Acknowledgment

Calcium Signaling in the Cardiovascular System

In virtually all cell types in the heart and vasculature, changes in the frequency and amplitude of calcium transients have been recognized as an important response to the changing workload [1,2]. Encoded in these calcium transients are signals that alter not only the immediate contractile response, as occurs with excitation contraction coupling, but also signals that initiate and maintain a remodeling response that adjusts cellular mass, ionic currents, kinetic properties of contractile proteins, and metabolic capacity [3]. Indeed, increased Ca^2 + loading inside the cell, resulting in prolonged Ca^2 + transients and elevated end diastolic Ca^2 + concentrations, may be one of the pathophysiological changes that occurs in myocytes of the heart (cardiomyocytes) and vasculature [4,5]. Whether the handling of Ca^2 + and the downstream signaling are the same in these different cell types would have important therapeutic implications. In fact, recent attention has begun to focus on transient receptor potential (TRP) channels because they may link mechanical activity to cell signaling in all muscle cell types [6]. Once considered the purview of only nonexcitable cells, TRP channels are now believed to be a key downstream target of activated G-protein coupled receptors (GPRCs) in excitable muscle cells. Hypertrophic agonists use GPRC signaling to change the activation of TRPC channels and promote calcium entry, thereby triggering growth signaling.

One of the key pathways thought to be a cornerstone in the development of pathological hypertrophy of cardiac and smooth muscle myocytes is the calcineurin-NFAT (nuclear factor of activated T-cells) pathway. Calcineurin is a Ca^2 +-sensitive serine/threonine phosphatase that dephosphorylates NFAT and causes its translocation to the nucleus. In the nucleus, NFAT works with the cardiac-restricted zinc finger transcription factor GATA4 to activate the transcription of hypertrophic response genes encoding transcription factors such as NFAT and myocyte enhancing factor 2 (MEF2) [7]. Activation of the Ca^2 +-calcineurin-NFAT pathway has been strongly associated with the development of ventricular hypertrophy and found to be both sufficient and necessary for this pathological process [8,9]. MEF2, which is regulated independently by the class II histone deacytelases, is also turned on by calcineurin-induced desphosphorylation. The downstream effects of these Ca^2 +-calcineurin-activated pathways result in a hypertrophic response that is mediated by growth factors such as transforming growth factor-β (TGF-β), connective tissue growth factor, and a switch to cardiac fetal gene expression patterns [10]. Many of the same signaling events contribute to the pathologic changes that occur in response to injury of the pulmonary artery. Recently, there has been increased interest in understanding these intracellular Ca^2 + transients, which could potentially have a bigger role in controlling these transcription pathways. How the cell discriminates between different Ca^2 + signals remains a poorly understood area of research, but nevertheless important in truly understanding how a ubiquitous second messenger has the ability to modulate a wide variety of intracellular mechanisms, both in physiological and pathological states.

TRP Channels and Cardiovascular Disease

TRP channels were the last superfamily of ion channels to be identified and represent a large family of nonselective cation channels that operate at the plasma membrane [11]. The individual channels in the family are unique in their biophysical and electrical properties and are activated by different mechanisms [12]. TRP channels are expressed in various cell types including cardiomyocytes, smooth muscle cells, and Purkinje fibers, but their role in cardiovascular disease is currently being defined [13–16]. There is a growing body of evidence that certain TRP subfamilies, such as TRPC and TRP melastatin (TRPM), are more critically important in various cardiovascular pathologies, from cardiac hypertrophy and arrhythmogenesis to pulmonary arterial hypertension (PAH) [17].

TRP channels contribute to Ca^2 + entry via their action at the plasma membrane as nonselective cation channels. Regulation of intracellular Ca^2 + by extracellular mechanisms occurs through several pathways, including Ca^2 + release from intracellular organelles, activation of voltage-gated channels, Ca^2 + entry through ligand-gated channels, and receptor-operated Ca^2 + entry (ROCE). One of the postulated mechanisms by which TRP channels control Ca^2 + is through store-operated Ca^2 + entry (SOCE), where a drop in sarcoplasmic reticulum (SR) Ca^2 + stores triggers the influx of Ca^2 + through channels like Orai1 and TRPs at the plasma membrane [18]. SOCE has been implicated as a distinct mechanism of regulating intracellular calcium apart from L-type calcium channel and sodium-calcium exchanger and has been demonstrated in cardiomyocytes at the embryonic and neonatal stages [19]. With the discovery of Stromal Interaction Molecule 1 (STIM1) and Orai1 and their role in executing SOCE, it is not completely clear how critical a role TRP channels play or if they represent a distinct form of SOC channels [18]. Because many TRP channels are activated by a rise in intracellular Ca^2 + levels, it has been difficult to distinguish channel gating between Ca^2 + versus store dependence. However, there are several studies postulating that TRPCs, STIM1, and Orai1 interact with each other at a subcellular level to control SOCE, suggesting that at least some of the members of the TRP family are important in this mechanism [20].

