Mitochondrial Coenzyme Q₁₀ Delivery Improves Energy Production in Rat Cardiac Myoblasts and Duchenne Muscular Dystrophy Model Rat Cardiomyocytes

Abstract

Enhancing cardiomyocyte mitochondrial function has been reported as a potential therapeutic approach for various diseases. However, this is technically difficult, and its practical use has not been described. Although treatments such as exon skipping for skeletal muscle have been established for Duchenne muscular dystrophy (DMD), no curative treatment has been developed for DMD cardiomyopathy, and only cardioprotective medications and symptomatic treatments are available. In this study, we attempted to activate cardiac myocyte mitochondria via direct drug delivery using lipid nanoparticles and investigated the application of this strategy to diseased cells. First, we delivered CoQ₁₀, a mitochondrial activator with cellular antioxidant capacity, into mitochondria in H9C2 cells using MITO-Porter, a mitochondria-directed nanoparticle. Cellular MITO-Porter uptake was measured using flow cytometry. Co-localization of mitochondria and MITO-Porter was confirmed using confocal laser microscopy. Mitochondrial respiratory capacity was measured using an extracellular flux analyzer. Furthermore, the concentration–response relationships of the amount of nanoparticles or CoQ₁₀ with mitochondrial energy production capacity were confirmed. Next, we examined the possibility of improving mitochondrial energy production capacity in diseased cells. Cells were isolated from the myocardium of DMD model rats generated using the CRISPR-Cas9 system. Mitochondrial energy production capacity was lower in DMD primary cardiomyocytes than in wild-type primary cardiomyocytes. CoQ₁₀ delivery to mitochondria in DMD primary cardiomyocytes using MITO-Porter improved mitochondrial energy production capacity. Thus, enhanced cardiomyocyte mitochondrial energy production might represent a potential treatment for cardiomyopathy.

INTRODUCTION

Cardiomyocytes have a higher number of mitochondria than all other human cell types. Ischemic cardiomyopathy is associated with decreased mitochondrial function and increased compensatory glycolytic energy production. Improving myocardial energy production capacity via mitochondrial activation has been described as a potential treatment strategy for cardiomyopathy.¹⁾ However, few studies have attempted to increase myocardial mitochondrial energy production capacity because of the difficulty of direct mitochondrial activation. The relationship between myocyte mitochondrial function and drug delivery, and the relationship between mitochondrial function and cardiac function are not well known.

Mitochondria-targeted therapy for cardiomyopathy might be useful for patients with secondary cardiomyopathy, such as those with Duchenne muscular dystrophy (DMD). Currently, patients with secondary cardiomyopathy are not candidates for heart transplantation. In recent years, mitochondrial dysfunction has been reported early in the onset of DMD-related cardiomyopathy, and improving myocardial mitochondrial function is expected to emerge as a treatment strategy for DMD-related cardiomyopathy.^2,3)

In our previous study, resveratrol was delivered to mitochondria using lipid nanoparticles to increase mitochondrial respiratory capacity in normal rat cardiomyocytes (H9C2 cells).⁴⁾ Previous studies have shown that naked CoQ₁₀ does not reach the mitochondria and it is difficult to improve mitochondrial function.⁵⁾ Furthermore, we have previously reported that substance delivery with mitochondria-undirected nanoparticles produced no activity in mitochondria.⁶⁾ Therefore, MITO-Porter, a mitochondria-directed nanoparticle, was used to ensure delivery of CoQ₁₀ into the mitochondria. No reports have described the use of the nanoparticle MITO-Porter⁷⁾ to target mitochondria in H9C2 cells. Our previous efforts in using H9C2 cells were unsuccessful. However, by using a microfluidic device, particles were decreased in size and made more homogeneous, and modification of some of the protocols for particle creation^8,9) permitted the use of MITO-Porter in H9C2 cells. In this study, we first examined the cellular uptake and intracellular dynamics of MITO-Porter at H9C2 cells. Next, MITO-Porter was used to deliver CoQ₁₀ to mitochondria in H9C2 cells, and changes in mitochondrial respiratory capacity were measured. The amounts of particles and CoQ₁₀ delivered were examined to determine to optimize the particle dosage and CoQ₁₀ delivery. To validate these results in diseased cells, we isolated primary cultured cells from the myocardium of DMD model rats with an excellent heart failure phenotype.¹⁰⁾ We evaluated mitochondrial energy production capacity in DMD cardiomyocytes and assessed the ability of CoQ₁₀ delivered using MITO-Porter to improve mitochondrial function. A schematic diagram of the study is presented in Fig. 1.

