# Mitochondrial permeability transition pore desensitization by a novel dispiranic derivative prevents cardiac reperfusion injury

## TL;DR

A new compound prevents heart damage during reperfusion by inhibiting mitochondrial pore opening without harming cell energy production.

## Contribution

A novel dispiranic compound (11d) is identified as a selective PTP desensitizer targeting ATP synthase c subunit.

## Key findings

- Compound 11d inhibits PTP opening by ~70% in human cardiomyocytes at low micromolar concentrations.
- 11d selectively binds to ATP synthase c-ring and reduces cell death and ROS in hypoxia/reoxygenation models.
- Ex vivo heart models show 11d improves cardiac recovery and reduces infarct cell death during reperfusion.

## Abstract

Mitochondrial permeability transition pore (PTP) opening is a major determinant of cardiac ischemia/reperfusion (I/R) injury, contributing to cardiomyocyte death and impaired cardiac function following revascularization. Despite extensive research, effective pharmacological strategies targeting PTP remain limited. Here, we report the identification and characterization of a novel class of dispiranic small molecules designed to inhibit PTP opening by targeting the c subunit of FO-ATP synthase. Using a multidisciplinary approach combining chemical synthesis, cellular and mitochondrial assays, ex vivo cardiac models and in silico analyses, we identified compound 11d as a potent and selective PTP desensitizer. In living human cardiomyocytes, 11d inhibited PTP opening by approximately 70% at low micromolar concentrations without impairing mitochondrial bioenergetics under basal conditions. Mechanistically, 11d selectively inhibited Ca2+-activated F1FO-ATP synthase activity, consistent with PTP modulation, while sparing Mg2+-dependent ATP synthesis and succinate oxidase activity. Molecular docking and molecular dynamics simulations revealed stable binding of 11d within the canonical oligomycin-binding pocket of the ATP synthase c-ring, a finding corroborated by cellular thermal shift assays and subcellular fractionation showing preferential mitochondrial accumulation. Functionally, 11d significantly reduced cell death, mitochondrial reactive oxygen species (ROS) production and PTP opening in cardiomyocytes and endothelial cells subjected to hypoxia/reoxygenation (H/R). Importantly, in an ex vivo Langendorff rat heart model, 11d administration at reperfusion markedly improved cardiac functional recovery, reduced infarct-associated cell death and preserved myocardial architecture. Collectively, these results identify 11d as a promising lead compound for cardioprotection and support targeting the ATP synthase c subunit as a viable therapeutic strategy against reperfusion injury.