MitoTEMPO (MT) and Visomitin (SKQ1) are regareded as mitochondria-targeted antioxidants, which inhibit production of mitochondrial reactive oxygen species (ROS). However, the differences in function between MT and SKQ1 remain unexplored. Herein, we investigated the differential potency of MT and SKQ1 in mitigating oxidative stress under different conditions. The results indicated that high levels of SKQ1 induced cell death. The appropriate concentrations of MT and SKQ1 can prevent or rescue cell damage triggered by hydrogen peroxide (H2O2) and menadione (MEN). MT and SKQ1 reduced ROS levels and reversed the down-regulation of antioxidant defence genes and enzymes. These effects can alleviate the damage to lipids, proteins, and deoxyribonucleic acid (DNA) caused by oxidative stress and restore adenosine 5' triphosphate (ATP) generation. Subsequently, we found that MT administration in ischemic reperfusion kidney injury in mice provided superior renal protection compared to SKQ1, as evidenced by reduced plasma levels of kidney injury markers, improved renal morphology, decreased apoptosis, restored mitochondrial function, and enhanced antioxidant capacity. Overall, our findings suggest that MT is safer and has greater potential than SKQ1 as a therapeutic agent to mitigate oxidative stress damage or oxidative renal injury.

Acute myocardial infarction (MI) is a sudden, severe cardiac ischemic event that results in the death of up to one billion cardiomyocytes (CMs) and subsequent decrease in cardiac function. Engineered cardiac tissues (ECTs) are a promising approach to deliver the necessary mass of CMs to remuscularize the heart. However, the hypoxic environment of the heart post-MI presents a critical challenge for CM engraftment. Here, we present a high-throughput, systematic study targeting several physiological features of human induced pluripotent stem cell-derived CMs (hiPSC-CMs), including metabolism, Wnt signaling, substrate, heat shock, apoptosis, and mitochondrial stabilization, to assess their efficacy in promoting ischemia resistance in hiPSC-CMs. The results of 2D experiments identify hypoxia preconditioning (HPC) and metabolic conditioning as having a significant influence on hiPSC-CM function in normoxia and hypoxia. Within 3D engineered cardiac tissues (ECTs), metabolic conditioning with maturation media (MM), featuring high fatty acid and calcium concentration, results in a 1.5-fold increase in active stress generation as compared to RPMI/B27 control ECTs in normoxic conditions. Yet, this functional improvement is lost after hypoxia treatment. Interestingly, HPC can partially rescue the function of MM-treated ECTs after hypoxia. Our systematic and iterative approach provides a strong foundation for assessing and leveraging in vitro culture conditions to enhance the hypoxia resistance, and thus the successful clinical translation, of hiPSC-CMs in cardiac regenerative therapies.

An analysis of the mitochondrial respiration function represented by the oxygen consumption rate is necessary to assess mitochondrial bioenergetics and redox function. This protocol describes two alternative techniques to evaluate mitochondrial respiration function in situ: (1) measure oxygen consumption rates via an electrode; (2) measure oxygen consumption rates via a seahorse instrument. These in situ approaches provide more physiological access to mitochondria to evaluate mitochondrial respiration function in a relatively integrated cellular system.

FTY720 is an FDA-approved sphingosine derivative drug for the treatment of multiple sclerosis. This compound blocks lymphocyte egress from lymphoid organs and autoimmunity through sphingosine 1-phosphate (S1P) receptor blockage. Drug repurposing of FTY720 has revealed improvements in glucose metabolism and metabolic diseases. Studies also demonstrate that preconditioning with this compound preserves the ATP levels during cardiac ischemia in rats. The molecular mechanisms by which FTY720 promotes metabolism are not well understood. Here, we demonstrate that nanomolar concentrations of the phosphorylated form of FTY720 (FTY720-P), the active ligand of S1P receptor (S1PR), activates mitochondrial respiration and the mitochondrial ATP production rate in AC16 human cardiomyocyte cells. Additionally, FTY720-P increases the number of mitochondrial nucleoids, promotes mitochondrial morphology alterations, and induces activation of STAT3, a transcription factor that promotes mitochondrial function. Notably, the effect of FTY720-P on mitochondrial function was suppressed in the presence of a STAT3 inhibitor. In summary, our results suggest that FTY720 promotes the activation of mitochondrial function, in part, through a STAT3 action.