Fig. 1. Schematic of the Study Design

In this study, H9C2 cells, a cardiomyoblast line derived from rats, were first used. (a) The extent of the increase in OCR was examined by adjusting the concentration of MITO-Porter (CoQ₁₀) and the amount of CoQ₁₀ loaded into MITO-Porter. (b) Cells were isolated and cultured from dystrophin gene knockout (DMD) rat myocardium using the CRISPR/Cas9 system, and the mitochondrial energy-producing capacity of the cells was assessed. We confirmed that OCR, one of the indicators of mitochondrial respiratory capacity, was reduced even before the reduction in cardiac contractility occurred. The reduction in mitochondrial respiratory capacity was improved by delivering CoQ₁₀, a mitochondrial activator, to mitochondria using MITO-Porter, a mitochondria-targeted lipid nanoparticle.

MATERIALS AND METHODS

Animals

The DMD model rats used in this study (13 weeks old, male) were created by knocking out the dystrophin gene using the CRISPR/Cas9 system in the laboratory of Professor Yamanouchi in the Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo.¹⁰⁾ Rat cardiac muscle cells were isolated from the right vehicle for use in this study. The use of rats and genetically modified animals was approved by the Ethics of Pharmaceutical Science Animal Committee of Hokkaido University (Approval No.: 20-0176 and 2020-024). All experiments were performed in accordance with the National University Corporation Hokkaido University regulations on animal experimentation and the ARRIVE guidelines. The body weight and cardiac function of the rats are presented in Supplementary Fig. S1.

Preparation of CoQ₁₀-Loaded MITO-Porter [Mito-Porter (CoQ₁₀)]

MITO-Porter loaded with different amounts of CoQ₁₀ (1.5, 3, or 5 mM) was prepared by mixing 4.2 mM lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine : sphingomyelin:1,2-dimyristoyl-sn-glycerol : methoxy polyethylene glycol 2000 : stearylated R8 DOPE : SM : DMG-PEG2000 : STR-R8 molar ratio: 9 : 2 : 0.33 : 1.1) in ethanol and phosphate-buffered saline [PBS (–)] using the iLiNP microfluidic device.⁸⁾ The resulting suspension was collected after 2–3 h of dialysis in PBS (–) using a dialysis membrane (molecular weight cutoff, 12000–14000 Da; Spectrum Laboratories, Rancho Dominguez, CA, U.S.A.).

Cellular Uptake of MITO-Porter

H9C2 cells were seeded into six-well plates at 1.0 × 10⁵ cells/2 mL/well and cultured for 48 h at 37°C in a 5% CO₂ atmosphere before measurement. After washing the cells with PBS (–), medium containing MITO-Porter (CoQ₁₀) labeled with DiI (0.5 µM) was added. The same amount of particles was added each time. After two washes with heparin solution (20 U/mL), cells were trypsinized and collected in 1.5-mL microtubes. The collected solution was centrifuged (700 × g, 4°C, 3 min) and the pellet was suspended in 500 µL of FACS buffer. The cell suspensions were filtered and measured by flow cytometry (Beckman Coulter Inc., Brea, CA, U.S.A.).

