Fibroblasts, electrophysiological changes, and atrial fibrillation: Focus on the arrhythmogenic properties of fibroblasts

Atrial fibrosis is one of the fundamental mechanisms for the pathogenesis of atrial fibrillation (AF), but the underlying electrophysiological changes involved are not completely understood. Aging, neurohumoral activation, and chronic atrial stretch due to structural heart disease activate various signaling pathways that lead to cellular hypertrophy, fibroblast proliferation, and complex alterations of the extracellular matrix (ECM), such as tissue fibrosis; these may lead to disruption of the electrical side-to-side junctions between muscle bundles [1], resulting in electrical dissociation. Cardiomyocytes and cardiac fibroblasts are 2 major myocardial cell types, which constitute <50% and 40–60% of the total cell population, respectively [2], [3]. In normal adult hearts, quiescent fibroblasts substantially outnumber myocytes, and in response to hemodynamic stress or injury, these fibroblasts differentiate into myofibroblasts that proliferate, secrete collagen, and synthesize new proteins such as α-smooth muscle actin (α-SMA), stretch-sensitive ion channels, and connexins [4]. Fibroblasts have arrhythmogenic properties such as those involved in blocking impulse propagation and differentiation into myofibroblasts, which have contractility, have electrical connectivity and stimulate electrical impulse. The alteration of fibroblast-induced electrical impulses depends on the number of fibroblasts: small numbers of fibroblasts lead to slow impulse conduction whereas large numbers of fibroblasts lead to conduction block [5]. Recently, the mechanism of cardiac fibrosis moved from fibroblast-induced electrical impulse propagation alteration to myofibroblasts, which is a key factor of the proarrhythmic mechanism. During embryonic development, mesenchymal cells migrate from the proepicardium to the epicardium, where they differentiate into epicardial-derived cells, which differentiate into fibroblast or myofibroblast phenotypes. Although phenotypically most of these epicardial-derived cells differentiate into fibroblasts, these cells can differentiate into myofibroblasts in response to injury or stress [6], [7]. The incidence of fibrosis increases after fibroblasts are transformed to activated fibroblasts or myofibroblasts [5]-[8]. Myofibroblasts have contractile proteins and α-smooth muscle actin (SMA), which is a smooth muscle cell marker with enhanced migratory and proliferative properties [2]. Transforming growth factor-β (TGF-β) has shown to induce differentiation of fibroblasts to myofibroblasts in cultures [8]. Fibroblasts also differentiated to myofibroblasts in connexin-abundant cultures, especially in cultures mimicking the post-myocardial infarction (MI) state [4], [9]. Askar et al. [10] reported that cardiomyocyte hypertrophy and fibrosis induced by myofibroblast proliferation have similar arrhythmogenicity. This study reported that prolonged action potential durations (APDs) and early after depolarization (EAD)-triggered activity observed in cultured hypertrophic and fibrotic tissue led to a high incidence of spontaneous re-entrant arrhythmias or focal arrhythmias [10]. In addition, they found that high myofibroblast contents in cardiac cultures were associated with depolarized membrane potentials in cardiomyocytes, prolonged APD80, and increased incidence of EADs that lead to arrhythmias [10]. Arrhythmogenicity may also be induced by paracrine factors secreted by fibroblasts. The paracrine effect of post-MI-activated fibroblasts prolongs APD and slows conduction velocity [6], [11]. Adult murine cardiomyocytes developed significant cellular hypertrophy when treated with fibroblast-conditioned media, and when co-cultured with cardiac fibroblasts, they secreted high levels of atrial natriuretic peptide [12]. Interestingly, conduction velocity increased and APD70 reduced in these cultures with MI heart myofibroblasts compared with cultures with normal heart fibroblasts [6], [11]. Here, mathematical models could be used for evaluating the myocyte–fibroblast electrical interaction. Fibroblasts can influence conduction velocity by obstructing, creating electrotonic load, and depolarizing myocytes. Since myofibroblasts have a less negative resting membrane potential, they can depolarize myocytes sufficiently to induce spontaneous pacemaking when the myofibroblast population exceeds 15% in co-cultures [13]. Fibroblasts can couple to cardiomyocytes and substantially affect their cellular electrical properties, including conduction, resting potential, repolarization, and excitability. Such a myofibroblast–myocyte coupling can trigger an arrhythmia such as EAD, and these mechanisms may contribute to the proarrhythmic risk in fibrotic hearts via spontaneous impulse formation and rotor-driven re-entry [14]. The increase in the resting membrane potential of cardiomyocytes to more depolarized fibroblasts by coupling can result in an increase of conduction velocity even at low fibroblast concentrations [6]. Similar to a low fibroblast number, even weak electrical coupling (gap junction connectivity) between myocytes and fibroblasts can slightly increase the resting membrane potential and conduction velocity of cardiomyocytes. At intermediate coupling levels, fibroblasts act as a current capacitor, resulting in slow conduction velocity and decreased maximum upstroke velocity with a decrease in sodium channel activation [6]. Cardiac fibroblasts express various ion channels, in particular voltage-gated K+ channels and nonselective cation channels of the transient receptor potential (TRP) family [15]. Both K+ and TRP channels are important determinants of fibroblast function, with TRP channels acting as Ca2+ entry pathways that stimulate fibroblast differentiation into secretory myofibroblast phenotypes that produce ECM proteins. A study investigated the activity of voltage-gated sodium channels in human atrial fibroblasts and myofibroblasts [16] and found that the TRP melastatin-related 7 (TRPM7) channel, a Ca2+/Mg2+-permeable channel, is strongly expressed in human atrial fibroblasts and likely plays an essential role in TGF-β-elicited fibrogenesis in human AF [17]. Recently, Harada et al. [18] showed that the Ca2+-permeable TRP canonical-3 (TRPC3) channels regulate cardiac fibroblast proliferation and differentiation, perhaps by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. They showed that TRPC3 expression was upregulated in atria of AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. Interestingly, the expression and function of TRPC3 channels disappeared after differentiation of fibroblasts to myofibroblasts under culture conditions, which suggests that TRPC3 controls proliferation and differentiation of fibroblasts but is downregulated in the end-cell myofibroblast. This negative feedback system may prevent excessive ECM remodeling. In contrast, TRPM7 expression remained high in myofibroblasts, implicating that TRPM7 is likely the dominant TRP channel in differentiated myofibroblasts [17]. TRPC3 channels act as a platform for protein kinase C associated with ERK-1/2 activation, which contributes to fibroblast function. Because angiotensin II increases the cellular production of diacylglycerol, which activates TRPC3 channels, angiotensin-induced increases in intracellular Ca2+ and protein kinase C activation synergistically contribute to fibroblast proliferation via the TRPC3 channel. Harada et al. [18] showed that a selective TPRC3 channel blocker, pyrazole-3, suppressed angiotensin II-induced Ca2+ influx, proliferation, and α-SMA protein expression in fibroblasts in addition to extracellular signal-regulated kinase phosphorylation and ECM gene expression. Knocking down TRPC3 by small hairpin RNA decreased canine atrial fibroblast proliferation. Harada et al. [18] also showed that microRNA-26 was downregulated in canine AF atria, and experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has nuclear factor of activated T cells (NFAT) binding sites in the 5′ promoter region, and microRNA-26 transcription was negatively regulated by NFAT. Because NFAT activation is increased in AF fibroblasts, AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, pyrazole-3 suppressed AF while decreasing fibroblast proliferation and ECM gene expression. Harada et al. [18] introduces the possibility of a novel potential therapeutic target that prevents fibroblast activation for the treatment of AF. The authors importantly showed that TRPC3 plays an important role in AF by promoting fibroblast pathophysiology as well as the mechanism of TRPC upregulation in AF via microRNA-26. The present findings point to TRPC3 as a candidate target for AF prevention. However, arguably, the main limitation of this study is that properties of fibroblasts were probably affected by cell isolation and culture conditions. Any extrapolation of these results to human disease should be made cautiously. Further, the discrepancy between atrial fibroblast density/signs of fibroblast activation and fibrous tissue content in AF dogs (7 days) showed that longer intervals (>3 months) are necessary for fibrosis development. However, myofibroblast–myocyte coupling as an arrhythmia trigger does not require enhanced fibrous tissue content; therefore, fibroblast proliferation and differentiation can promote AF. Fibrosis, inflammation, and oxidative stress appear strongly interconnected in the pathogenesis of remodeling-induced abnormalities. Moreover, insights into the ECM network strongly suggested an active cross-talk within the cardiac microenvironment, which should be further investigated as a promising “anti-remodeling” approach. In addition, the excess ECM proteins produced by fibroblasts can interrupt cardiomyocyte-bundle continuity, leading to local conduction disturbances and re-entrant arrhythmias, resulting in “electric-structural remodeling.” Therefore, a better understanding of the electrical properties of fibroblasts could lead to an improved comprehension of AF pathophysiology, and pharmacological modulation of ionic targets associated with atrial fibrosis would be an area of interest in preventive strategy in AF. There is no conflict of interest related to this article.