Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow

Abstract

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 mmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% 6 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca 21 concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 mmol/l) maximally reduced volumetric lymph flow to 34% 6 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% 6 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

The Development and Treatment of Lymphatic Dysfunction in Cancer Patients and Survivors

“…Such lymphatic dysfunction can occur prior to treatments of surgical dissection and/or RT, suggesting that cancer progression, particularly LN metastasis, contributes to lymphatic dysfunction in cancer patients prior to surgery and RT. While there is evidence that chemotherapy may potentially contribute to the development of cancer-acquired LE [66][67][68][69], further longitudinal studies are needed to understand all the contributing factors that can cause lymphatic dysfunction. Nonetheless, recent results in Figure 7 confirm the results of other studies showing subclinical edema measured with bioimpedance of water content in the arms in breast cancer patients prior to first-line treatment [70,71].…”

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

“…DOX appears to impair lymph flow by tonically activating ryanodine receptors (RYRs) to mediate Ca 2+ release from the sarcoplasmic reticulum of LMCs, thereby causing "calcium leak" and establishing persistently elevated levels of cytosolic free calcium ([Ca 2+ i ]). Because rhythmic contractions of LMCs rely on the cyclic elevation of [Ca 2+ i ] to mediate contraction, followed by restoration of resting levels of [Ca 2+ i ] to permit relaxation, the tonic "calcium leak" induced by DOX disrupts rhythmic contractions and impairs lymph flow (Stolarz et al, 2019). Importantly, we noted that rhythmic contractions in isolated LVs can be restored by blocking RYRs using high concentrations of ryanodine or therapeutic concentrations of dantrolene (DANT).…”

“…In this respect, we recently reported that DOX acutely disrupts the rhythmic contractions of isolated rat mesenteric LVs and attenuates lymph flow in vivo at clinically achievable plasma concentrations (Stolarz et al, 2019). DOX appears to impair lymph flow by tonically activating ryanodine receptors (RYRs) to mediate Ca 2+ release from the sarcoplasmic reticulum of LMCs, thereby causing "calcium leak" and establishing persistently elevated levels of cytosolic free calcium ([Ca 2+ i ]).…”

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema

“…A single LV was visualized, and increasing concentrations of cromakalim or minoxidil sulfate (0.01–10 μmol/l; log units) were added to the superfusate bathing the mesentery for 15 minutes at each drug concentration. Customized software developed in collaboration with our institution’s Nanomedicine Center was used to track individual lymph cells in flow as described earlier in detail ( Sarimollaoglu et al, 2018 ; Stolarz et al, 2019 ). Using these data files, flow velocity and positive volumetric flow were calculated to evaluate the effect of KCOs on lymph flow in vivo.…”

“…Origin 9.1 software (OriginLab Corporation, Northampton, MA, USA) was used to analyze the following contractile parameters: end diastolic diameter (EDD), end systolic diameter (ESD), frequency of contraction, and amplitude of contraction. Flow per unit length in a single lymph vessel was calculated using the equation: π/4(EDD 2 -ESD 2 )F, as previously described (Stolarz et al, 2019).…”