Main Research Findings

1) Treatment with SkQ1was found to reduce cancer-induced muscle wasting in male mice and enhance muscle contractility in tumor-bearing female mice.

2) Following hemorrhagic shock SkQ1 was shown to protect mitochondria within myocardial tissue by reducing levels of inflammation.

 

Selected Data

1) The study completed by the research team of Tsitkanou et al investigated the efficacy of the mitochondria-targeted antioxidant SkQ1 in mitigating cancer cachexia (CC) in both biological sexes, utilizing a comprehensive array of methods to assess physiological, molecular, and functional outcomes. The animal model consisted of 80 Balb/c mice, equally divided into 2 groups of 10 males and 10 females. Mice were housed in a temperature-controlled environment with a 12:12-hour light-dark cycle and provided ad libitum access to standard rodent chow and water. At 8 weeks of age, mice were bilaterally injected subcutaneously with either Colon-26 Carcinoma (C26) cells or an equal volume of sterile PBS as a sham control [1].

Seven days post-tumor induction, SkQ1, at a dose of ~250 nmol/kg body wt/day, was administered dissolved in drinking water to treatment groups, while control groups received normal drinking water. This timing for SkQ1 administration was chosen based on prior work showing increased mitochondrial reactive oxygen species (ROS) emission at this stage, positioning it as a preventative treatment strategy. Tumors were allowed to develop for 25 days, which marked the study’s endpoint. This experimental design resulted in four groups per sex: PBS CON (PBS-injected, no SkQ1), PBS SkQ1 (PBS-injected, SkQ1-treated), C26 CON (C26-injected, no SkQ1), and C26 SkQ1 (C26-injected, SkQ1-treated). At endpoint, skeletal muscle tissues from the gastrocnemius, tibialis anterior (TA), plantaris, soleus, extensor digitorum longus (EDL), quadriceps, organ tissue from the liver, spleen, heart, gonadal fat, and tumors were collected, weighed, snap-frozen, and stored for subsequent analysis. Tibia length was also measured as a surrogate for body size.

In vivo muscle contractility of dorsiflexors (TA and EDL) was assessed two days before the experimental endpoint to allow for recovery prior to tissue collection. Mice were anesthetized with isoflurane and positioned on a heated platform. Torque-frequency curves were generated by stimulating the left peroneal nerve with needle electrodes at increasing frequencies from 10 to 300 Hz, with 60-second rest intervals between stimuli. Fatigability was assessed by 120 submaximal contractions at 40 Hz, followed by a recovery stimulus. Torque data were normalized by tumor-free body weight [1].

For molecular analyses, total RNA was extracted from gastrocnemius, TA, and plantaris muscles using TRIzol reagent and a commercial kit. RNA quantity and purity were determined by fluorometry. Reverse transcription was performed to synthesize cDNA, which was then used for quantitative real-time PCR to measure mRNA content of various genes related to anabolic repressors (Deptor, Redd1), catabolic markers (Murf1, Atrogin, Ubc, Gadd45a, Mapk14), mitochondrial markers (Opal, Bnip3, Pgc1a, Drp1, Fis1), neuromuscular junction degeneration (Ncam1, Runx1), and calcium channel regulators (Ryr1, Serca1, Serca2a). The 18S gene served as the housekeeping control.

Mitochondrial oxygen consumption was evaluated in permeabilized plantaris and medial head of gastrocnemius fibers using polarography (Oxygraph+). Muscle fibers were prepared by removing connective tissue, separating into near-single bundles, and permeabilized with saponin. State 2 respiration was stimulated with malate and pyruvate, state 3 with ADP, and state 4 with oligomycin. The mitochondrial respiratory control ratio (RCR) was calculated as the ratio of state 3 to state 4. To measure fractional protein synthetic rate (FSR), mice received an intraperitoneal injection of 99.9% deuterium oxide of 20 µL/g body wt, 24 hours prior to tissue collection, with drinking water supplemented with 4% D2O. FSR in the gastrocnemius was determined using gas chromatography-mass spectrometry (GC-MS), involving protein hydrolysis, derivatization of alanine, and measurement of 2H labeling. Deuterated plasma enrichment was also assessed via GC-MS after plasma sample preparation. Statistical analyses included two-way ANOVAs, followed by Tukey’s post hoc tests and unpaired t-tests where interactions were significant [1].

