Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and β-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (β1 + β2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (β1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (β2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating β1- and α1-ARs in both human and rabbit, with a β2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.


Introduction
The catecholamine noradrenaline (NA) is released by sympathetic (adrenergic) post-ganglionic nerves terminating on cardiac myocytes. It is substantially involved in regulating cardiac excitation-contraction and the fight-orflight response, and sometimes in the generation of cardiac arrhythmias including the most common, atrial fibrillation (AF) [39,46]. The generation of AF by NA probably involves its marked effect to increase atrial L-type Ca 2+ current, I CaL , as shown in human [4,6] and rat [3] atrial myocytes, in turn contributing to triggered activity from atrial spontaneous depolarisations or afterdepolarisations [9,19,44,46]. This I CaL increase results, in large part, from activation of cell surface beta-adrenoceptors (β-AR), supported by numerous studies showing marked effects of the synthetic broad action (β 1 -, β 2 -and β 3 -AR) agonist isoprenaline (ISO) on human atrial I CaL (e.g. [4,21,31]). Furthermore, ISO infusion in patients produced AF [29]. β-AR antagonists are used in the pharmacological treatment of patients with AF, primarily for controlling the associated rapid ventricular rates (rate control), but they may also be effective in suppressing AF (rhythm control) when adrenergic tone is elevated, e.g. β 1 -AR sub-type antagonists in patients with postoperative AF (bisoprolol, metoprolol) [8] or with heart failure (metoprolol) [37]. However, NA activates α-as well as β-ARs, and each main AR sub-type has been identified in human atrial myocardium [46]. Moreover, since the mixed α 1 -, β 1 -, β 2 -AR antagonist, carvedilol, was more effective at preventing postoperative AF than the β 1 -antagonists 1 3 metoprolol or atenolol [15,23], this suggests the possibility of identifying specific mixed AR sub-type antagonism profiles for optimising rhythm control drug efficacy during adrenergic AF.
To do so, however, requires an improved understanding of the contributions of activation of the individual AR subtypes to the effect of NA on human atrial I CaL . So far, this has been addressed using AR sub-type selective agonists, with β 2 -agonism (salbutamol) increasing human atrial I CaL [50], β 3 -agonism (BRL37344) having no effect [5] and selective β 1 -agonism not yet studied for human atrial I CaL . Reports of α-AR agonism in human atrium are so far restricted to contraction, e.g. positive inotropic effect of the α 1 -agonist phenylephrine [14], although I CaL has been studied in other species, with a marked increase in the current by phenylephrine in cat atrial myocytes [41], and no effect of phenylephrine or methoxamine in rabbit or rat atrial myocytes [12,18]. Mixed effects of α-AR agonists have also been reported for ventricular I CaL [36]. It is important, however, to investigate the AR sub-type contribution to the I CaL response when using the naturally occurring catecholamine, NA, because this will stimulate all the ARs, simultaneously as would occur in vivo if desired, with consequent physiologically relevant relative activation levels amongst the different AR sub-types, as well as physiologically relevant interactions amongst their associated signalling pathways.
However, there are no reports, to our knowledge, of studies investigating the independent contributions to the I CaL response of the different AR sub-types in this way, i.e. using NA in the presence of AR sub-type selective antagonists, in human atrial myocytes. Potential species-differences in I CaL responses to NA and AR antagonists should also be considered, in order that data from animal species used in models of AF from adrenergic stimulation and/or altered pathology can be adequately compared with those from human. Rabbits have been studied previously to investigate atrial cellular electrophysiological mechanisms of AF promotion by β-AR stimulation with ISO [20], but I CaL responses to NA with AR antagonists have yet to be studied in this species.
The aim, therefore, is to investigate effects, on NA-stimulated I CaL , of various broad-action and sub-type-specific α-and β-AR antagonists, alone or in combination, in human and rabbit atrial myocytes.

