Canonical Transient Receptor Potential Channels and Their Link with Cardio/Cerebro-Vascular Diseases.

The canonical transient receptor potential channels (TRPCs) constitute a series of nonselective cation channels with variable degrees of Ca2+ selectivity. TRPCs consist of seven mammalian members, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7, which are further divided into four subtypes, TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7. These channels take charge of various essential cell functions such as contraction, relaxation, proliferation, and dysfunction. This review, organized into seven main sections, will provide an overview of current knowledge about the underlying pathogenesis of TRPCs in cardio/cerebrovascular diseases, including hypertension, pulmonary arterial hypertension, cardiac hypertrophy, atherosclerosis, arrhythmia, and cerebrovascular ischemia reperfusion injury. Collectively, TRPCs could become a group of drug targets with important physiological functions for the therapy of human cardio/cerebro-vascular diseases.

INTRODUCTION

In 1969, Cosens and Manning (1969) discovered that Drosophila with mutations in a peculiar gene was defective and displayed transient light-induced receptor potentials (TRPs) in response to continuous light exposure, causing visual impairment in photoreceptor cells. This phenomenon was explained by a deletion in ion channels, and led to the discovery of “TRP genes” that were named TRP channels. To date, the TRP channels superfamily contains 28 members in mammals and is subdivided into six subfamilies: TRPA, TRPC, TRPML, TRPM, TRPN, TRPV and TRPP, all of which permeate cations (Montell, 2005). The canonical transient receptor potential channels (TRPCs) are the first encoded TRP gene family in mammals and are the most dominating non-voltage-gated, Ca2+-permeable cation channels in various cells (Zhu ). TRPCs fall into four groups in terms of their amino acid homology and similarities in function: TRPC1, TRPC2 (as a pseudogene in humans), TRPC4/5, and TRPC3/6/7 (Table 1) (Nilius and Voets, 2005; Minke, 2006). The seven subtypes have an invariant sequence in common in the C-terminal tail called a TRP box (Philipp ) and include three to four ankyrin-like repetitive sequences in the N-terminus (Montell ). Many subunits of TRPCs are able to coassemble. There exist heteromultimeric channels that consist of heterologously expressed and endogenous TRPC monomers (Nilius ). Indeed, TRPC1, TPRC4 and TRPC5 can form heteromers. Similarly, TRPC3, TRPC6, and TRPC7 form heteromers. In terms of activation mechanisms, members of the TRPC3, TRPC6 and TRPC7 subtypes can be stimulated by diacylglycerol (DAG) (Hofmann ), which is the phospholipase C (PLC)-derived production regulating their physiological activation. In contrast, the TRPC1/4/5 subgroups are completely insensitive to DAG, which is still a controversial mechanism (Venkatachalam ).

Table 1.

The properties of the TRPC family members

Category	Tissue distribution	Structure	Activation mechanism	Proposed regulation	Reference
TRPC1	Heart, Cartilage, Pituitary gland, Cerebellum, Caudate nucleus, Amygdala.	Six transmembrane spanning domains, TRP box in the C-terminus and three to four ankyrin-like repetitive sequences in the N-terminus	PKC-dependent phosphorylation	Store-operated, Store depletion	Riccio et al., 2002; Nilius and Voets, 2005; Minke, 2006; Xia et al., 2014
TRPC3	Pituitary gland, Cerebellum, Caudate nucleus, Putamen, Striatum.	Ibid ibidem	PKC-independent mechanism	DAG, Store-operated, Store depletion	Riccio et al., 2002; Welsh et al., 2002; Minke, 2006
TRPC4	Prostate, Bone. Parahippocampus.	Ibid ibidem	G-protein-coupled agonists	Store-operated, Store depletion?	Schaefer et al., 2000; Riccio et al., 2002; Plant and Schaefer, 2003
TRPC5	Cerebellum, Middle frontal gyrus, superior frontal gyrus	Ibid ibidem	G-protein-coupled agonists	Store-operated, Store depletion?	Schaefer et al., 2000; Riccio et al., 2002; Plant and Schaefer, 2003
TRPC6	Heart, Kidney, Adipose, Prostate, Cerebellum, Cingulate gyrus.	Ibid ibidem	PKC-independent mechanism	DAG, Receptor-operated	Riccio et al., 2002; Welsh et al., 2002; Winn et al., 2005; Xia et al., 2014
TRPC7	Pituitary gland, Kidney, Intestine, Prostate, Brain, Testis, Spleen, Cartilage.	Ibid ibidem	PKC-independent mechanism	DAG, Store depletion	Okada et al., 1999; Riccio et al., 2002
TRPC2	Only expressed in rodent,	Ibid ibidem	PLC-dependent mechanism	DAG, Store depletion?	Leypold et al., 2002; Stowers et al., 2002

