Abnormal Ca2+ handling or cycling in cardiac tissues is the main cause for arrhythmia (Ter Keurs & Boyden, 2007). As briefly introduced above, diastolic Ca2+ release (or leak) from the SR is a key event to initiate arrhythmogenic premature depolarizations DAD. Presently, the mechanism whereby DAD is generated by the Ca2+ release can be accounted for by the activation of three ionic conductances which can generate Iti near the resting membrane potential; an electrogenic forward-mode NCX1 current which allows the influx of three Na+ ions in exchange of one Ca2+ efflux after the repolarization has completed; and Ca2+-activated Cl- and nonselective cation channel (CAN) currents. Although the most accepted hypothesis suggests that Iti is carried exclusively by enhanced forward-mode NCX1 inward current in failing ventricle (Pogwizd, et al. 2001; Pogwizd, 2003; Wu and Anderson, 1999), there is also experimental evidence suggesting the involvement of Ca2+-activated Cl- current (Han et al., 1996; Laflamme et al., 1996) and CAN (Hill et al., 1988; Kass et a., 1978; Wu & Anderson, 2000) in the genesis of Iti.
CAN constitutes a large heterogenous family of cation channels with varying unitary conductances, Ca2+-sensitivities, voltage dependence and regulations, and distributes broadly in neurons, smooth muscle, heart, exocrine and endocrine glands, and other epithelial tissues (Siemen, 1993). The most frequently recorded CAN from cardiac tissues, first identified in a cultured rat neonatal ventricular myocyte (Colquhoun, 1981), is a 20-40pS channel with almost equal selectivity over monovalent cations and poor permeability to divalent cations (Guinamard et al, 2006). Albeit rather great variations, these cardiac CANs are activated by the micro- to mili-molar range of Ca2+ and voltage-dependent. The molecular identification that best fits this type of CAN is now thought to be TRPM4 protein (Launay et al., 2002). Expressed TRPM4 channel shows a ~25pS conductance (in near-physiological ionic milieu), is monovalent cation-selective, and, in cell-free conditions, undergoes Ca2+-dependent activation and depolarization-dependent enhancement of open probability only in their unphysiologically high ranges. However, it has been found that the Ca2+- and voltage-sensitivities of TRPM4 rapidly shift to their much higher ranges immediately after the excision of patch membrane (‘desensitization’ or ‘rundown’), and this is substantially prevented by pretreatment with MgATP, protein kinase C activators, maneuvers replenishing phosphatidylinositol 4,5-bisphosphate and increased temperature (Nilius, 2006). These findings suggest that, in vivo, TRPM4 may be more effectively regulated by physiological ranges of [Ca2+]i and membrane potential. Expressed TRPM4 channel also undergoes negative regulation of free adenosine nucleotide phosphates (efficacy; ADP > ATP = AMP), and is subject to spermine block, so that severe ischemic conditions might affect this channel activity (Nilius et al., 2004).
In human, cardiac expression of TRPM4 is ubiquitous in the order of abundance; Purkinje fibers, septum, atrium, left and right ventricles (Kruse et al., 2009). Guinamard et al (2002) found that expression of TRPM4-like CAN, which was virtually absent at the time of cell isolation, was progressively enhanced in rat dedifferentiating ventricular myocytes with the time of culture. Studies from the same group also found that purinergic receptor activation by ATPyS, PKC activators and DAG, all of which act as interventions to facilitate cardiac remodeling, enhanced the occurrence of TRPM4-like channel in cultured ventricular myocytes. This led to the postulation that remodeling of ventricular myocytes in response to excessive hypertrophic signals may enhance the activity of TRPM4 thereby increasing arrhythmogenic propensity mostly likely by increasing DAD. Consistent with this, in the hypertrophic heart of spontaneously hypertensive rat, the elongation of Q-T interval in electrocardiogram has been reported together with increased expression of TRPM4 protein and density of CAN (Guinamard et al., 2006). Recent reports indicate that TRPM4 is more abundantly expressed in atrium and sinoatrial node (Demion et al, 2007; Guinamard, 2004; Kruse et al., 2009), where DAD-based tachyarrhythmias have been well established (Dobrev, 2010; Guinamard 2006; Nattel 2007; Raves, 2010). However, TRPM4-deficient mice showed no obvious abnormalities in heart rate, cardiac output, ejection fraction and cardiac contractility at basal conditions questioning a vital role of TRPM4 in physiological regulation of cardiac functions (Mathar et al., 2009). Rather unexpectedly, the genetic deletion of TRPM4 caused postnatal development of high blood pressure due to increased sympathetic catecholamine secretion. These seemingly paradoxical observations might point to the pathological contribution of TRPM4 to arrhythmogenicity in diseased remodeling heart rather than to normal rhythm formation, in which the expression and activity of TRPM4 are enhanced.