Data and statistical analysis
Data are expressed as mean ± SEM. Comparisons amongst three or more groups were made using (for matched, parametric data) repeated measures one-way ANOVA or (for un-matched, parametric data) ordinary one-way ANOVA, each followed by Tukey's multiple comparisons test or (for matched, non-parametric) Friedman test and Uncorrected Dunn's test. Comparisons between two groups of un-matched data were made using either an un-paired t-test (for parametric data) or Mann-Whitney (non-parametric). P < 0.05 was regarded as statistically significant. All statistical and curve fitting analyses were done using Graphpad Prism 7.00.

Noradrenaline increases human atrial L-type Ca 2+ current in a concentration-dependent manner
In human atrial myocytes, NA produced a marked, concentration-dependent increase in I CaL , shown by the original current traces and concentration-response curve in Fig. 1A and B, respectively. At maximally effective concentration, NA increased I CaL ~ 2.5-fold (Fig. 1B)

Effects of broad action βand α-adrenoceptor antagonists on NA-stimulated I CaL in human and rabbit atrial myocytes
Broad-action β-, and α-, AR antagonism of I CaL -responses to this NA concentration was studied using propranolol, and phentolamine, applied in a step-wise cumulative fashion, in atrial cells from patients, and also from rabbits for direct comparison (Fig. 2). Following a control period to allow for stabilisation of the normal rate of I CaL run-down (timedependent decrease following cell rupture), NA superfusion caused a rapid and substantial increase in peak I CaL in all cells studied, with the response stabilising within ~ 1-1.5 min (e.g. Figure 2A and C). In two representative human atrial cells ( Fig. 2Ai and ii), propranolol, still in the presence of NA, caused a rapid and substantial decrease in I CaL to below the NA-stimulated response, and subsequently applied phentolamine caused a rapid, and relatively smaller, decrease in I CaL to below the NA + propranolol response. In one of these cells (Fig. 2Aii), propranolol and phentolamine were simultaneously washed off; the resulting increase in I CaL (itself reversible) shows that the NA-stimulatory effect had been preserved throughout the preceding superfusion of the antagonists. In each of nine human atrial cells studied in this way, propranolol decreased, then phentolamine further decreased, the NA-stimulated I CaL . The mean data (Fig. 2B) show that both propranolol and phentolamine significantly decreased I CaL , and that the degree of reduction from phentolamine was significantly smaller than that from propranolol. In rabbit atrial cells, NA also produced a rapid and significant increase in I CaL , and propranolol then caused a rapid, substantial and significant decrease in NA-stimulated I CaL , in each of 7 cells studied ( Fig. 2C and D). However, by contrast with the human atrial cells, phentolamine (following propranolol) produced a mixed response, either decreasing (e.g. Figure 2Ci) or increasing (e.g. Figure  The bi-exponential time course of I CaL inactivation was also examined. NA (310 nM) had no significant effect on the fast (τ 1 ) or slow (τ 2 ) time constants in either species: in human, control τ 1 and τ 2 were 9.77 ± 1.26 and 112.1 ± 23.2 ms, respectively, vs 7.30 ± 0.64 and 236.9 ± 66.4 ms with NA (P = 0.087 and 0.126, respectively; n = 9 cells); in rabbit: control τ 1 and τ 2 were 12.58 ± 4.40 and 91.9 ± 20.1 ms, vs 13.54 ± 2.96 and 88.7 ± 12.4 ms with NA (P = 0.781 and 0.797; n = 7 cells).

Comparison of independent anti-adrenergic effects of propranolol and phentolamine
In rabbit atrial cells, effects of broad-action α-and β-antagonism were also studied independently of one other, by using phentolamine in the absence of propranolol (for α-antagonism without concurrent β-antagonism) and, in a different group of cells, vice versa. Propranolol alone again caused a consistent, marked and significant decrease in NAstimulated I CaL (Fig. 3Ai and Bi). However, phentolamine alone ( Fig. 3Aii and Bii), by contrast with phentolamine in the continued presence of propranolol ( Fig. 2C and D), also caused a consistent (i.e. in each of 9 cells studied), marked and significant decrease in NA-stimulated I CaL . Furthermore, the degree of the inhibitory effect of phentolamine was not significantly different from that of propranolol.

