Encapsulated mitochondria to reprogram the metabolism of M2-type macrophages for anti-tumor therapy.

M2-type macrophages (M2Φ) play a pro-tumorigenic role and are closely associated with tumor development, where metabolic dysregulation exacerbates the immunosuppressive tumor microenvironment and fosters tumor growth. Mitochondria serve as the regulatory center of cellular metabolism, yet effective methods to modulate M2Φ mitochondria within the tumor microenvironment remain lacking. In this study, we developed a technique utilizing the bio-encapsulation of mitochondria in Zeolitic Imidazolate Framework-8 (ZiF-8), referred to as Mito@ZiF-8. Our findings demonstrated that this coating protects intact mitochondria and preserves their bioactivity over an extended period after isolation. We successfully delivered Mito@ZiF-8 into M2Φ, which inhibited the secretion of pro-inflammatory factors, promoted the release of anti-inflammatory factors, and reprogrammed M2Φ metabolism. This innovative approach has the potential to reduce breast cancer cell metastasis and enhance sensitivity to chemotherapy drugs such as 6-thioguanine, cisplatin, and doxorubicin (Dox). Mito@ZiF-8 aims to reprogram the M2Φ microenvironment to support anti-tumor therapies, offering a novel strategy for improving the effectiveness of breast cancer treatment.

Mitochondrial Raf1 Regulates Glutamine Catabolism.

Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.

Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma.

Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

Metabolic reprogramming by histone deacetylase inhibition preferentially targets NRF2-activated tumors.

The interplay between metabolism and chromatin signaling is implicated in cancer progression. However, whether and how metabolic reprogramming in tumors generates chromatin vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor aberrant activation of the NRF2 antioxidant pathway, which drives aggressive and chemo-resistant disease. Using a chromatin-focused CRISPR screen, we report that NRF2 activation sensitizes LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDACs). This association is observed across cultured cells, mouse models, and patient-derived xenografts. Integrative epigenomic, transcriptomic, and metabolomic analysis demonstrates that HDAC inhibition causes widespread redistribution of H4ac and its reader protein, which transcriptionally downregulates metabolic enzymes. This results in reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest NRF2 activation as a potential biomarker for effective repurposing of HDAC inhibitors to treat solid tumors.

Epigenetic priming targets tumor heterogeneity to shift transcriptomic phenotype of pancreatic ductal adenocarcinoma towards a Vitamin D susceptible state.

As a highly heterogeneous tumor, pancreatic ductal adenocarcinoma (PDAC) exhibits non-uniform responses to therapies across subtypes. Overcoming therapeutic resistance stemming from this heterogeneity remains a significant challenge. Here, we report that Vitamin D-resistant PDAC cells hijacked Vitamin D signaling to promote tumor progression, whereas epigenetic priming with glyceryl triacetate (GTA) and 5-Aza-2'-deoxycytidine (5-Aza) overcame Vitamin D resistance and shifted the transcriptomic phenotype of PDAC toward a Vitamin D-susceptible state. Increasing overall H3K27 acetylation with GTA and reducing overall DNA methylation with 5-Aza not only elevated the Vitamin D receptor (VDR) expression but also reprogrammed the Vitamin D-responsive genes. Consequently, Vitamin D inhibited cell viability and migration in the epigenetically primed PDAC cells by activating genes involved in apoptosis as well as genes involved in negative regulation of cell proliferation and migration, while the opposite effect of Vitamin D was observed in unprimed cells. Studies in genetically engineered mouse PDAC cells further validated the effects of epigenetic priming for enhancing the anti-tumor activity of Vitamin D. Using gain- and loss-of-function experiments, we further demonstrated that VDR expression was necessary but not sufficient for activating the favorable transcriptomic phenotype in respond to Vitamin D treatment in PDAC, highlighting that both the VDR and Vitamin D-responsive genes were prerequisites for Vitamin D response. These data reveal a previously undefined mechanism in which epigenetic state orchestrates the expression of both VDR and Vitamin D-responsive genes and determines the therapeutic response to Vitamin D in PDAC.

Serine starvation silences estrogen receptor signaling through histone hypoacetylation.

