CARDIAC PACEMAKING: PATHOPHYSIOLOGY AND PHARMACOLOGY OF THE IF CURRENT

During my Ph.D. I focussed my experiments on the investigation of physiopathological and pharmacological role of the cardiac sinoatrial If since it has a key role in the generation and modulation of cardiac pacemaker activity. The identification of pharmacological agents able to reduce sinus heart rate has a strong interest in the clinic since they could be useful in the treatment of ischemic heart disease. Despite longstanding and intense investigation at present there is only one such agent (ivabradine) that has reached therapeutic application since all other compounds tested have shown undesired side-effects. I therefore investigated the effect of Tong Mai Yan Xin (TMYX), a drug currently used in China as a cardiac regulator of both brady- and tachy-cardic condition. Electrophysiological experiments performed on rabbit SA node cells have shown a dose-dependent slowing effect of TMYX on pacemaking rate, associated with a reduction of the early part of the pacemaker depolarization and with a moderate prolongation of APD50. The investigation of the effects of TMYX on the If current, the major contributor of diastolic depolarization phase, has revealed a dual effect: TMYX causes a leftward-shift of the activation current curve and an increase of the channel conductance. At physiological potential the bradycardic action (leftward shift) strongly prevails, thus confirming the effect on the spontaneous automaticity observed in SA node cells. The effect of TMYX was also evaluated in freely moving mice implanted with ECG transmitters. Preliminary experiments surprisingly show an increment of heart rate after the i.p. injection of the drug, while when TMYX was delivered during pharmacological blockade of only sympathetic or both sympathetic and parasympathetic autonomic system, a deep bradycardia was observed. Given the importance of the If current in the pacemaking process, it is important characterize the functional role of mutations in the HCN4 channels, the molecular constituent of If current, that are associated with clinically relevant modification of heart rate. So far all mutations reported in the literature are associated with tachycardic conditions. During my study I had the opportunity to investigate the properties of the R524Q mutation which is located in the first portion of C-linker, a region connecting the S6 transmembrane domain to the CNBD; this mutation cosegregates in familial members affected by inappropriate sinus tachycardia. Electrophysiological analysis carried out on HEK 293 cells showed an increment of cAMP sensitivity in the R524Q channels resulting in a rightward shift of the activation curve that mimic the effect of β-adrenergic stimulation. When transfected with mutant rather than wild-type HCN channels newborn ventricular myocytes, an excitable cellular model used to test the effect of mutations on spontaneous rate, showed a faster pacemaking rate. Finally I carried out some experiments to test the quantitative relevance of the If current during pacemaking. The study was carried out by means of an indirect approach based on mathematical models of SA node action potentials combined with the dynamic-clamp technique. I compared the models developed by Maltsev-Lakatta and Severi-DiFrancesco. Both these formulations describe the SA node automaticity, but they have differences in quantitative contribution of If current. My experiments allowed to validate the Severi-DiFrancesco model, that is associate with a higher contribution of If current during the diastolic depolarization than Maltsev-Lakatta formulation. In conclusion, using independent and separate approaches, my experiments confirm that alterations of the amount of the If current flowing during the diastolic depolarization leads to robust effect on automaticity of the SA node.