Comparison of β-AR sub-type contributions to I CaL -stimulation by NA, between human and rabbit atrial myocytes
Having established a substantial β-AR contribution to the stimulatory effect of NA on atrial I CaL , the relative contributions to this of the main β-AR subtypes (β 1 and β 2 ) were investigated using CGP20712A (CGP) and ICI118551 (ICI), respectively, again applied in a step-wise cumulative fashion and compared between species. In each of 6 human atrial cells studied (e.g. Figure 4Ai and ii), CGP caused a rapid and marked decrease in NA-stimulated I CaL , with a significant average inhibitory effect (Fig. 4B) similar to that from propranolol (β 1 + β 2 -antagonist) earlier (Fig. 2B). In rabbit atrial cells, CGP had similar effects, both in terms of the comparison with human ( Fig. 4C and D vs A and B) and with propranolol (Figs. 4C and D vs 2 C and D). However, the effects of ICI on NA-stimulated I CaL differed substantially, both when compared with CGP, and between species. Thus, amongst 5 human atrial cells studied with ICI, there was a mixed response: a reversible (upon ICI-washout) increase in 3 cells (e.g. Figure 4Ai), by 12, 35 and 37% (Fig. 4B), and a marked and reversible decrease in 2 cells (e.g. Figure 4Aii), by 72 and 78% (Fig. 4B). There was no significant effect of ICI on average, contrasting with the consistent and significant inhibitory effect of CGP in the same cells (Fig. 4B). In the rabbit atrial cells, by contrast with the human atrial cells under the same conditions, ICI consistently and reversibly (in each of 5 cells studied) increased I CaL (e.g. Figure 4Ci and ii), an effect which was significant on average (Fig. 4D). The degree of I CaL increase by ICI was not significantly different (P = 0.391) to the degree of I CaL decrease by CGP in these cells.

α-AR sub-type contributions to NA-stimulation of I CaL in human and rabbit atrial myocytes
The relative contributions of the main α-AR subtypes (α 1 and α 2 ), to the broad α-AR contribution to the stimulatory effect of NA on I CaL , were investigated using prazosin and yohimbine, respectively, again compared between the two species. In each of 6 human atrial cells studied (e.g. Figure 5Ai and ii), prazosin decreased NA-stimulated I CaL , and with a significant effect on average (Fig. 5B). By contrast, yohimbine (still in the presence of NA + prazosin) produced a mixed I CaL response: a moderate decrease in 4 cells (e.g. Figure 5Ai), by 19, 36, 43 and 49% (Fig. 5B); a marked increase in one cell (Fig. 5Aii), by 78%, and no effect in the other cell. There was no significant effect of yohimbine on average, contrasting with the consistent and significant inhibitory effect of prazosin in the same cells (Fig. 5B). The degree of reduction in NA-stimulated I CaL by prazosin in these human atrial cells was significantly smaller (P = 0.002) than that observed with CGP earlier (compare Fig. 5B with Fig. 4B). In rabbit atrial cells, similar to human, prazosin consistently (in each of 7 cells studied) decreased NA-stimulated I CaL (e.g. Figure 5Ci and ii), also significant on average (Fig. 5D). The degree of the I CaL -decrease by prazosin was not significantly different (P = 0.073) from that by CGP earlier (compare Fig. 5D with Fig. 4D). Yohimbine, by contrast with prazosin (and also similarly to the finding in human), produced a mixed I CaL response: a decrease in 6 of these 7 cells (e.g. Figure 5Ci), an increase in the other (Fig. 5Cii) and no significant effect on average (Fig. 5D).

