Advancing a sensitive method for measuring mitochondrial ATP production in small muscle biopsy samples

Area of Science:

• Biochemistry
• Cellular Biology
• Human Physiology

Background:

• Mitochondrial dysfunction is implicated in numerous diseases.
• Accurate measurement of mitochondrial ATP production rate (MAPR) is crucial for diagnosis and research.
• Existing methods can be time-consuming or require large sample volumes.

Purpose of the Study:

• To present an optimized, high-throughput luminometric assay for measuring muscle mitochondrial ATP production rate (MAPR).
• To establish a reference dataset for MAPR across a wide age range.
• To demonstrate the assay's utility in diagnosing mitochondrial disorders and understanding age-related changes in mitochondrial function.

Main Methods:

• Adaptation of a luminometric assay to a 96-well microplate format for MAPR quantification.
• Use of 12+ substrate combinations with 10 μL of isolated mitochondria for rapid ATP production measurement.
• Collection and analysis of a reference dataset from 92 individuals and case studies of three patients with known genetic variants.

Main Results:

• The assay provides accurate and precise MAPR measurements within 15 minutes, with a validated range of 0.3-70 nmol/min/L.
• Reference data from 92 individuals support clinical interpretation.
• Patients with pathogenic variants in mitochondrial DNA (ATP6, MT-TL1) or nuclear PDHA1 gene showed decreased MAPR.
• Adults exhibit 60-80% higher maximal oxidative ATP production capacity than young children, while fatty acid-supported MAPR remains constant with age.

Conclusions:

• The optimized MAPR assay is a robust, efficient, and high-throughput tool for clinical diagnostics and research into mitochondrial disorders.
• The assay effectively identifies decreased MAPR in patients with specific genetic defects.
• Significant age-dependent differences in muscle oxidative ATP production capacity were observed, highlighting developmental changes in mitochondrial function.