Authors

Calum J. McMullen, Susan Chalmers, Rachel Wood, Margaret R. Cunningham, and Susan Currie

Affiliations

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow (United Kingdom)

Topic

Medical Research

Abstract

"Background: Tyrosine kinase inhibitors (TKIs) have dramatically improved cancer treatment but are known to cause cardiotoxicity. The pathophysiological consequences of TKI therapy are likely to manifest across different cell types of the heart, yet there is little understanding of the differential adverse cellular effects. Cardiac fibroblasts (CFs) play a pivotal role in the repair and remodeling of the heart following insult or injury, yet their involvement in anti-cancer drug induced cardiotoxicity has been largely overlooked. Here, we examine the direct effects of sunitinib malate and imatinib mesylate on adult rat CF viability, Ca2+ handling and mitochondrial function that may contribute to TKI-induced cardiotoxicity. In particular, we investigate whether Ca2+/calmodulin dependent protein kinase II (CaMKII), may be a mediator of TKI-induced effects.

Methods: CF viability in response to chronic treatment with both drugs was assessed using MTT assays and flow cytometry analysis. Calcium mobilization was assessed in CFs loaded with Fluo4-AM and CaMKII activation via oxidation was measured via quantitative immunoblotting. Effects of both drugs on mitochondrial function was determined by live mitochondrial imaging using MitoSOX red.

Results: Treatment of CFs with sunitinib (0.1–10 μM) resulted in concentration-dependent alterations in CF phenotype, with progressively significant cell loss at higher concentrations. Flow cytometry analysis and MTT assays revealed increased cell apoptosis and necrosis with increasing concentrations of sunitinib. In contrast, equivalent concentrations of imatinib resulted in no significant change in cell viability. Both sunitinib and imatinib pre-treatment increased Angiotensin II-induced intracellular Ca2+ mobilization, with only sunitinib resulting in a significant effect and also causing increased CaMKII activation via oxidation. Live cell mitochondrial imaging using MitoSOX red revealed that both sunitinib and imatinib increased mitochondrial superoxide production in a concentration-dependent manner. This effect in response to both drugs was suppressed in the presence of the CaMKII inhibitor KN-93.

Conclusions: Sunitinib and imatinib showed differential effects on CFs, with sunitinib causing marked changes in cell viability at concentrations where imatinib had no effect. Sunitinib caused a significant increase in Angiotensin II-induced intracellular Ca2+ mobilization and both TKIs caused increased mitochondrial superoxide production. Targeted CaMKII inhibition reversed the TKI-induced mitochondrial damage. These findings highlight a new role for CaMKII in TKI-induced cardiotoxicity, particularly at the level of the mitochondria, and confirm differential off-target toxicity in CFs, consistent with the differential selectivity of sunitinib and imatinib.

DOI: https://dx.doi.org/10.3389%2Ffcvm.2020.630480

Extract

MitoSOX fluorescence was recorded on a Nikon TiE inverted epifluorescence microscope with 40 × 1.3 NA oil immersion objective, illuminated with 515 nm excitation light (Pe4000 multi-LED; CoolLED, Andover, UK) and emitted light >550 nm selected by dichroic mirror and captured on an ORCA Flash 4.0 CMOS camera (Hamamatsu, Welwyn Garden City, UK)

Product Associated Features

The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, which covers a wide variety of fluorophores such as MitoSOX.

Product Type

pE-4000

Journal

Front Cardiovasc Med.

Year of Publication

2021

Country of Publication

UK