Loss of estrogen receptor (ER) pathway activity promotes breast cancer progression, yet how this occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in breast cancer, represses estrogen receptor alpha (ERα) signaling by reprogramming glucose metabolism and epigenetics. Using isotope tracing and time-resolved metabolomic analyses, we demonstrate that serine is required to maintain glucose flux through glycolysis and the TCA cycle to support acetyl-CoA generation for histone acetylation. Consequently, limiting serine depletes histone H3 lysine 27 acetylation (H3K27ac), particularly at the promoter region of ER pathway genes including the gene encoding ERα, ESR1. Mechanistically, serine starvation impairs acetyl-CoA-dependent gene expression by inhibiting the entry of glycolytic carbon into the TCA cycle and down-regulating the mitochondrial citrate exporter SLC25A1, a critical enzyme in the production of nucleocytosolic acetyl-CoA from glucose. Consistent with this model, total H3K27ac and ERα expression are suppressed by SLC25A1 inhibition and restored by acetate, an alternate source of acetyl-CoA, in serine-free conditions. We thus uncover an unexpected role for serine in sustaining ER signaling through the regulation of acetyl-CoA metabolism.

Mitochondrial Uncoupling Inhibits Reductive Carboxylation in Cancer Cells.

When the electron transport chain (ETC) function is impaired, cancer cells rely on reductive carboxylation (RC) to convert α-ketoglutarate (αKG) to citrate for macromolecular synthesis, thereby promoting tumor growth. Currently, there is no viable therapy to inhibit RC for cancer treatment. In this study, we demonstrate that the mitochondrial uncoupler treatment effectively inhibits RC in cancer cells. Mitochondrial uncoupler treatment activates the ETC and increases the NAD+/NADH ratio. Using U-13C-glutamine and 1-13C-glutamine tracers, we show that mitochondrial uncoupling accelerates the oxidative tricarboxylic acid (TCA) cycle and blocks RC under hypoxia, in von Hippel-Lindau (VHL) tumor suppressor-deficient kidney cancer cells, or under anchorage-independent growth condition. Together, these data demonstrate that mitochondrial uncoupling redirects α-KG from RC back to the oxidative TCA cycle, highlighting that the NAD+/NADH ratio is one key switch that determines the metabolic fate of α-KG. Inhibiting RC could be a key mechanism by which mitochondrial uncouplers inhibit tumor growth. IMPLICATIONS: Mitochondrial uncoupling is a novel strategy to target RC in cancer.

Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors.

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

Differential effects of SARM1 inhibition in traumatic glaucoma and EAE optic neuropathies.

Optic neuropathy is a group of optic nerve (ON) diseases with progressive degeneration of ON and retinal ganglion cells (RGCs). The lack of neuroprotective treatments is a central challenge for this leading cause of irreversible blindness. SARM1 (sterile α and TIR motif-containing protein 1) has intrinsic nicotinamide adenine dinucleotide (NAD+) hydrolase activity that causes axon degeneration by degrading axonal NAD+ significantly after activation by axon injury. SARM1 deletion is neuroprotective in many, but not all, neurodegenerative disease models. Here, we compare two therapy strategies for SARM1 inhibition, antisense oligonucleotide (ASO) and CRISPR, with germline SARM1 deletion in the neuroprotection of three optic neuropathy mouse models. This study reveals that, similar to germline SARM1 knockout in every cell, local retinal SARM1 ASO delivery and adeno-associated virus (AAV)-mediated RGC-specific CRISPR knockdown of SARM1 provide comparable neuroprotection to both RGC somata and axons in the silicone oil-induced ocular hypertension (SOHU) glaucoma model but only protect RGC axons, not somata, after traumatic ON injury. Surprisingly, neither of these two therapy strategies of SARM1 inhibition nor SARM1 germline knockout (KO) benefits RGC or ON survival in the experimental autoimmune encephalomyelitis (EAE)/optic neuritis model. Our studies therefore suggest that SARM1 inhibition by local ASO delivery or AAV-mediated CRISPR is a promising neuroprotective gene therapy strategy for traumatic and glaucomatous optic neuropathies but not for demyelinating optic neuritis.

Small-molecule toosendanin reverses macrophage-mediated immunosuppression to overcome glioblastoma resistance to immunotherapy.

T cell-based immunotherapy holds promise for treating solid tumors, but its therapeutic efficacy is limited by intratumoral immune suppression. This immune suppressive tumor microenvironment is largely driven by tumor-associated myeloid cells, including macrophages. Here, we report that toosendanin (TSN), a small-molecule compound, reprograms macrophages to enforce antitumor immunity in glioblastoma (GBM) in mouse models. Our functional screen of genetically probed macrophages with a chemical library identifies that TSN reverses macrophage-mediated tumor immunosuppression, leading to enhanced T cell infiltration, activation, and reduced exhaustion. Chemoproteomic and structural analyses revealed that TSN interacts with Hck and Lyn to abrogate suppressive macrophage immunity. In addition, a combination of immune checkpoint blockade and TSN therapy induced regression of syngeneic GBM tumors in mice. Furthermore, TSN treatment sensitized GBM to Egfrviii chimeric antigen receptor (CAR) T cell therapy. These findings suggest that TSN may serve as a therapeutic compound that blocks tumor immunosuppression and circumvents tumor resistance to T cell-based immunotherapy in GBM and other solid tumors that warrants further investigation.

