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Mitochondrial Function - Metabolism Assay

Cardiotoxicity is a major cause of drug attrition in preclinical and clinical drug development. Our Mitochondrial Function Service offers a structural cardiotoxicity assay based on oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and morphology of mitochondrial network of human iPSC-derived cardiomyocytes.

What are your benefits?

1.

2.

3. Enabling multiplexed assays
Compatible with a wide range of electrophysiology assays such as MEA, impedance or Ca2+-flux

Introduction

Cardiotoxicity occurring as a result of acute or chronic exposure to medications is one of the most common adverse clinical side-effects, placing a significant burden on both pharmaceutical companies and healthcare providers.

To maintain their physiological function, cardiomyocytes need to possess an abundant network of mitochondria and require a constant supply of respiratory substrates to meet their high energy demand. Disruption in the ATP-generating pathways (oxidative phosphorylation and glycolysis) or the state of mitochondrial network would significantly impact on cardiac health. Therefore, monitoring drug-induced dysfunction in these processes can be a significant contributor to evaluating cardiotoxicity.

Service specifications

Cell type

Human iPSC-derived cardiomyocytes

Service type

Metabolism assay

Format

96 wells

Time points

24 hours after compound addition

Compound concentration*

0.01, 0.1, 1, 10 µM

Positive control

Doxorubicin

DMS-986094

Vehicle control

0.1% DMSO

Parameters

Oxygen consumption rate (OCR)

Extracellular acidification rate (ECAR)

Morphology of mitochondrial network

Timelines

 

Deliverable

A study protocol will be agreed before study initiation. Results will be presented in a study report.

* Suggested concentrations, to be agreed with client

Case Study

Monitoring of mitochondrial function and health in Ncyte hiPSC-derived cardiomyocytes.

Methods

The number, function and morphology of mitochondria are critical markers of hiPSC-CM maturity and function. Therefore, we first studied culture-conditions enabling these fetal-like cells to expand their oxidative capacity towards an adult-like state by promoting a shift from glycolysis to fatty acid oxidation. hiPSC-derived ventricular-like cardiomyocytes (Ncyte CMs) manufactured using Ncardia’s proprietary protocols were cultured according to manufacturer’s instructions. Oxygen consumption rate (OCR) indicative of oxidative phosphorylation and extracellular acidification rate (ECAR), a marker of glycolysis, were measured using Agilent Ph-Xtra and MitoXpress kits. For signal detection a BMG plate reader in Dual-read Ratiometric Time Resolved Fluorescent mode was utilized. Mitochondria were stained with MitoTracker Red CMX Ros for morphological assessment. High-magnification images (40x) were acquired using an ImageXpress Micro Confocal microscope and image analysis performed in MetaXpress software version 6.6.

Results

First, OCR and ECAR as well as mitochondrial fluorescence signal readouts were used to facilitate selection of hiPSC-CM culture medium compositions that resulted in mitochondrial function and morphology changes indicative of maturation. OCR and ECAR assays were standardized using a small set of validation compounds consisting of known inhibitors of the electron transport chain and glycolysis. Subsequently, changes induced by two known cardiotoxic compounds (Doxorubicin and BMS-986094) that are closely associated with mitochondrial dysfunction were studied.

Conclusions

Our results show the diverse applicability of standardized metabolic assays to monitor mitochondrial function in hiPSC-CMs. This set of assays can aid in gaining a deeper understanding of metabolic dysfunction as a mechanism for drug-induced cardiotoxicity.

Graph showing the effect of known cardiotoxic compounds on mitochondrial function of iPSC-derived cardiomyocytes

Figure: Doxorubicin, an anthracycline with well-known metabolic effects on cardiomyocytes, decreased OCR and increased ECAR per cell in a dose-dependent manner, indicating cellular stress. It also significantly decreased cell numbers at concentrations >0.1 µM. BMS-986094, a compound withdrawn from the clinic due to cardiac safety concerns, decreased OCR starting at 1 µM in a dose-dependent manner, whereas it only reduced ECAR at 100 µM, and did not affect cell numbers.

Workflow

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