Intracellular Dynamics by Confocal Laser-Scanning Microscopy (CLSM)

H9C2 cells were seeded in 3.5-cm glass dishes (IWAKI, Tokyo, Japan) at 2.0 × 10⁴ cells/3 mL/dish and incubated for 24 h (37°C, 5% CO₂). MITO-Porter (CoQ₁₀), empty MITO-Porter, or PBS (–) were added to each dish and incubated for 24 h. MitoTracker Deep Red (1.2 µL/dish) was added and incubated for 30 min before observation. After the medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM) F-12, each dish was observed by CLSM. Cells were excited with an LD laser at 473 and 635 nm. A Nikon A1 microscope (Nikon Instruments Inc., Tokyo, Japan) equipped with a water immersion objective (UPlanSApo 60 × /NA. 1.2) and a dichroic mirror (DM405/473/559/635) was used. Fluorescence detection channels (Ch) were set to the following filters: Ch1, 560/50 (green) DiI-labeled MITO-Porter; and Ch2, 660/50 (red) Mito-Tracker Deep Red.

Mitochondrial Respiratory Capacity Following CoQ₁₀ Delivery to Mitochondria

H9C2 cells or DMD primary cardiomyocytes were seeded into each well of Seahorse Mito Stress Kit plates (Agilent Technologies, Santa Clara, CA, U.S.A.) at a density of 1.0 × 10⁴ cells/well and incubated for 24 h. Then, 20 µL of MITO-Porter (CoQ₁₀) or PBS (–) and 80 µL of DMEM [fetal bovine serum (FBS) (–)] were added to each well and incubated for 1 h. Next, 100 µL of DMEM (20% FBS) were added to each well and incubated for 24 h. The nanoparticles were added at the same concentration. After the medium was replaced with running medium and incubated in a 37°C, CO₂-free incubator for 1 h, mitochondrial respiratory capacity was measured using an extracellular flux analyzer (Agilent Technologies). The mitochondrial oxygen consumption rate (OCR), a measure of energy production in mitochondria, was corrected to OCR/cell based on the cell count. All incubations not mentioned were performed at 37°C in 5% CO₂ incubator.

Other details are provided in the supplementary materials.

RESULTS

Physical Properties of MITO-Porter

The physical properties of MITO-Porter are listed in Table 1. The average particle size, polydispersity index (PDI), and ζ-potential of the 3 MITO-Porter (CoQ₁₀) constructs were approximately 60 nm, 0.25, and 15 mV, respectively. These results were similar to those reported previously.^5,8) No obvious difference in properties was observed according to the concentration of CoQ₁₀ loaded. The CoQ₁₀ recovery rate and CoQ₁₀ concentration were measured using HPLC. The CoQ₁₀ contents of MITO-Porter loaded with 1.5, 3.0, and 5.0 mM CoQ₁₀ were approximately 70, 140, and 260 µM, respectively, giving a recovery rate of approximately 65% for all three constructs (Table 1).

Table 1. Physicochemical Properties of the Nanoparticles

	CoQ10 concentration in lipid	Diameter (nm)	PDI	ζ-Potential (mV)	CoQ10 recovery rate (%)	CoQ10 concentration (µM)
MITO-Porter (CoQ10)	1.5 mM	53.6 ± 7.5	0.222 ± 0.06	14.1 ± 2.2	64.8 ± 1.97	66.4 ± 2.02
MITO-Porter (CoQ10)	3.0 mM	65.1 ± 25.2	0.243 ± 0.02	14.1 ± 0.7	69.9 ± 15.0	143.0 ± 30.6
MITO-Porter (CoQ10)	5.0 mM	56.9 ± 1.7	0.283 ± 0.05	16.0 ± 2.4	76.1 ± 7.73	257.0 ± 26.1

Data are presented as the mean ± standard deviation (S.D.) (N = 3). CoQ₁₀ recovery rate (%) = measured concentration/theoretical concentration ×100. PDI: polydispersity index.