Another mechanism by which TRP channels are activated to regulate intracellular Ca^2 + is receptor-mediated Ca^2 + entry. Agonist binding to a membrane receptor that is distinct from the TRP channel stimulates the production of intracellular second messengers that then lead to the activation of TRP channels to cause Ca^2 + influx [21]. Most literature suggests that TRP channels function downstream of GPCRs, which then activate phospholipase C and a downstream cascade of second messengers. Diacylglycerol (DAG) has been postulated as one of the downstream signals responsible for direct activation of TRP channels in receptor-mediated Ca^2 + entry. Some TRP channels have been proposed to be more involved in SOCE, and some are thought to be more receptive to receptor-mediated activation [22]. Within the TRPC subfamily, TRPC1/4/5 are thought to be activated through SOCE whereas TRPC3/6/7 are thought to be activated mainly by second messenger DAG [11] (Figure 20.1).

TRP Channels in Cardiac Hypertrophy

Pathological cardiac hypertrophy is a typically maladaptive state of cardiac muscle that is associated with a variety of disease states, including hypertension, ischemic injury, heart failure, and valvular disease. Once cardiac hypertrophy has developed, the risks of adverse cardiac events increase along with associated morbidity and mortality [23]. Understanding the pathophysiology behind cardiac hypertrophy is critical to any pharmacological approach that attempts to alter disease mechanisms in the heart [24]. There is a growing amount of evidence that the canonical TRP subfamily (TRPC) is involved in mediating the development of cardiac hypertrophy, especially in disease models of pressure overload and neurohormonal excess [17,25,26].

Overexpression of TRPC channels (especially TRPC1/C3/C6) has been associated with an exaggerated response of cardiac myocytes to stressors such as pressure overload and stimulation via neuroendocrine agonists. TRPC3 is overexpressed in neonatal rat ventricular myocytes subjected to phenylephrine (PE) infusions, suggesting that TRPC channels are activated in pathological states [27]. TRPC3 transgenic mice also show an age-dependent cardiomyopathy resulting from cardiac hypertrophy [28]. This cardiomyopathy also proved itself to be dose dependent, with high expressing TRPC3 transgenic mice developing pathology much earlier than the corresponding low-dose TRPC3 mouse line. Especially when subjected to agonists such as PE and angiotensin II (ATII) infusions for prolonged periods of time and transverse aortic constriction (TAC) to simulate pressure overload situations, mice overexpressing TRPC3 developed greater cardiac hypertrophy compared to wild-type controls. In certain studies, merely overexpressing TRPC3 was insufficient to stimulate the expression of genes associated with hypertrophy such as BNP (brain natriuretic peptide), but costimulation with hypertrophic agonists like PE did produce this response [27]. This finding suggests that even if altering TRPC channel expression at baseline does not induce pathological hypertrophy, the heart may become more susceptible to stresses such as pressure overload and neurohormonal excesses. ATII, which has been shown to induce the nuclear translocation of NFAT, also activates TRPC3 and TRPC6 channels through DAG, which results in the Ca^2 + influx that is necessary in activating the calcineurin-NFAT pathway leading to cardiac hypertrophy [29]. In addition, combined deletion of TRPC3 and TRPC6 was protective against cardiac hypertrophy, suggesting a role for pharmacological inhibitors of these channels [30].

Similar studies were conducted in mouse hearts with transgenic expression of TRPC6. The transgene dosage of TRPC6 in mouse hearts directly correlated with the heart weight/birth weight ratios in these mice, with the highest dosage of TRPC6 inducing the earliest and greatest signs of cardiac hypertrophy. Even though the transgenic mice with the lowest expression of TRPC6 showed no evidence of hypertrophy or heart failure at an adult stage, subjecting them to pressure overload by TAC immediately induced an increase in heart weight/birth weight ratios and a decrease in systolic function [31]. There is also evidence for the role of TRPC1 in the development of cardiac hypertrophy. Unlike TRPC3/6, which are DAG activated, TRPC1 may operate as a SOCE channel. Looking at TRPC1^−/− mice after subjecting them to pressure overload via TAC, we found that mice lacking TRPC1 only showed a modest increase in cardiac mass and had relatively preserved cardiac function when compared to wild type (WT) mice subjected to the same stresses. TRPC1^−/− mice also showed decreased NFAT activation post-TAC compared to their WT counterparts, suggesting that the absence of TRPC1^−/− was cardioprotective in the pathological situation of pressure overload [32]. Another study showed that TRPC1 channels stimulated the calcineurin/NFAT pathway through serotonin receptors in cardiomyoblasts, lending more support to the importance of TRPC1 in the development of cardiac hypertrophy [33].