A recent genetic analysis of human families with lethal conduction failure has found a gain-of-function mutation (c.19G^A in exon 1 or p.E7K) in the N-terminal domain of TRPM4 channel (PFHBI; progressive familial heart block type I) (Kruse et al., 2009). More recently, other TRPM4 mutations with very similar biological impacts (p.R164W, p.A432T, p.G844D) have also been assigned to autosomal dominant isolated cardiac conduction block (ICCD) of Lebanese and French families (Liu et al., 2010). Biochemical and immunocytochemical data suggested that these mutations increase the cell surface expression of TRPM4 protein due to desregulated SUMOylation/deSUMOylation process which results in impaired endocytotic protein degradation. The accompanying electrophysiological changes for these mutations are consistent with the biochemical/immunicytochemical data; the density of whole-cell CAN current was increased with little noticeable changes in macroscopic Ca2+ sensitivity and voltage dependence or in unitary conductance and open probability at single channel level. These results suggest that the number of functional channels were increased without altered biophysical properties. The precise pathogenic mechanism for these mutations to cause conduction block remains unclear. However, RT-PCR analysis indicated the highest expression of TRPM4 in Purkinje fiber, and detailed immunohistochemical examination showed strongly TRPM4-positive subendocardial bundles of Purkinje fibers branching and penetrating toward subepithelial layer (Kruse et al., 2009; Liu et al., 2010). These morphological findings, combined with a broadened QRS complex in ECG in both PFHBI and ICCD patients, led the authors to speculate that elevated TRPM4 expression may increase the membrane leak conductance thereby disabling action potential propagation along the Purkinje fibers. There are several previous studies showing that Ca2+ overload causes membrane oscillations in Purkinje fibers due to increased spontaneous Ca2+ release when Na+ extrusion via Na+/K+-ATPase was pharmacologically inhibited (Kass et al., 1978; Kass & Tsien, 1982; Lederer & Tsien, 1976). Considering that TRPM4 may also act as a Na+ entry pathway in response to GPCR stimulation (Launay, et al., 2002), the resultant increase in [Na+]i might then elevate [Ca2+]i via the reversed mode operation of NCX1 in Purkinje fiber cells (Bers, 2006; Pogwizd, 2003). This would not only facilitate Ca2+ overload and DAD generation due to increased TRPM4 channel activation, but could also induce cell death leading to cardiac injury/remodeling if the elevation of [Ca2+] would persist. Fibrotic replacement observed in the His-Purkinje fiber system of a PFHBI patient (Kruse et al., 2009) may support the latter possibility. Thus, the depolarizing and Na+-permeating properties of TRPM4 channel could bring about both acute (induction of DAD) and chronic (conduction block due to remodeling) pro-arrhythmic effects.