2) This study conducted by the research team of Jia et al investigated the protective effects of the mitochondrial antioxidant SkQ1 against myocardial damage and inflammation following hemorrhagic shock (HS). The primary animal model utilized was male Sprague-Dawley rats, weighing between 240-310g. Anesthesia was induced using 1%-3% vaporized isoflurane for all surgical interventions. The hemorrhagic shock model involved a 40% fixed-blood-loss, induced by sterile placement of a polyethylene 50 tube into the femoral artery and controlled blood withdrawal. Sham group rats underwent femoral cannulation without hemorrhage. SkQ1 was administered intraperitoneally at a dose of 1 µM/kg immediately after HS surgery in the treatment group, while the HS + vehicle group received the same dose of vehicle. Samples, including whole blood and heart tissue, were collected 12 hours post-HS for comprehensive analysis [2].

To further elucidate cellular mechanisms, neonatal rat cardiomyocytes were isolated and cultured using a detailed protocol. Ventricular myocytes from postnatal 1-2 day old SD rats were digested with HBSS containing trypsin and collagenase, followed by a pre-plating step to remove fibroblasts. Purified cardiomyocytes were then cultured for 48 hours in DMEM with 10% FBS, with 0.1 µM 5-bromo-2′-deoxyuridine added to inhibit fibroblast proliferation. Immunofluorescence staining with alpha-actinin confirmed cardiomyocyte purity. An oxygen-glucose deprivation (OGD) model was established in these cultured cardiomyocytes to mimic the cellular hypoxia of HS. Cells were switched to glucose-free DMEM without FBS and incubated in a hypoxia chamber for 6 hours, with normal glucose and oxygen conditions serving as control.

Myocardial ultrastructure was assessed using transmission electron microscopy (TEM). Fresh rat heart tissues were fixed overnight in glutaraldehyde and then in osmium tetroxide, followed by dehydration and embedding. Digital images were acquired using a JEM-1230 high-contrast TEM. Quantification of mitochondrial morphology was meticulous, involving ImageJ software to measure mitochondrial volume density, length, width, perimeter, area, and circularity index. Crucially, cristae morphology was evaluated by cristae volume density and a qualitative cristae score, ranging from 0 for no sharply defined cristae to 4 for many regular cristae, with at least 200 mitochondria analyzed per group from 5 rats [2].

Mitochondrial DNA (mtDNA) copy number in peripheral blood was quantified as a marker of mitochondrial damage. Whole blood was collected 12 hours post-HS, and genomic and mitochondrial DNA were extracted. Relative copy number ratios of mtDNA to genomic DNA were determined via RT-PCR, using cytochrome B (Cyt B) as the mtDNA marker and Gapdh as the genomic DNA reference. ROS content was detected in both myocardial tissue and cultured cardiomyocytes. Myocardial ROS levels were measured using a high-quality living-tissue oxidative stress ROS fluorescence assay kit. In cardiomyocytes, mitochondrial ROS were identified using MitoSOX Red, and cytoplasmic ROS with DCFH-DA, with fluorescence intensity quantified using ImageJ after imaging with an immunofluorescence microscope.

For broad molecular profiling, mRNA sequencing was performed on rat heart tissues. Total mRNA was extracted using TRIzol, treated with DNase I to remove genomic DNA, and then used for library construction with an RNA sample preparation kit. The libraries were sequenced on an Illumina HiSeq xten/NovaSeq 6,000 platform, generating 2 × 150 bp read length reads. Differential expression analysis involved aligning reads to the reference genome using HISAT2. Gene expression levels were quantified, and differential expression was determined using edgeR. Gene Ontology (GO) and Reactome enrichment analyses were conducted to identify significantly enriched biological processes and pathways, particularly focusing on mitochondrion-related, inflammation-related, and ROS-related terms. Furthermore, inflammatory response gene sets from the rat gene database RGD were used for comparative analysis. Finally, protein levels of key inflammatory factors, TNF-α, IL-6, and MCP-1, in peripheral blood were measured by ELISA. All statistical data were expressed as mean ± SEM, with significance determined by unpaired t-tests or one-way ANOVA followed by Student-Newman-Keuls analysis [2].

