Search bioRxiv⌕ Search

Biology subjects

Santos, W. L.

Publications and source records attributed to Santos, W. L..

2 recordsLinked to original sources

Mitochondrial uncoupler BAM15 improves skeletal muscle function and mitochondrial respiration in Sarcopenia

BackgroundAgeing is accompanied by progressive declines in skeletal muscle mass and strength, culminating in sarcopenia, a condition that contributes to frailty, multimorbidity, and mortality. Age-related changes to mitochondria lead to oxidative damage and dysfunction and are proposed to occur early in the trajectory of sarcopenia, supporting the candidacy of mitochondrial-protective therapies. Here, we test the efficacy of mitochondrial uncoupler BAM15 in age-dependent sarcopenic mouse models. MethodsMale and female MitoQC mice aged 24 months received either standard chow or chow supplemented with BAM15 (0.033% mg/g) ad libitum for eight weeks (n=13-14/group). Young (3-month-old) mice served as reference controls (n=8/group). Muscle mitochondrial respiration was assessed in permeabilized fib res, and contractile function was measured in isolated extensor digitorum longus and soleus muscles. Mitophagy was quantified by immunofluorescence confocal microscopy. Data were analyzed using one-or two-way ANOVA followed by Dunnetts or Bonferronis multiple comparison tests. ResultsAged male and female mice exhibited reduced gastrocnemius muscle mass relative to body mass compared with young controls (p<0.05; [~]18% and [~]32% loss, respectively). BAM15 did not alter muscle size but reversed the age-related loss of contractile function in EDL muscles, to that of the young reference controls in both sexes (p<0.05; [~]33% in males, [~]16% in females). In male mice, BAM15 improved mitochondrial efficiency, evidenced by restoration of Complex I-linked respiration and decreased proton leak ([~]52% improvement; p<0.05), and normalized protein levels of oxidative stress marker 4 -HNE, without changes in mitophagy or mitochondrial content. In females, BAM15 did not improve mitochondrial parameters, which may be, in part, due to aged female muscle exhibiting unchanged Complex I leak and 4-HNE protein abundance, alongside lower complex I subunit (NDUFB8) protein abundance. ConclusionsBAM15 improved skeletal muscle mitochondrial efficiency and contractile function in aged male mice, supporting the potential of mitochondrial uncoupling as a therapeutic strategy for sarcopenia.

physiology↗

Saccharomyces cerevisiae as a platform for assessing sphingolipid kinase inhibitors

Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with rank order of potencies of existing inhibitors.

biochemistry↗