Role of TRP Channels in Cardiac Hypertrophy Signaling Pathways

The pathological changes that occur in cardiac hypertrophy along with the alteration of the functional properties of the myocardium are likely mediated by altered gene expression patterns. Pathological cardiac hypertrophy is a result of cardiac remodeling and a change in the functional properties of the myocardium that can only be a result of altered gene expressions. Altered Ca^2 + signaling directly affects signaling pathways involved in the development of cardiac hypertrophy [24]. Because many of these pathways engage in cross talk with each other, understanding the choreographic details of these pathways is important if effective pharmacological approaches are to be developed in the future. It is beyond the scope of this chapter to discuss all the signaling mechanisms that are involved in the development of cardiac hypertrophy. We will focus the discussion on the pathways involved in the development of cardiac hypertrophy with which TRP channels have been associated.

Several studies have implicated the importance of the calcineurin-NFAT pathway in the development of cardiac hypertrophy [10]. Calcineurin (Ca^2 +/calmodulin-dependent protein phosphatase 2B) links changes in intracellular Ca^2 + to altered gene expression through NFAT. It is a serine/threonine phosphatase that is activated by increases in intracellular Ca^2 +. Once activated, it causes NFAT to become dephosphorylated and translocated to the nucleus. NFAT in the nucleus activates the transcription of several genes mediating cardiac hypertrophy through the cardiac-restricted zinc finger transcription factor GATA4 [34]. Fetal cardiac genes such as beta-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and BNP are all transcribed by the activation of the calcineurin-NFAT signaling pathway, with which TRP channels have been associated [35]. TRPC3 transgenic mice showed greater increases in the expression of a NFAT-luciferase transgene, especially when subjected to pressure overload and agonist stimulation, indicating that NFAT activation occurs downstream of TRPC channel activity. TRPC3 overexpression also stimulated translocation of NFAT to the nucleus, and TRPC6 was found to be a positive regulator of the calcineurin-NFAT pathway resulting in β-MHC expression [31,36]. TRPC3 and TRPC6 are directly activated by DAG, which links receptor activation to Ca^2 + influx through the TRPC channels and subsequently to the activation of the calcineurin-NFAT pathway [37].

TRP channels have also been linked to regulation by antihypertrophic pathways. ANP and BNP are antihypertrophic cardiac fetal proteins that are up-regulated by calcineurin-NFAT activation, possibly to modulate the complex process of pathological cardiac hypertrophy. ANP and BNP have a common receptor, guanylyl cyclase-A (GC-A), which leads to the activation of protein kinase G (PKG) by synthesis of cyclic guanosine monophosphate (cGMP). Initially, only the antiadrenergic effects of ANP, nitric oxide (NO), and cGMP through the inhibition of β and α adrenergic effects in cardiofibroblasts and myocytes were described [38]. In addition, the negative inotropic and growth-inhibiting effects of ANP were partly attributed to its ability to inhibit Ca^2 + influx by blocking L-type Ca^2 + channels [38]. Recently, the ANP-GC-A pathway has been better described in cardiac remodeling, opening up potential therapeutic avenues [39]. In mice with cardiomyocyte-specific deletion of (GC)-A, the heart weight/body weight ratios are increased, suggesting cardiac hypertrophy, and biomarkers of cardiac hypertrophy such as ANP and β-MHC are up-regulated [40]. These effects are even more exaggerated once the mice are subjected to pressure overload, indicating that preserving the ANP-GC-A pathway is important for prevention of cardiac hypertrophy. Studies are now showing that PKG, which is downstream of this pathway, catalyzes the phosphorylation of highly conserved threonine and serine residues on TRPC3/6, which attenuates the activities of these TRPC channels [41]. GC-A may even form a complex with TRPC3/6 in mice, suggesting that the pathway may be activated by TRPC channels independent of ANP [42]. Given that cardiac hypertrophy involves up-regulation of TRPC3/6, which contributes to NFAT activation, ANP/BNP may be acting as in vivo inhibitors of TRPC3/6 to produce an antihypertrophic effect [43,44]. In cultured neonatal rat ventricular myocytes, it was found that endothelin-1-activated NFAT expression was suppressed by ANP both at baseline and in models of TRPC6 overexpression [45]. Furthermore, when the PKG phosphorylation site, Thr69, on TRPC6 was mutated, the inhibitory effects of ANP were completely abolished, suggesting that ANP acts on TRPC channels through PKG-mediated phosphorylation of these conserved residues [45]. Thus, the calcineurin-NFAT pathway, which is prohypertrophic, and the ANP-GC-A-cGMP-PKG pathway, which is antihypertrophic, may cross talk with each other through TRPC channels to regulate cardiac remodeling [44].

