A plasma signature of human mitochondrial disease revealed through metabolic profiling of spent media from cultured muscle cells

Abstract

Mutations in either the mitochondrial or nuclear genomes can give rise to respiratory chain disease (RCD), a large class of devastating metabolic disorders. Their clinical management is challenging, in part because we lack facile and accurate biomarkers to aid in diagnosis and in the monitoring of disease progression. Here we introduce a sequential strategy that combines biochemical analysis of spent media from cell culture with analysis of patient plasma to identify disease biomarkers. First, we applied global metabolic profiling to spotlight 32 metabolites whose uptake or secretion kinetics were altered by chemical inhibition of the respiratory chain in cultured muscle . These metabolites span a wide range of pathways and include lactate and alanine, which are used clinically as biomarkers of RCD. We next measured the cell culture-defined metabolites in human plasma to discover that creatine is reproducibly elevated in two independent cohorts of RCD patients, exceeding lactate and alanine in magnitude of elevation and statistical significance. In cell culture extracellular creatine was inversely related to the intracellular phosphocreatine:creatine ratio suggesting that the elevation of plasma creatine in RCD patients signals a low energetic state of tissues using the phosphocreatine shuttle. Our study identifies plasma creatine as a potential biomarker of human mitochondrial dysfunction that could be clinically useful. More generally, we illustrate how spent media from cellular models of disease may provide a window into the biochemical derangements in human plasma, an approach that could, in principle, be extended to a range of complex diseases.

Fig. 1.

Metabolic profile of RC dysfunction in culture media. (A) Thirty-two metabolites were altered significantly (P < 0.05) with rotenone, antimycin, or both inhibitors. Metabolites are ordered by the degree of change from fresh media in the control (DMSO) condition. Each condition was tested in 8 biological replicates. (B) Discordance between complex I and complex III profiles in pathways coupled to the RC. Dashed arrows represent electron flow. Red and green arrows denote trends with rotenone and antimycin, respectively. I, complex I; III, complex III; DHODH, dihydroorotate dehydrogenase; Q, coenzyme Q; UMP, uridine monophosphate.

Fig. 2.

Differences between patient and control groups in plasma metabolites derived from the cellular model. Significance and magnitude of differences between plasma levels of patients and controls in cohort 1, for all 26 metabolites detected in human plasma out of the 32 metabolites altered in cell culture media (Fig. 1A). Metabolites marked in red exhibited a significant difference (P < 0.001).

Fig. 3.

Plasma levels of lactate, alanine, and Cr in two human cohorts. Levels are expressed as chromatographic peak area (arbitrary units). The magnitude (in percentage) and significance (denoted “p”) of the difference between the patient and control groups are shown for each metabolite. Magnitude is calculated based on patient group median relative to control group median.

Fig. 4.

Correlation of Cr release with the intracellular energetic state in cultured muscle cells. (A) The creatine phosphate energy shuttle. ADP, adenosine diphosphate; ATP, adenosine triphosphate; CyCK, cytosolic creatine kinase; MtCK, mitochondrial creatine kinase; OXPHOS, oxidative phosphorylation. (B) Mean PCr, Cr, and total Cr concentrations in C2C12 myotube extract 8 h after treatment with rotenone or DMSO (control). Each condition was measured in eight biological replicates. Error bars indicate SD. (C) Cr release as a function of the intracellular PCr/Cr ratio. Each circle represents an individual culture plate treated with rotenone or DMSO.