“?” indicates that the proposed regulation is not completely confirmed.

Most TRPCs are inserted in the plasma membrane (PM) and can be hindered by blockers (Zhang ). Generally speaking, G protein-coupled receptors (GPCRs) have important roles in the regulation of TRPCs. In some cases, lipid signals can regulate the signals from GPCRs to TRPCs (Kukkonen, 2011).

A cytosolic Ca2+ change may be induced by activation of specific GPCRs, including an initial transient increase resulting from release of calcium ions from the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) and a subsequent sustained plateau phase via receptor-operated channels (ROCs) (Berridge ). This latter manner of Ca2+ entry is named “receptor-operated Ca2+ entry” (ROCE) (Soboloff ; Inoue ). Another manner of Ca2+ entry has been termed “store-operated Ca2+ entry” (SOCE) via store-operated channels (SOCs) (Shi ). SOCE occurs linked to depletion of intracellular Ca2+ stores (Putney, 1986; Ng and Gurney, 2001). Ca2+ refills depleted intracellular Ca2+ storages, directly accessing the SR/ER via SOCE. Although the exact functional relationship between TRPC and SOCE/ROCE is still indistinct, it is clear that TRPCs are the main channels of SOCs and ROCs. In recent years, SOCs and ROCs have gained increased attention for their role in mediating Ca2+ influx in response to cell function and disease. Previous studies suggested that TRPC family members, except TRPC2, are detectable at the mRNA level in the whole heart, vascular system, cerebral arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) (Yue ). TRPCs may participate in most cardio/cerebro-vascular diseases (Table 2) and play important roles in reactive Ca2+-signaling in the cardio/cerebro-vascular system (Fig. 1).

Table 2.

TRPC channels may participate in most cardio/cerebro-vascular diseases

Disease	Related TRPCs	Cells	Reference
Hypertension	TRPC1,TRPC3,TRPC6	SMCs, Monocytes	Dietrich et al., 2006; Chen et al., 2010; Dietrich et al., 2010; Inoue et al., 2009; Fuchs et al., 2010; Eder and Molkentin, 2011; Gopal et al., 2015
Pulmonary hypertension	TRPC1,TRPC3,TRPC6	PASMCs	Liu et al., 2007a, 2009; Edwards et al., 2010; Iwasaki et al., 2011; Liu et al., 2012; Loga et al., 2013; Malczyk et al., 2013; Maier et al., 2015
Cardiac hypertrophy	TRPC1,TRPC3,TRPC6, TRPC7	Cardiomyocytes	Piper et al., 2004; Montell, 2005; Minke, 2006; Onohara et al., 2006; Nakashima and Kumagai, 2007; Ohba et al., 2007; Rosenbaum et al., 2015
Atherosclerosis	TRPC1,TRPC3,TRPC4, TRPC5,TRPC6	Platelets, VSMCs, Monocytes/Macrophages, Endothelial cells	Short et al., 1993; Satoh et al., 2007; Shan et al., 2008; Smedlund and Vazquez, 2008; Smedlund et al., 2010
Arrhythmia	TRPC3,TRPC6	Myocardial cells, Fibroblast	Wang et al., 2006; Takahashi et al., 2007; Thilo et al., 2009; Tauseef et al., 2016
Ischemia-reperfusion	TRPC3,TRPC6	Myocardial cells	Xu and Beech, 2001; Wu et al., 2010; Zhang et al., 2014

Fig. 1.