Pharmacological Developments Targeting TRP Channels in Cardiac Hypertrophy

Using TRP channels as a therapeutic target is potentially useful given its role in the pathophysiology of cardiac hypertrophy, but it presents several pharmacological challenges. Defining a single-channel TRPC current has proven difficult, as TRPC channels work in a highly heterogeneous system and are present in vivo as homo and heterotetramers. Within the TRPC subfamily, the individual properties of each channel have not been well defined. In addition, TRPC channels most likely act in concert with STIM1 and Orai1 to regulate SOCE [20,46]. Understanding how these channels function collectively presents a significant challenge in finding antagonists to specific TRPC channels. Gene deletion could result in compensatory overexpression of other channels involved in SOCE and ROCE in ways that are not yet well understood. Aside from these challenges, there has been a relative paucity of selective small-molecule inhibitors of TRP channels. Kaneko and Szallasi provide a comprehensive review of the various TRP agonists and antagonists that have been implicated as potential therapeutic options [47]. BTP2 (N-[4-(3,5-bis(trifluoromethyl)-1H– pyrazol-1-yl)phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide) has been known to inhibit both TRPC and calcium release activated channels (CRAC), and SKF96365, which has been used in many of the studies investigating the physiology of TRPC channels, is an inositol triphosphate 3 (IP3) receptor that blocks both TRPC and T-type Ca^2 + channels [48–50].

For many years, only nonselective inhibitors of TRPC channels were available. One of the earliest TRPC inhibitors, known as BTP2, was initially identified as an inhibitor of SOCE current in T lymphocytes [50] but was eventually shown to be an inhibitor of TRPC3,5,6 and possibly even TRPM channels in human embryonic kidney (HEK293) cells [51]. Given the relative potency of BTP2 for TRPC channels and its ability to modify SOCE, it was a definite advance in the search for selective blockers of TRPC channels [52]. This discovery led to the development of other TRPC channel blockers by looking at the structural moieties that made up the family of BTPs. In 2008, a pyrazole compound, ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)- 1H-pyrazole-4-carboxylate was identified and named Pyr3 [53]. By stimulating HEK293 cells with carbachol and DAG to activate TRPC channels, the study showed that application of Pyr3 inhibited the activity of the TRPC3 channel with some selectivity when compared to Pyr1, Pyr2, and Pyr4. Looking at rat neonatal cardiomyocytes subjected to ATII infusions, it was found that treatment with Pyr3 decreased the translocation of NFAT and the expression of BNP, both of which are implicated in the process of pathological cardiac remodeling. When subjected to TAC, chronic treatment with Pyr3 resulted in attenuation of both concentric and dilated cardiomyopathy, as well as a decrease in ANP expression [53]. Pyr3 represents one of the first compounds showing relatively increased selectivity for TRPC3 channels compared to others in its class. However, because it blocks TRPC3 selectively and leaves other channels like TRPC6 and TRPC1 relatively untouched, it remains to be seen whether it can accomplish sufficient SOCE current blockade to prevent the development of cardiac hypertrophy. Other studies have suggested that individual TRPC channel blockade may not be enough to overcome the compensation by other members of the TRPC subfamily mediating the same pathological processes [30]. On the other hand, because TRPC channels function in heteromers, individual channel blockade may be sufficient to disrupt the normal functioning of the other channels making up the heteromer. Pyr3 has been shown to block Orai1 as well at concentrations that are approachable to the ones used for TRPC3 blockade, suggesting that it may not be as selective as predicted [54].

As part of the search for selective TRP channel inhibitors, a high throughput screen of piperidine and isoquinolone analogs based on an aniline-thiazide core helped identify high-potency analogs that blocked the increased TRPC3 and TRPC6 current in HEK cells when they were challenged with carbachol and a DAG analog [55]. Two of the novel compounds identified through this screen were used to model dual inhibition of TRPC3/6 in mouse and rat myocytes [30]. This study was unable to look at in vivo effects of the novel compounds GSK503A and GSK255B because of pharmacodynamic challenges in both animal models. However, when looking at cultured myocytes, it showed that the calcineurin-NFAT activation seen on stimulation by endothelin-1 was blunted by GSK503A in a dose-dependent manner. On subjecting mice to TAC to simulate pressure overload and then stimulating the cultured myocytes with ET-1, it showed increases in expression of TRPC3, TRPC6, and fetal gene markers of cardiac hypertrophy as well as increased NFAT activation. All these changes were attenuated when the myocytes were coincubated with GSK503A. Interestingly, when GSK503A was present, the overexpression of TRPC1 was also prevented, suggesting that blocking TRPC3/6 might prevent expression of other TRPC channels in the same subfamily. The results of this study go back to the original question of single versus dual inhibition of TRPC channels in future pharmacological approaches. Their single knockout models of TRPC3 and TRPC6, respectively, did not show any attenuation in cardiac hypertrophy when stimulated, suggesting that the methodologies of future studies modeling single TRPC channel inhibition will have to be scrutinized for their use of dominant negative proteins versus gene deletion models. The method of inhibition may be significant in the extrapolation of the data to the pharmacological potential of the compounds under study.

