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Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs), which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens, including spontaneously contracting lymphatics, were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution, and treated with HCN channels inhibitors cesium chloride (CsCl), ivabradine, and ZD-7288. Compared with control, exposure to 10 mM CsCl reduced (-65%, n = 13, P &lt; 0.01) the contraction frequency (F L ) and increased end-diastolic diameter (D L-d , +7.3%, P &lt; 0.01) without changes in end-systolic diameter (D L-s ). Ivabradine (300 μM) abolished contraction and increased D L-d (-14%, n = 10, P &lt; 0.01) or caused an incomplete inhibition of F L (n = 3, P &lt; 0.01), leaving D L-d and D L-s unaltered. ZD-7288 (200 μM) completely (n = 12, P &lt; 0.01) abolished F L , while D L-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channel isoforms, likely arranged in different configurations, in LMCs. Hence, all together, data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs.

Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics