The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Mitochondrial bioenergetic function is a central component of cellular metabolism in health and disease. Mitochondrial oxidative phosphorylation is critical for maintaining energetic homeostasis, and impairment of mitochondrial function underlies the development and progression of metabolic diseases and aging. However, measurement of mitochondrial bioenergetic function can be challenging in human samples due to limitations in the size of the collected sample. Furthermore, the collection of samples from human cohorts is often spread over multiple days and locations, which makes immediate sample processing and bioenergetics analysis challenging. Therefore, sample selection and choice of tests should be carefully considered. Basic research, clinical trials, and mitochondrial disease diagnosis rely primarily on skeletal muscle samples. However, obtaining skeletal muscle biopsies requires an appropriate clinical setting and specialized personnel, making skeletal muscle a less suitable tissue for certain research studies. Circulating white blood cells and platelets offer a promising primary tissue alternative to biopsies for the study of mitochondrial bioenergetics. Recent advances in frozen respirometry protocols combined with the utilization of minimally invasive and non-invasive samples may provide promise for future mitochondrial research studies in humans. Here we review the human samples commonly used for the measurement of mitochondrial bioenergetics with a focus on the advantages and limitations of each sample.

The ketogenic diet (KD) has demonstrated benefits in numerous clinical studies and animal models of disease in modulating the immune response and promoting a systemic anti-inflammatory state. Here we investigate the effects of a KD on systemic toxicity in mice following SARS-CoV-2 infection. Our data indicate that under KD, SARS-CoV-2 reduces weight loss with overall improved animal survival. Muted multi-organ transcriptional reprogramming and metabolism rewiring suggest that a KD initiates and mitigates systemic changes induced by the virus. We observed reduced metalloproteases and increased inflammatory homeostatic protein transcription in the heart, with decreased serum pro-inflammatory cytokines (i.e., TNF-α, IL-15, IL-22, G-CSF, M-CSF, MCP-1), metabolic markers of inflammation (i.e., kynurenine/tryptophane ratio), and inflammatory prostaglandins, indicative of reduced systemic inflammation in animals infected under a KD. Taken together, these data suggest that a KD can alter the transcriptional and metabolic response in animals following SARS-CoV-2 infection with improved mice health, reduced inflammation, and restored amino acid, nucleotide, lipid, and energy currency metabolism.