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Molecular Analysis of Mitochondrial Compromise in Rodent Cardiomyocytes Exposed Long Term to Nucleoside Reverse Transcriptase Inhibitors (NRTIs)

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Abstract

Despite the highly effective impact of NRTI therapy in patients infected with the human immunodeficiency virus type 1 (HIV-1), long-term treatment has revealed cardiotoxicity, considered to be due to mitochondrial dysfunction. To evaluate mitochondrial damage, and design therapeutic interventions, we established cultures of rat H9c2 and mouse HL-1 cardiomyocytes and exposed them to the NRTIs zidovudine (AZT), and AZT plus didanosine (ddI). Proliferation assays showed that H9c2 cells grew well in 50 μM AZT and 50 μM AZT/50 μM ddI and that HL-1 cells grew well in 10 μM AZT and 10 μM AZT/10 μM ddI. Both types of cells were exposed to the drugs for 39 passages (P), and mitochondrial integrity in the form of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was examined by Seahorse XF24 analyzer. In NRTI-exposed H9c2 cells at most passages, OCR was reduced, in both the basal and uncoupled states, compared to unexposed controls (P < 0.05). NRTI-exposed HL-1 cells showed a different pattern of mitochondrial compromise, with inhibition of OCR, in basal and uncoupled cells, occurring largely before P14 and after P17 (P < 0.05). The ECAR response in uncoupled cells of both types was unchanged at early passages, but increased after P18 (P < 0.05). Evaluation of mitochondrial biogenesis in H9c2 cells revealed reduction before P29, no change at P29, and reduction at P39 in NRTI-exposed cells, compared to unexposed cells (P < 0.05). Western blotting of transcription factors critical for mitochondrial biogenesis, PGC-1α, Nrf-1 and mtTFA, showed downregulation in NRTI-exposed H9c2 cells compared to unexposed controls. In addition, electron microscopy (EM) revealed increasing mitochondrial morphological damage in H9c2 cells over passages. For both cell types, AZT/ddI was more damaging than AZT alone. These studies demonstrate progressive mitochondrial compromise in cardiomyocytes-exposed long term, and the model will be used to evaluate potentially protective intervention strategies.

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Abbreviations

HIV-1:

Human immunodeficiency virus type 1

ARV:

Antiretroviral

ATP:

Adenosine-5′-triphosphate

AZT:

Zidovudine, 3′-azido-3′-deoxythymidine

ddI:

Didanosine, 2′, 3′-dideoxyinosine

NRTI:

Nucleoside reverse transcriptase inhibitor

FCCP:

Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone

OLIGO:

Oligomycin

ROT:

Rotenone

HL-1:

Mouse cardiomyocytes

H9c2:

Rat cardiomyocytes

OCR:

Oxygen consumption rate

ECAR:

Extracellular acidification rate

EM:

Electron microscopy

COX-1:

Cytochrome c oxidase-1

SDH-A:

Succinate dehydrogenase-A

mtDNA:

Mitochondrial DNA

nDNA:

Nuclear DNA

OXPHOS:

Oxidative phosphorylation

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Acknowledgments

We wish to thank Dr. Aleksandra Michalowski for statistical consultation. We would also like to thank Dr. Kevin Bittman and Tony Nardei of Seahorse Bioscience Inc. for their help. This study was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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  1. Carcinogen-DNA Interactions Section, Laboratory of Cancer Biology and Genetics, CCR, National Cancer Institute, Bldg 37 Rm 4032, NIH, 37 Convent Drive, MSC-4255, Bethesda, MD, 20892-4255, USA

    Yongmin Liu, Phuonggiang Nguyen, Tara Z. Baris & Miriam C. Poirier

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  1. Yongmin Liu
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  2. Phuonggiang Nguyen
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Liu, Y., Nguyen, P., Baris, T.Z. et al. Molecular Analysis of Mitochondrial Compromise in Rodent Cardiomyocytes Exposed Long Term to Nucleoside Reverse Transcriptase Inhibitors (NRTIs). Cardiovasc Toxicol 12, 123–134 (2012). https://doi.org/10.1007/s12012-011-9148-5

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