The ability of native proteins to regulate TRPC channels is also being discovered. A type I membrane protein called Klotho produced in the kidney, parathyroid glands, and the choroid plexus has been shown to inhibit TRPC6 and thus act as a potentially cardioprotective agent at baseline [56]. Murine hearts that were stressed by isoproterenol infusions showed exaggerated cardiac hypertrophy and cardiac fibrosis in Klotho deficient mouse lines. Even though underexpression or overexpression of Klotho produced no changes at baseline, under isoproterenol stress, overexpressing Klotho was cardioprotective.

Targeting the ANP-GC-A-cGMP-PKG-TRPC pathway is another strategy currently under study. Several mouse lines that were deficient in endothelial nitric oxide synthase (eNOS) demonstrated the expected phenotype of arterial hypertension but no cardiac hypertrophy [43]. In another study, the eNOS^−/− mouse line had increased expression of ANP in left ventricular myocytes, suggesting that the increase in ANP might be protecting the heart against hypertrophic processes [43]. When GC-A was knocked out in these eNOS^−/− mice, the cardioprotective effect against hypertrophy was lost. Given that the production of NO increases the synthesis of cGMP, thus activating the pathway that eventually results in TRPC6 inhibition, this is a therapeutic target that could potentially be exploited to mitigate the prohypertrophic signaling mechanisms governed by the TRPC channels [57,58]. Sildenafil is a cGMP specific phosphodiesterase 5 (PDE5) inhibitor clinically used for the treatment of pulmonary hypertension with new pharmacological roles in the attenuation of cardiac remodeling [39]. In experiments that looked at how sildenafil could affect TRPC activity, it was found that PDE5 inhibition led to PKG activation and subsequent phosphorylation of TRPC6 at Thr69. This phosphorylation by PKG was found to be necessary to suppress the activity of TRPC channels because altering the Thr69 residue abolished this effect [45,59]. When cultured cardiomyocytes stimulated by endothelin were treated with sildenafil, NFAT activation was suppressed, and the overexpression of TRPC1/3/6 was attenuated as well [60]. Using sildenafil to prevent cardiac hypertrophy is a tempting pharmacological approach, given that PDE5 inhibitors are already in clinical use. There is suggestion in the literature that phosphorylation mediated by PDE3 inhibitors like cilostazol may also impact the activity of TRPC channels [57].

TRP Channels and Cardiac Arrhythmias

The role of TRP channels in various cardiac conduction abnormalities is now being described. Several studies showed the presence of TRP channels in excitable tissue across the cardiac conduction system, from sinoatrial node cells to Purkinje cells to atrial myocytes [61,62]. In 2006, a Ca^2 +-activated, nonselective cation channel current was described in human atrial myocytes, which was similar to the current described in rat ventricular myocytes 2years earlier [63,64]. The fact that this channel was activated by micromolar concentrations of intracellular Ca^2 + led to the hypothesis that it might be involved in the development of cardiac arrhythmias such as atrial flutter and fibrillation in conditions like heart failure, where Ca^2 + homeostasis is disrupted. Furthermore, the biophysical properties of this channel were found to be similar to that of TRPM4 and TRPM5, which suggested that these channels might be one and the same [65]. Since then, there has been increased evidence from genetic studies of families with conduction disease and arrhythmias that the TRPM channels are indeed involved in abnormal cardiac arrhythmias [66].

TRPM4 and Cardiac Conduction Disease

TRPM4 is a member of the melastatin TRP subfamily. Unlike other TRP channels that are permeable to Ca^2 + in varying degrees, the TRPM4 channel is Ca^2 + impermeable and is only activated by an increase in intracellular Ca^2 +. It is only permeable to monovalent cations, being the most permeable to Na⁺ and K⁺. It is closely related to TRPM5 in these properties [65]. The presence of TRPM4 has been detected in various cardiac tissues, whether directly or indirectly. Ca^2 +-activated nonselective cation channel currents have been observed in the sinoatrial node whose properties are very similar to the TRPM4 current [67,68]. Ventricular myocytes have also shown evidence of a TRPM4-like current, even though detecting TRPM4 mRNA levels in the ventricle has proved to be challenging [64]. Staining for TRPM4 has shown in both atrial myocytes and the Purkinje fibers, suggesting its role in the normal physiology of the cardiac conduction system [69]. In fact, there is speculation that TRPM4 currents could be responsible for the phenomenon of supernormal conduction in the excitable tissue of the atria and His bundles of the heart [35,70].