Arrhythmia associated with ischemia and oxidative stress
In the acute phases of myocardial infarction, many arrhythmogenic substances are released (see above). ATP and UTP are amongst these, being released from cardiomycytes during ischemia and may promote the occurrence of ventricular tachyarrhythmias (Dutta et al., 2004; Kuzmin et al., 1998). In vitro, ATP can induce DAD-based arrhythmic depolarizations in single cadiomyocytes when combined with Ca2+ increasing agents such as catecholamines (Song & Belardinelli, 1994). In voltage-clamped adult ventricular myocytes, ATP/UTP can activate a sustained inward current via P2Y2 receptor and enhance the opening of 14 and 23pS single channel activities in a PLC-dependent manner. TRPC3 and TRPC7 proteins from rat ventricular myocytes immunocoprecipitated, and the macroscopic current induced by
ATP was suppressed by intracellular application of anti-TRPC3 antibody from the patch pipette (Alvarez et al., 2008). These findings are collectively interpreted as indicating that ATP/UTP may cause arrhythmia by activating TRPC3/TRPC7 heterotetrameric Ca2+ and Na+ entry channels whereby to facilitate intracellular Ca2+ overloading and trigger electrical activities. Consistent with this scenario, the same researchers’ group has recently shown that flash photolysis of caged ATP can evoke one or even a train of extrasystolic contractions. Further, intraperitoneal injection of creatine, which is capable of buffering a sudden ATP/UTP release and clinically exhibits anti-arrhythmic effects, markedly reduced ventricular tachyarrhythmias and early death events in a rat coronary-ligature myocardial infarction model. These ameliorating effects of creatine were defective in an inactive creatine analogue P-guanidinopropionate (Vassort et al., 2010). Cell surface expression of TRPC3 was found tightly controlled by vesicular trafficking, cytoskeletal actin dynamics and interaction with caveolar or subsarcolemmal proteins and molecules (Groschner K, et al., 2005). And PLC stimulation by AngII in cardiomycytes was reported to facilitate the recruitment of TRPC3 complexed with NCX1 to cell membrane, resulting in the reverse mode Ca2+ entry through NCX1 (Eder et al., 2007). Thus, although the precise connection between TRPC3 (and possibly TRPC7) channel activation by ATP and arrhythmogenicty is unclear, the above mechanism may have particular pathophysiological significance in Ca2+-dependent arrhythmogenesis during postinfaction remodeling period or sudden ischemic insult of hypertrophied heart, where enhanced expression of TRPC3 and sympathetic nerve activity play pivotal roles. Seemingly in line with these results, a recent independent study has reported that cardiomyocytes from TRPC3-overexressing mice show an increased susceptibility to apoptotic death due to Ca2+ overload when subjected to hypoxia/reoxgenation (Shan et al., 2007).
Oxidative stress causes tissue damage in a variety of pathological states including aging, cancer, neurogenerative disorders, autoimmune diseases, atherosclerosis and ischemia/reperfusion injury of myocardium (Chandra et al., 2000; Langley et al., 2004; Misra et al., 2009). Many reactive oxygen spices (ROS; O2"-,-OH, H2O2, NO, -ONOO-) are generated as the result of ischemia and reoxygenation and may confer the susceptibility to arrhythmia in direct and indirect ways (via modifying autonomic nerve activity (Danson et al., 2006; Misra et al., 2009). Several TRP members including TRPM2, TRPM7, TRPC5 and a TRPC3/TRPC4 heterooligomer are known to be activated by oxidative stresses (Miller & Zhang, 2011; Naylor et al., 2011; Poteser et al., 2006). TRPM2 is the first reported ROS-sensitive Ca2+-permeable nonselective cation channel activated by H2O2, ADP-ribose, TNFa, and P-amyloid peptide, and is implicated in both physiological functions and pathophysiology including oxidant (H2O2)-induced cell damage/death in pancreatic P-cells and neurons. TRPM2 is positively regulated by intracellular Ca2+, and its cell damaging action appears to be mediated at least in part by sustained [Ca2+]i elevation (Takahashi et al., 2011). In myocardial infarction, reperfusion injury and congestive heart failure, elevated plasma levels of TNFa have been reported (Sack et al., 2000). TNFa can activate both cytotoxic and cytoprotective signaling pathways in many cell types including mitochondrial ROS production through the recruitment of caspase-8 into the death-inducing signaling complex (Kroemer et al., 2007; Zhu et al., 2006). Further, altered redox state changes the activity of cardiac ion channels and transporters regulating Ca2+ dynamics in the cardiomyocyte, i.e. L-type Cav, RyR2, NCX1 and SERCA2a (Zima & Blatter, 2006). In keeping with these observations, activation of TRPM2 by oxidative stress (H2O2) has been found to cause apoptotic/necrotic changes in single cardiomyocytes with ultrastructural changes characteristic of cardiac ischemia/reperfusion injury (Yang et al, 2006). Although whether these changes are pro-arrhythmic remains to be explored, it is possible that sustained [Ca2+]i elevation through activated TRPM2 channel would disturb basal Ca2+ homeostasis underlying rhythmic excitations in cardiomyocytes thereby inducing Ca2+-dependent arrhythmogenicity. The arrhythmogenic effect of TRPM2 may not necessarily be restricted to direct cell-damaging actions via sustained [Ca2+]i elevation. It could occur indirectly through the production of inflammatory cytokines from ambient immune cells, as was demonstrated in an animal model of inflammatory bowel syndrome (Yamamoto et al., 2008).
