The mitochondrial-targeted peptide therapeutic elamipretide improves cardiac and skeletal muscle function during aging without detectable changes in tissue epigenetic or transcriptomic age

Abstract

Aging-related decreases in cardiac and skeletal muscle function are strongly associated with various comorbidities. Elamipretide (ELAM), a novel mitochondrial-targeted peptide, has demonstrated broad therapeutic efficacy in ameliorating disease conditions associated with mitochondrial dysfunction across both clinical and pre-clinical models. ELAM is proposed to restore mitochondrial bioenergetic function by stabilizing inner membrane structure and increasing oxidative phosphorylation coupling and efficiency. Although ELAM treatment effectively attenuates physiological declines in multiple tissues in rodent aging models, it remains unclear whether these functional improvements correlate with favorable changes in molecular biomarkers of aging. 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 are 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 heart failure 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.

Figure 1:. Effect of 2-months ELAM treatment on frailty, cardiac function, and muscle force.

A. Experimental design. B. Baseline frailty index (FI) of young (Yng) and old mice. C. Delta (Δ, Post-pre) FI at midpoint (4 weeks) and endpoint (8 weeks) for old mice. D. Combined Δ frailty index at endpoint for control (Con) and elamipretide (ELAM) treated male and female old mice. E. Baseline ejection fraction determined and F. Global longitudinal strain (GLS) from strain analysis of parasternal long axis images of the left ventricle (LV) of young and old mice. G. Δ ejection fraction and H. GLS determined using strain analysis at endpoint for old mice. I. Baseline in vivo muscle force normalized to body mass during repeating fatigue stimulations in young and old mice. J. Δ in vivo muscle force normalized to body mass during repeating fatigue stimulations at endpoint for old mice. 4–5-month-old (young) female and male (n=9–10) and 23–24-month-old (old) female (n=23–24) and male were compared for each measurement at baseline. ELAM treatment effects were compared in control and ELAM-treated old mice (n=11–12) using Δ measurements. Only mice that survived to study endpoint were included in the analysis. Statistical significance was determined by two-way ANOVA with Tukey’s post-hoc test, except for in vivo muscle fatigue and ΔFI which were determined by three-way ANOVA. Combined ΔFI significance determined by student’s t-test. Significant ANOVA factors written in text with selected Tukey’s post hoc test comparisons on graphs. Error bars represent sample means ± standard deviations; error bars omitted from I., J. for clarity.

Figure 2:. Effect of 2-months ELAM treatment on the transcriptome of young and old mouse heart and skeletal muscle tissues.

A. Principal components analysis (PCA) of mRNA-seq samples. PCA was performed following filtering of genes with low numbers of reads and RLE normalization. B. Number of differentially expressed genes following ELAM treatment. Differentially expressed genes were determined using edgeR separately for males and females. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg method, and the False Discovery Rate (FDR) was set at 5%. C. Gene set enrichment analysis (GSEA) of pathways affected by signatures of aging, mortality, lifespan-extending interventions, and ELAM treatment. Normalized enrichment scores (NES) of aging-related pathways affected by ELAM treatment (blue), lifespan-increasing interventions (green), and signatures of aging and mortality (red). GH: grown hormone deficiency, CR: caloric restriction. ‘adjusted p-value < 0.1, *adjusted p-value < 0.05, **adjusted p-value < 0.01, ***adjusted p-value < 0.001. D. Correlation analysis of gene expression changes induced by ELAM treatment at the level of enriched pathways. Spearman correlation between normalized enrichment scores (NES) from gene set enrichment analysis (GSEA) performed for signatures of ELAM treatment (blue), lifespan-extending interventions (green), and mammalian aging and mortality (red). ‘adjusted p-value < 0.1, *adjusted p-value < 0.05, **adjusted p-value < 0.01, ***adjusted p-value < 0.001. E. Effect of ELAM treatment on predicted transcriptomic age (tAge) of mouse heart and skeletal muscle tissues. Mouse multi-tissue transcriptomic clocks of relative chronological age (left) and mortality (right) have been applied. Benjamini-Hochberg adjusted p-values comparing young vs. old animals, and sex- and age-matched control vs. ELAM treated mice are shown in text.

Figure 3:. Network analysis of upregulated GObp terms following 2-months ELAM treatment in old mouse hearts.

A. Females. B. Males. Gene Ontology biological process (GObp) terms that were significantly upregulated (FDR < 0.05) were first consolidated using REVIGO before visualization using Cytoscape.

Figure 4:. Effect of 2-months ELAM treatment on biomarkers of lifespan in young and old mouse hearts.

A. PCA of DNA methylation (DNAm) microarray samples. PCA was performed following data normalization in SeSAMe and filtering of failed probes. B. Mean DNAm levels. Mean DNAm was estimated by taking the means of the filtered beta values for each sample. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Lines represent sample means. C. Effect of ELAM treatment and aging on epigenetic age (DNAmAge) of mouse hearts. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Error bars represent sample means ± standard deviations. D. Effect of ELAM treatment and aging on protein expression of cap-independent translation (CIT) targets. Upper panels: Western blot images of CIT targets Hsp70, TFAM, and MGMT alongside a loading control (β-actin). Lower panels: Quantification of the protein levels of CIT targets. P-values were determined by two-way ANOVA and Tukey’s post-hoc test. P-values for the most relevant comparisons are shown in text. Error bars represent sample means ± standard deviations.