SS-31

Abstract

Mitochondrial dysfunction contributes to impaired myocardial energetics and performance in heart failure with preserved ejection fraction (HFpEF). Elamipretide (Ela) enhances mitochondrial bioenergetics in preclinical models, yet its relevance in HFpEF remains unclear. This study examined the effects of Ela on cardiac mitochondrial function, structure, and cardiovascular performance in a rodent HFpEF model. Female obese ZSF1 rats received vehicle or Ela for 12 weeks, with age-matched lean rats as controls. Cardiac function and hemodynamics were assessed by echocardiography and pressure-volume analysis. Mitochondrial respiration was measured in permeabilized fibers and ultrastructure evaluated by transmission electron microscopy. Molecular and histological analyses included cardiolipin lipidomics and mRNA/protein profiling of hypertrophic, fibrotic, and inflammatory markers. Ela modestly improved complex I and II respiration, whereas mitochondrial ultrastructure, cardiolipin composition, and tafazzin expression were unchanged. Diastolic dysfunction persisted, reflected by unchanged E/é, ventricular stiffness factor β, and titin phosphorylation. Compared to untreated HFpEF, systolic performance showed a mild decline, with small reductions in LV ejection fraction and end-systolic elastance. Accordingly, cardiac remodeling, including hypertrophy, fibrosis, and inflammatory activation, remained unaltered. Vascular stiffness slightly increased, while carotid reactivity and morphology were preserved. In conclusion, despite enhanced mitochondrial respiration following Ela treatment, no functional or structural benefits were observed in experimental HFpEF, suggesting limited therapeutic efficacy once HFpEF is established.

Abstract

Mitochondrial trifunctional protein (TFP) deficiency is an inherited disorder of long-chain fatty acid β-oxidation (FAO). TFP is a heteromeric enzyme composed of two α and two β-subunits. Despite early detection and dietary treatment, TFP deficiency patients often develop hypoglycemia, rhabdomyolysis, cardiomyopathy, and peripheral neuropathy. Degenerative retinopathy and milder peripheral neuropathy occur in patients with an isolated deficiency of the αTFP subunit of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity. Triheptanoin treatment improves most complications, but not peripheral neuropathy and retinopathy. Notably, TFP also carries a fourth enzymatic function involved in cardiolipin remodeling, which we previously found to be impaired in TFP/LCHAD deficiency. We therefore tested whether elamipretide, a synthetic cardiolipin-binding peptide, could improve mitochondrial function and cardiolipin levels in βTFP-deficient mice and patient-derived fibroblasts. Mice were treated with elamipretide delivered by osmotic minipump and challenged with treadmill exercise or cold stress after fasting. βTFP-deficient mice treated with elamipretide showed improved exercise endurance, but cold tolerance was not altered. Liver mitochondria from male βTFP-deficient mice demonstrated improved FAO-ETC enzyme activities. However, cardiolipin content and composition were unchanged. In patient fibroblasts, elamipretide produced a possible genotype-dependent increase in mitochondrial bioenergetics and a reduction in ROS. These results support a mechanism in which elamipretide stabilizes between FAO enzymes and ETC complexes, thereby improving mitochondrial function independently of changes in cardiolipin levels. Elamipretide thus emerges as a potential therapeutic agent for TFP/LCHAD deficiency, warranting further preclinical studies.

Abstract

Several studies have used molecular dynamics (MD) simulations to examine the relationship between transmembrane potentials (ΔΨm) and electroporation; however, research on how this relationship presents in complex membranes with heterogeneous lipid compositions is limited. Here, we use all-atom, double-bilayer MD simulations with explicitly modeled ΔΨm voltages to compare how membranes with homogeneous vs heterogeneous lipid compositions respond to varying degrees of electrochemical stress. These two bilayer systems were also exposed to a mitochondria-targeted peptide named Elamipretide, which has been shown experimentally to modulate membrane electrostatics. Additionally, we used Computational Electrophysiology (CompEL) MD simulations to analyze how lipid composition and peptide exposure influence rates of passive transmembrane ion flux during electroporation events. The addition of cardiolipin (CL) and Elamipretide increased the membrane capacitance, decreasing the ΔΨm for a given transmembrane charge imbalance (δTM) and protecting bilayers against electroporation. These results expand our understanding of how more complex, biologically relevant bilayers respond to electrochemical stress.