Evaluation of the Cellular Uptake of MITO-Porter in H9C2 Cells

The uptake of MITO-Porter (CoQ₁₀) by H9C2 cells was evaluated by fluorescently labeling MITO-Porter with DiI. The level of uptake was evaluated at 1, 6, and 24 h after the addition of MITO-Porter. As presented in Fig. 2, MITO-Porter, with or without CoQ₁₀, exhibited stronger fluorescence intensity than the control (PBS) at 1 h after addition. This finding indicated that the incorporation of MITO-Porter into cells had already begun at 1 h after administration. A further increase in uptake was observed at 6 h after addition, but there was no difference in fluorescence intensity at 6 and 24 h after addition. These results indicate that most of the MITO-Porter attached to the cell surface is fully incorporated into the cell within approximately 6 h after addition, and it persists in the cells for 24 h.

Fig. 2. Evaluation of the Cellular Uptake of MITO-Porter

Fluorescence intensity was measured using flow cytometry at 1, 6, and 24 h after the administration of DiI-labeled MITO-Porter. Adequate cellular uptake of MITO-Porter (CoQ₁₀) and empty MITO-Porter was observed in H9C2 cells.

Observation of the Intracellular Dynamics of MITO-Porter in H9C2 Cells

To confirm the intracellular dynamics of MITO-Porter in H9C2 cells, we observed the intracellular localization of MITO-Porter fluorescently labeled with 0.5% DiI using CLSM. In H9C2 cells, MITO-Porter (green) co-localized with mitochondria (red). The results for the control group [PBS (–)] are presented in Figs. 3a–3c, those for the empty MITO-Porter group are presented in Figs. 3d–3f, and those for the MITO-Porter (CoQ₁₀) group are presented in Figs. 3g–3i. We have previously confirmed that MITO-Porter (CoQ₁₀) remains stable in culture medium for 24 h.⁵⁾ The results indicated that MITO-Porter co-localizes with mitochondria with or without CoQ₁₀.

Fig. 3. Intracellular Dynamics of MITO-Porter

Intracellular dynamics of MITO-Porter (CoQ₁₀) labeled with 0.5% DiI was observed by CLSM in H9C2 cells. (a–c) Images of the PBS (–) group. (d–f) Images of the empty MITO-Porter group. (g–i) Images of the MITO-Porter (CoQ₁₀) group. Mitochondria were stained with MitoTracker Deep Red (red) before intracellular observation. DiI-labeled particles co-localized with mitochondria, as indicated by yellow fluorescence (f and i). Bar: 20 µm.

Evaluation of the Mitochondrial Respiratory Capacity of H9C2 Cells Following CoQ₁₀ Delivery Using MITO-Porter

To determine the effect of MITO-Porter (CoQ₁₀) on the mitochondrial respiratory capacity of H9C2 cells and the relationship between the CoQ₁₀ delivery and mitochondrial respiratory capacity, we evaluated mitochondrial respiratory capacity using OCR. OCR measurements were performed at 24 h after administration. Basal respiratory capacity, which is the steady-state respiratory capacity of mitochondria, and maximal respiratory capacity, in which mitochondrial respiration is maximally activated using FCCP, were evaluated.

Evaluation of OCR Based on the Concentration of MITO-Porter (CoQ₁₀)

First, the contribution of MITO-Porter (CoQ₁₀) to the increase in OCR was evaluated. MITO-Porter (CoQ₁₀ 1.5 mM) was added to H9C2 cells at nanoparticle concentrations of 10, 20, and 40 µM, and OCR was measured. Concerning both basal and maximal respiratory capacity, OCR increased with increasing concentrations of MITO-Porter (CoQ₁₀) (Fig. 4a). The time course and measurement results are presented in Supplementary Fig. S2. These results suggest that OCR increases proportionally with the concentration of MITO-Porter for both basal and maximal respiration.

