Myogenic tissue nanotransfection improves muscle torque recovery following volumetric muscle loss

Abstract

This work rests on our non-viral tissue nanotransfection (TNT) platform to deliver MyoD (TNT_MyoD) to injured tissue in vivo. TNT_MyoD was performed on skin and successfully induced expression of myogenic factors. TNT_MyoD was then used as a therapy 7 days following volumetric muscle loss (VML) of rat tibialis anterior and rescued muscle function. TNT_MyoD is promising as VML intervention.

Fig. 1. TNT_MyoD transfects deep into the dermis and causes myogenic reprogramming.

a Image of TNT_2.0 device. b SEM images of TNT device showing needle array projections. Scale, 400 µm. c Schematic diagram showing TNT set up for skin. d Distribution of FAM-labeled plasmids immediately after TNT using various voltages on mouse dorsal skin. Scale bar = 40 µm. e Visualization of eGFP signal 24 h after TNT with the eGFP-MyoD plasmid, showing expression of eGFP in epidermis and dermis. No eGFP fluorescence was detected in skin transfected with mock plasmids. The white dashed line indicates the dermal-epidermal junction. Scale, 50 µm. f MyoD expression in skin 24 h and day 10 post-TNT (n = 7,6). g Immunofluorescence staining of MF20 (myosin heavy chain) at 10 days post-TNT in the dermis. The white dashed line indicates the dermal-epidermal junction. The sections were co-stained with DAPI. Scale bar = 20 µm, 5 µm (h) transcript abundance of myogenic genes compared to mock-transfected skin (n = 9). All data are expressed as mean ± SD. Data analyzed by Student’s t-test. Figure c was created with BioRender.com.

Fig. 2. TNT_MyoD improves torque recovery in volumetric muscle loss.

a Image of TNT being performed on tibialis anterior muscle 1 week after VML injury. b Gene expression of MyoD in VML injured muscle 24 h post-TNT (n = 4). c Image of rat undergoing muscle functional testing. d Maximum dorsiflexion torque produced by VML affected limb. e Dorsiflexion torque produced at different frequencies of direct tibialis anterior stimulation 4 weeks after VML(n = 7). f At 4 weeks after VML injury, dorsiflexion torque is produced after repeated electrical stimulation of the tibialis anterior muscle at 70 Hz. Stimulations occurred every 3 s (n = 7). Regression line equations for data between 60–120 stimulations: 7 = −0.0056*x + 2.387 (TNT_mock) and y = −0.0086*x + 3.2345 (TNT_MyoD). g Images of VML affected tibilais anterior and synergist extensor digitorium longus and their contralateral counterparts 4 weeks post-injury (3 weeks after TNT). h Quantification of muscle weights 4 weeks after VML (n = 7). i Immunohistochemistry of eMyHC and quantification per area of regenerating myofiber at the injury site post-TNT with either mock or MyoD plasmid. Scale, 500 µm (n = 5). j Immunohistochemistry of laminin and quantification per area of regenerating myofiber at the injury site post-TNT with either mock of MyoD. Scale, 100 µm (n = 7,6). Data are expressed as mean ± SD in b, h, and i. *p < 0.05. Data expressed as individual data points with mean trend lines in d and e. Data in b, i, and j were analyzed by Student’s t-test. Data in d, e, f, and h were analyzed by ANOVA.