Molecular mechanism underlying cardiovascular diseases associated with the changing of intracellular Ca2+ through TRPCs. GPCRs, releasing DAG and IP3 via PIP2 with the subsequent activation of PLC, were stimulated by Ang II and PE, which were hypertrophic stimuli. DAG stimulated ROCs, including TRPC3 and TRPC6, resulting in extracellular Ca2+ influx. IP3 activated SOCE in response to depletion of intracellular Ca2+ stores by Ca2+ release in the SR/ER and subsequently activated TRPCs. The sustained TRPC-mediated Ca2+ entry directly activated the calcineurin-NFAT pathway, subsequently resulting in the activation of hypertrophic gene expression, including TRPC1, TRPC3 and TRPC6. Simultaneously, after activating, NFAT might activate TRPC gene expression through a positive feedback mechanism. TRPCs interacted with the LTCC through membrane depolarization, playing a role in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility. Mechanical stretch caused arrhythmia through the activation of SACs to elevate cytosolic Ca2+ levels. Fibroblast regulated by Ca2+-permeable TRPCs might be associated with AF, and fibroblast proliferation and differentiation are a central feature in AF-promoting remodeling. TRPCs maintained adherens junction plasticity and enabled EC-barrier destabilization by suppressing SPHK1 expression to induce endothelial hyperpermeability, leading to atherosclerosis. In addition, the omission of extracellular Ca2+ with channel blockers (SKF96365, Pyr3) reduced monocyte adhesion and ATP-induced VCAM-1 and also relieved the progress of atherosclerosis. The rise of cytosolic [Ca2+]i promoted SMC proliferation. TRPC channels associated with vascular remodeling caused hyperplasia of SMCs. Moreover, TRPCs participated in blood pressure regulation due to receptor-mediated and pressure-induced changes in VSMC cytosolic Ca2+. Signaling via cGKI in vascular smooth muscle, by which endothelial NO regulated vascular tone, caused VSMC contraction. Activated TRPCs can activate downstream effectors and CREB proteins that have many physiological functions; TRPCs activated in neurons are linked to numerous stimuli, including growth factors, hormones, and neuronal activity through the Ras/MEK/ERK and CaM/CaMKIV pathways. GPCRs, G protein-coupled receptor; Ang II, Angiotensin II; PE, phenylephrine; ROCs, receptor-operated channels; SOCE, store-operated Ca2+ entry; LTCC, L-type voltage-gated calcium channel; SACs, stretch-activated ion channels; AF, atrial fibrillation; SPHK1, sphingosine kinase 1; VCAM-1, Vascular cell adhesion molecule-1; SMCs, smooth muscle cells; VSMC, vascular smooth muscle cells; cGKI, cGMP-dependent protein kinase I; CREB, cAMP/Ca2+-response element-binding.

Role of TRPCs in hypertension

Hypertension is a chronic cardiovascular disease characterized by persistently elevated blood pressure and is a major risk factor for coronary artery disease, stroke, heart failure, and peripheral vascular disease. In recent years, numerous studies have focused on the relationship between primary hypertension and TRPCs (Fuchs ). In pathological states, some signaling factors are involved in the transition of SMCs into the proliferative phenotype, leading to an excessive growth of SMCs (Beamish ). Abnormal over-growth of SMCs is implicated in various vascular diseases, including hypertension (Beamish ). Previous studies have convincingly suggested that several TRPC members are involved in hyperplasia of SMCs. TRPC1/3/6 all have been involved in enhanced proliferation and phenotype switching of SMCs (Dietrich ; Takahashi ; Koenig ). Kumar suggested that TRPC1 was up-regulated in rodent vascular injury models and in human neointimal hyperplasia after vascular damage. In coronary artery SMCs, upregulation of TRPC1 results in angiotensin-II (Ang II)-mediated human coronary artery SMC proliferation (Takahashi ). Moreover, other studies found that the visible whole-cell currents were triggered by passive depletion of Ca2+ storages in vascular smooth muscle cells (VSMCs) in wild type mice, but not in Trpc1−/− mice (Shi ), suggesting TRPC1 contributed to the alteration of whole-cell currents in VSMCs (Shi ).

