Purpose: The aim of the present study was to investigate the electrical activity of the cardiac tissue in the rat SVC and AZV.Materials and methods: Electrically evoked or spontaneous action potentials (AP) and resting membrane potential (RPM) were recorded in multicellular isolated Tirode perfused preparations of left or right atria (LA, RA), SVC or AZV with use of standard microelectrode technique in control conditions and under application of noradrenaline (NA, 10 lM), isoproterenol (ISO, 10 lM) or phenylephrine (PHE, 10 lM).All preparations were dissected from Wistar rats (male, 200-250g).All experiments were approved by local bioethical committee.Results: The atrial-like action potentials (AP) were observed in various sites of both rat SVC and AZV under continuous steady-state electrical pacing (CEP).The AP duration (APD) in the isolated paced SVC and AZV were similar to those in LA, but was significantly shorter compared to the RA myocardium.RMP in the SVC and AZV was depolarized under CEP in comparison to the level in atrial myocardium.Unlike to the atrial myocardium the termination of electrical pacing in both SVC and AZV resulted in significant spontaneous positive shift of the RMP.However, no spontaneous AP were observed in quiescent veins preparation without adrenergic stimulation in both veins preparations.NA induced significant additional depolarization, while ISO caused hyperpolarization of the RMP in both quiescent veins preparations.RMP alteration was smaller in SVR compered to AZV.PHE induced depolarization of the RMP in quiescent AZV preparations, but failed to alter RMP in SVC preparations.Spontaneous AP in form of repetitive quasy-periodic bursts were observed in 30% quiescent AZV preparations in response to the NA application.Spontaneous AP were observed in 20% SVC preparations under application of NA, ISO and PHE. Conclusion:In conclusion, the electrical activity in SVC and AZV under a regular excitation resembles those in atrial myocardium, rare adrenergic automaticity has been revealed in non-paced cardiac tissue in the rat SVC and AZV despite RMP instability.

Purpose: Understanding the mechanism underlying atrial electrical remodelling in AF is of fundamental importance for the prevention and treatment of AF. We have recently found that neuronal nitric oxide synthase (nNOS) activity is dramatically reduced in atrial myocytes from patients with AF. Whether loss of nNOS activity contributes to AF-induced atrial electrical remodelling remains to be established. Methods: Whole-cell patch clamp was used to record action potentials (APs) and ion currents in human and murine right atrial myocytes. AF was induced in isoflurane anaesthetised mice by using trans-oesophageal electrical stimulation. N=number of patients or mice, n= number of myocytes. Results: Inhibition of nNOS by S-methylthiocitrulline (SMTC, 100 nM), induced a significant reduction in APD at 20 (38%), 50 (39%) and 90 (30%) percent of repolarization in atrial myocytes from patients in sinus rhythm (SR, N=8, n=38 control vs. N=8, n=31 in the presence SMTC, p<0.001) and suppressed APD rate-dependent adaptation (from 0.5 to 3 Hertz, N=6, n=24 control vs. N=6, n=17 cells with SMTC, p<0.05). In mice, nNOS inhibition or gene deletion reduced APD50 by 46% and 29%, respectively. (N=9, n=35 from nNOS-/- & N=6, n=11 WT plus SMTC vs. N=10, n=28 WT control, p<0.001 for the effect of nNOS dysruption). By contrast, SMTC had no effect on APD in atrial myocytes from patients with AF or nNOS-/- mice. In agreement with these findings, nNOS-/- mice displayed a 2-fold increase in AF inducibility in response to burst stimulation (p<0.05 vs. WT littermates, N=18 per genotype). Investigations of the ionic changes underlying the effect of nNOS dysruption on APD in RA myocytes from patients in SR (N=7, n=31 control vs. N=7, n=35 in SMTC) or WT mice with SMTC (N=9, n=19 control via N=6, n=9 in SMTC) showed an increase in Ito with SMTC in both species with no change in IK1 or IKr. Ito blockade with 4-AP (0.5 mM) significantly prolonged APD20 (by 19% in SR and by 153% in SMTC), APD50 (by 19% in SR and by 55% in SMTC) and, to a lesser extent, APD90 (by 7% in SR and by 13% in SMTC) in atrial myocytes from patients in SR. 4-AP also recovered the APD rate dependent adaptation that was suppressed by SMTC, confirming a role of Ito in the nNOS regulation of APD in human atrial myocytes. Conclusions: In mammalian atrial myocytes, nNOS-derived NO plays an important role in the regulation of APD and its rate-dependent adaptation by modulating Ito. These findings suggest that the marked loss of nNOS protein and activity in the fibrillating atrial myocardium has potentially important implication for AF-induced electrical remodelling.

Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) confer distinct primary alterations of cardiac contractility. We have shown that altered Ca2+-buffering by mutant thin filaments leads to altered Ca2+ handling and results in stimulation of Ca2+-dependent signalling pathways. To do this we have used adenoviral mediated expression of cTnT R92Q, cTnI R145G and α-TM D175N in adult guinea pig cardiomyocytes at a ratio of 1:1 with the endogenous protein. Simultaneous measurement of unloaded sarcomere-shortening and Ca2+ transients using fura-2 loading, showed the HCM mutations caused a significant decrease in the basal sarcomere length coupled with an increase in the diastolic Ca2+ concentration. The mechanism of alterations to EC-coupling was also investigated using tetracaine and caffeine challenging combined with simultaneous whole cell patch clamping. HCM mutant cells displayed reduced SR load (~1.4fold), slowed NCX calcium extrusion (~2.5fold), unchanged SERCA2 activity, increased ryanodine receptor leak (~5fold) and increased calcium buffering (~3fold). This was coupled to an increase in Ca2+ dependent NFAT nuclear localisation. Further studies using the green tea catechin, epigallocatechin gallate (EGCg) and sister compound epicatechin-3-gallate (ECG), have shown that the compounds can partiality reverse the increase in diastolic Ca2+ observed in cardiomyocytes containing HCM causing mutations. The mechanism is thought to be via an interaction with cTnC (measured using intrinsic cTnC tyrosine fluorescence) which in turn causes a reduction of in vitro thin filament Ca2+ affinity (measured using cTnC labelled with IAANS fluorophore at Cys 35). Data acquired so far suggests that catechins and their derivative compounds may provide a possible therapeutic approach for correcting Ca2+ regulation and Ca2+ dependent remodelling in HCM. We have recently obtained library of 37 EGCg analogues which we have screened for cTnC affinity and thin filament Ca2+ affinity. We have now identified several catechins with potentially greater efficacy than the parent compound, and plan to test their ability to rescue the cellular HCM phenotype characterised above.