Fig. 4. Relationship between the Nanoparticle Concentration in H9C2 Cells and Mitochondrial Respiratory Capacity

(a) OCR was plotted after administration of 0, 10, 20, or 40 µM MITO-Porter (CoQ₁₀). Increases in basal and maximal respiratory capacity in proportion to the MITO-Porter (CoQ₁₀) concentration was observed. The concentration indicates the measured concentration of lipid components. OCR was normalized to the number of cells. (b) The maximal respiratory capacity (OCR) relative to the amount of CoQ₁₀ loaded in MITO-Porter was plotted. The change in OCR is presented relative to the findings in the control group (PBS-treated group). OCR increased with increases in the CoQ₁₀ content.

Evaluation of OCR Based on CoQ₁₀ Content in MITO-Porter

Next, we examined the contribution of CoQ₁₀ itself to OCR after excluding the effect of MITO-Porter particles on OCR. MITO-Porter loaded with different amounts of CoQ₁₀ (1.5, 3, and 5 mM) was prepared. OCR was measured after the addition of the particles. The physical properties of these particles are presented in Table 1. Both basal and maximal respiratory capacity were higher in the MITO-Porter (CoQ₁₀ 3.0 mM) and MITO-Porter (CoQ₁₀ 5.0 mM) groups than in the MITO-Porter (CoQ₁₀ 1.5 mM) group. The relationship between the amount of CoQ₁₀ delivered and maximal OCR is presented in Fig. 4b. The time course and measurement data are presented in Supplementary Fig. S3. These results suggest a proportional relationship between the amount of CoQ₁₀ delivered and the increase in OCR. Thus, increasing the amount of CoQ₁₀ loaded per nanoparticle increases OCR.

Evaluation of Energy-Producing Capacity in DMD Model Rat Cardiomyocytes

To examine the energy-producing capacity of DMD model rat cardiomyocytes, ATP production in DMD and wild-type primary cardiomyocytes was measured to assess glycolysis and oxidative phosphorylation. An extracellular flux analyzer (Seahorse XF, Agilent) was used for the measurements. The total ATP production in wild-type primary cardiomyocytes was approximately 140 pmol/min/1 × 10⁴ cells, whereas it was significantly lower in DMD primary cardiomyocytes (80 pmol/min/1 × 10⁴ cells). The results indicate that DMD primary cardiomyocytes can produce approximately 50% as much energy as wild-type primary cardiomyocytes (Fig. 5a). The extracellular acidification rate (ECAR), reflecting energy production in the glycolytic system, did not differ between wild-type and DMD cardiomyocytes (Fig. 5b). DMD primary cardiomyocytes displayed a significant decrease in OCR (Fig. 5c). These results suggest that the reduction in energy production in DMD primary cardiomyocytes is attributable to mitochondrial dysfunction.

Fig. 5. Evaluation of ATP Production in Wild-Type and DMD Rat Primary Cardiomyocytes

(a) Mitochondrial respiratory capacity was assessed in wild-type and DMD primary cardiomyocytes using the Seahorse XFp analyzer (p < 0.01). (b–c) Energy production rates were measured separately in the glycolytic system (b) and mitochondria (c) (p < 0.01). OCR and ECAR were normalized to the cell count. Significant OCR reduction was observed in DMD rat primary cardiomyocytes compared with the findings in wild-type rat primary cardiomyocytes. Data are presented as the mean ± S.D. (N = 4).

Effects of CoQ₁₀ Delivery to DMD Primary Cardiomyocytes Using MITO-Porter on OCR

As OCR was decreased in DMD primary cardiomyocytes (Fig. 5), we investigated the effect of CoQ₁₀ delivery to diseased cell mitochondria on OCR. We previously reported that diseased cells can accumulate MITO-Porter intracellularly and internalize it into mitochondria.¹¹⁾ Microscopic images of the intracellular dynamics of MITO-Porter in DMD primary cardiomyocytes are presented in Supplementary Fig. S4. The effect of CoQ₁₀ accumulation in DMD cardiomyocyte mitochondria on mitochondrial respiratory capacity was evaluated by measuring OCR at 24 h after MITO-Porter (CoQ₁₀) administration. OCR was measured in 2 phases, namely basal and maximum respiratory capacity (Fig. 6). Both basal (Fig. 6a) and maximal (Fig. 6b) respiratory capacity were increased in the CoQ₁₀ delivery group. These results suggest that CoQ₁₀ delivery using MITO-Porter can improve OCR in DMD primary cardiomyocytes. The time course and raw data of OCR for Fig. 6 are presented in Supplementary Figs. S5 and S6.

