Pacemaking and pacemaker current in cardiac and neuronal cells: a multi-method electrophysiological approach for evaluating innovative blockers and modulatory signals

Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are membrane proteins, which mediate a Na+/K+ current (If/Ih), that is activated by hyperpolarization and regulated by intracellular cAMP/cGMP levels. HCN channel family is composed by four isoforms (HCN1-4) that are expressed in the heart and the nervous system, where they are involved in pacemaker activities, excitability and pain perception. Alterations of expression and/or function of these channels are associated to pathological conditions such as heart hypertrophy and failure, neuropathic pain and specific form of epilepsy. For these reasons, HCN channels represent valuable targets to uncover novel pharmacological molecules and endogenous mediators capable to modulate their function or dysfunction in cardiac and neuronal physiology and pathology. Ivabradine is the first and only HCN blocker employed in current clinical use as specific bradycardic agent in patients with stable angina and failure, intolerant or not-responsive to -blockers. Furthermore, the function of distinct HCN isoforms in the central and peripheral nervous system has opened to new applications of HCN blockers. In line with this, a recent clinical study has indicated ivabradine as effective drug for neuropathic pain relief, further confirming the potentials of HCN blockers as innovative drugs for neurological disorders. However, ivabradine is unable to discriminate among the four channel isoforms, thereby lacking tissue specificity, similarly to other HCN antagonists such as zatebradine and cilobradine. During the last decade, the research group I joined during my PhD has identified novel zatebradine derivatives among which MEL57A and MEL55A display higher selectivity toward HCN1/HCN2 isoforms over HCN4 and have been successfully tested in animal models of neuropathic pain; differently, EC18 displays higher selectivity toward HCN4 isoform and has proved effective against specific forms of experimental epilepsy. Despite main physiological signals involved in HCN channel modulation and control of pacemaker function have been thoroughly characterized over the years, much less information is available on the effect of inflammatory signals, including those of the cytokine IL-6. The latter is growingly emerging as multichannel modulator in the heart, where different arrhythmic disturbance have been associated to its elevate plasma levels. This notion consolidated the hypothesis that IL-6 may play a role as a direct modulator of cardiac pacemaker activity leading us to test IL-6 in appropriate models of pacemaking. Following on these premises, in this thesis I studied the effects of two new zatebradine analogues, PK9 and PK19, synthesized by Prof. Romanelli and co-workers (Department of NEUROFARBA, University of Florence) as HCN channel blockers; furthermore, in the setting of inflammatory mediators, I characterized the effects of the human cytokine IL-6 on cardiac pacemaker activity. To these purposes, I used a multimethod approach that enabled me to accomplish different tasks. The electrophysiological effects of PK19 and PK9 on HCN-mediated current were studied by using single-cell patch-clamp recordings (whole-cell configuration) on two cell models: HEK293 cells expressing recombinant homo-tetrameric mouse HCN1, mouse HCN2 and human HCN4 channel isoforms, and rat Dorsal Root Ganglia (rDRG) neurons in primary culture that express functional homo- and hetero-tetrameric HCN channels. In order to study the spontaneous electrical activity of human cardiomyocyte monolayer derived from pluripotent cells (hiPSCs), thanks to a fruitful collaboration with Prof. Sacconi, I developed the optical High Throughput System MULTIPLE, which allowed to assess the effects of both zatebradine analogues and of human IL-6. Finally, to further enhance my knowledge and expertise on hiPSCs derivation and differentiation, I spent a six-month research activity at the University Medical Center of Utrecht (The Netherlands), where, under the supervision of Prof. Stillitano, I developed and characterized six novel hiPSC lines using most updated cell culture biology and molecular techniques. Results on properties of novel zatebradine derivatives demonstrated that PK19 and PK9 have a stronger selectivity and a higher potency toward HCN2 and HCN4 isoforms over HCN1 compared to the previous derivatives ivabradine, MEL55A and MEL57A. For HCN2- and HCN4-currents measured at -80 mV IC50 were 0.32 and 0.24 μM, while at -120 mV IC50 were 0.12 and 0.11 μM, respectively. Moreover, at -80 mV, 5 μM PK19 was more effective on HCN2- and HCN4-mediated currents that were reduced by 90 and 86%, respectively, compared to HCN1-current, which was reduced by 58%. In the same conditions, PK9 reduced HCN2- and HCN4-currents by 100% and 65%, respectively, and HCN1-current by 61%. These results are in line with those obtained in rDRG neurons, where HCN2 and HCN1 are the prevalent isoforms. In this setting PK9 was 5-fold more potent than PK19 at physiological potential (IC50 0.36 and 1.52 μM, respectively), although reduction of current conductance at 5 μM was comparable (92% by PK9, 88% by PK19). Finally, PK9 and PK19 showed a rate-lowering action on frequency of spontaneous action potentials in hiPSC-CMs, displaying a 15 to 30-fold higher potency, respectively, compared to ivabradine, which was taken as reference drug. Taken together, these results suggest PK9 and PK19 may represent leading compounds to further investigate the physiological and pathological role of HCN2/4 isoforms in cardiac and neuronal tissues and develop additional compounds with HCN2 or HCN4 inhibitory function. Future experiments are necessary to explore the effects of PK19 and PK9 in appropriate animal models of neuropathic pain and cardiac disease. Data on acute effects of IL-6 on cardiomyocytes differentiated from hiPSCs demonstrated that the cytokine dose-dependently reduces the frequency of the spontaneous action potentials and the slope of diastolic depolarization, uncovering a typical hallmark of HCN channel blockade. The reduction of spontaneous activity is partially prevented by Tocilizumab, an IL-6 receptor antagonist. These results demonstrate that IL-6 acts as negative modulator of cardiac pacemaking mainly through interaction with its cognate receptor. The finding uncovers a novel modulatory signal of cardiac pacemaking that provides a basis to better understand the ionic mechanisms underlining human heart rate modifications in clinical conditions related to inflammatory diseases, immune response and aging.