INVESTIGATION ON PHARMACOLOGICAL AND AGE-INDUCED MODULATIONS OF CARDIAC PEACEMAKING AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF A COMPOUND MUTATION IN THE CARDIAC SODIUM CHANNEL

Abstract: During my Ph.D. internship at the ‘PaceLab’, I have been involved in several scientific projects that, although involving different experimental models and with different aims, share the common background of cardiac pacemaker modulation and ion channels activity. In this sec-tion are briefly described the three main research lines in which I took part; a more complete discussion of the data will be presented later in this thesis. Notably, by the time I am writing this proposition, two of these studies have been submitted to scientific journals. - Identification of the bradycardic molecule contained in the traditional Chinese medi-cine drug Tongmai Yangxin: The identification of new bradycardic agents able to specifically bind HCN channels, rises great interest in the scientific community since a decreased cardiac pacemaker cur-rent would lead to an general heart rate lowering without side-effects. To date, despite a long and intensive investigation, only one pure bradycardic agent (Ivabradine)1-3 is used in the clinical setting. Ivabradine is considered virtually free of negative side-effects even though, in some rare cases, it can elicit minor optical limitations. A few years ago, our research group started a collaboration with a traditional Chinese medicine (TMC) drug producer (Le Ren Tang Pharmaceutical Factory, Tianjin, China) with the aim of unravelling the molecular mechanism of one of their products: Tongmai Yangxin (TMYX). This drug is currently used in China for the treatment of several car-diac diseases like coronary artery disease, palpitation, heart failure and angina4 and, like other TCM compounds, it is a mixture of botanical and animal products. Remarkably, recent investigations highlighted its ability to reduce cardiac metabolic disorders, oxida-tive stress and inflammation on stable angina patients4-6. Our previous studies explored the bradycardic action of this drug; in particular, its ef-fects were evaluated on freshly isolated rabbit SAN myocytes. TMYX displayed a dose dependent and fully reversible rate slowing of the spontaneous action potential (AP) ac-tivity, specifically acting on the pacemaker current by shifting the activation curve of HCN channels towards more negative potentials; furthermore, its efficacy appeared to be strictly correlated to the intracellular cAMP concentration. Detailed analysis demon-strate that this drug acts as a functional cAMP surmountable competitive antagonist, competing for the CNBD of f-channels according to a mode of action never observed before for the regulation of this current. Still, the nature of the bio-active molecule is un-known. With the aim of identify this principle, TMYX was divided into 4 fractions (F1-4) by our Chinese colleagues at the Pharmaceutical Informatics Institute of Zhejiang University (Hangzhou, China) according to the solubility of its components, and the ability of each preparation to modulate spontaneous rate and the If current was evaluated by patch-clamp experiments in rabbit SAN myocytes. Data clearly demonstrate that only the most hydrophilic fraction (F1) displayed features similar to the total drug, decreasing the AP rate up to ~20% specifically acting on the pacemaker current. To further narrow down the number of molecules to examine, F1 was subsequently di-vided into 4 sub-fractions (F1.1-1.4) and, as for the previous preparations, their effects were examined on rabbit SAN myocytes. The results pointed out the presence of the target molecule both in F1.1 and F1.2 since they were able to reduce the spontaneous AP firing by ~25 and ~20%, respectively, with a specific action on If current. Eventually, HPLC data revealed the presence of uridine in both these sub-fraction, sug-gesting that this molecule (or one of its derivates) could be involved in the bradycardic process. Therefore, a preliminary experiment was carried out to analyze its possible car-diac rate regulation properties on rabbit SAN myocytes. Surprisingly, the perfusion of uridine (1 μM) generated a small increase in the AP rate (+3.45%) but a decrease in the pacemaker current at -65mV. Additional experiment are requested in order to shed more light on the properties of this molecule, however, given that uridine, and in partic-ular its cyclic nuclidic form (cUMP), have been reported to interact with some isoforms of the HCN channels family7-9, it appears to be a good starting point for the identifica-tion of the active principle of TMYX. - Age-related changes in cardiac autonomic modulation and heart rate variability in mice: The incidence of mortality caused by age-associated cardiovascular diseases is increas-ing dramatically and it will represent a serious clinical issue in the next decades10,11. In humans, the natural process of aging is associated with progressive changes in cardi-ac autonomic nervous system (ANS) regulation that may predispose to higher cardiac risks. Animal models of aging are needed to gain insights into the relation between the aging of the ANS and cardiac pathophysiology. Specifically, the aim of this study is to verify the translational relevance of mouse models for further in-depth evaluation of the link between cardiac ANS regulation and increased arrhythmic risk with advancing age. Therefore, heart rate and time- and frequency-domain indexes of HRV were calculated from ECG recordings in two groups of conscious C57BL6/J male mice of different ages (4- and 19-months-old) during daily undisturbed conditions following peripheral β‐adrenergic (atenolol), muscarinic (methylscopolamine), and β‐adrenergic + muscarinic blockades and β‐adrenergic (isoprenaline) stimulation. Eventually, vulnerability to ar-rhythmias was evaluated during daily, undisturbed conditions and following β‐adrenergic stimulation. HRV analysis and heart rate responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect did not however affect basal heart rate, since it compensated for the lower intrinsic heart rate observed in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain indexes of HRV were reduced fol-lowing muscarinic, but not β‐adrenergic, blockade, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnera-bility to both spontaneous and isoprenaline-induced arrhythmias. These results reveal the presence of a reduced cardiac vagal modulation and HRV asso-ciated with an increased vulnerability to cardiac arrhythmias in older mice, which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart. - Electrophysiological characterization of a SCN5A compound mutation (K1578N-G1866fs) discovered in a young patient affected by sinus node disfunction, atrial flut-ters and drug-induced long QT syndrome: Given its high relevance in the generation of the cardiac AP event, alterations in the ge-netic sequence encoding for NaV1.5 channel (SCN5A) are often related to severe dis-function in the heart function12. In this situation, a compound mutation (K1578N/G1866fs) has been reported in a child affected by severe bradycardia, atrial flutter and drug-induced QT prolongation. Nota-bly, the parents, who present a heterozygous mutation each, did not suffer of any cardi-ac problem and their ECG signals were unremarkable. Following clinical examinations, the diagnosis was sinus node disfunction and the patient was implanted with a pace-maker13. With this study we intended to characterize the electrical properties of the Na+ current carried by the mutated NaV1.5 channels in order to better understand the impact of these alteration and explore whether the patient will benefit from a specific pharmaco-logical treatment. According to the literature14,15, SCN5A gene can undergo a series of alternative splicing events, among which the inclusion of different exon 6 sequences that identify the neona-tal and the adult NaV1.5 isoform. Consequently, the electrical difference between these two isoforms was assessed by patch-clamp experiments on HEK-293 cells transfected with a vector containing their genetic sequence. The results displayed no differences in the current density compared to the adult isoform (HP: -120 mV) while a positive shift of both activation and inactivation curves were detected (5.1 mV and 8.8 mV, respec-tively). The impacts of the mutations on the Na+ current were then evaluated on the same mod-el both in the patient (compound mutation K1578N/G1866fs in the neonatal isoform) and in the parents (heterozygous expression in the adult isoform) conditions. For what concerns the parents, the data collected are reasonably in agreement with the clinical in-vestigation indicating no pathologic conditions. On the other hand, the transfection of both the mutations in the neonatal SCN5A isoform caused a dramatic reduction in the current density associated to a rightward shift of the activation curve (6.5 mV), com-pared to the corresponding WT isoform. In addition, since the time at which the neonatal to adult isoform switch occurs has still not been clearly identify, the compound mutation was also inserted in the WT adult vector. However, no changes were detected in the current density while a leftward shift of the activation curve (7.5 mV), which can be ascribed to the isoform change, suggested that the alterations have a similar effect on the activation kinetics regarding of the iso-form in which are expressed. Eventually, no changes in the recovery from inactivation process were found between all the conditions investigated. All in all, these data show an important loss-of-function of NaV1.5 channels in the pa-tient’s condition, suggesting that the two mutations (K1578N–G1866fs), when ex-pressed together, generate a far worst phenotype than their single heterozygous expres-sion. Furthermore, even when incorporated in the adult SCN5A isoform, the effects caused by the alterations did not change suggesting that the neonatal to adult isoform switch will probably grant no benefits for the patient.