Discussion
Investigation of independent AR sub-type contributions to NA's effect on human atrial I CaL first required establishing the NA-I CaL concentration-response relationship, to select a suitable NA concentration for testing with the AR subtype selective antagonists. We found NA to have a marked, concentration-dependent stimulatory effect on I CaL , with an EC 50 of 156 nM, comparable with that in another human atrial study (200 nM) [4], although a markedly higher value has also been reported [6]. Whilst NA circulates in the subto low-nanomolar range in humans [35], it is expected to be substantially more concentrated at the adrenergic nerve endings and in cardiac tissues [51]. We selected our EC 75 for use in all subsequent experiments (in human and rabbit for their direct comparison) because whilst near maximally effective, this would not saturate the stimulatory response, therefore permitting the antagonists to readily exert their effects. Whilst NA consistently increased I CaL , its subsequent "rundown" (line graphs, Figs. 2, 3, 4 and 5), an accepted limitation of the ruptured-patch technique (due to "a decrease in channel activity with time during recording in dialyzed cells" [43]), required the antagonist responses to be normalised with respect to the previous intervention (bar graphs, Figs. 2, 3, 4 and 5) to compensate for this rundown and thus adequately assess average antagonist effects. Broad action β-AR antagonism (with propranolol) revealed a substantial and consistent contribution to NA's stimulatory effect on human atrial I CaL from either β 1 -or β 2 -ARs or both (since β 3 -ARs are not expected to be involved in this response [5,24]). This is congruent with numerous studies in which the broad action AR agonist ISO substantially increased human atrial I CaL [4,21,31], although no previous atrial I CaL study could be found in which propranolol was applied following either ISO or NA. In the continued presence of NA plus propranolol, i.e. with the β 1 -and β 2 -ARs still antagonised and the α-ARs thus adrenergically activated and solely (independently) amenable to antagonism, broad action α-AR antagonism with phentolamine revealed a substantial and consistent contribution to NA's stimulatory effect on human atrial I CaL from either α 1 -or α 2 -ARs or both. Furthermore, we found that the α-AR contribution to the stimulatory effect of NA on I CaL was significantly smaller (at 37%) than that of the β-AR contribution (at 60%), in human atrial cells. Use of the same protocol in the rabbit atrial cells, i.e. stepwise cumulative addition of NA, propranolol and phentolamine, revealed important species similarities, but also a curious difference regarding the contribution of α-ARs. Thus, whilst propranolol consistently, markedly and significantly antagonised NA's stimulatory effect on rabbit as well as human atrial I CaL , in rabbit, by contrast with human, phentolamine had a mixed response following propranolol, producing increases in I CaL in some cells, as well as the decreases as seen in human. These I CaL increases by phentolamine were clear, marked and reversible and occurred in approximately half of the rabbit atrial cells studied. By contrast, no I CaL increase was produced by phentolamine in any of the nine human atrial cells studied in this way. Since only the α-ARs were noradrenergically activated at this point in these experiments (β-AR activation prevented by propranolol in both species), such I CaL increases by the α-AR antagonist indicate an inhibitory contribution of independent α-AR activation to the effect of NA on I CaL in those rabbit atrial cells, i.e. attenuating, but not overcoming, the overall effect of NA to increase I CaL . The reason for this mixed effect of phentolamine in the rabbit atrial cells is unknown, but the resulting net (average) absence of effect, as presumably would occur in the syncytium (multicellular), suggests a potentially important species difference that whilst noradrenergic activation of human atrial I CaL involves a significant contribution from α-ARs, this may not be the case in rabbit, at least when the α-ARs are activated independently of the β-ARs. To assess the α-AR contribution to NA's effect on rabbit atrial I CaL , this time in the presence of simultaneously activated β-ARs, phentolamine was applied in the absence of propranolol and, in a different group of cells, propranolol in the absence of phentolamine for comparison. In this case, we found either α-or β-AR antagonism to consistently (in every cell), markedly and significantly decrease (and by a similar degree between α-and β-) NA-stimulated I CaL , suggesting that the attenuating influence of independent α-AR activation on the stimulatory influence of NA on I CaL as seen above is prevented when α-and β-ARs are simultaneously activated. This finding likely relates to the highly complex interactions which can occur between α-and β-ARs and their signalling pathways [48]. It also highlights another complex, potentially limiting, yet intriguing, aspect of this type of study, the relevance of the order of application of AR-antagonist(s) following NA.
