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

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 Cloride, ivabradine and ZD-7288. Compared to control, exposure to 10 mM CsCl reduced (- 65%, n=13, p<0.01) the contraction frequency (FL) and increased end-diastolic diameter (DL-d, + 7.3 ± 0.7%, p<0.01) without changes in end-systolic (DL-s) diameter. 300 µM ivabradine abolished contraction and increased DL-d (≈ 14%, n=10, p<0.01) or caused an incomplete inhibition of FL (n=3, p<0.01), leaving DL-d and DL-s unaltered. 200 µM ZD-7288, completely (n=12, p<0.01) abolished FL, while DL-d decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channels 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.