# Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models

> **NIH NIH R35** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $445,000

## Abstract

ABSTRACT.
Complex V (CV, or ATP synthase) of the electron transport chain is the central enzyme of cellular energy
capture. CV synthesizes ATP driven by the proton gradient generated by the electron transport chain. The
advent of clinical gene sequencing has highlighted the devastating effects of deficiency of this critical enzyme.
Pathogenic variants in CV subunits give rise to multi-system disease, including strokes, neuropathy, ataxia,
retinopathy, and cardiomyopathy. Genetic variation in CV is frequent, and the inability to distinguish pathogenic
mutations from the variants of unknown significance is a clinical challenge preventing understanding of
prognosis and rational approach to management. However, no clinical test for CV function exists. This thwarts
our ability to classify genetic variants. Our Goal is to develop a biochemical approach to evaluating CV function
and ultimately predicting the clinical significance of CV variants. We previously demonstrated that basal ATP
levels are normal with CV deficiency while the rate of ATP synthesis can be low, suggesting that clinical
symptoms result from an inability of CV to accommodate an increased metabolic demand. Direct enzymatic
testing of ATP synthesis by CV is impossible, as the substrate for CV is the proton motive force, which is
dissipated when the enzyme is purified. We therefore propose to assay ATP flux in our human cell (fibroblast,
transmitochondrial cybrid) models of diverse CV genetic variants, including novel candidate genes. We have
observed that the biochemical effects of CV variants result in diverse biochemical sequelae; therefore, we will
also assay oxygen consumption, mitochondrial membrane potential, matrix pH, CV assembly, and
mitochondrial cristae structure and correlate results with clinical manifestations. We anticipate that this
approach will furnish a biochemical and morphologic profile that informs the pathogenicity of the variant. We
hypothesize that the observation that steady-state ATP levels are normal in CV deficient cell-lines despite low
enzymatic flux implies that CV function is responsive to cellular metabolic state. We will introduce a series of
provocative (stimulus-response) testing procedures that involve modulation of nutrient levels (glucose, αKG)
and exposure to cell stress (galactose, lipopolysaccharide). By rigorously investigating the biochemical
consequences of CV deficiency including in dynamic models of cellular stress, we will establish the foundation
on which to develop clinical diagnostic assays to confirm CV mutation pathogenicity and treatment response.
The Central Hypothesis of this proposal is: pathogenic variants in CV subunit genes evoke changes in CV
bioenergic function resulting in diverse downstream biochemical defects that predict clinical presentation.
Further, we propose that the clinical manifestations of Complex V deficiency severity are influenced by the
biochemical and nutritional milieu in which the genetic deficiency find...

## Key facts

- **NIH application ID:** 10883600
- **Project number:** 5R35GM151098-02
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Rebecca Ganetzky
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $445,000
- **Award type:** 5
- **Project period:** 2023-07-15 → 2028-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10883600

## Citation

> US National Institutes of Health, RePORTER application 10883600, Biochemical and Physiological Phenotypes of CV Dysfunction In Human Cell Models (5R35GM151098-02). Retrieved via AI Analytics 2026-07-01 from https://api.ai-analytics.org/grant/nih/10883600. Licensed CC0.

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