Novel pharmacological options for cardiac hypertrophy: a functional study in human myocardium

Cardiac hypertrophy is a pathological condition characterized by the enlargement of the cardiac walls, occurring in both hypertrophic cardiomyopathy (HCM) and aortic stenosis (AoS). HCM is a genetically determined disease recognized as one of the leading causes of sudden cardiac death in the young population. In HCM, large cardiac remodelling begins with thickening of the left ventricle, which impairs diastolic filling and leads to arrhythmias. This disease is characterized by significant electrophysiological abnormalities, Ca2+ handling alterations and myocardial fibrosis. Treatment involves invasive interventions such as myectomy, or drug therapy with β-blockers, Ca2+ channel blockers, class IA antiarrhythmic drugs, or K+ channel blockers. Early diagnosis and treatment are important to avoid lethal arrhythmias but a specific treatment to reverse the cardiac remodelling is still missing. AoS is a condition characterized by the calcification of a congenitally bicuspid or normal aortic valve. The calcification progressively reduces the aortic valve orifice, impeding the normal unidirectional flow of blood from the left ventricle to the arterial system. Like HCM, AoS is also associated with an increased risk of arrhythmia, and its symptoms are like those of the aforementioned pathology. AoS treatment consist of surgical replacement or transcatheter implantation of the aortic valve. If these treatments are impossible to perform or refused, therapy includes diuretics and ACE inhibitors. Despite advances, new pharmacological strategies for both diseases are urgently needed to improve patient quality of life and disease progression. Cibenzoline is a class IA antiarrhythmic drug used in Japan and Korea to manage obstructive HCM. In this thesis we explored its electrophysiological properties on human cardiomyocytes and its potential role as therapeutic agent for HCM patients. Using isolated human cardiomyocytes, patch clamp experiments and Ca2+ handling analysis, our study showed that Cibenzoline exerts different positive effects at cellular level that may contribute to explain its therapeutic value and safety profile. Cibenzoline shows that QT interval is preserved after treatment highlighting the cardiac safety of the drug; moreover, it reduces late Na+ current and shows a lower inhibitory activity on muscarinic receptors compared to other drugs of the same class. Avoidance of anticholinergic side effects, such as dry mouth and constipation, makes Cibenzoline more suitable for long life treatment of HCM patients. Beside Cibenzoline, we also investigated the properties of a sodium glucose co-transporter 2 (SGLT2) inhibitor, Dapagliflozin, in preventing or restoring the cardiomyocyte remodelling associated to HCM or AoS. Recent clinical trials have highlighted the beneficial effects of this drug in different cardiovascular pathologies, despite the underlying mechanistic explanation still remain unclear. For this reason, our primary goal was to test the effect of Dapagliflozin on the electrophysiological alterations of human cardiomyocytes isolated from HCM and AoS patients. Recordings on human ventricular cardiomyocytes from HCM or AoS patients revealed that the drug, at 1 μM and 10 μM concentrations, was able to decrease action potential duration stimulated at different frequencies (0.2Hz, 0.5Hz and 1Hz). This effect is dose-dependent, but not frequencydependent. Moreover, Dapagliflozin proved able to reduce late Na+ current without affecting the peak Na+ current. This selective activity on the late current could be important to reduce the risk of triggering lethal arrhythmic events. Cardiac remodelling that occurs in many cardiac conditions, including in HCM, involves not only electrical abnormalities but also structural alterations that modify the cardiac architecture through induction of fibrosis. For this reason, in parallel with the study of new specific treatments for managing HCM, we investigated the mechanisms behind fibroblast activation that lead to myofibroblast formation and fibrotic formation. In particular, we examined the role of channels responsible for Ca2+ influx into cells that mediates myofibroblast activation. Using fluorescence imaging, we analysed Ca2+ fluxes induced by increasing extracellular Ca2+ concentration in cultured fibroblasts isolated from control or HCM cardiac samples, proving that in HCM-fibroblasts the entity of Ca2+ influx is larger than in control fibroblasts. Aiming to identify the channels responsible for the modifications of Ca2+ influx, we used Lacidipine, an L-Type Ca2+ channel blocker and SKF, a TRPC channels blocker. Both blockers gave similar results in HCM or control fibroblasts, suggesting that the enhanced Ca2+ influx observed in HCM fibroblasts is independent from L-Type or TRP Ca2+ channel activity. Our future objective aims to further investigate the mechanisms underlying the increased Ca2+ influx in HCM compared to control and better understand the implications for fibroblast activation and its potential as novel therapeutic target in HCM remodelling.