Abstract

Barth syndrome (BTHS) is a rare X-linked mitochondrial disorder caused by tafazzin mutations that impair cardiolipin remodeling, leading to mitochondrial dysfunction and symptoms such as cardiomyopathy, myopathy, and neutropenia. On September 19, 2025, the U.S. Food and Drug Administration (FDA) granted accelerated approval to elamipretide, the first therapy directly targeting the mitochondrial etiology of BTHS. Elamipretide binds to cardiolipin on the inner mitochondrial membrane, stabilizing respiratory chain supercomplexes, enhancing electron transport efficiency, and reducing reactive oxygen species production. In a randomized, double-blind, placebo-controlled, crossover trial, elamipretide resulted in no significant improvement in the 6-minute walk test or fatigue scores; however, sustained benefits were observed during a 168-week open-label extension. The most common adverse events were mild injection-site reactions. As a condition of accelerated approval, a confirmatory trial is required. Elamipretide represents a promising therapy addressing an unmet medical need in BTHS and provides a foundation for future mitochondria-targeted treatments.

Abstract

Imeglimin, a novel oral antidiabetic drug, has been suggested to affect mitochondrial functions in some types of cells and tissues, however, it has never been investigated whether aging has an impact on its pharmacological effects in the brain. In this study, we investigated whether imeglimin directly affects functions of brain mitochondria and whether its intraperitoneal injection protects against ischemic brain injury in young, middle-aged and aged Wistar rats. We found that direct addition of imeglimin to mitochondria isolated from young and middle-aged rat brains suppressed oxidative phosphorylation and activities of mitochondrial Complexes I and IV. The opposite, stimulating effect on Complex II activity was observed within the same groups. Injection of imeglimin 24 h before simulated brain ischemia in vitro reduced infarct size only in young and middle-aged rat groups. In the aged rat group, imeglimin did not reduce cerebral infarct size nor directly modulate mitochondrial respiration and activities of the complexes. In conclusion, we provided novel evidence on potential effects of imeglimin in the brain by demonstrating a direct stimulating effect on mitochondrial Complex II activity and age-dependent protective effects against brain injury under in vitro simulated ischemia.

Abstract

Patients with idiopathic carpal tunnel syndrome (CTS) exhibit impaired mitochondrial function in the subsynovial connective tissue (SSCT) of the carpal tunnel. We hypothesized that Imeglimin, a compound known to enhance mitochondrial function, may improve mitochondrial activity in SSCT from these patients. This study aimed to evaluate the effects of Imeglimin on mitochondrial function in SSCT-derived cells obtained from patients with idiopathic CTS. SSCT samples were collected from 15 patients (mean age: 67.5  ±  9.7 years) who underwent carpal tunnel release surgery between April 2022 and March 2024. The cells were cultured under control conditions (Dulbecco's Modified Eagle Medium alone) or with 100 µM Imeglimin for 24 h. Mitochondrial function was assessed using multiple assays, including cell proliferation, superoxide dismutase (SOD) activity, apoptosis rate, mitochondrial volume, membrane potential, reactive oxygen species (ROS) production, gene expression related to mitochondrial biogenesis and antioxidant capacity, mitochondrial permeability transition pore opening, and ultrastructure by transmission electron microscopy. Statistical analyses were performed using the Mann-Whitney U test, one-way ANOVA, Kruskal-Wallis test, and Fisher's protected least significant difference test, with p  <  0.05 considered significant. Compared with the control group, the Imeglimin-treated group showed significantly increased cell proliferation, SOD activity, mitochondrial membrane potential, mitochondrial volume, cristae density, and expression of genes related to mitochondrial biogenesis and antioxidant defense. Apoptosis and mitochondrial ROS production were significantly reduced (p <  0.05). These findings suggest that Imeglimin may enhance mitochondrial function in SSCT-derived cells from patients with idiopathic CTS, offering a potential therapeutic strategy for mitochondrial dysfunction in CTS.

