Figure 5.
Figure 5. Myogenic potential of Sgcb-null cardiac progenitors is abolished by miR669a overexpression in vivo. (A) H&E staining of ctx-injured TA muscle sections. (B) Sirius red staining of fibrotic area in muscle sections from ctx-treated TA muscles. (C) H&E-stained coronal sections of ctx-injured muscles sham-operated (left), transplanted with Sgcb-null cells (H4 KO/GFP; middle), and with Sgcb-null cells transduced with LV-CMV-EGFP-miR669a2× (H4 KO/GFP/miR669; right). (D–M) Immunofluorescence analysis on the ctx-treated sham-operated muscle (D, G–G′′, and K), ctx-treated muscle after a single intramuscular injection of 5 × 105 H4 KO/GFP cells (E, H–H′′, and L), and ctx-treated muscle after a single intramuscular injection of 5 × 105 H4 KO/GFP/miR669 (F, I–I′′, and M). (D–F) TA muscles were analyzed for the expression of laminin and GFP. GFP-positive cells were specifically localized and integrated with muscle fibers in H4 KO/GFP–injected muscles (E). On the contrary, GFP-positive cells are restricted to interstitial compartment in H4 KO/GFP/miR669-injected muscles (F). As expected, no GFP-positive cells were found in shame-operated muscles (D). Sgcb-null cardiac progenitors transplanted into ctx-damaged TA participate in muscle regeneration. Higher magnification GFP-positive fibers are reported in the inset. (G–I) Muscle sections from untreated (G) and treated (H and I) mice were stained with anti-MyHC and anti-GFP. GFP-positive fibers were differently fused with skeletal muscle fibers in H4 KO/GFP transplanted muscles (H) and shown at a higher magnification in H′ and H′′. MyHC/GFP double-positive fibers were never detected in H4 KO/GFP/miR669 transplanted muscles, in which the only GFP-positive cells were localized in the interstitial and vessel compartment as shown in I–I′′. MyHC/GFP double-positive fibers or GFP-positive cells were not found in the sham-operated muscles (G–G′′). (K–M) MyoD expression was analyzed in untreated (K) and treated (L and M) TA muscles. MyoD was strongly expressed in H4 KO/GFP transplanted muscle fibers (L) and extremely reduced in untreated (K) and in H4 KO/GFP/miR669 (M) transplanted muscle fibers. Arrows indicate MyoD-positive nuclei. (N) Cell counts of GFP/MyHC double-positive cells were performed on muscle and cardiac sections from transplanted mice. Six mice per each group of treatment were independently and statistically analyzed using Student’s t test (P < 0.05). Sk.Mu, skeletal muscle; Ven, ventricle. Error bars show means ± SEM. Bars: (A, B, and D–I) 50 µm; (G′–M) 100 µm.

Myogenic potential of Sgcb-null cardiac progenitors is abolished by miR669a overexpression in vivo. (A) H&E staining of ctx-injured TA muscle sections. (B) Sirius red staining of fibrotic area in muscle sections from ctx-treated TA muscles. (C) H&E-stained coronal sections of ctx-injured muscles sham-operated (left), transplanted with Sgcb-null cells (H4 KO/GFP; middle), and with Sgcb-null cells transduced with LV-CMV-EGFP-miR669a2× (H4 KO/GFP/miR669; right). (D–M) Immunofluorescence analysis on the ctx-treated sham-operated muscle (D, G–G′′, and K), ctx-treated muscle after a single intramuscular injection of 5 × 105 H4 KO/GFP cells (E, H–H′′, and L), and ctx-treated muscle after a single intramuscular injection of 5 × 105 H4 KO/GFP/miR669 (F, I–I′′, and M). (D–F) TA muscles were analyzed for the expression of laminin and GFP. GFP-positive cells were specifically localized and integrated with muscle fibers in H4 KO/GFP–injected muscles (E). On the contrary, GFP-positive cells are restricted to interstitial compartment in H4 KO/GFP/miR669-injected muscles (F). As expected, no GFP-positive cells were found in shame-operated muscles (D). Sgcb-null cardiac progenitors transplanted into ctx-damaged TA participate in muscle regeneration. Higher magnification GFP-positive fibers are reported in the inset. (G–I) Muscle sections from untreated (G) and treated (H and I) mice were stained with anti-MyHC and anti-GFP. GFP-positive fibers were differently fused with skeletal muscle fibers in H4 KO/GFP transplanted muscles (H) and shown at a higher magnification in H′ and H′′. MyHC/GFP double-positive fibers were never detected in H4 KO/GFP/miR669 transplanted muscles, in which the only GFP-positive cells were localized in the interstitial and vessel compartment as shown in I–I′′. MyHC/GFP double-positive fibers or GFP-positive cells were not found in the sham-operated muscles (G–G′′). (K–M) MyoD expression was analyzed in untreated (K) and treated (L and M) TA muscles. MyoD was strongly expressed in H4 KO/GFP transplanted muscle fibers (L) and extremely reduced in untreated (K) and in H4 KO/GFP/miR669 (M) transplanted muscle fibers. Arrows indicate MyoD-positive nuclei. (N) Cell counts of GFP/MyHC double-positive cells were performed on muscle and cardiac sections from transplanted mice. Six mice per each group of treatment were independently and statistically analyzed using Student’s t test (P < 0.05). Sk.Mu, skeletal muscle; Ven, ventricle. Error bars show means ± SEM. Bars: (A, B, and D–I) 50 µm; (G′–M) 100 µm.

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