Transplantation of MITO cells, mitochondria activated cardiac progenitor cells, to the ischemic myocardium of mouse enhances the therapeutic effect

Abstract

Given the potential for myocardial stem cell transplantation as a promising treatment for heart failure, numerous clinical trials have been conducted and its usefulness has been clearly confirmed. However, the low rate of engraftment of transplanted cells has become a clinical problem, and this needs to be improved in the case of transplanting cells to the heart. To address this issue, we report on attempts to prepare mitochondria-activated stem cells (MITO cells) for use in transplantation. MITO cells, which is cardiac progenitor cells (CPCs) activated by the mitochondrial delivery of resveratrol with an anti-oxidant and mitochondrial activation effects were successfully prepared using a mitochondrial targeting nanocarrier (MITO-Porter). The purpose of this study was to validate the therapeutic effect of cell transplantation by the MITO cells using a mouse model of myocardial ischemia-reperfusion. Mouse CPCs were used as transplanted cells. The transplantation of CPCs and MITO cells were conducted after myocardial ischemia-reperfusion, and the therapeutic effect was determined. The MITO cells transplanted group showed increase in postoperative weight gain, improve cardiac function and inhibition of fibrosis compared to the non-transplanted group and the CPC group. The transplantation of MITO cells to the ischemic myocardium showed a stronger transplantation effect compared to conventional CPC transplantation.

Figure 1

Experimental protocol for evaluating the therapeutic effect with respect to the ischemic cardiomyopathy mouse model after the transplantation of MITO cells. Evaluation of treatment of heart failure by transplanting cells to an ischemic lesion of ischemic cardiomyopathy mouse model (see the Movies 1, 2 for the operation).

Figure 2

Schematic image of mitochondrial activation in CPC by RP/S2-MITO-Porter. The MITO-Porter (S2 peptide & RP aptamer dual ligand system) could be taken up by the cells (1st step), followed by mitochondrial targeting and a membrane fusion process with mitochondria membrane (2nd step). Finally, mitochondria are activated by mitochondrial delivery of RES to construct MITO-cells. Chol-RP cholesteryl RP aptamer, DOPE dioleoyl-sn-glycero-3-phosphatidyl ethanolamine, RES resveratrol, SM sphingomyelin, STR-S2 stearylated S2 peptide.

Figure 3

Intracellular observation of MITO-Porter (RES). (A) Cellular uptake of RP/S2-MITO-Porter (RES), S2-MITO-Porter (RES) and DOPE/SM-LP (RES) labeled with NBD-lipids were evaluated by flow cytometry, using CPCs. The quantitative analysis of cellular uptake using the mean fluorescence intensity (MFI) of the carriers. Data are represented as the mean with S.D. (n = 3). The significant differences were calculated using one-way ANOVA followed by SNK test (**p < 0.01). (B) Intracellular observation of DOPE/SM-LP (RES) (a–c), S2-MITO-Porter (RES) (d–f) and RP/S2-MITO-Porter (RES) (g–i) using CLSM. When green labeled carriers (green color) were co-localized with red stained mitochondria, yellow signals were observed in the merged image. Lines indicate the edges of the cells. Scale bars 20 μm.

Figure 4

Evaluation of the mitochondrial function of MITO cells by measuring extracellular flux analyzer. The extracellular flux analyzer provides a continuous quantitative assessment of mitochondrial respiration in CPCs by measuring oxygen consumption. The Seahorse FXp was used to measure the mitochondrial oxygen consumption rate (OCR) of CPCs based on the protocol shown in (A)(a). Basic OCR, ATP-linked OCR, maximum OCR and non-mitochondrial OCR were measured. These parameters were estimated by the sequential addition of oligomycin (an inhibitor of ATP synthesis), carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone (FCCP) (a mitochondrial inner membrane decarboxylator that maximizes the mitochondrial electron transfer system), and rotenone and antimycin A (the complete inhibition of mitochondrial respiration) and the OCRs were measured (B). Significant differences were calculated using one-way ANOVA followed by the SNK test (**p < 0.01) (n = 3).

Figure 5

Evaluation of therapeutic effect after cell transplantation. (A) Body weight of mice after ischemic perfusion. This figure shows the change in body weight at 30 days after transplantation of each group (PBS treated group (n = 7), CPC group (n = 6), MITO-cell group (n = 6)) in an ischemia–reperfusion mouse model. Data are represented as the mean with S.D. (n = 6–7). *Significant differences were calculated by one-way ANOVA, followed by SNK test (p < 0.05). (B) Evaluation of cardiac function of ischemic myocardium. (a) Echocardiographic image in 30 days after cell transplantation in ischemia reperfusion model mice. (i) Sham operation mice, (ii) PBS treated, (iii) CPC transplanted and (iv) MITO cell transplanted ischemic reperfusion model mice. Scale bars 2 mm. (b) Comparison of fraction shortening among these groups. Fraction shortening (%) was estimated by two-dimension (see “Methods” section for the detail). Sham; Sham operation mice (n = 11), PBS; PBS treated (n = 5), CPC CPC transplanted (n = 6) and MITO cell MITO cell transplanted (n = 6) ischemic reperfusion model mice group. Data are represented as the mean ± S.D. Significant differences (*p < 0.05, **p < 0.01) were calculated by one-way ANOVA, followed by SNK test.

Figure 6

HE and Masson trichrome straining in ischemic myocardium after cell transplantation. HE staining (A) and Masson trichrome staining (B) in sections of ischemic myocardium 30 days after treatment in each group (PBS group (a), CPC group (b), MITO-cell group (c)). Left panels are magnified images of interest of region. Scale bar is 200 μm. (C) The photographic images shown in (B) were analyzed to determine the rate of fibrosis using Image-pro Plus 7.0. PBS, PBS treated; CPC, CPC transplanted; MITO cell, MITO cell transplanted. Data are represented as the mean ± S.D (n = 5). Significant differences (**p < 0.01) were calculated by one-way ANOVA, followed by the SNK test.

Figure 7

Detection of oxidative stress of ischemic myocardium after cell transplantation. (A) CLSM observation of oxidative stress of ischemic myocardium 3 days after treatment in each group using DHE (a red dye for reactive oxygen spices). (a) Sham group, (b) PBS group, (c) CPC transplantation group and (d) MITO-cell transplantation group. Scale bars, 200 μm. (B) Quantification of relative DHE positive region ratio was estimated using CLSM images. Data are represented as the mean ± S.D (sham group (n = 6), PBS group (n = 4), CPC transplantation group (n = 4) and MITO-cell transplantation group (n = 4)). Significant differences (*p < 0.05, **p < 0.01) were calculated by one-way ANOVA, followed by SNK test. Scale bar is 200 μm. Detection of mitochondrial membrane potential of ischemic myocardium after cell transplantation. (C) CLSM observation of mitochondrial membrane potentials of ischemic myocardium 3 days after treatment in each group using TMRM (a red dye for mitochondrial membrane potential). (a) Sham group, (b) PBS group, (c) CPC transplantation group and (d) MITO-cell transplantation group. Scale bars 200 μm. (D) Quantification of relative TMRM positive region ratio was estimated using CLSM images. Data are represented as the mean ± S.D (sham group (n = 6), PBS group (n = 5), CPC transplantation group (n = 5) and MITO-cell transplantation group (n = 5)). Significant differences (*p < 0.05, **p < 0.01) were calculated by one-way ANOVA, followed by SNK test.