PHGDH-mediated endothelial metabolism drives glioblastoma resistance to chimeric antigen receptor T cell immunotherapy.

The efficacy of immunotherapy is limited by the paucity of T cells delivered and infiltrated into the tumors through aberrant tumor vasculature. Here, we report that phosphoglycerate dehydrogenase (PHGDH)-mediated endothelial cell (EC) metabolism fuels the formation of a hypoxic and immune-hostile vascular microenvironment, driving glioblastoma (GBM) resistance to chimeric antigen receptor (CAR)-T cell immunotherapy. Our metabolome and transcriptome analyses of human and mouse GBM tumors identify that PHGDH expression and serine metabolism are preferentially altered in tumor ECs. Tumor microenvironmental cues induce ATF4-mediated PHGDH expression in ECs, triggering a redox-dependent mechanism that regulates endothelial glycolysis and leads to EC overgrowth. Genetic PHGDH ablation in ECs prunes over-sprouting vasculature, abrogates intratumoral hypoxia, and improves T cell infiltration into the tumors. PHGDH inhibition activates anti-tumor T cell immunity and sensitizes GBM to CAR T therapy. Thus, reprogramming endothelial metabolism by targeting PHGDH may offer a unique opportunity to improve T cell-based immunotherapy.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the NAD+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate/2-hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and ß-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and ß-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma. SIGNIFICANCE: Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

The magic bullet: Niclosamide.

The term 'magic bullet' is a scientific concept proposed by the German Nobel laureate Paul Ehrlich in 1907, describing a medicine that could specifically and efficiently target a disease without harming the body. Oncologists have been looking for a magic bullet for cancer therapy ever since. However, the current therapies for cancers-including chemotherapy, radiation therapy, hormone therapy, and targeted therapy-pose either pan-cytotoxicity or only single-target efficacy, precluding their ability to function as a magic bullet. Intriguingly, niclosamide, an FDA-approved drug for treating tapeworm infections with an excellent safety profile, displays broad anti-cancer activity in a variety of contexts. In particular, niclosamide inhibits multiple oncogenic pathways such as Wnt/ß-catenin, Ras, Stat3, Notch, E2F-Myc, NF-κB, and mTOR and activates tumor suppressor signaling pathways such as p53, PP2A, and AMPK. Moreover, niclosamide potentially improves immunotherapy by modulating pathways such as PD-1/PDL-1. We recently discovered that niclosamide ethanolamine (NEN) reprograms cellular metabolism through its uncoupler function, consequently remodeling the cellular epigenetic landscape to promote differentiation. Inspired by the promising results from the pre-clinical studies, several clinical trials are ongoing to assess the therapeutic effect of niclosamide in cancer patients. This current review summarizes the functions, mechanism of action, and potential applications of niclosamide in cancer therapy as a magic bullet.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

A stromal Integrated Stress Response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumour progression.

Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis.

ß-Cell Succinate Dehydrogenase Deficiency Triggers Metabolic Dysfunction and Insulinopenic Diabetes.

Mitochondrial dysfunction plays a central role in type 2 diabetes (T2D); however, the pathogenic mechanisms in pancreatic ß-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the tricarboxylic acid cycle and electron transport chain. Using samples from human with diabetes and a mouse model of ß-cell-specific SDH ablation (SDHBßKO), we define SDH deficiency as a driver of mitochondrial dysfunction in ß-cell failure and insulinopenic diabetes. ß-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential collapse, thereby compromising glucose-stimulated ATP production, insulin secretion, and ß-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mammalian target of rapamycin (mTOR) complex 1-regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human ß-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive ß-cell failure of diabetes and identify mTOR complex 1 inhibition as a potential mitigation strategy.

NMNAT2 is downregulated in glaucomatous RGCs, and RGC-specific gene therapy rescues neurodegeneration and visual function.

The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse γ-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline, and that modulating RGC-intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell-Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium.

BACKGROUND: Mitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary. OBJECTIVES: The purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics. METHODS: Human-induced pluripotent stem cell-derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model. RESULTS: Electron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV-derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 108/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 108/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 108 M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis. CONCLUSIONS: M-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.