Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation.

Serena Van,Nancy J Rusch,Brittney R Garner,Kate Steed,Vijayalakshmi Sridharan,Shengyu Mu,Soumiya Pal,Amanda J Stolarz

doi:10.3389/fphar.2021.727526

Serena Van, Nancy J Rusch + Show 6 more

Open Access

https://doi.org/10.3389/fphar.2021.727526

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Abstract

Background and Purpose: Doxorubicin (DOX) is a risk factor for arm lymphedema in breast cancer patients. We reported that DOX opens ryanodine receptors (RYRs) to enact “calcium leak,” which disrupts the rhythmic contractions of lymph vessels (LVs) to attenuate lymph flow. Here, we evaluated whether dantrolene, a clinically available RYR1 subtype antagonist, prevents the detrimental effects of DOX on lymphatic function.Experimental Approach: Isolated rat mesenteric LVs were cannulated, pressurized (4–5 mm Hg) and equilibrated in physiological salt solution and Fura-2AM. Video microscopy recorded changes in diameter and Fura-2AM fluorescence tracked cytosolic free calcium ([Ca2+ i]). High-speed in vivo microscopy assessed mesenteric lymph flow in anesthetized rats. Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs).Key Results: DOX (10 μmol/L) increased resting [Ca2+ i] by 17.5 ± 3.7% in isolated LVs (n = 11). The rise in [Ca2+ i] was prevented by dantrolene (3 μmol/L; n = 10). A single rapid infusion of DOX (10 mg/kg i.v.) reduced positive volumetric lymph flow to 29.7 ± 10.8% (n = 7) of baseline in mesenteric LVs in vivo. In contrast, flow in LVs superfused with dantrolene (10 μmol/L) only decreased to 76.3 ± 14.0% (n = 7) of baseline in response to DOX infusion. Subsequently, expression of the RYR1 subtype protein as the presumed dantrolene binding site was confirm in isolated mesenteric LMCs by flow cytometry.Conclusion and Implications: We conclude that dantrolene attenuates the acute impairment of lymph flow by DOX and suggest that its prophylactic use in patients subjected to DOX chemotherapy may lower lymphedema risk.

Highlights

Doxorubicin (DOX) is an anthracycline chemotherapeutic agent used as a mainstay treatment for nearly a third of breast cancer patients (Giordano et al, 2012; McGowan et al, 2017)
We reported earlier that DOX disrupted rhythmic contractions in cannulated lymph vessels (LVs), and this effect was partially prevented by DANT (Stolarz et al, 2019)
Rat mesenteric LVs were exposed to increasing bath concentrations of DOX (0.5–20 μmol/L) either in the absence of DANT or 20 min after the addition of DANT (3 μmol/L) to the physiological salt solution (PSS)-filled perfusion chamber

Summary

Introduction

Doxorubicin (DOX) is an anthracycline chemotherapeutic agent used as a mainstay treatment for nearly a third of breast cancer patients (Giordano et al, 2012; McGowan et al, 2017). The search continues for a medication that could protect lymph flow and prevent lymphedema after DOX chemotherapy. In this regard, normal lymph flow relies on the spontaneous and rhythmic contractions of collecting lymph vessels (LVs), which “pump” lymph fluid from the interstitial space to the bloodstream of the systemic circulation to avoid fluid accumulation in peripheral tissues. Disruption of the rhythmic contractions of lymph vessels elevates intraluminal pressure, which can permanently damage the endothelial cells, lymphatic muscle cells (LMCs) and valves that comprise the delicate LVs. The end result is compromised lymph flow (lymphostasis) and development of the clinical manifestation of lymphedema (Zawieja et al, 1991; Ji and Kato, 2001; Nipper and Dixon, 2011; Choi et al, 2015). Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs)

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