Lentivirus-mediated Wnt11 gene transfer enhances Cardiomyogenic differentiation of skeletal muscle-derived stem cells

Abstract

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Figure 1

Wnt11 expression in sorted mouse muscle-derived stem cells (MDSCs). (a) Schematic illustration of feline leukemia virus (FLV)-based lentiviral vectors used in our experiments. The vectors contain human cytomegalovirus (CMV) immediate-early enhancer/promoter, enhanced green fluorescent protein (eGFP) as an expression reporter and neomycin (Neo) as a selection marker, and Wnt11 gene was inserted into the downstream of eGEP gene. (b) Immunofluorescent (IF) staining of Wnt11 revealed a significant increase in the expression of Wnt11 in MDSCs transduced with Lv-Wnt11 at 2 weeks after infection, whereas low levels of Wn11 expression was also detected in the control cells (magnification: ×200). (c) Semiquantitative RT-PCR analysis showed that Lv-Wnt11 transduction increased the expression of Wnt11 mRNA in MDSCs 2 weeks after infection. Fifty nanogram of RNA extracted from MDSCs by Trizol reagent was used as the template for reverse transcription (RT)-PCR.

Figure 2

Morphology of lentivirus-transduced and green fluorescent protein (GFP)-sorted mouse muscle-derived stem cells (MDSCs). High GFP expression (>90%) was shown in the GFP-sorted mouse MDSCs transduced with the Lv-GFP control- or Lv-Wnt11 groups (upper panel). The transduction of lentivirus carrying Wnt11cDNA enables the MDSCs to undergo morphological changes including an increase in size and elongation 20 days after transduction, whereas the control cells remained small and round (magnification: ×200).

Figure 3

Wnt11 promotes the early cardiac differentiation of sorted mouse muscle-derived stem cell (MDSCs). (a) Immunofluorescent (IF) staining for cardiac-specific markers showed that the expression of Wnt11 in sorted MDSCs led to an increase in the expression of the early cardiac marker NK2 transcription factor related 5 (Nkx2.5) and GATA-binding protein 4 (GATA4) and the gap junction protein Connexin43 (Cx43) and promoted cardiac differentiation of the MDSCs as revealed by the morphological changes of cells (magnification: ×400). (b) Percentages of Nkx2.5, GATA4, and Cx43 positive cells in MDSCs 3 weeks following transduction with Lv-Wnt11 or control Lv-GFP lentiviruses (*P < 0.05 versus control). (c,d) The semiquantitative analysis of Nkx2.5 and Cx43 by reverse transcription (RT)-PCR and western blot analysis also showed significantly increased expression of Nkx2.5 and Cx43 in Lv-Wnt11-transduced MDSCs at the level of mRNA and protein when compared to the control Lv-GFP transduced cells (50 ng of RNA was used for RT-PCR and about 10 µg of protein was loaded for western blot analysis). The results are a representative of three independent experiments.

Figure 4

Wnt11 enhances the expression of late-stage cardiac markers in sorted mouse muscle-derived stem cells (MDSCs). (a) The semiquantitative analysis of α, β-myosin heavy chain (MHC) and brain natriuretic protein (BNP) mRNAs by reverse transcription (RT)-PCR revealed a significant increase in expression in the Lv-Wnt11-transduced MDSCs compared to the control Lv-green fluorescent protein (GFP)-transduced cells. The results are a representative of three independent experiments. (b) Relative mRNA expression of late cardiac marker genes by quantitative real-time PCR analysis using SYBR detect system. mRNA expression of α, β-myosin MHC and BNP was remarkably increased in Lv-Wnt11-transduced MDSCs compared to Lv-GFP transduced MDSCs(*P < 0.01). Data are mean ±SEM of three independent experiments. (c) Immunofluorescent (IF) staining for the cardiac-specific markers showed that the expression of Wnt11 in the sorted MDSCs led to an increase in the expression of late-stage cardiac markers such as MHC and Troponin I (TnI) and promoted cardiac differentiation of the MDSCs (magnification: ×100). (d) Troponin I positive cells displayed three types of cellular shapes including: spindle, triangular, and round (×200). (e) Percentages of TnI and MHC positive cells were quantified (*P < 0.01), respectively.

Figure 5

Wnt11 expression increases c-Jun-terminal kinase (JNK) activity in muscle-derived stem cells (MDSCs). (a) The expression levels of JNK1 and phosphorylated JNK (Thr183 /Tyr185) in Lv-GFP- or Lv-Wnt11-transduced MDSCs were detected by immunoblotting and increased JNK activity was evidenced by higher levels of phosphorylated JNK after Wnt11 expression. (b) Densitometric analyses of JNK and phosphorylated-JNK signals normalized to actin and expressed as the percentage of the actin control, showing relative JNK activities from three independent experiments. Error bars represent mean ± SEM (*P < 0.01).

Figure 6

Wnt11 enhances the survival and cardiac differentiation of mouse muscle-derived stem cells (MDSCs) in vivo. (a) Troponin I (TnI) staining (red) of frozen sections from remote area (left) and infarct border area (right) of myocardium injected with Lv-Wnt11 or Lv-green fluorescent protein (GFP) transduced MDSCs, 3 weeks after acute myocardial infarction and cell transplantation (magnification: ×100). Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue). (b) Representative double immunofluorescent staining for GFP (green) and TnI (red) inside the infarct area (magnification: ×100). More cells inside infracted hearts injected with Lv-Wnt11-transduced cells survived than the hearts injected with Lv-GFP transduced cells (as shown by GFP staining). Markedly increased cardiomyocytic differentiation occurred in the Lv-Wnt11-transduced cell group as revealed by GFP and TnI colocalization. (c) A greater magnification of the colocalization of GFP and TnI from a frozen section of a heart injected with Lv-Wnt11transduced cells (magnification: ×400), indicating in vivo cardiomyocyte differentiation.