In addition, expression of TRPC4 and TRPC5 was found to increase 2-3-fold in cultured rat cardiomyocytes subjected to downregulation of SERCA by siRNA technology which mimics the remodeling process of diseased heart (Seth et al., 2004). TRPC5 is known to be activated by many noxious stimuli such as H2O2 and LPC (Flemming et al., 2006; Naylor et al., 2011), and thus could act as a pro-arrhythmic mediator in ischemic remodeling heart. This possibility will be an intriguing subject of future investigation, together with other TRP isoforms implicated in the pathogenesis of cardiomyopathy (TRPV2, TRPC7; Iwata et al., 2003; Satoh et al., 2007).
Arrhythmia related to fibrosis
Cardiac fibroblasts occupy about 70% of whole cardiac cell populations, and, once placed in pathological states such as myocardial injury, oxidative stress and excessive mechanical stretch, start to proliferate and differentiate into the active phenotype ‘myofibroblast’. Activated myofibroblasts secrete extracellular matrix proteins (e.g. collagen), matrix metaloproteinases, cytokines and growth factors thereby promoting the pathological restructuring of diseased heart with fibrotic replacement of damaged myocardial tissue (Souders, et al., 2009). Cardiac fibroblasts have a shallow resting membrane potential (-30 – -10mV) and are non-excitable themselves, but show mechanically-induced depolarizations well synchronized with spontaneous contractions of myocardium (Kamkin et al, 2000b; Kamkin et al, 2003). These electrophysiological properties allow them, by coupling electrically to myocytes as a leaky capacitor, to modify the electrical properties of myocyte action potential and its propagation (Yue et al., 2011). In contrast, fibrotic tissues generated by myofibroblasts act as a physical barrier insulating the spread of electrical currents between the bundles of cardiomyocytes. Thus, disturbances in the myocyte-fibroblast coupling and alterations in myocardial architecture due to spatially inhomogenous fibrosis can lead to cardiac arrhythmias associated with reentry or conduction block. It has been known that fibrotic remodeling is a fundamental process underlying the perpetuation of AF. Atrial fibrosis is dependent on Ca2+, but what source of Ca2+ is involved therein had been elusive. A recent study of Du et al. (2010) demonstrated that upregulation of TRPM7, a constitutively active Ca2+/Mg2+ entry channel (Wu et al, 2010), and resultant Ca2+ influx was crucial for the progression of atrial fibrosis in AF patients. This was supported by the following evidence; (1) human atrial fibroblasts express TRPM7 protein abundantly, and show basal Ca2+ influx and spontaneously active Ca2+-permeable inward currents with the fingerprint features of heterologously expressed TRPM7 channels [potentiation by low pH and inhibition/potentiation by low/high concentrations of 2-aminoethoxydiphenyly boroate (2-APB) respectively: Li et al., 2006, 2007]; (2) TRPM7-like current and accompanying Ca2+ influx were strikingly increased in parallel with the upregulation of TRPM7 expression in differentiated fibroblasts from AF patients; (3) all these changes were largely eliminated by knockdown of TRPM7 expression with small hairpin RNA; and (4) in vitro promotion of myofibroblastic differentiation by TGF-P1, a major stimulator of atrial fibrosis, was correlated with the upregulation of TRPM7. Although in this study a direct linkage between Ca2+ influx through TRPM7 channel, the fibrogenesis and arrhythmogenesis in AF have not been explored, the above results may provide a new therapeutic target against the progression of AF, by disclosing an essential role of TRPM7-asscociated Ca2+ influx in fibroblast proliferation and differentiation. In rat neonatal cardiac fibroblasts, endothelin-1 (ET-1) or AngII can induce myofibroblastic differentiation and collagen synthesis via the Ga12/13 signaling. ET-1 (via ETA receptor) and AngII can also selectively enhance the expression of TRPC6, thereby increasing basal Ca2+ influx in the fibroblasts. Importantly, increased TRPC6 expression is causally correlated with anti-fibrotic effects via calcineurin/NFAT pathway (Nishida et al., 2007). The upregulation of TRPC6 likely occurs through the G12/13- (Rac/NOX)-ROS-JNK signaling, which is reminiscent of TRPC6-mediated abnormal proliferation of pulmonary artery smooth muscle cells induced by platelet-derived growth factor, a pathogenic model of pulmonary artery hypertension (Yu et al., 2003). The concentrations of ET-1 or AngII required to upregulate TRPC6 are significantly higher than those for the differentiation of fibroblasts (Nishida et al., 2007). Thus, in an intriguing contrast to the pro-fibrotic role of TRPM7, the anti-fibrotic effects of enhanced TRPC6 channel activity may serve as a negative feedback mechanism to limit excessive fibrogenesis via ET-1/AngII signaling during cardiac remodeling. It remains to be determined whether this mechanism works beneficially in human AF patients or other fibrosis-associated arrhythmias.
Arrhythmia related to autonomic imbalance
The cardiac rhythm is under tight control of the autonomic nervous system. For instance, baroreceptors monitor blood pressure fluctuations and transmit the information to the brainstem vasomotor center via parasympathetic afferents, which then modulates the cardiac pumping force and rate via sympathetic efferents. The renin-angiotensin-aldosterone system is another well-established system centrally controlled via sympathetic nerves (via ^-adrenoceptor). It fulfills a long-term control of body fluid and electrolyte balance, thereby effectively regulating the cardiac output (Guyenet, 2006). Any disturbances in these centrally-mediated autonomic regulations could therefore become the substrates for cardiac arrhythmias (Danson, et al., 2006; Janse 2004; Nattel, et al., 2007). Accumulating evidence however suggests that nociceptive reflexes via respiratory sensory neurons (e.g. C-fibers in nose and lung) have also some pathophysiological impact on cardiac functions through centrally-mediated autonomic mechanisms, as exemplified by air pollutant-induced changes in ‘repolarization’ parameters of ECG (Henneberger et al., 2005). The sensory nerve endings in respiratory airways contain two TRP members sensing noxious stimuli, i.e. TRPV1 and TRPA1 which likely participate in airway chemosensation and inflammation (Bessac & Jordt, 2008). It has thus been postulated that these TRP isoforms may be involved in nociceptive signaling in the respiratory system (Vay et al., 2011). Two recent studies have revealed intriguing associations between respiratory sensory TRP channels and arrhythmogenicity. In one experimental model, activation of respiratory sensory neurons by inhalation of concentrated ambient particles significantly affected the cardiac rhythm with decreased heart rate, and shortened QT interval and P wave duration. Abrogation of these cardiac effects by a selective TRPV1 antagonist capsazepine suggested that TRPV1-mediated autonomic reflexes play a central role therein (Ghelfi et al., 2008). In another study employing a gaseous pollutant (Hazari et al., 2011), one-day exposure of rats to diesel exhaust gas resulted in increased heart rate with pro-arrhythmic ECG changes of prolonged action potential and shorted repolarization. Notably, these rats showed much heightened sensitivity to aconitine challenge that can induce moderate to lethal ventricular tachyarrhythmias dose-dependently. This pro-arrhythmic change was prevented by pretreatment with a TRPA1 antagonist HC-030031 or by sympathetic blockade with guanethidine, suggesting the involvement of increased sympathetic drive. In addition, partial involvement of TRPV1 in moderate aconitine-induced tachyarrhythmias was also suggested by pharmacological inhibition with a selective TRPV1 antagonist SB-366791. Although detailed mechanisms underlying remain unclear, these results clearly point to the pro-arrhythmic risk of environmentally-induced autonomic imbalance in which sensory chemosensing channels TRPA1 and TRPV1 may play a pivotal role.
