue] and AlexaFluor 647 [red] stains, 3-micron-thick section; original magnification ×40). Representative images from (A) control group, (B) lipopolysaccharide (LPS-RS) group, (C) Toll-like receptor 4 (TLR4)−/− group, and (D) IgG (negative) control. (B) Inflammatory cell quantification in hematoxylin and eosin (H&E)-stained ischemic gastrocnemius muscle showed less inflammatory cell infiltration in both TLR4−/− and LPS-RS groups when compared with the control group at each time point (P < .05; Kruskal-Wallis test; n = 6). (C) Representative H&E staining of ischemic gastrocnemius muscle on postoperative days 3, 7, and 21 (scale bar, 50 μm).
Fig 7
Fig 7
Schematic showing the potential link between ischemia-induced Toll-like receptor 4 (TLR4) activation and the resulting tissue damage. Ischemia leads to muscle damage with the release of inflammatory cytokines. Upon activation of TLR4, adaptor proteins (MyD88 and TRIF) and transcription proteins (P38, JNK, and nuclear factor κB [NF-κB]) are recruited to activate the inflammatory gene transcription.
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