Abstract

Traumatic brain injury (TBI) is a major clinical problem because of the high incidence and the severity of the subsequent sequelae. Despite extensive efforts, there are no therapeutic drugs clinically approved for treating acute TBI patients. To address this unmet need, we assessed the activity of the tetrapeptide, CAQK, in mice. When administered intravenously shortly after moderate or severe TBI, CAQK accumulates in the injured brain in mice and pigs. CAQK binds to an extracellular matrix glycoprotein complex that is upregulated in injured brain. Treatment of TBI mice with CAQK resulted in reduction in the size of the injury compared to control mice. There was reduced upregulation of the glycoprotein complex, less apoptosis, and lower expression of inflammatory markers in the injured area, indicating that CAQK alleviates neuroinflammation and the ensuing secondary injury. CAQK treatment also improved functional deficit in TBI mice, with no overt toxicity. Our findings suggest that CAQK may have therapeutic applications in TBI.

Abstract

Acute kidney injury (AKI) continues to pose a significant clinical burden, characterized by high morbidity and mortality rates. Emerging evidence has established mitochondrial dysfunction as a central driver in the pathogenesis of AKI, encompassing deficits in bioenergetics, excessive production of reactive oxygen species, and disruption of mitochondrial dynamics. Therapeutic interventions targeting mitochondrial pathways-most notably peptide-based agents such as SS-31-have demonstrated promising results in preclinical models. Recent discoveries have identified phospholipid scramblase 3 (PLSCR3) as an essential mediator of SS-31's mitochondrial protective effects, positioning it as a novel therapeutic target. This review synthesizes current mitochondrial-directed approaches for AKI, with a particular emphasis on the mechanistic role of PLSCR3 in maintaining mitochondrial homeostasis and injury responses. Despite encouraging data, mitochondrial therapies face several translational hurdles, including limited bioavailability, challenges in establishing effective dosing regimens, incomplete mechanistic understanding, and variability in efficacy across different experimental models. Moreover, concerns regarding cost, accessibility, and long-term safety remain unresolved, contributing to inconsistent outcomes in clinical trials. Herein we evaluate the emerging role of PLSCR3 as a potentially druggable mitochondrial target, supported by recent genetic, biochemical, and in vivo evidence, and discuss translational strategies that may bridge the gap between experimental promise and clinical application.

Abstract

To elucidate the effects of the new antidiabetic agent, imeglimin (Ime, 2 mM), on high-glucose-induced cellular stress in cardiac cells, its effects were compared with those of the conventional antidiabetic agent metformin (Met, 2 mM) based on various cellular pathophysiological functions. H9c2 cardiomyoblasts were cultured under normal-glucose (5.5 mM, N-Glu) or high-glucose (50 mM, H-Glu) conditions. Cellular metabolic function was evaluated using a Seahorse XFe96 Bioanalyzer, along with measurements of reactive oxygen species (ROS) production, expression levels of the autophagy-related marker LC3, and intercellular adhesion properties measured based on transepithelial electrical resistance (TEER). Cells cultured under H-Glu conditions showed enhanced mitochondrial and glycolytic activities, which were suppressed by Met or Ime. Under H-Glu conditions, total cellular ROS (t-ROS) levels were significantly increased. Met had little effect on t-ROS under H-Glu conditions, whereas Ime markedly reduced both t-ROS and mitochondrial ROS (m-ROS) levels under H-Glu conditions. The LC3-II/LC3-I ratio, a marker of autophagic activity, decreased under H-Glu conditions; however, this reduction was not significantly affected by treatment with either Met or Ime. Regarding intercellular adhesion properties, TEER values were elevated under H-Glu conditions compared to N-Glu conditions, and those under H-Glu conditions were further increased by Ime but not Met. In support of these results, the mRNA levels of cell-adhesion-related molecules, including β-catenin and N-cadherin, were also altered by Ime. Collectively, Ime modulated high-glucose-induced alterations in the biological properties of H9c2 cardiomyoblasts, independent of changes in autophagic activity.

