Table 1.
List of primers and sequences.
Fig 1.
Myotrauma is resolved faster in Phd2-hypomorphic mice.
(A) H&E staining of skeletal muscle at indicated time points after myotrauma (6 h, 24 h, 72 h, 96 h und 168 h) and of sham-treated wild type (WT) and Phd2-hypomorphic (knockdown, KD) mice. Loss of muscle fiber architecture, haemorrhage and vacuole formation occur early after trauma and are followed by increased immune cell invasion and tissue reorganisation. Regenerating muscle cells are characterised by central nuclei and marked with asterisks (*). Shown are slides of 1 μm thickness (200x magnification; scale bar: 200 μm). Magnification image of 168h: regenerating (*) and non-regenerating (#) muscle cells are displayed exemplarily. Due to the magnification of central trauma areas, genotype differences may not be visible in these images (scale bar: 20μm). (B) Relative numbers of regenerating muscle cells (in % of all intact muscle cells) are significantly increased in KD animals compared to WT; n = 4–6. (C) Relative numbers of traumatic muscle cells (in % of all muscle cells) decrease faster in KD mice compared to WT; n = 4–6. * P < 0.05, *** P < 0.001. (D) Representative images of traumatized muscles in full section. Indicated are traumatic areas. Due to contusion artefacts, areas may appear separated. We observed no difference in acute trauma size between the genotypes indicating a standardized impact procedure. (scale bar: 500μm).
Fig 2.
Skeletal muscle repair is enhanced in Phd2-hypomorphic mice.
(A/C/E) Immunofluorescence staining of Pax7, MyoD and myogenin in skeletal muscle of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice (time periods after trauma application are indicated). Stained are Pax7 (red), nuclei (DAPI; blue), intrinsic fluorescence (green). Colours have been adjusted for visualization in a linear fashion. The myogenin signals in (E) are cellular signals, red-stained myofibers are artificial due to technical reasons. Shown are slides of 1 μm thickness (200x magnification; scale bar: 20 μm). (B/D/F) Pax7-, MyoD-, and myogenin-positive nuclei per 0.128 mm2 area of trauma were counted from the stained slides. Time courses of induction of Pax7, MyoD and myogenin reveal subsequent activation of the named factors of the myogenic program. Inductions are significantly higher in Phd2-hypomorphic mice. Mean ± SD; n 4–6; sham: n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 3.
HIF-1α protein stabilization in myotrauma is comparable between WT and KD.
(A) Immunohistochemical staining of HIF-1α in skeletal muscle of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice. Cell nuclei positive for HIF-1α at early time points are restricted to invaded immune cells. Over time there are increasing numbers of HIF-1α positive myonuclei. Shown are slides of 1 μm thickness (200x magnification; 600x magnification; scale bar: 200 μm). (B) Quantification of presented immunohistochemical staining (% of DAB-positive cells) of regenerating muscle cells. (D) Immunohistochemical staining of PHD2 in skeletal muscle of traumatic and sham treated wildtype (WT) and Phd2-hypomorphic (KD) mice. Seven days after myotrauma, Phd2-hypomorphic mice exhibited reduced numbers of positively stained myonuclei in representative regenerating areas compared to wild type mice (arrows). Shown are slides of 1 μm thickness (200x magnification; scale bar: 200 μm). (C) qPCR analysis (whole muscle tissue) of Phd2 Exon 1 (E1)—Exon 2 (E2) for knockdown control (E) analysis of DAB-positive cells in areas containing almost exclusively regenerating muscle cells revealed lower PHD2 staining; mean ± SD; n = 4–8; sham: n = 3. * P < 0.05.
Fig 4.
VEGF expression after myotrauma is enhanced in Phd2-hypomorphic mice.
(A) Immunohistochemical staining of VEGF of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice. Early after trauma, VEGF staining can be detected in areas of invaded immune cells. At all time points staining is much more intensive in KD mice compared to WT. Shown are slides of 1 μm thickness (200x magnification; scale bar: 200 μm). (B-D) qPCR analysis (whole muscle tissue) of key growth factors in myotrauma (B) hepatocyte growth factor, Hgf; (C) insulin-like growth factor, Igf1; (D) vascular-endothelial growth factor, Vegf. Hgf and Vegf expression are significantly induced in KD mice 6h after myotrauma. Significant induction of Igf1 expression in KD mice 24 h after trauma (mean ± SD; n = 4–6; sham: n = 3). * P < 0.05, *** P < 0.001.
Fig 5.
Chemokine/Cytokine induction in skeletal muscle trauma is higher in Phd2-hypomorphic mice.
qPCR analysis (whole muscle tissue) of selected chemokines and cytokines in myotrauma of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice. (A) stroma cell-derived factor 1, Sdf1; (B) CXC-motive chemokine receptor 4, Cxcr4; (C) C-C ligand 2, Ccl2; (D) chemokine receptor 2, Ccr2; (E) cyclooxygenase 2, Cox2). Sdf1 expression is significantly induced in KD mice at later time points (72 and 96 hours after myotrauma), Ccr2 expression is significantly induced in sham treated mice and 6 h and 96 h, whereas Cox2 expression is significantly induced in KD mice 6 h and 168 h after myotrauma (mean ± SD; n 4–6; sham: n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 6.
Macrophage recruitment to traumatic areas starts earlier in Phd2-hypomorphic mice.
(A) Immunohistochemical staining of F4/80 of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice. Shown are slides of 1 μm thickness (200x magnification; scale bar: 200 μm). 72 hours after trauma, macrophage invasion is significantly higher in KD animals (B). (C) Bone marrow derived macrophages of KD mice show a 50% reduction in Phd2 Exon 1 (E1)—Exon 2 (E2) in qPCR analysis (mean ± SD; n = 5). (D) Flow cytometric analysis of bone marrow derived macrophages of WT and KD cells reveals a significantly reduced expression of the surface marker CD206, which characterizes anti-inflammatory macrophages, in KD cells (mean ± SD; n = 5). (E) Gating strategy of F4/80+CD206+ bone marrow derived macrophages from WT and KD animals. Mean percentages (± SD; n = 5) of this population are indicated in the upper right corner * P < 0.05, *** P < 0.001.
Fig 7.
Genes of the glucose and lactate metabolism after trauma are enhanced in Phd2-hypomorphic mice.
qPCR analysis (whole muscle tissue) of selected factors of glucose and lactate metabolism in myotrauma of traumatic and sham treated wild type (WT) and Phd2-hypomorphic (KD) mice. (A) glucose transporter 3, Glut3; (B) phosphofructokinase muscle type, Pfkm; (C) monocarboxylate transporter 3; Mct3). Glut3 is significantly induced in SHAM KD mice and 168h after trauma. There is a significant induction of Pfkm 168h after trauma in WT mice while Pfkm expression is reduced in KD mice. We observed a significantly induced Mct3 expression in sham treated KD mice and 72h after myotrauma (mean ± SD; n = 4–6; sham: n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.