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Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors - PubMed

Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors

Leonardo Martins et al. Inflamm Res. 2023 Aug.

Abstract

Objective and design: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing.

Material and methods: To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated.

Results: Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals.

Conclusions: We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.

Keywords: Contusion; Fibrosis; Injury; Kinin; Myogenesis; Skeletal muscle.

© 2023. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

Lack of kinin B2 but not B1 receptors increases damage of injured muscle. A Representative photos from H&E staining of Tibial anterior (TA) muscles for all animal groups at indicated days after injury showing an inflammatory area (*), fibers centrally nucleated (arrow) and necrotic cells (arrowhead) and B and C injured area delimited (*). (Scale bars = 100 μm). (n = 5, all groups). D TA injury area normalized to cross-sectional area (square micrometer per square micrometer). Data are represented as mean ± SD. *P < 0.05 KO groups vs WT control group. #P < 0.05 B1KO vs other KOs. E Number of inflammatory cells within the areas of injury. High-power fields (HPF). Data are represented as mean ± SD. *P < 0.05 KO groups vs WT control group. #P < 0.05 B1KO vs others KOs. F Percentage of centronucleated cells within the areas of injury. High-power fields (HPF). Data are represented as mean ± SD. *P < 0.05 KO groups vs WT control group. # P < 0.05 all KO groups vs KO groups

Fig. 2
Fig. 2

Lack of kinin B2 receptors but not B1 receptor increases fibrosis of injured muscle. A Representative immunofluorescence analyses of Col1A in the injured skeletal muscle at indicated days after injury and non-injured tissue (0 day) in each group (n = 5, for all groups). Col1A (red), DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) B Collagen deposition area. High-power fields (HPF). Data are represented as mean ± SD. *P < 0.05 KO groups vs WT control group. #P < 0.05 B1KO vs other KOs. C Relative expression of Col1a1 mRNA was detected at lower levels in B1KO group and high levels in B2KO group at 8th day after injury (n = 5 mice per group) compared with each other (#, P < 0.05.) and only in the B1KO group compared to WT control (*, P < 0.05). D Representative Sirius red staining combined with polarized light microscopy for collagen fiber structure. Polarized images of the Picrosirius Red stained sections were quantified to evaluate collagen differentiation by fiber length, width, and angle. Yellow–red strong birefringence (type I collagen) and greenish color with thinner fibers (type III collagen) (white arrow) (Scale bars = 100 μm.) E Type III collagen area by High-power fields (HPF). Data are represented as mean ± SD. *P < 0.05. KO groups vs WT control group. #P < 0.05 all KO groups vs KO groups (color figure online)

Fig. 3
Fig. 3

Lack of B2 enhances the activation of the Tgf-β/Smad pathway, whereas the lack of B1 stimulates the production of inhibitory Smad (SMAD7). A Representative immunofluorescence analyses of CD69 + cells (green, arrow), p-SMAD2/3 (red, arrowhead) (Top), and SMAD7 (red, arrowhead) (Bottom) in the injured skeletal muscle at indicated days after injury in each group (n = 5 per group). CD69 (green), p-SMAD2/3 (red, arrowhead, top), and SMAD7 (red, arrowhead, bottom). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) B p-SMAD2/3 quantification in the injured skeletal muscle after injury. Data are expressed as percentage of p-SMAD2/3-expressing cells in each group (mean ± SE) (*, P < 0.05). C SMAD7 quantification in the injured skeletal muscle at 4, 8 and 15 days after injury. Data are expressed as percentage of Smad7-expressing cells in each group (mean ± SE) (*, P < 0.05). No positive markings were found on days 0 and 30. D, E, F and G Relative expressions of Tgfβ1, Smad7, Smad2 and Mmp9 mRNA were quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA, (n = 5 mice per group) compared KO group vs WT (*P < 0.05, **P < 0.01, ***P < 0.001) or each other (#, P < 0.05) (color figure online)