Fig. 6. Evaluation of the Mitochondrial Respiratory Capacity of DMD Primary Cardiomyocytes after the Administration of MITO-Porter (CoQ₁₀)

(a) Basal respiratory capacity. (b) Maximal respiratory capacity. OCR was expressed as the extent of the change in basal respiratory capacity relative to the control group (set at 100%). The OCR tended to increase in the MITO-Porter (CoQ₁₀) group compared with that in the control group. OCR was normalized to the cell count. Statistical analysis by t-test showed (A) basic respiratory capacity: p = 0.06, (B) maximal respiratory capacity: p < 0.01.

DISCUSSION

As described in the “Introduction”, few reports have described myocardial-specific drug delivery, and studies of delivery to organelles inside cardiomyocytes are even rarer. Although the need to functionally activate cardiac mitochondria has been discussed, it is well known that drug delivery to cardiac mitochondria is technically challenging.¹²⁾ In vivo, various strategies have been reported for drug delivery to myocardial mitochondria, such as targeting cell surface receptors expressed in damaged myocardium (e.g., angiotensin II type 1 receptor¹³⁾) and utilizing factors that accumulate in the myocardium (e.g., vascular endothelial growth factor¹⁴⁾). However, compared with the number of reports on cancer, there are considerably fewer reports of myocardial-specific drug delivery.¹⁵⁾ Although there are reports of treatment involving cardiac stem cell transplantation, the low delivery rate to the myocardium following stem cell transplantation therapy limits its use.^16,17) Myocardial-specific drug delivery holds great promise for practical application, but further research is necessary.¹⁸⁾

CoQ₁₀ is a cofactor for respiratory chain complexes I and II of the electron transfer system, and it is involved in ATP production in mitochondria. In this report, we demonstrated that CoQ₁₀ delivery to mitochondria in H9C2 cells increased the maximum mitochondrial respiratory capacity. In addition, increasing the amount of nanoparticles administered or the concentration of CoQ₁₀ loaded into MITO-Porter increased mitochondrial respiratory capacity. These results suggest that the increase in OCR is positively correlated with the amount of CoQ₁₀ delivered into the mitochondria (Fig. 4). In the lipid dose range used in this study, no issues were observed¹⁹⁾; however, as a general consideration, increasing the nanoparticle dose may raise concerns regarding lipid-induced cytotoxicity. This report has demonstrated that OCR increased by a dose-dependent CoQ₁₀. Therefore, so far, an approach that maintains a constant lipid amount of the MITO-Porter while increasing its CoQ₁₀ loading is considered to be a valuable and promising strategy.

The mechanism of CoQ₁₀-induced OCR increase in mitochondria was previously reported by metabolomic analysis.⁵⁾ In that previous report, activation of the electron transfer system, the main source of energy production in mitochondria and activation of the trichloroacetic acid cycle, the source of reduced nicotinamide adenine dinucleotide to the electron transfer system were observed. Furthermore, activation of the glutathione pathway, one of the antioxidant pathways was observed.⁵⁾ We considered that similar pathway activation occurred in the present results.

Reduced mitochondrial energy production in cardiomyocytes has been reported in many pathological conditions, including ischemic cardiomyopathy and chronic heart failure.¹⁾ In recent years, reduced mitochondrial energy production has been described from the early stages of DMD-related cardiomyopathy^2,3) and skeletal disfunction.¹¹⁾ Several strategies for improving cardiac targeting using nucleic acids and nanoparticles with enhanced myocardial selectivity through the use of targeting factors expressed only in damaged myocardium such as ischemia were previously reported,^13,14) but therapeutic strategies targeting intracellular organelles within myocardial cells have not been described.¹²⁾ Myocardial muscle is the most mitochondria-rich tissue in the human body because it requires constant energy to support its constant movement. Most of the energy required by the myocardium is supplied by mitochondria in myocardial cells. Activating mitochondria in cardiomyocytes, as performed in this study, represents an effective therapeutic strategy for many cardiac diseases and cardiomyopathies.