 

Discussion

1) The results of the study conducted by Tsitkanou et al revealed significant sex-specific differences in the effects of SkQ1 on cancer cachexia, muscle wasting, and contractility. SkQ1 treatment itself did not alter tumor-free body weight in either sex, but C26 tumor inoculation significantly decreased tumor-free body weight in both males and females. Tumor-bearing mice also exhibited altered organ weights, including increased liver (females only) and spleen (both sexes), and reduced gonadal fat (both sexes). Critically, tumor-bearing mice showed significant reductions in the mass of various hindlimb muscles, including plantaris, gastrocnemius, TA, EDL, quadriceps, and heart, confirming the induction of cachexia. In males, SkQ1 treatment successfully attenuated cancer-induced gastrocnemius atrophy, preserving muscle weight compared to untreated tumor-bearing mice. Conversely, in females, SkQ1 treatment led to a decrease in plantaris muscle mass compared to non-treated tumor-bearing mice, indicating an unexpected induction of atrophy in this specific muscle [1].

 

Figure 1: Changes in muscle weight and tibia length in male and female mice.

Analysis of fractional protein synthetic rate (FSR) in the gastrocnemius showed a significant interaction in males: SkQ1-treated tumor-bearing males exhibited a ~2.2-fold higher FSR compared to untreated tumor-bearing males, suggesting enhanced muscle protein synthesis. This anabolic effect in males was corroborated by molecular findings; SkQ1 treatment inhibited the C26-induced upregulation of the anabolic repressor Redd1 and the catabolic marker Atrogin in the gastrocnemius.

Furthermore, SkQ1-treated male tumor-bearing mice showed lower levels of Bnip3 and Pgc1a mRNA, markers associated with mitochondrial function. In stark contrast, female gastrocnemius FSR showed no significant differences across groups. Molecularly, female tumor-bearing mice generally had higher Redd1, and SkQ1-treated females specifically exhibited elevated Murf1, Ubc, and Gadd45a, all catabolic markers [1]. SkQ1-treated female gastrocnemius also showed higher Bnip3 and lower Drp1 mRNA levels, alongside a generalized lower mitochondrial RCR in various C26 and SkQ1 groups compared to healthy controls.

The detrimental effects of SkQ1 on female plantaris muscle mass were linked to specific molecular changes. SkQ1-treated female tumor-bearing mice had significantly higher Redd1, Murf1, and Atrogin content in the plantaris, indicating an increase in catabolic signaling. This was coupled with lower mitochondrial oxygen consumption in SkQ1-treated female plantaris. In males, SkQ1 treatment lowered Atrogin, Ubc, Gadd45a, and Mapk14 content in the plantaris, suggesting a reduction in catabolic processes.

Regarding muscle contractility, male tumor-bearing mice displayed reduced peak torque and lower torque production across a range of frequencies from 100-300 Hz in dorsiflexors. However, in females, SkQ1 administration yielded a remarkable beneficial effect: SkQ1-treated tumor-bearing mice exhibited higher peak torque and improved torque production at multiple frequencies including 40, 80, 100, 125, 150, 200, 250, and 300 Hz, compared to untreated tumor-bearing females. This enhanced contractility in females was associated with molecular adaptations in the TA muscle. While male tumor-bearing mice had lower Runx1 and SkQ1 increased Ncam1, female tumor-bearing mice showed higher Runx1, Ryr1, and Serca1. Notably, SkQ1 treatment further elevated Ryr1, Serca1, and Serca2a mRNA content in the TA of female tumor-bearing mice, indicating a potential improvement in calcium handling crucial for muscle contraction [1].

2) The study by researchers Jia et al comprehensively demonstrated that HS inflicted significant damage to myocardial mitochondria and triggered a systemic inflammatory response, both of which were substantially attenuated by SkQ1 treatment. Initial findings confirmed the successful establishment of a 40% fixed-blood-loss HS rat model, characterized by widespread myocardial mitochondrial ultrastructural damage observed 12 hours post-HS via TEM. Mitochondria in HS rats showed clear signs of swelling and disruption, with increased length, width, perimeter, area, and circularity compared to sham controls, indicating abnormal morphology. Critically, the internal cristae structures were severely compromised, manifested by a reduced cristae volume density and a lower cristae score, signaling a breakdown in the functional integrity of mitochondria. This structural degradation was paralleled by a significant increase in the peripheral blood content of mtDNA, specifically the Cyt B marker, suggesting the release of endogenous damage-associated molecular patterns (DAMPs) from damaged mitochondria into circulation. Increased myocardial ROS content was also confirmed in HS rats, highlighting oxidative stress as a key component of HS-induced pathology [2].

