Forskolin treatment enhances muscle regeneration and shows therapeutic potential with limitations in Duchenne muscular dystrophy

Abstract

Background: Duchenne Muscular Dystrophy (DMD) is a progressive neuromuscular disorder characterized by impaired muscle repair. Forskolin (FSK), an adenylyl cyclase activator, has shown potential in enhancing muscle regeneration and limiting muscle stem cell senescence. This study aimed to evaluate the effects of FSK on muscle repair, fibrosis, inflammation, and long-term muscle function in DMD using a preclinical rat model.

Methods: BaCl₂-induced muscle injury was performed on 6-month-old DMD (R-DMDdel52) and wild-type (WT) rats. FSK was supplied via short-term and long-term administration. Muscle tissues were harvested 14 days post-injury for histological analysis, including hematoxylin and eosin and Sirius red staining. Immunofluorescence was used to assess fibroadipogenic progenitors (FAPs), regeneration, muscle stem cells, and macrophage phenotypes. Moreover, we performed a study by chronically administering FSK to DMD rats from 1 to 7 months of age, either intraperitoneally (IP) or subcutaneously (SC). Functional assessments included grip strength test, in vivo muscle force measurements, plethysmography and electrocardiograms. Post-sacrifice, Tibialis anterior, diaphragm and heart tissues were histologically analyzed, to evaluate muscle architecture, fibrosis, and histopathological indices.

Results: FSK treatment significantly improved muscle histology and reduced fibrosis in both uninjured and injured DMD muscles by decreasing the number of FAPs. Long-term FSK treatment in the acute injury model enhanced muscle regeneration, increased MuSC proliferation, and reduced senescence. FSK also modulated inflammation by reducing pro-inflammatory macrophages and promoting a shift to a restorative phenotype. However, despite these histological improvements, FSK treatment from 1 to 7 months resulted in limited functional benefits and worsened ventricular histology in the heart.

Conclusions: FSK shows promising results in improving muscle regeneration and reducing fibrosis in DMD, but concerns remain regarding its limited chronic functional benefits and potential adverse effects on cardiac tissue. Our results highlight the need for optimized adenylyl cyclase activators for therapeutic use in DMD patients.

Keywords: Cellular senescence; Duchenne muscular dystrophy; Fibrosis; Forskolin; Muscle regeneration; Muscle stem cells.

Fig. 1

Morphological analysis of uninjured and injured muscles upon FSK treatments. A Scheme of FSK administration on DMD rats for a short (4-day) or long (12-day) treatment starting at 2 days post-injury (DPI). FSK was daily administered by IP injection at 25 mg/kg. B, C H&E (B) and SR (SR) on TA harvested at 14 DPI from WT, DMD, DMD treated with FSK short term (FSK S-T) or long term (FSK L–T). Scale bar 20 µm. D Quantification of SR positive area. E Quantification of the number of PDGFRa + cells per mm². F–H Quantifications of FAP proliferation (F), Col I-Col III (G) and αSMA (H) positive area

Fig. 2

Assessment of muscle regeneration and MuSC senescence in injured and control muscles upon FSK treatments. A Immunofluorescence for Laminin (red) and eMHC (green) on TA harvested at 14 DPI from WT, DMD, DMD treated with FSK short term (FSK S-T) or long term (FSK L–T). Scale bar 20 µm. B Quantification of the number of eMHC + fibers per mm². C Immunofluorescence for KI67 (red), PAX7 (green) and DAPI on TA harvested at 14 DPI from WT, DMD, DMD treated with FSK short term (FSK S-T) or long term (FSK L–T). Scale bar 25 µm. D Quantification of the number of PAX7 + cells per mm². E Quantification of the percentage of PAX7 + : KI67 + cells. F) Quantification of the percentage of PAX7 + : γH2AX + cells

Fig. 3

Evaluation of macrophages infiltration in injured and control muscles upon FSK treatment. A Immunofluorescence for CD68 (red) and CD206 (green) on TA harvested at 14 DPI from WT, DMD, DMD treated with FSK short term (FSK S-T) or long term (FSK L–T). Scale bar 50 µm. B Quantification of the number of CD68 + cells per mm². C Quantification of the percentage of CD68 + : CD206- and CD68 + : CD206 + cells. D, E qPCR analysis of Tnfα (D) and Il10 (E) on BMDM from both WT and DMD rats upon treatment with FSK

Fig. 4

Physiological and functional outcomes following chronic FSK treatment. A Scheme of rat injection plan. Forskolin (FSK) was weekly administrated for two months (25 mg/kg) followed by 2 months of wash-out up to 5 months of age. The rats were then injected for another month with FSK (2,5 mg/kg) up to 6 months and then sacrificed at 7 months. FSK was delivered by intraperitoneal (IP) or subcutaneous (SC) injection. B Body weight. C TA, heart and EDL weights normalized on the cubic tibial length at the end of the treatment. D Maximal force normalized on the cubic tibial length by grip test. E Normalized Maximal torque force. F) Normalized Maximal torque force as function of muscle stimulation frequency

Fig. 5

Histopathological evaluation of TA muscles. A H&E (upper panels) and SR (lower panels) on TA from WT, DMD vehicle and DMD treated with FSK by IP or SC administration. Scale bar 50 μm. B Quantification of the histopathological index. C Quantification of fibrosis and SR positive area. D Percentage of centrally nucleated fibers

Fig. 6

Histopathological evaluation of Diaphragms. A H&E (upper panels) and SR (lower panels) on diaphragms from WT, DMD vehicle and DMD treated with FSK by IP or SC administration. Scale bar 50 μm. B Quantification of the histopathological index. C Quantification of fibrosis and SR positive area. D Plethysmography analysis of tidal volume (TV, D) and end expiratory pause (EEP, E)

Fig. 7

Histopathological evaluation of left and right ventricles. A H&E on heart sections from WT, DMD vehicle and DMD treated with FSK by IP or SC administration. Scale bar 600 μm. B SR staining on heart sections from WT, DMD vehicle and DMD treated with FSK by IP or SC administration. Scale bar 600 μm. C Quantification of the histopathological index. D Quantification of fibrosis and SR positive area. E ECG evaluation of QTpc value that corresponds to the interval from the Q wave to the peak of the T wave (QTp) corrected with the Bazette’s formula normalized to the average rat RR (QTpc = QTp(eak) / (RR / f)1/2, f = 150 ms)