Interestingly, many mutations in TRPM4 are being identified in several types of familial conduction disease. Progressive familial heart block type I (PFHBI) is a type of autosomal dominant cardiac conduction disease that has been genetically mapped to the locus on chromosome 19 that encodes TRPM4 [71]. The families with PFHBI show initial right bundle branch blocks (RBBB) that progress to bifascicular block and eventually complete heart block (CHB). Liu et al. recently described three families from Lebanon and France with mutations in the region on chromosome 19 that spans TRPM4 presenting with RBBB, left axis deviation, right axis deviation, and atrioventricular (AV) blocks [69]. The inheritance pattern of this isolated cardiac conduction disease (ICCD) was autosomal dominant with incomplete penetrance. Both studies found that TRPM4 was expressed at higher levels at the cell surface in the mutants compared to the wild types. They also proposed that defects in post-translational processes such as endocytosis and Small Ubiquitin MOdifier Conjugation (SUMOylation) caused the increased trafficking of TRPM4 to the plasma membrane. Endocytotic packaging of proteins and the balance between SUMOylation/deSUMOylation affect the degree of protein expression and degradation, ultimately impacting the functional intracellular protein levels. Both ICCD and PFHBI suggest that increased TRPM4 levels lead to an increase in the supernormal conduction, causing delays in normal conduction pathway impulse propagation, expressed clinically as RBBB, CHB, or AV blocks on the electrocardiogram. Another six novel mutations in TRPM4, including both amino acid substitutions and in-frame deletions, were discovered in both familial and sporadic cases of cardiac conduction disease by Stallmeyer et al., bringing the total number of known TRPM4 mutations linked with cardiac conduction abnormalities to 10 [72]. These mutations also resulted in conduction abnormalities such as RBBB and varying degrees of AV block.

Given that TRPM4 channelopathies are being discovered in families with cardiac conduction disease, TRPM4 may become a potentially useful therapeutic target for prevention of cardiac arrhythmias. In fact, various compounds with TRPM4 inhibitory and activation properties have already been described. 9-Phenanthrol, a benzo(c)quinolizinium derivative, has been described as an inhibitor of TRPM4 with high selectivity for this channel. 9-Phenanthrol was found to decrease the frequency of early after depolarizations induced by hypoxia and reoxygenation, suggesting that its antiarrhythmic activity may be linked to its direct inhibition of TRPM4 [73,74]. MPB-104 is another compound in the same pharmacological family as 9-phenanthrol, which is also known to have effects on the cystic fibrosis transmembrane conductance regulator [75]. Flufenamic acid is the TRPM4 inhibitor that has been most widely used in experimental models, but is also fairly nonselective, with actions on Ca^2 + activated Cl⁻ channels as well [76]. BTP2, which is an inhibitor of TRPC3 and TRPC5 as well as other CRAC channels, is thought to be a TRPM4 inhibitor. All these compounds are potential pharmacological targets that need to be further investigated for their applicability in the cardiac conduction diseases associated with TRPM4. In addition, the discovery that endocytic dysregulation is associated with the increased TRPM4 expression in both PFHBI and ICCD is raising interest about the possibility of regulation of ion channel density by controlling the mechanisms of endocytosis as a novel pharmaceutical approach. It remains to be seen whether any of these drugs can make it to the market as potential antiarrhythmics.

TRPM7 Channels and Atrial Fibrillation

Abnormal Ca^2 + handling is thought to be one of the mechanisms underlying the development of atrial fibrillation (AF). Increased delay after depolarizations due to ryanodine receptor dysregulation causing increased SR Ca^2 + load and subsequent Ca^2 + leak is thought to be one of the arrhythmogenic factors resulting in paroxysmal AF [74,77]. These Ca^2 + handling abnormalities can result in electrical and structural remodeling that leads to chronic AF [78]. The role of Ca^2 +-permeable channels in maintaining Ca^2 + homeostasis in cardiomyocytes is obviously important for prevention of arrhythmogenesis. However, another important pathological process underlying the development of AF is cardiac fibrosis. The degree of fibrotic changes in the atrium of the heart correlates directly with the persistence of AF [79]. Atrial fibrosis is one of the key structural arrhythmogenic changes that underlie the development of AF [77]. Fibrosis in the myocardium is usually triggered by insults like ischemia, oxidative stress, hypertension, and so on. In response to these stimuli, fibroblasts undergo proliferation and differentiation and adversely remodel the extracellular matrix incident to fibrosis. Evidence from multiple sources suggests that Ca^2 + has an important role to play in fibroblast growth and proliferation [80]. Because fibroblasts are nonexcitable and lack voltage-gated Ca^2 + channels, TRP channels have been suggested as one of the candidates regulating Ca^2 + entry into fibroblasts [81]. Given that TRP channels might play a part in mediating Ca^2 + homeostasis in conduction tissue and in the pathophysiology of cardiac fibrosis, their role in AF is being explored.