Abstract

Ischemia-reperfusion (IR)-induced acute kidney injury (AKI) is a complex pathophysiological process involving inflammation, oxidative stress, and apoptosis. Asprosin (ASP), a fasting-induced glucogenic hormone, has been shown to influence oxidative and apoptotic pathways in various tissues. This study investigated the potential renoprotective effects of ASP in a murine model of IR-induced AKI. Thirty-two male Balb/c mice were randomly assigned to four groups (n = 8): Control, IR, ASP1 (1 µg/kg ASP), and ASP10 (10 µg/kg ASP). While the control group received no treatment. Vehicle and ASP (1 or 10 µg/kg) were administered intravenously five minutes before ischemia to the IR and ASP-treated groups, respectively. Renal ischemia was induced for 22 min, followed by a 24-h reperfusion period. Renal function markers, inflammatory cytokines, oxidative stress parameters, and caspase-3 expression were evaluated. Histopathological alterations were assessed using hematoxylin-eosin staining. IR significantly increased BUN, creatinine, IL-1β, TNF-α, MDA levels, and caspase-3 expression, while reducing antioxidant enzymes (SOD, CAT). ASP pretreatment effectively reversed these changes (p < 0.05), as reflected by improved renal function, reduced inflammation and oxidative stress, and decreased apoptotic activity. These functional and molecular improvements were also supported by histological evidence showing reduced kidney damage following ASP treatment. Collectively, the findings suggest that ASP protects against IR-induced AKI by alleviating inflammation, oxidative stress, and apoptosis.

Abstract

Mitochondria play a pivotal role as therapeutic targets in a range of disorders, including metabolic and neurodegenerative diseases. SS31, a peptide engineered to target mitochondria, offers potent antioxidant activity, positioning it as a promising therapeutic option. Nevertheless, the hydrophilic profile of SS31 poses challenges such as reduced stability, suboptimal delivery, and poor mitochondrial localization in clinical applications. This study was designed to develop a mitochondria-targeted liposomal carrier by conjugating SS31KRKC to the liposome surface (SS31-LP) and to investigate its biological effects in vitro.

Abstract

Cryopreservation of rooster sperm is a vital technique in avian reproductive management; however, it is often hindered by oxidative stress induced by reactive oxygen species (ROS) that negatively impact sperm quality during the freezing-thawing process. The present study aimed to investigate the impact of elamipretide, a mitochondria-targeted peptide, on sperm quality post-thaw. Sperm samples from 32-week-old broiler breeder roosters were cryopreserved using a Lake extender buffer with glycerol as the cryoprotectant. Four different concentrations of elamipretide (0, 6, 9, and 12 µmol/L) were tested in combination with the extender. Post-thaw sperm quality was evaluated by assessing motility, kinematic parameters, membrane integrity, mitochondrial activity, ROS levels as a direct marker of oxidative stress, lipid peroxidation, cell viability, and antioxidant enzyme activities (SOD, GPx and TAC). Sperm motility increased significantly at the 6 µmol/L (60.33 ± 1.54) and 9 µmol/L (64.96 ± 1.96) concentrations compared to the control, with the highest straight-line velocity observed at 9 µmol/L (21.21 ± 0.59). Membrane integrity also improved significantly at 9 µmol/L (61.78 ± 2.70) compared to lower doses (36.30 ± 1.64) and decreased at 12 µmol/L (49.57 ± 1.63). ROS production was significantly lower at 6 µmol/L (2.88 ± 0.07). Mitochondrial activity peaked at 9 µmol/L (60.21 ± 1.92), reflecting enhanced cell vitality and function. However, the effects were diminished at 12 µmol/L, indicating toxicity at higher concentrations. This study demonstrates the potential of elamipretide to improve rooster sperm cryopreservation outcomes by mitigating oxidative damage and preserving sperm quality post-thaw.

Abstract

Sepsis-associated encephalopathy (SAE) is a prevalent and significant neurological complication that arises following sepsis, for which there is currently no effective treatment. Environmental enrichment (EE) has been shown to exert a neuroprotective effect through various mechanisms, including the promotion of neurogenesis, enhancement of neuroplasticity, and the inhibition of inflammatory processes. However, the precise mechanisms underlying these effects remain poorly understood. Utilizing RNA sequencing data, our research demonstrated that energy metabolism facilitated by the mitochondrial inner membrane is crucial for the neuroprotective effects associated with EE. LPS exposure resulted in cognitive deficits, particularly characterized by diminished working memory. Mechanistically, LPS treatment in mice was associated with disrupted function of the mitochondrial inner membrane and altered mitochondrial energy metabolism within the hippocampus. Importantly, the administration of SS-31 was found to maintain mitochondrial integrity, enhance the functionality of the mitochondrial inner membrane, and ultimately mitigate both synaptic and cognitive deficits. Our research indicates that focusing on impaired mitochondrial inner membrane function could serve as a potentially effective preventive or therapeutic approach for SAE.

