Elsevier

Journal of Controlled Release

Volume 269, 10 January 2018, Pages 177-188
Journal of Controlled Release

Cardiac progenitor cells activated by mitochondrial delivery of resveratrol enhance the survival of a doxorubicin-induced cardiomyopathy mouse model via the mitochondrial activation of a damaged myocardium

https://doi.org/10.1016/j.jconrel.2017.11.024Get rights and content

Abstract

It has been reported that transplanting native cells would lack efficiency without producing artificial cell-tissue, due to the exaggerated oxidative stress in doxorubicin-induced cardiomyopathy. We attempted to activate cardiac progenitor cells (CPCs) by delivering resveratrol to mitochondria using a mitochondrial drug delivery system (MITO-Porter system). We first evaluated the viability of H9c2 cells (a cardio myoblast cell line) after doxorubicin treatment, where H9c2 cells were co-cultured with or without the mitochondria activated CPCs (referred to herein as MITO cell). We next evaluated the survival rate of doxorubicin treated mice, with or without the injection of MITO cells into the myocardium. Finally, we examined the molecular mechanism of the cell therapy by detecting oxidative stress and the induction of apoptosis in addition to quantification of the mRNA and protein levels about oxidative phosphorylation (OXPHOS). The MITO cell transplanted mice lived significantly longer than the conventional CPC transplanted ones. Oxidative stress and massive cell death were both significantly reduced in the MITO cell transplanted hearts, in which the expression levels of OXPHOS protein and gene were also higher than the control group. In doxorubicin-induced cardiomyopathy, the transplantation of MITO cells, which possess activated mitochondria, is more efficient compared to conventional CPC transplantation.

Introduction

Acramone reported that doxorubicin is a novel cytotoxic drug in 1967 [1] and it is currently one of the most widely used anticancer drugs in the world. Twelve years after this discovery, Von Hoff et al., in a clinical study, reported on the myocardial toxicity of doxorubicin. Since this report, numerous attempts have been made to reduce the myocardial toxicity of doxorubicin. One possibility for achieving this would be to limit the dosage of doxorubicin, to include an alternative less toxic drug such as a liposomal drug, and apply a combination therapy with dexrazoxan [2]. Unfortunately, such a strategy would have significant clinical limitations, from the viewpoint of its effectiveness or cost-benefit ratio, and it has been recently reported that many cancer survivors are now at risk of developing residual cardiovascular damage [3]. There are two major mechanisms responsible for doxorubicin-induced cardiomyopathy. The first group is from oxidative phosphorylation (OXPHOS), mitochondrial biogenesis, and antioxidant-related DNA damage via the inhibition of cardiac topoisomerase β [4], and the second involves the accumulation of iron in mitochondria as the result of injury to the mitochondrial membrane [5]. The final result is the development of a pathophysiological state with exaggerated oxidative stress in the myocardium as the result of mitochondrial injury [6].

Cardiac progenitor cells (CPCs) have cardiac stemness [7] and are clinically currently used in cardiac regenerative therapies [8], [9]. It has been reported that cardiac stem cell therapies hold some promise for dealing with doxorubicin-induced cardiomyopathy [10]. They have also been reported to have some advantages in, not only the ischemic myocardium, but also in doxorubicin related cardiomyopathy [10] and to confer resistance against oxidative stress in the ischemic heart [11]. However transplanting native cells would not be efficient, without the production of artificial cell-tissue due to the exaggerated oxidative stress that develops in the host-myocardium [11].

Resveratrol, originally characterized by Takaoka in 1930, has a stilbene structure, and has been reported to prolong the longevity of nematodes or obese mice due to its ability to improve lipid-metabolism [12]. Numerous mechanistic studies have been reported: activation of the nicotinamide adenine dinucleotide (NAD+)- sirtuine1 (Sirt1)- peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) axis [13] and the AMP-activated protein kinase (AMPK) pathway [13], the role of an mitochondrial electron transport agonist [14] or antagonist [15], and the inhibition of mitochondrial phosphodiesterase [16]. While many of these reports support the conclusion that resveratrol improves mitochondrial biogenesis, a significant efficacy has never been demonstrated in clinical trials [17]. Naked resveratrol might be not efficiently delivered into mitochondria of a cell in the absence of a drug delivery system (DDS).