RNA sequencing analysis of myocardial tissues further unveiled the molecular landscape of HS-induced damage. A total of 3,468 differentially expressed genes (DEGs) were identified 12 hours post-HS. Reactome and GO enrichment analyses revealed that these DEGs were predominantly enriched in mitochondrial pathways, including the Tricarboxylic Acid (TCA) cycle, mitochondrial RNA metabolic processes, electron transport chain, protein import, biogenesis, and translation. Furthermore, ROS-related pathways, such as the regulation of cellular response to oxidative stress, were also significantly enriched, underscoring the critical role of mitochondrial dysfunction and oxidative stress in HS pathophysiology.

A pivotal finding of the study was the protective effect of SkQ1. Treatment with SkQ1 significantly attenuated the HS-induced increase in myocardial ROS, demonstrating its potent antioxidant capacity. This effect was consistent at the cellular level, where SkQ1 effectively reduced both mitochondrial and cytoplasmic ROS in cardiomyocytes subjected to OGD, a cellular model of HS. SkQ1 treatment also remarkably preserved myocardial mitochondrial ultrastructure. TEM images of SkQ1-treated HS rats showed a reversal of HS-induced mitochondrial swelling and rounding, with restored mitochondrial length, width, perimeter, area, and circularity values. Crucially, SkQ1 protected the cristae morphology, leading to a significant increase in cristae volume density and cristae score compared to untreated HS animals. These structural improvements were accompanied by a significant reduction in peripheral blood mtDNA (Cyt B) levels in SkQ1-treated HS rats, indicating less mitochondrial damage and DAMPs release [2].

At the transcriptional level, SkQ1 significantly modulated the gene expression profile. Out of 2,770 DEGs between SkQ1-treated and untreated HS groups, 1,623 were increased and 1,147 decreased. Importantly, SkQ1 treatment maintained enrichment in mitochondrial and ROS-related pathways, but critically, it reversed many of the HS-induced gene expression changes. Four specific pathways that were significantly enriched in HS including regulation of stress-activated MAPK cascade, cellular response to oxidative stress, inflammatory response, and positive regulation of leukocyte migration, were no longer significantly enriched after SkQ1 treatment, highlighting SkQ1’s ability to ameliorate key aspects of the HS-induced stress response.

Beyond mitochondrial protection, SkQ1 demonstrated a profound anti-inflammatory effect. Overlapping DEGs with rat inflammatory response gene sets revealed that SkQ1 significantly reversed the transcription levels of 56.5% of inflammation-related genes that were altered by HS. Specifically, SkQ1 treatment increased the expression of anti-inflammatory genes, Adoral, Lrfn5, Foxp3, Pla2g5, Ppara, Gpx1, and Fem1a, that were reduced by HS, and conversely, decreased the expression of pro-inflammatory genes, Fabp4, Tgm2, RT1-S3, Gpr4, Tnfrs1a, Hyal2, Ace, Trpv4, Tlr6, Lbp, and Il33, that were elevated by HS. These transcriptional changes translated into significant reductions at the protein level. ELISA measurements showed that SkQ1 treatment significantly reversed the HS-induced increases in key systemic inflammatory factors, including TNF-α, IL-6, and MCP-1 in rat peripheral blood. Collectively, these results strongly suggest that SkQ1 protects myocardial mitochondria from HS-induced damage, leading to a reduction in mtDNA release and a significant improvement in both local and systemic inflammatory responses [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Tsitkanou S, Morena da Silva F, Cabrera AR, et al. Mitochondrial antioxidant SkQ1 attenuates C26 cancer-induced muscle wasting in males and improves muscle contractility in female tumor-bearing mice. Am J Physiol Cell Physiol. 2024;327(5):C1308-C1322. doi:10.1152/ajpcell.00497.2024

[2] Jia B, Ye J, Gan L, et al. Mitochondrial antioxidant SkQ1 decreases inflammation following hemorrhagic shock by protecting myocardial mitochondria. Front Physiol. 2022;13:1047909. Published 2022 Nov 16. doi:10.3389/fphys.2022.1047909

 

SKQ1 is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.