Abstract

Imeglimin, a novel oral antidiabetic drug, enhances glucose-stimulated insulin secretion, improves insulin sensitivity, and reduces mitochondrial reactive oxygen species (ROS) generation. Diabetic neuropathy is driven by oxidative stress caused by hyperglycemia, with mitochondrial ROS overproduction playing a central role. Hypoglycemia also contributes to oxidative stress. This study evaluates the effects of imeglimin on Schwann cells under high- and low-glucose conditions.

Abstract

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of vision loss. Short peptides, such as di-, tri-, and tetrapeptides, have various beneficial activities, including antioxidant, antimicrobial, and anti-inflammatory effects. This study aims to test the hypothesis that the antioxidant effect of the synthetic tetrapeptide AEDG (Ala-Glu-Asp-Gly, Epitalon) improves the delayed healing process associated with hyperglycemia in DR, using a high glucose (HG)-injured human retinal pigment epithelial cell line (ARPE-19). We found that HG exposure delayed wound healing in ARPE-19 cells and increased intracellular levels of reactive oxygen species (ROS), while decreasing antioxidant gene expression. HG also induced epithelial-mesenchymal transition (EMT) and upregulated fibrosis-related genes, suggesting that HG-induced EMT contributes to subretinal fibrosis, the end-stage of eye diseases, including proliferative DR. The antioxidant Epitalon restored impaired wound healing in HG-injured ARPE-19 cells by inhibiting hyperglycemia-induced EMT and fibrosis. These findings support using the antioxidant agent Epitalon as a promising therapeutic strategy for DR to improve retinal wound healing compromised by hyperglycemia. More mechanistic investigations are needed to confirm Epitalon's benefits and safety. Developing ophthalmic forms of Epitalon may enhance its delivery directly to the retina, potentially improving its therapeutic efficacy.

Abstract

Volumetric muscle loss (VML) is characterized by contractile weakness, dysfunctional mitochondrial bioenergetics, and poor rehabilitation plasticity. A hyperpolarized mitochondrial membrane potential is one attribute of the dysfunction bioenergetics and can lead to excessive reactive oxygen species (ROS) emissions. The primary objective of this study was to define the role of acute ROS emissions after VML injury. Male C57BL/6J mice were randomized into experimental and control groups. A time course of ROS emissions and antioxidant buffering capacity (AoxBC) for VML-injured muscles was established across the first 60 days postinjury (dpi). SS-31, a mitochondrial-targeted peptide, was administered subcutaneously (8 mg/kg/day) for upto 14 dpi, and specific electron transport chain complex ROS emissions and mitochondrial bioenergetics were investigated. SS-31 and wheel running were combined in a regenerative rehabilitation model to determine whether attenuating acute ROS emissions improved adaptive capability of the remaining muscle. Lipidomic and proteomic analyses were conducted to explore mechanisms of SS-31 benefit after VML. ROS emissions were greater and AoxBC was less during the first 14 dpi and this was associated with dysfunctional mitochondrial bioenergetics regardless of carbohydrate or fat fuel substrate. Complexes I, II, and III were identified as the primary sources of ROS emissions. SS-31 attenuated ROS emissions at both 7 and 14dpi and led to greater mitochondrial respiratory conductance and efficiency out to 30 dpi. Regenerative rehabilitation did not produce greater contractile adaptations, but there was modest evidence of greater metabolic adaptations compared with rehabilitation alone. Lipidomic and proteomic analyses suggest that SS-31 contributes to redox protein abundance alterations after VML injury.NEW & NOTEWORTHY Volumetric muscle loss (VML) impairs mitochondrial bioenergetics, causing hyperpolarization, reduced respiratory conductance, and elevated reactive oxygen species (ROS). A mitochondrial-targeted peptide, SS-31, improved mitochondrial efficiency, lowered ROS, and boosted antioxidant buffering in VML-injured muscle. Combining SS-31 with rehabilitation slightly enhanced metabolism but not contractile function. This suggests oxidative stress is not the sole factor in contractile dysfunction after VML injury and underscores the need for multifaceted therapies to restore muscle after VML.

Abstract

The aim of this research was to investigate if the mitochondria- targeting peptide SS-31 could serve as a protective measure against bleomycin-induced pulmonary fibrosis in mice.