We recently reported that some mitochondrial functional molecules, such as bongkrekic acid [18], gentamicin [19] or coenzyme Q10[20], have excellent pharmaceutical effects in vitro and in vivo, after being encapsulated in a MITO-Porter, which is a liposomal DDS for mitochondrial delivery. In this study, we hypothesized that delivering resveratrol into mitochondria in stem-cells for cell therapy could strengthen mitochondrial biogenesis and confer resistance to oxidative stress after cell transplantation.

We first prepared a RES–MITO-Porter, in which resveratrol was dispersed in the lipid phase of a MITO-Porter, and examined the physicochemical properties of the preparation. We next added the RES-MITO-Porter into CPCs, and found that the RES-MITO-Porter caused the mitochondrial enzymatic function to be activated and membrane potential to be elevated, as the result of the delivery of resveratrol. We refer to the mitochondria activated CPC as a MITO cell, and validated its utility for transplantation in an attempt to treat doxorubicin-induced cardiomyopathy using in vitro and in vivo doxorubicin-treated models. In this study, we used a doxorubicin-induced cardiomyopathy mouse model, that had previously been reported by S. Zhang et al. [4]. This model is a severely toxic model. Because of this, we were not able to transplant MITO cells to a heart damaged by doxorubicin, because all of the model mouse died immediately after the transplantation operation. To circumvent this problem, we transplanted MITO cells to the heart first and then damaged the myocardium by the administration of doxorubicin, and then evaluated the therapeutic effects. Finally, we analyzed the in vivo molecular mechanism of the cell therapy by evaluating the extent of oxidative stress and the induction of apoptosis in the damaged myocardium. We also quantified the mRNA levels of OXPHOS and mitochondrial biogenesis related genes in the heart. A schematic showing the experimental flow of this study is shown in Fig. 1.

Section snippets

2.1 materials

Resveratrol (RES) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 1,2-Dioleoyl-sn-glycero-3-phosphatidyl ethanolamine (DOPE) and DOPE-N-(7-nitro-2-1,3-benzoxadiazole-4-yl) (NBD-DOPE) were purchased from Avanti Polar Lipids (Alabaster, Alaska). Sphingomyelin (SM) was purchased from Sigma (St. Louis, Missouri). STR-S2 (stearyl-Dmt-d-Arg-FK-Dmt-d-Arg-FK-NH2) was obtained from the Toray Research Center, Inc. (Tokyo, Japan). The H9c2 rat embryonic cardiac myoblast cell line

Experimental design

A schematic of the experimental is shown in Fig. 1. We first obtained primary cultures of CPCs from two male mice (C57BL6, 8–10 weeks of age) (Fig. 1A). The procedure for this preparation of CPCs involved collecting of the heart from mice, treatment of the heart tissue with collagenase, separating cardiac myocytes to crude CPCs and purification of the CPCs using MACS for Sca-1, a stem cell antigen, as shown in Fig. 1A. The Sca-1 positive CPC lineage was similar to the reported one (Fig. S1) [21]

Discussion

CPC transplantation methods using naked CPCs [21], CPCs with genetic recombination [11], or CPCs treated with chemical agents without DDS have been reported [26]. In this study, we prepared MITO cells, in which CPC is activated by the mitochondrial delivery of resveratrol using mitochondrial DDS, a RES-MITO-Porter, and then transplanted MITO cells in an attempt to treat doxorubicin induced-cardiomyopathy. The RES-MITO-Porter contains the S2-peptide, which permits cellular uptake and

Conclusion

Using MITO-Porter system, we call CPC activated via mitochondrial delivery of resveratrol as “MITO cell”. We confirmed that the MITO cell transplantation could improve survival via mitochondrial biogenesis and OXPHOS compared with the conventional CPC transplantation in doxorubicin-induced cardiomyopathy mouse model. This provides a demonstration that MITO-Porter system represents a potentially carrier for use in cardiac stem-cell therapy.

Acknowledgements

This work was supported, in part by, a Grant-in-Aid for Scientific Research (B) (grant 26282131 to Y.Y.) and a Grant-in-Aid for Challenging Exploratory Research (grant 25560219 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), the Mochida Memorial Foundation for Medical and Pharmaceutical Research. We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.

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