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. 2022:2497:207-220.
doi: 10.1007/978-1-0716-2309-1_13.

Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy

Chowdhury S Abdullah  1 , Richa Aishwarya  2 , Mahboob Morshed  1 , Naznin Sultana Remex  2 , Sumitra Miriyala  3 , Manikandan Panchatcharam  3 , Md Shenuarin Bhuiyan  4   5
Affiliations

Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy

Chowdhury S Abdullah et al. Methods Mol Biol. 2022.

Abstract

Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.

Keywords: Doxorubicin-induced cardiomyopathy; Mitochondrial morphology; Mitochondrial respiration; Oxygen consumption rates.

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Figures

Fig. 1
Fig. 1
Monitoring mitochondrial network organization and morphology in cardiomyocytes. Representative confocal fluorescence microscope images demonstrating Dox alone (a), MitoTracker® Red alone (b), and MitoTracker® Green (c, d) labeled neonatal rat cardiomyocytes. (a) DOX (red) accumulates at subcellular locations as appeared as fluorescent dots in the cytosol and diffuse accumulation in the nucleus of cardiomyocytes following DOX-treatment (10 μM, 24 hours). (b) MitoTracker® Red staining reveals mitochondrial network (red) as appeared in a blend of fragmented, short tubular, and long tubular forms. Hence, we do not recommend the use of MitoTracker Red dyes to use in DOX-treated cells to avoid potential overlap between them in captured fluorescence images which may lead to erroneous interpretation of the data (please see Note 5 for explanation). (c, d) MitoTracker® Green staining reveals a mixture of a fragmented, short, and long tubular network of mitochondria (green) in vehicle (c) treated cardiomyocytes while DOX treatment (10 μM, 24 hours) resulted in hyperfused tubular mitochondrial network in cardiomyocytes
Fig. 2
Fig. 2
Measurement of mitochondrial respiration in DOX-treated neonatal rat cardiomyocytes. Examples of oxygen consumption rates (OCR) tracing in vehicle and DOX-treated (10 μM, 24 h) cardiomyocytes. Oligomycin (1 μM), FCCP (4 μM), Rotenone (0.5 μM) and Antimycin A (0.5 μM) were sequentially injected at indicated time points (black arrows). OCR values are represented as pmol/min/μg of protein. Numerical values indicate data points where average values from replicate wells at particular time points are analyzed and plotted for OCR profiles in vehicle and DOX-treated cardiomyocyte wells
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