Abstract

Mitochondria are cellular hubs integral for metabolism, signaling, and survival. Mitochondrial dysfunction is centrally involved in the aging process and an expansive array of disease states. Elamipretide is a novel mitochondria-targeting peptide that is under investigation for treating several disorders related to mitochondrial dysfunction. This review summarizes recent data that expand our understanding of the mechanism of action (MOA) of elamipretide. Elamipretide is a potential first-in-class therapeutic that targets the inner mitochondrial membrane. Despite initial descriptions of elamipretide's MOA involving reactive oxygen species scavenging, the last ten years have provided a significant expansion of how this peptide influences mitochondrial bioenergetics. The cardiolipin binding properties of elamipretide have been corroborated by different investigative teams with new findings about the consequences of elamipretide-cardiolipin interactions. In particular, new studies have shown elamipretide-mediated modulation of mitochondrial membrane electrostatic potentials and assembly of cardiolipin-dependent proteins that are centrally involved in mitochondrial physiology. These effects contribute to elamipretide's ability to improve mitochondrial function, structure, and bioenergetics. In animal studies, elamipretide-mediated amelioration of organ dysfunction has been observed in models of cardiac and skeletal muscle myopathies as well as ocular pathologies. A number of clinical trials with elamipretide have been recently completed, and a summary of the results focusing on Barth syndrome, primary mitochondrial myopathy, and age-related macular degeneration, is also provided herein. Elamipretide continues to show promise as a potential therapy for mitochondrial disorders. New basic science advances have improved understanding of elamipretide's MOA, enabling a better understanding of the molecular consequences of elamipretide-cardiolipin interactions.

Abstract

Spinal cord injury (SCI) affects millions globally, leading to severe motor and sensory deficits with no effective clinical treatment. Cardiolipin (CL), a mitochondria-specific phospholipid, plays a critical role in bioenergetics and apoptosis. Emerging evidence suggests that CL alterations contribute to secondary SCI pathology, but their precise role and underlying mechanisms remain fully understudied. In this study, we investigated the protective effects of SS-31 on CL alteration, neuronal death, tissue damage, and behavioral recovery after SCI using both in vitro and in vivo models, lipidomics analysis, histological evaluation, and behavioral assessments. In vitro investigations used primary spinal cord neuron cultures, challenged with either rotenone or glutamatergic excitotoxicity, with protective capabilities measured via cell death assays and neurite morphological analysis. In vivo investigations used female adult C57Bl/6 mice, challenged with a contusive SCI. The results showed that SS-31 reduced rotenone- and glutamate-induced mitochondrial dysfunction and neuronal death in a dose-dependent manner in vitro. Additionally, SS-31 attenuated rotenone- and glutamate-induced neurite degeneration in vitro. Lipidomics analysis revealed a reduction in CL at 24 h post-SCI in adult mice, which was attenuated by SS-31 in a dose-dependent manner. Consistent with this effect, SS-31 improved behavioral recovery after SCI in adult mice, although it had no significant effect on tissue damage. These findings suggest that CL alteration may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary SCI.

Abstract

Cerebral microhemorrhages (CMHs, also known as cerebral microbleeds) contribute to vascular cognitive impairment and dementia (VCID), with aging and hypertension being key risk factors. Mitochondrial oxidative stress is a hallmark of cerebrovascular aging, leading to endothelial dysfunction. This study tests the hypothesis that increased mitochondrial oxidative stress contributes to age-related CMH susceptibility and evaluates the mitochondrial-targeted antioxidative peptide SS-31 (elamipretide) as a potential protective agent in an aged, hypertensive mouse model. Concurrently, we developed a high-throughput, machine learning-driven imaging pipeline to enhance CMH quantification and facilitate the screening of anti-aging vasoprotective interventions. To detect CMHs, brain sections were labeled with diaminobenzidine (DAB) and digitized using a slide scanner-based imaging platform. We developed multiple quantification tools, including color space transformation for enhanced contrast separation and a supervised machine-learning approach utilizing a random forest algorithm to generate whole-brain 3D reconstructions and precisely localize CMHs. We optimized a semi-automated detection method integrating color space transformation and machine learning, benchmarking it against traditional manual counting and color deconvolution-based approaches. While SS-31 treatment did not significantly mitigate hypertension-induced CMH burden in aged mice, our high-throughput imaging pipeline provided a reliable, scalable, and unbiased approach to CMH detection, reducing processing time while improving accuracy. This methodological advancement paves the way for future preclinical studies evaluating therapeutic strategies for cerebrovascular protection in aging. Our findings underscore the need for multi-targeted interventions to mitigate CMH-related neurovascular impairments and prevent VCID.