Conversely, in cardiomyopathy resulting from systemic diseases such as DMD, mitochondrial dysfunction is also expected to be systemic. In fact, decreased cardiac function, possibly attributable to decreased mitochondrial energy production capacity, has been reported.²⁰⁾ In cardiomyopathy caused by systemic diseases such as DMD-related cardiomyopathy, selectively activating mitochondria in target organs is an ongoing challenge. Because of the difficulty of directly approaching mitochondria, there are no therapeutic agents that selectively target cardiac mitochondria. We believe that direct mitochondrial therapy through drug delivery to myocardial mitochondria will facilitate the treatment of many myocardial diseases.

In conclusion, we investigated drug delivery to cardiomyocyte mitochondria using the lipid nanoparticle MITO-Porter. The amount of CoQ₁₀ delivered was related to OCR. Next, isolated and cultured cardiomyocyte mitochondria from DMD model rat myocardium exhibited decreased energy production capacity. We then delivered CoQ₁₀ to the mitochondria of DMD primary cardiomyocytes using the same method investigated in H9C2 cells and observed improvements in OCR. Cardiac muscle is rich in mitochondria. Improving energy production via direct drug delivery to cardiomyocyte mitochondria might represent a therapeutic strategy for diseases associated with reduced mitochondrial energy production, such as cardiomyopathy and heart failure.