Having established a substantial broad β-AR contribution to NA's stimulatory effect on atrial I CaL in both species, we then dissected the β 1 -versus β 2 -AR involvement, using CGP and ICI, respectively, and showed β 1 -AR activation to mediate a consistent, substantial and significant contribution to noradrenergic activation of human and rabbit atrial I CaL . The similarity in the magnitude of effect of CGP with that of propranolol, in both species, indicated the prominence of the β 1 -AR involvement. By contrast, we found β 2 -AR activation, amongst human atrial cells, to have a mixed, and on average negligible, involvement in the overall β-adrenergic activation of I CaL . This mixed response could relate to stimulatory and inhibitory responses known to result from β 2 -activation, via G s and G i signalling pathways, respectively [45]. In the only similar human atrial I CaL study, in which a synthetic agonist rather than NA was used to activate β 2 -ARs [50], salbutamol increased the current, which would suggest a stimulatory contribution of β 2 -activation to its adrenergic activation under their conditions. We found an important species difference regarding β 2 , since in each of the rabbit atrial cells, independent β 2 -AR antagonism with ICI (since β 1 -AR activation prevented by CGP in both species) produced a consistent, reversible, substantial and on average significant increase in I CaL . This indicated a significant inhibitory contribution of β 2 -AR activation to the effect of NA on rabbit (but not human) I CaL , attenuating the overall effect of NA to increase I CaL , presumably relating to a relatively enhanced G i signalling response to β 2 -AR activation [45]. Consistent with this, in rat atrial tissues, β 2 -antagonism (butoxamine) potentiated the effect of ISO to produce spontaneous contractions [2]. Furthermore, and also in line with the present data, recent studies comparing effects of β 1 -and β 2 -AR agonism on rat ventricular I CaL , intracellular Ca 2+ -cycling and action potentials found that initial β 2 -AR stimulation suppressed most of the well-characterised changes of cardiac excitation-contraction coupling commonly seen when adding a β 1 -AR agonist [27,49].
Dissection of the respective α 1 -versus α 2 -AR involvement in NA's effect on human atrial I CaL (with prazosin and yohimbine) revealed α 1 -AR activation to mediate a consistent, substantial and significant contribution to noradrenergic activation of the current, but an overall negligible contribution from α 2 -AR activation. The stimulatory contribution from this α 1 -AR activation was, nevertheless, significantly smaller (at 37%) than that observed from the β 1 -AR activation (at 71%). Although no studies of effects of synthetic α-AR agonists on human atrial I CaL could be found, the α 1 -AR agonist phenylephrine had positive inotropic effects on human atrial muscle strips [14]. These could potentially be explained, at least in part, by the presently observed stimulatory contribution of α 1 -AR activation on I CaL . However, it should be noted that such inotropic effects could also be due, at least in part, to inhibition of repolarising K + current, as shown with phenylephrine for human atrial I K1 , I TO and I Kur [33], or to increased IP 3 -dependent sarcoplasmic reticular Ca 2+ release [41]. No human atrial I CaL studies using prazosin or yohimbine could be found, although there are reports of attenuation by prazosin of NA-induced positive inotropy [32], again congruent with the observed effects of prazosin on NA-stimulated I CaL . In the rabbit atrial cells, we also found a consistent, substantial and significant stimulatory contribution of α 1 -AR activation to the NA-stimulation of I CaL and a negligible contribution from α 2 -AR activation. Previous atrial I CaL studies, using synthetic α 1agonists rather than NA, showed either no effect (in rabbit [12] and rat [18]), or a stimulatory effect, in cat [41]. In mice, NA-induced AF was inhibited by prior injection of the α 1 -antagonist prazosin [34]. Both NA and α 1 -agonism inhibit rabbit atria I TO [12], carried prominently by Kv1.4 [42]. We blocked I TO using 4-AP, to avoid contaminating I CaL recordings. However, in vivo, I TO decrease from α 1stimulation could exert an action potential prolonging influence additional to that from the present I CaL increase, and other effects of α-stimulation, including pre-synaptic, should also be considered.
Taking our results together, we find that stimulation of atrial I CaL by NA is mediated, based on responses to AR subtype-antagonists (applied in a set order: sub-type 1 , followed by sub-type 2 ), mainly by activating β 1 -and α 1 -ARs, in both human and rabbit. Whilst α 2 -AR involvement was negligible in both species and β 2 -AR involvement negligible in human, in rabbit, β 2 -activation can attenuate the stimulatory effect of NA on I CaL . Finally, in human (but not rabbit), the contribution of β 1 -activation to the I CaL stimulatory response to NA was larger than that of α 1 -activation. An overview of these AR sub-type contributions, with a qualitative estimation of their relative weights, and differences between human and rabbit, is given in Table 2.