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation, pain, and progressive joint damage. Current treatments, while effective, are limited by their potential side effects, particularly in long-term use. This study introduces a novel nanoemulsion-based therapeutic approach combining rapamycin, an mTOR inhibitor, with SS31, a mitochondrial-targeting antioxidant peptide. The rapamycin-SS31 conjugate (RS31) is encapsulated within a nanoemulsion (RS31@NEs) designed to selectively release its components in response to elevated Caspase-3 levels, prevalent in inflamed joints. In vitro and in vivo studies using zymosan-induced arthritis (ZIA) and collagen-induced arthritis (CIA) mouse models demonstrated that RS31@NEs effectively reduced pro-inflammatory cytokines, mitigated oxidative stress, and improved immune modulation by enhancing regulatory T and B cell functions. These findings highlight RS31@NEs as a promising dual-action therapy for RA, combining anti-inflammatory and mitochondrial protective effects while minimizing systemic toxicity.

Abstract

Aging-related decreases in cardiac and skeletal muscle function are strongly associated with various comorbidities. Elamipretide (ELAM), a novel mitochondria-targeted peptide, has demonstrated broad therapeutic efficacy in ameliorating disease conditions associated with mitochondrial dysfunction across both clinical and pre-clinical models. Herein, we investigated the impact of 8-week ELAM treatment on pre- and post-measures of C57BL/6J mice frailty, skeletal muscle, and cardiac muscle function, coupled with post-treatment assessments of biological age and affected molecular pathways. We found that health status, as measured by frailty index, cardiac strain, diastolic function, and skeletal muscle force, is significantly diminished with age, with skeletal muscle force changing in a sex-dependent manner. Conversely, ELAM mitigated frailty accumulation and was able to partially reverse these declines, as evidenced by treatment-induced increases in cardiac strain and muscle fatigue resistance. Despite these improvements, we did not detect statistically significant changes in gene expression or DNA methylation profiles indicative of molecular reorganization or reduced biological age in most ELAM-treated groups. However, pathway analyses revealed that ELAM treatment showed pro-longevity shifts in gene expression, such as upregulation of genes involved in fatty acid metabolism, mitochondrial translation, and oxidative phosphorylation, and downregulation of inflammation. Together, these results indicate that ELAM treatment is effective at mitigating signs of sarcopenia and cardiac dysfunction in an aging mouse model, but that these functional improvements occur independently of detectable changes in epigenetic and transcriptomic age. Thus, some age-related changes in function may be uncoupled from changes in molecular biological age.

Abstract

Mitochondria serve an essential metabolic and energetic role in cellular activity, and their dysfunction has been implicated in a wide range of disorders, including cardiovascular conditions, neurodegenerative disorders, and metabolic syndromes. Mitochondria-targeted therapies, such as Elamipretide (SS-31, MTP-131, Bendavia), have consequently emerged as a topic of scientific and clinical interest. Elamipretide has a unique structure allowing for uptake in a variety of cell types and highly selective mitochondrial targeting. This mitochondria-targeting tetrapeptide selectively binds cardiolipin (CL), a lipid found in the inner mitochondrial membrane, thus stabilizing mitochondrial cristae structure, reducing oxidative stress, and enhancing adenosine triphosphate (ATP) production. Preclinical studies have demonstrated the protective and restorative efficacy of Elamipretide in models of heart failure, neurodegeneration, ischemia-reperfusion injury, metabolic syndromes, and muscle atrophy and weakness. Clinical trials such as PROGRESS-HF, TAZPOWER, MMPOWER-3, and ReCLAIM elaborate on preclinical findings and highlight the significant therapeutic potential of Elamipretide. Further research may expand its application to other diseases involving mitochondrial dysfunction as well as investigate long-term efficacy and safety of the drug. The following review synthesizes current knowledge of the structure, mechanisms of action, and the promising therapeutic role of Elamipretide in stabilizing mitochondrial fitness, improving mitochondrial bioenergetics, and minimizing oxidative stress.