Fig. 4
Fig. 4

A Representative immunofluorescence analyses of CD69 + cells (green, white arrows) in the injured skeletal muscle at indicated days after injury (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) B Number of CD69 + cells in injured skeletal muscle. Data are expressed by the number of CD69-expressing cells in each group per High-power fields (HPF) (mean ± SE) (*, P < 0.05). C Representative immunofluorescence analyses of F4/80 (green, white arrows) in the injured skeletal muscle at indicated days after injury (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) D Number of F4/80 + cells in injured skeletal muscle. Data are expressed by the number of F4/80-expressing cells in each group per High-power fields (HPF) (mean ± SE) (*, P < 0.05). E Relative expression of Il-6 mRNA was increased on the 4th day after the injury only in the B2KO group, compared to the other groups. Relative expression of Il-6 mRNA was quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA (n = 5 mice per group) (*, P < 0.05) (color figure online)

Fig. 5
Fig. 5

A Representative immunofluorescence for endothelial cells (CD31 +) (red, arrow) in the injured skeletal muscle at indicated days after injury (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) B Relative expression of Vegfa mRNA was increased on the 4th day after the injury only in the B1KO group, compared to the other groups (#, P < 0.05.). Relative expression of Vegfa mRNA were quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA (n = 5 mice per group) (***, P < 0.001). C Representative immunofluorescence for αSMA (red) in the injured skeletal muscle at indicated days after injury (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) D Relative expression of Ncl mRNA was increased on the 4th day after the injury only in the B1KO group, compared to the other groups (#, P < 0.05.). Relative expression of Ncl mRNA was quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA (n = 5 mice per group) (***, P < 0.001). E Number of CD631 + capillaries per HPF in injured skeletal muscle. Data as expressed mean ± SE (n = 5 per group) (*, P < 0.05). F αSMA-positive vessel (< 50 m outside diameter) in injured skeletal muscle. Data as expressed mean ± SE (n = 5 per group) (*, P < 0.05) (color figure online)

Fig. 6
Fig. 6

A Representative immunofluorescence for Myod (red, arrowhead) in the injured skeletal muscle at indicated days after injury (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm.) B Relative expression of Myod1 mRNA was increased on the 4th day after the injury in the B1KO group as observed in WT group, compared to the other groups. Relative expression of Myod1 mRNA was quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA (n = 5 mice per group) (*P < 0.05, **P < 0.01, ***P < 0.001) or each other (#, P < 0.05) C Number of Myod + cells per HPF in injured skeletal muscle. Data as expressed mean ± SE (n = 5 per group) (*, P < 0.05). D Density of the regeneration marker MyHC-emb in injured skeletal muscle. Data as expressed mean ± SE (n = 5 per group) (*, P < 0.05). E Immunostaining for the regeneration marker MyHC-emb (red). There was no detectable expression of MyHC-emb in uninjured TA muscle of either genotype at 0 day (supplementary). By contrast, the expression of MyHC-emb was clearly evident on the 8-day-injured TA muscle sections (n = 5 per group). DAPI, 4’,6-diamidino-2-phenylindole stain (blue). (Scale bars = 100 μm) F Relative expression of Pax7 mRNA was increased on the 4th day after the injury in the B1KO group, compared to the other groups. Relative expression of Pax7 mRNA was quantified by RT-qPCR. Values (mean ± SD) normalized to 18 s rRNA (n = 5 mice per group) (*P < 0.05, **P < 0.01, ***P < 0.001) or each other (#, P < 0.05) (color figure online)

Fig. 7
Fig. 7

Relative expression of A Bdkrb2 (B2R) and B Bdkrb1 (B1R) mRNA in the injured skeletal muscle in the WT group. Relative expression of B2R mRNA for B1KO vs WT (C–F) and relative expression of B1R mRNA for B2KO vs WT (G–J). Relative expression of Bdkrb2 and Bdkrb1 mRNA were quantified by RT-qPCR. Values (mean ± SE) normalized to 18 s rRNA (n = 5 mice per group) (*, P < 0.05)

Fig. 8
Fig. 8

Treadmill running ability (A) and climbing strength/endurance test combined with incremental mass series (B) were tested in all groups in functional analyzes. (n = 10 mice per group) compared KO group vs WT (*P < 0.05) or each other (#, P < 0.05). Data presented as mean ± SD

Fig. 9
Fig. 9

Scheme of the main differential effects observed in knockout animals for kinin Bdkrb1 and Bdkrb2 receptors. CNF, centronucleated fibers

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