Acknowledgments

This work was partially supported by a Grant-in-Aid for Scientific Research (A) (Grant No. 23H00541 to Y.Y.) provided by the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT), JST FOREST (Grant No. JPMJFR203X to Y.Y.) sanctioned by the Japan Science and Technology Agency (JST), and AMED under Grant No. JP25ym012684 (to Y.Y.). This work was supported by the CASIO SCIENCE PROMOTION FOUNDATION (J42-02) funds (to Y.Y.). We are grateful to Dr. Manabu Tokeshi and Dr. Masatoshi Maeki for providing the microfluidic device (iLiNP device). We thank Joe Barber Jr., PhD, for editing a draft of this manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Honka H, Solis-Herrera C, Triplitt C, Norton L, Butler J, DeFronzo RA. Therapeutic manipulation of myocardial metabolism: JACC State-of-the-Art review. J. Am. Coll. Cardiol., 77, 2022–2039 (2021).
• 2)  Kang C, Badr MA, Kyrychenko V, Eskelinen EL, Shirokova N. Deficit in PINK1/PARKIN-mediated mitochondrial autophagy at late stages of dystrophic cardiomyopathy. Cardiovasc. Res., 114, 90–102 (2018).
• 3)  Meyers TA, Townsend D. Cardiac pathophysiology and the future of cardiac therapies in duchenne muscular dystrophy. Int. J. Mol. Sci., 20, 4098 (2019).
• 4)  Tsujioka T, Sasaki D, Takeda A, Harashima H, Yamada Y. Resveratrol-encapsulated mitochondria-targeting liposome enhances mitochondrial respiratory capacity in myocardial cells. Int. J. Mol. Sci., 23, 112 (2021).
• 5)  Sato I, Hibino M, Takeda A, Harashima H, Yamada Y. Activation of mitochondrial oxygen consumption rate by delivering coenzyme Q₁₀ to mitochondria of rat skeletal muscle cell (L6). J. Pharm. Sci., 113, 1836–1843 (2024).
• 6)  Satrialdi, Munechika R, Biju V, Takano Y, Harashima H, Yamada Y. The optimization of cancer photodynamic therapy by utilization of a pi-extended porphyrin-type photosensitizer in combination with MITO-Porter. Chem. Commun., 56, 1145–1148 (2020).
• 7)  Yamada Y, Akita H, Kamiya H, Kogure K, Yamamoto T, Shinohara Y, Yamashita K, Kobayashi H, Kikuchi H, Harashima H. MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim. Biophys. Acta Biomembr., 1778, 423–432 (2008).
• 8)  Hibino M, Yamada Y, Fujishita N, Sato Y, Maeki M, Tokeshi M, Harashima H. The use of a microfluidic device to encapsulate a poorly water-soluble drug CoQ₁₀ in lipid nanoparticles and an attempt to regulate intracellular trafficking to reach mitochondria. J. Pharm. Sci., 108, 2668–2676 (2019).
• 9)  Hibino M, Filosi T, Carrion LL, Porcu E, Malhotra A, Lüdtke-Buzug K, Ibrahim SM, Yamada Y, Hirose M. An effective approach to modulate mitochondrial function in murine primary macrophages by a mitochondria-targeted nanocapsule, MITO-Porter. Biomed. Pharmacother., 186, 118019 (2025).
• 10)  Nakamura K, Fujii W, Tsuboi M, Tanihata J, Teramoto N, Takeuchi S, Naito K, Yamanouchi K, Nishihara M. Generation of muscular dystrophy model rats with a CRISPR/Cas system. Sci. Rep., 4, 5635 (2014).
• 11)  Sato I, Sasaki D, Abe J, Yamanouchi K, Takeda A, Yamada Y. Improvement of mitochondrial function in Duchenne muscular dystrophy skeletal muscle cells by coenzyme Q10 delivery to mitochondria. Biochem. Biophys. Res. Commun., 771, 152003 (2025).
• 12)  Yamada Y, Satrialdi, Hibino M, Sasaki D, Abe J, Harashima H. Power of mitochondrial drug delivery systems to produce innovative nanomedicines. Adv. Drug Deliv. Rev., 154-155, 187–209 (2020).
• 13)  Lomis N, Sarfaraz ZK, Alruwaih A, Westfall S, Shum-Tim D, Prakash S. Albumin nanoparticle formulation for heart-targeted drug delivery: in vivo assessment of congestive heart failure. Pharmaceuticals, 14, 697 (2021).
• 14)  Carlsson L, Clarke JC, Yen C, et al. Biocompatible, Purified VEGF-A mRNA improves cardiac function after intracardiac injection 1 week post-myocardial infarction in swine. Mol. Ther. Methods Clin. Dev., 9, 330–346 (2018).
• 15)  Sahoo S, Kariya T, Ishikawa K. Targeted delivery of therapeutic agents to the heart. Nat. Rev. Cardiol., 18, 389–399 (2021).
• 16)  Tang J, Su T, Huang K, et al. Targeted repair of heart injury by stem cells fused with platelet nanovesicles. Nat. Biomed. Eng., 2, 17–26 (2018).
• 17)  Cheng K, Shen D, Hensley MT, Middleton R, Sun B, Liu W, De Couto G, Marbán E. Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting. Nat. Commun., 5, 4880 (2014).
• 18)  Sato I, Shiraishi M, Norota K, Shiraki K, Yamada Y. Mitochondrial activation and therapeutics: innovations in cell- and organelle-specific medicine. Biol. Pharm. Bull., (2025), in press.
• 19)  Maruo Y, Shiraishi M, Hibino M, Abe J, Takeda A, Yamada Y. Activation of mitochondria in mesenchymal stem cells by mitochondrial delivery of coenzyme Q₁₀. Biol. Pharm. Bull., 47, 1415–1421 (2024).
• 20)  Fujikura Y, Kimura K, Yamanouchi K, Sugihara H, Hatakeyama M, Zhuang H, Abe T, Daimon M, Morita H, Komuro I, Oishi K. A medium-chain triglyceride containing ketogenic diet exacerbates cardiomyopathy in a CRISPR/Cas9 gene-edited rat model with Duchenne muscular dystrophy. Sci. Rep., 12, 11580 (2022).