The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

Abstract

Background: Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT.

Method: Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury.

Results: Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1.

Conclusion: Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

Keywords: Angiogenesis; Li-SWT; Myoblasts; Skeletal muscle regeneration; Vascularization.

Fig. 1

Quantifications of cell death, total cell counts, BrdU incorporation in cell cultures and gene expressions of TGFB1, SMAD3, SMAD7, KI67, CYCLIN D1, P21, P53 and CASP3 during myoblast proliferation. a Viability testing by tryphan blue at 300500, 1000 and 1500 impulses (0.1 mJ/mm², 5hz) with 3 human cell cultures showed a small, but insignificant, increase in cell death after Li-SWT. b The myoblast proliferation was assessed in controls and after Li-SWT by detaching and counting the cells every day for 4 days. Variation between cell cultures was found, but no effect of Li-SWT was observed. c Proliferation was further investigated by BrdU incorporation followed by immunostainings, which revealed a 2% increase in proliferation on day 1 after Li-SWT. d Gene expression study during proliferation revealed that TGFB1 expression was significant lower at 12 h after Li-SWT while SMAD7 was higher expressed in Li-SWT compared to controls. P21 was significant different at 12 h, 24 h and 72 h while SMAD3, KI67, CYCKLIND1, P53 and CASP3 showed no difference between treatment groups.n = 3, * p < 0.05

Fig. 2

In vitro gene expression of PAX3, PAX7, MYF5, MYOD1, MYOG, MEF2A, HGF and MET during proliferation in control and shockwave treated myoblasts. MEF2A was significantly increased at 12 h after Li-SWT, while the expression of PAX3, PAX7, MYF5, MYOD1, MYOG, HGF and MET did not differ between treatment groups.n = 3, * p < 0.05

Fig. 3

The number of MYOG positive myofibers and the fraction of MYOG positive cells during myoblast differentiation after Li-SWT. Myoblasts were treated with shockwaves and cultured in differentiation medium and harvested 2, 4 and 6 days after Li-SWT. a We found no difference in the number of myofibers (>2 nuclei) in control and Li-SWT myoblasts, thus cells had maintained their capacity to differentiate after Li-SWT. b The fraction of MYOG positive nuclei was significantly lower in Li-SWT cultures on day 2 during differentiation. However on day 4 and 6, the MYOG fractions were not changed by Li-SWT. c Immunohistochemical stainings for MYOG on day 2 after Li-SWT.n = 3, * p < 0.05

Fig. 4

Quantifications of adherence of explants, outgrowth visualisations, cell adhesion and phalloidin staining of cells after shockwave treatment. a In explant cultures treated with 300 impulses of 0.1 mJ/mm² 46% of the treated explants adhered compared to 92% in controls (n = 48), however (b) the number of cells grown out from the explants and their expression of NCAM did not differ. c The experiment was then performed with human myoblasts isolated by enzyme dissociation. The cells were cultured on coverslips and harvested every 30 min the first 2 h; no difference in adherence was observed. (d) However, when the harvested coverslips were stained for F-actin with phalloidin, the shockwave treated cells displayed a much lower expression of F-actin, indicating a delayed actin assembly. The shown images are from cells harvested 90 min after culture.n = 3, * p < 0.05

Fig. 5

Expressions of Bax, Bcl2, Casp3, Il1a, Il1b, Il6, Ccl2, Ccr2 and Tnfα and CD45+ cells during muscle regeneration in control and shockwave treated mice. a Bax, Bcl2 and Casp3 were highly expressed in the early phase of regeneration, and significantly different in the Li-SWT group on day 2, while the apoptotic potential, Bax/Bcl2 seemed lower in the Li-SWT group on day 3. Similarly Il1a and Tnfα were significantly higher expressed in the early phase of regeneration. The expression of Il1b, Il6, Ccl2 and Ccr2 showed similar expression pattern with highest levels from day 2–5 with no difference between the control and Li-SWT group. b Quantifications of CD45+ cells showed increased lymphocyte infiltration on day 2 and 3, with the only significant change in the number of CD45+ cells on day 21 in the SWT group. c Immunohistochemical stainings for CD45 on day 3. n = 3 in gene expressions and n = 4 in quantifications, * p < 0.05

Fig. 6

In vivo gene expression of Pax7, Myf5, Myod1, Myog, Mstn, Myf6, Hgf, Met, Mef2c, Tgfb1 and protein expression of PAX7 and MYOG. a Pax7, Myf5 were significantly increased on day 2 in the Li-SWT group, while Met was significantly increased on day 3 in the Li-SWT group. No difference was found between treatment groups in the expression of Myod1, Myog, Myf6, Mstn, Hgf, Mef2c and Tgfb1. b Immunohistochemical staining for PAX7 was made for Li-SWT and controls and the expression on day 3 is shown. c The PAX7 stainings were quantified, and the highest number of satellite cells (PAX7+) was found as expected in regenerating areas of the muscle, however no difference was observed in the number of PAX7+ cells in Li-SWT compared to control. d The number of MYOG+ cells peaked on day 3 but no difference was observed between the Li-SWT and control group. MYOG stainings are shown for day 3. n = 3 in gene expressions and n = 4 in quantifications, * p < 0.05

Fig. 7

Expression of Angpt1, Angpt2, eNOS, iNOS, VEGFa, Pecam1, Kdr, CD34, Fgf2 and Fgfr1 and vessel density during muscle regeneration in control and Li-SWT mice. a Angpt2, eNOS and iNOS were high expressed on day 2 in Li-SWT, with eNOS being significantly higher expressed in the Li-SWT group. Angpt1, eNOS, iNOS, VEGFa and Pecam1 were significant up regulated on day 21 in the Li-SWT group. Thus Li-SWT induced an early and late angiogenic signal. No effect of Li-SWT was found in expression of Kdr, CD34, Fgf2, but Fgfr1 was significantly increased in the Li-SWT group on day 2. b and (c) Blood vessels were visualized by immunostainings for vWF and the vessel density (number of vessels/cm²) peaked on day 3 in regenerating areas before decreasing again. No effect of Li-SWT on vessel density was found in either regenerating- or normal muscle areas. c Immunostainings for vWF are shown for day 3 in Li-SWT and controls.n = 3 in gene expressions and n = 4 in quantifications, * p < 0.05