These findings have relevance to the electrophysiological mechanisms and potential inhibition of NA-induced AF. Delayed afterdepolarisations (DADs) were produced by catecholamines in dog atria [19], identified as such by their rate-dependent increase in amplitude and decrease in coupling interval [19,44]. Furthermore, afterdepolarisations of various types were produced or facilitated by ISO in human atrial tissues or cells [28,31,40]. DADs are caused by increased inward Na + /Ca 2+ exchange current (I Na/Ca ) associated with increased intracellular Ca 2+ loading and Ca 2+ waves [10], and it may be argued that NAinduced increase in I CaL could contribute to such Ca 2+ loading and thus facilitate DADs. In support, in human atrial myocytes, β-AR stimulation (ISO) increased intracellular Ca 2+ spark frequency [25], systolic intracellular [Ca 2+ ] and Ca 2+ transient amplitude [6,38], and Ca 2+ waves occurred when intracellular [Ca 2+ ] was elevated by increasing extracellular [Ca 2+ ] [25]. In dog atrial cells, ISO also increased the number of pacing-induced spontaneous Ca 2+ transients [7]. Furthermore, NA, which dose-dependently increased the duration of pacing-induced AF in mice [34], also increased intracellular Ca 2+ leak and spontaneous sarcoplasmic reticular Ca 2+ release in the isolated atrial myocytes in the same study. Perhaps such mechanisms also contribute to an observed concentration-dependent increase in arrhythmic contractions by NA in human [6] and rat [2] atrial tissues. The low [Ca 2+ ] i -buffering used here should allow assessment of NA effects on the atrial I CaL bi-exponential inactivation time course including any influence of Ca 2+ -induced inactivation of I CaL . We found that NA (310 nM) had no significant effect on either τ 1 or τ 2 in human or rabbit. No previous studies of NA on atrial I CaL inactivation τs could be found, although ISO was tested in human atrial cells [30]. Despite relatively high [Ca 2+ ] i -buffering (10 mM [EGTA] i ) and low temperature (22 °C), τ 1 and τ 2 were comparable with the present study and, also in agreement, ISO (1 μM) had no significant effect on either [30].
The present data suggest that potential therapeutic targeting of AR sub-types as a means of inhibiting NA-evoked atrial arrhythmias should be most effective with β 1 -AR antagonism, and potentially more effective with concurrent α 1 -AR antagonism. This would be consistent both with the clinical use of β 1 -AR antagonists for preventing postoperative AF [8], and the observation that carvedilol (α 1 -, β 1 -, β 2 -AR-antagonist) was more effective at preventing this arrhythmia than β 1 -AR antagonists [15,23], although extra-AR actions of carvedilol [11] might also contribute.
However, since α 1 -AR activation might exert various cardioprotective effects, α 1 -AR antagonism should nevertheless be considered with caution [52]. Furthermore, potentially therapeutic targeting of selected AR sub-types must be considered in the context of highly complex, dynamic and pathology-dependent interactions between each of the various AR sub-types and their associated signalling pathways [48].
Funding This work was supported by British Heart Foundation Project Grant (PG/16/42/32142).

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate Ethical approval number: REC 17/WS/0134. Written, informed consent was obtained from all patients. Procedures and experiments involving human atrial myocytes were approved by West of Scotland Research Ethics Service (REC: 17/ WS/0134). Written, informed consent was obtained from all patients. The investigation conformed to the principles outlined in the Declaration of Helsinki. Procedures and experiments involving rabbit left atrial myocytes (UK Project Licence: 70/8835) were approved by Glasgow University Ethics Review Committee and conformed to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.
Consent for publication All authors agree with the content and all gave explicit consent to submit and obtained consent from the responsible authorities at the institute where the work was carried out.

Competing interests The authors declare no competing interests.
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