Both TRPM4 and TRPM7 are expressed in human atrial myocytes. However, TRPM4 is not Ca^2 + permeable, and there is currently no evidence linking its function to the development of AF. TRPM7 belongs to the same TRP melastatin subfamily as TRPM4, but it is permeable to divalent cations like Ca^2 + and is constitutively active [82]. In fact, immunoblotting has shown that not only is TRPM7 present in atrial myocytes along with TRPM4, but also its protein levels are highly up-regulated in patients with AF, whereas the TRPM4 levels remain unchanged [83]. TRPC1 is also expressed in both atrial myocytes and atrial fibroblasts, but although a functional TRPC1 current was discovered in atrial myocytes, the same could not be found in atrial fibroblasts, suggesting that the TRPC1 in fibroblasts does not contribute to the Ca^2 + entry and subsequent signaling mechanisms controlling the proliferation of fibroblasts [16,35]. Fibroblasts from AF patients tended to undergo increased differentiation into myofibroblasts compared to patients with normal sinus rhythm. Knocking down TRPM7 decreased this differentiation process [83]. TRPM7 expression was also found to be responsive to TGF-β1 treatment suggesting that TRPM7 and TGF-β1 work in concert to induce the differentiation process. There is also supportive evidence that TRPM7 is important in the early stages of cardiogenesis, and deletions of TRPM7 can result in abnormal ventricular structure and conduction properties [84].

TRPM7 is inhibited by Mg^2 +, both intracellularly and extracellularly, but there are currently not many pharmacological strategies targeting TRPM7 to prevent the arrhythmias associated with this channel. La^3 + and 2-aminoethoxydiphenylborate (2-APB) have been used as inhibitors of the TRPM7 channel in biological systems such as hepatic stellate cells and gastric adenocarcinoma cell lines, but none have been used to evaluate their effect on prevention of AF [85–87]. Because atrial fibrosis is also a process mediated by the renin-angiotensin system and the oxidative stress pathway apart from the TGF-β1 signaling pathway, it is not certain whether therapeutic approaches targeting TRPM7 will be sufficient for the abolishment of arrhythmias and the prevention of pathological atrial fibrosis. Research efforts are being directed toward identifying molecular players through high-throughput assays to search for effective TRPM7 inhibitors [88]. However, like other channels in the TRP family, TRPM7 is expressed in a variety of tissue types, and there is much more work to be done before targeting TRPM7 specifically for the purpose of AF therapy.

TRPC Channels in Pulmonary Hypertension

PAH is a panvasculopathy, with pathology found in every layer of the blood vessel. Histological findings include intimal hyperplasia, medial hypertrophy, distal muscularization of peripheral pulmonary arteries, adventitial thickening, and thrombosis in situ. Increased proliferation, decreased apoptosis, and hypertrophy of pulmonary artery smooth muscle cells (PASMC), sustained pulmonary vasoconstriction, and increased apoptosis of endothelial cells converge to create the obstruction and obliteration of the vascular lumen (reviewed in Ref. [89]). A major trigger for pulmonary vasoconstriction is an elevation in cytoplasmic free calcium concentration [Ca^2 +]i. Intracellular calcium is required for phosphorylation of myosin, a necessary step in the actin-myosin binding that results in cell contraction and ultimately pulmonary vasoconstriction. In addition, calcium serves as a second messenger in cell signaling, gene expression, and cell proliferation through nuclear transcription factors such as NFAT [90]. NFAT is a Ca^2 +-calcineurin-dependent transcription factor that promotes PASMC proliferation [91]. As such, alterations in intracellular calcium homeostasis regulate pulmonary vascular tone and PASMC growth [92–96]. Through their role in non-voltage-dependent calcium entry channels, TRPC channels have recently been implicated to support PASMC function and the development of PAH [97].

TRPC1 and TRPC6 are the two major TRPC channels expressed in human pulmonary arteries and PASMCs [94,96,98,99], with major contributing roles from TRPC3 and TRPC4 as well. In addition, mRNA and protein levels of TRPC1 and TRPC6 were found to be higher in distal than proximal pulmonary arteries (PAs) of rats, suggesting some heterogeneity of distribution (and its role in hypoxic vasoconstriction; Ref. [100]). In PASMCs derived from idiopathic PAH patients, TRPC6 and TRPC3 show excessive levels of expression compared to normal controls as well as patients with secondary (World Health Organization (WHO) group 2–5) pulmonary hypertension (PH) [98]. This finding suggests a unique role for the TRPC family in the pathogenesis of primary PAH as differentiated from the greater PH group. TRPC1 also plays a critical role in proliferation of human PASMCs [92,94,101]. Down-regulation of TRPC1 using antisense oligonucleotide inhibited the enhancement in cell proliferation by 50% and lowered the amplitude of capacitative calcium entry induced by serum and growth factors [94]. Experimental animal models of PAH show increased expression of TRPC1 and TRPC4 [102]. Increased expression/activity of platelet-derived growth factor receptor, an important mitogen involved in the vascular pathology of PAH [103], mediates cell proliferation at least partially by up-regulating TRPC6 expression [95]. Bosentan and sildenafil, oral antagonist of endothelin receptors and PDE5 inhibitor, respectively, both suppress PASMC proliferation as well as TRPC expression [101,104]. Capacitative calcium entry through TRPC family encoded receptors appears to represent a common downstream signaling pathway for many molecular mechanisms implicated in PAH.