Conclusions and therapeutic implications
The evidence presented above has disclosed that the unique activation profile (e.g. stretch-and Ca2+-sensitivities, neurohormonal activation, sensitivity to noxious stimuli) and permeability to Ca2+ and Na+ (TRPM4/TRPM5 are however virtually Ca2+-impermeable) render several TRP channels contribute to the acquired arrhythmogenesis during cardiac remodeling and other pathological processes. The examples so far available implicates, at least, TRPC1 and TRPC6 (and possibly their homologues) in stretch-induced arrhythmias in both acute and chronic manners, TRPM4 in Ca2+-dependent arrhythmia and familial conduction block, TRPC3 and TRPM2 in ischemia-induced arrhythmia, TRPA1 and TRPV1 in autonomic imbalance-induced arrhythmia, and TRPM7 and TRPC6 in fibrosis-related arrhythmogenicity, especially in AF, as pro-fibrotic and anti-fibrotic factors respectively. Although the mechanism of the actions of each TRP isoform appears variable in different pathophysiological settings, these new lines of evidence have certainly put forward our essential understanding about the pathogenesis of commonly observed arrhythmias associated with cardiac diseases accompanying structural and electrical remodeling. Simultaneously it may open an avenue toward exploiting an entirely new generation of anti-arrhythmic drugs for increased mechanosensitivity, abnormal Ca2+ handling, oxidative stress, or nociception.
When considering the actual strategy for developing such drugs, however, there are at least two not easily tractable problems. The serious lack of structure-based chemical knowledge about the selectivity and efficacy for most TRP members is the first obvious one, although substantial progress is going on for analgesic therapy based on TRPV1 pharmacology (Szallasi et al., 2007). Virtually all drugs so far shown to be effective for cardiovascular TRP isoforms appear to be non-specific or have only narrow ranges of concentrations for their relatively selective actions, as exemplified by a pyrazole compound Pyr-3 for TRPC3 which reportedly inhibits experimentally-induced cardiac hypertrophy (Kiyonaka et al., 2009). The second type of difficulty for exploiting TRP-selective drugs is that connecting one TRP isoform to one end-function is almost always irrelevant oversimplification. This is because signaling pathways linked to TRPs appear to form a complex interwoven network with high degrees of divergence and convergence and with numerous feed-back and -forward regulations. In addition to this, activation of TRP channels depends intimately on local lipid dynamics in the cell membrane, which dramatically changes by lipid composition, voltage, temperature, membrane stretch, and enzyme-assisted catalysis (Hardie, 2007; Inoue et al., 2009b). In the current absence of good knowledge about lipid physiology/pathophysiology for membrane proteins, these disadvantages would make it difficult to pharmacologically manipulate the gating of TRP channels. In this context, recent observations that activation of the cGMP-PKG signaling by nitric oxide, atrial natriuretic peptides and their structural and functional mimetics attenuate hypertrophic cardiac remodeling partly via inhibition of TRP channels (Inoue et al., 2010; Tsai & Kass, 2009) may provide a promising alternative maneuver to ameliorate the acquired arrhythmogenicity. Activation of this pathway is also known to improve myocardial damage after myocardial infarction (Burley et al., 2007; Garcia-Dorado et al., 2009). Obviously, there is much to be learned about the pathogenic roles and therapeutic potential of cardiovascular TRP channels for cardiac arrhythmias.