At least 6% of PAH cases occur within a familial context [105], and greater than 70% of them display loss-of-function mutations in bone morphogenetic protein receptor type 2 that promote cell proliferation [106]. These mutations have also been detected in idiopathic cases without an obvious family history. Epigenetic mechanisms of inheriting PAH or influencing disease susceptibility also exist. Single nucleotide polymorphism (SNP) variants in genes invoked in PAH have been found in the serotonin transporter, Kv1.5, as well as TRPC6 [99,107,108]. A gain-of-function − 254(C → G) SNP within the regulatory regions of the TRPC6 gene is statistically associated with idiopathic PAH. Specifically, this SNP creates a binding sequence in TRPC6 that activates the ubiquitously expressed, inflammatory transcription factor, nuclear factor-kB (NF-kB). NF-kB then translocates into the nucleus, where it up-regulates TRPC6 expression and enhances calcium entry in idiopathic PAH PASMCs with the − 254G allele. Inhibition of nuclear translocation of NF-kB attenuates TRPC6 expression [99]. NF-kB regulates cellular responses activated by inflammation, oxidative stress, and response to pathogens by controlling other transcription factors [109]. In this manner, the − 254G allele primes the PASMCs in idiopathic PAH to an exaggerated response to NF-kB, creating a mechanistic link between PAH and the heightened inflammation described in its pathophysiology [89]. Allele carriers who are exposed to inflammatory triggers in the lung (e.g., collagen vascular autoimmune conditions, schistosomiasis, HIV) may have increased risk in developing vascular remodeling and PAH.

Chronic hypoxia is a common cause of secondary pulmonary hypertension. Physiologically, pulmonary arteries constrict under conditions of alveolar hypoxia to divert blood flow away from poor- to well-ventilated areas of the lung. This phenomenon of hypoxic pulmonary vasoconstriction (HPV) occurs in an acute phase within seconds and a sustained phase over hours. If hypoxia becomes chronic, it induces pulmonary vascular remodeling that will elevate pulmonary vascular resistance; it is a common experimental model of WHO group 3 PH. Animal pulmonary arteries develop medial hypertrophy, but there is no intimal fibrosis or plexiform lesions, unlike in PAH. PASMCs show proliferation and extend to distal small pulmonary arteries [110]. Hypoxia inhibits potassium current through the voltage-dependent Kv1.5 channel, which depolarizes the cell and increases intracellular calcium through L-type voltage-operated calcium channels as well as TRPC channels [111–113]. Indeed, following exposure to chronic hypoxia, PASMCs showed twofold expression of TRPC1 and TRPC6 [114–116]. In TRPC6^−/− mice, the acute phase of HPV was completely absent. However, the second sustained phase of vasoconstriction was not affected at all. In addition, when exposed to chronic hypoxia for 3weeks, TRPC6 deficient mice developed the same vascular remodeling and PH as wild-type mice [115,117]. TRPC6 appears only indispensible for acute HPV and not essential for non-hypoxia-induced vasoconstrictor response and remodeling. In contrast, TRPC1^−/− mice had suppression of PH after chronic hypoxia [115]. There was less PASMC migration and muscularization of distal vessels, but a similar degree of right ventricular hypertrophy (RVH) compared to wild-type mice [115,116]. In combined TRPC1 and TRPC6 knockout mice, however, markers such as PA pressure, RVH, and muscularization of distal vessels were all further suppressed compared to single TRPC1 knockout mice [115]. These studies show how specific TRPC isoforms can act in a temporal manner to influence vascular remodeling. Future development of therapeutic strategies will need to take into account how these channels intersect (Table 20.1).

Table 20.1

Selectivity of the inhibitors of various TRP channels

TRP channel	Inhibiting compound	Selectivity	References
TRPC1	PDE5 inhibitors	Nonselective	[59,60]
TRPC3	Pyr3	Selective	[53]
	BTP2	Nonselective	[50,52,118]
	PDE5 inhibitors	Nonselective	[59,60]
	GSK503A	Selective	[30,55]
TRPC5	BTP2	Nonselective	[50–52]
TRPC6	BTP2	Nonselective	[50–52]
	GSK503A	Selective	[30,55]
	PDE5 inhibitors	Nonselective	[59,60]
	Klotho	Selective	[56]
TRPM4	9-Phenanthrol	Selective	[73,74]
	MPB-104	Nonselective	[75]
	Flufenamic acid	Non selective	[76]
	BTP2	Nonselective	[50–52]
TRPM7	La3 +	Nonselective	[86,87]
	2-APB	Nonselective	[87,119–121]

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Like this:

Like Loading...

Related

Related posts:

1. The Role of TRPV1 in Acquired Diseases: Therapeutic Potential of TRPV1 Modulators
2. Conclusions
3. Clinical and Preclinical Experience with TRPV1 Antagonists as Potential Analgesic Agents
4. Potential Therapeutic Applications for TRPV4 Antagonists in Lung Disease

Stay updated, free articles. Join our Telegram channel

Join