In addition, TRPC3 also plays a pivotal role in Ca2+ signaling and a pathophysiological role in hypertension. The previous studies suggested TRPC3 levels were elevated in patients with hypertension as well as in the pressure-overload rat and the spontaneous hypertensive rat (SHR) models (Liu ; Onohara ; Thilo ). In monocytes, DAG-, thapsigargin-and Ang II-induced Ca2+ influxes were elevated in response to pathological state in SHR. However, further studies proved that downregulating TRPC3 by siRNA or applying with Pyrazole-3 (Pyr3), a highly selective inhibitor of TRPC3, reduced DAG-, thapsigargin- and Ang II-induced Ca2+ influx in monocytes from SHR (Liu ; Chen ), prevented stent-induced arterial remodeling, and inhibited SMC proliferation (Yu ; Schleifer ). Similarly, compared with normotensive patients, increased expression of TRPC3 and a subsequent increase in SOCE has been noticed in monocytes from hypertension patients (Liu , 2007b). These data show a positive association between blood pressure and TRPC3, indicating an underlying role for TRPC3 in hypertension.

TRPC6 is a ubiquitous TRPC isoform expressed in the whole vasculature, which plays a pivotal role in blood pressure regulation because of its physiological importance in both receptor-mediated and pressure-induced increases of cytosolic Ca2+ in VSMCs (Toth ). Studies suggested that cGMP-dependent protein kinase I (cGKI), which was implicated in the regulation of smooth muscle relaxation, inhibited the activity of TRPCs in SMCs (Kwan ; Takahashi ; Chen ; Dietrich ) and regulated vascular tone via endothelial nitric oxide (NO) (Loga ). However, the knockout of TRPC6 might injure endothelial cGKI signaling and vasodilator tone in the aorta (Loga ). Although deletion of TRPC6 decreases SMC contraction and depolarization induced by pressure in arteries, the basal mean arterial pressure in Trpc6−/− mice is about more than 7 mm Hg higher than that in wild type mice (Welsh ; Dietrich ), indicating that TRPC6 participated in smooth muscle contraction. Similarly, in deoxycorticosterone acetate (DOCA)-salt-hypertensive rats, overexpression of TRPC6 strengthened agonist mediated VSMC contractility companied with increased mean blood pressure (Bae ). Additionally, mineralocorticoid receptor-induced TRPC6 mRNA level was elevated in the aldosterone-treated rat A7r5 VSMCs, suggesting that heightened TRPC6 expression importantly participates in increased VSM reactivity (Bae ).

Role of TRPCs in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by a thickening of the pulmonary arterial walls, which can cause right heart failure (Yu ). Increased pulmonary vascular resistance is a primary factor in the progression of PAH. Ca2+ entry from the extracellular space, acting as a crucial mediator, is implicated in vasoconstriction (through its pivotal effect on pulmonary artery smooth muscle cells (PASMCs) contraction) and vascular remodeling (through its stimulatory effect on PASMC proliferation) (Kuhr ; Weber ). The most frequently expressed isoforms of TRPC in VSMCs are TRPC1, TRPC4, and TRPC6; TRPC3, TRPC5, and TRPC7 are less frequently detected (Inoue ; Maier ). Studies showed that Ca2+ entry improved the level of cytosolic Ca2+ through SOCs and ROCs (which is formed by TPRCs), and sufficient Ca2+ in the SR induced VSMC proliferation (Birnbaumer ; Golovina ; Bergdahl ; Satoh ; Seo ).

TRPC1, TRPC4 and TRPC6 are involved in hypoxic pulmonary vasoconstriction, which is related to increased SOCE. Additionally, SOCE contributes to basal intracellular Ca2+ concentration ([Ca2+]i) and the proliferation and migration of PASMCs in rat (Lu ). Malczyk demonstrated that TRPC1 played an important role in hypoxia-induced PAH, as hypoxia-induced PAH is alleviated in Trpc1−/− mice. Xia found that TRPC1/6 are crucial for the regulation of neo-muscularization, vasoreactivity, and vasomotor tone of pulmonary vasculatures; the combined actions of the two channels have a distinctly larger influence using Trpc1−/−, Trpc6−/− and Trpc1−/−/Trpc6−/− mice. Significantly, another study confirmed the upregulation of TRPC1/6 expression in murine chronic hypoxia PAH models (Wang ). Silence of TRPC1 and TRPC6 specifically attenuated thapsigargin-and 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced cation entries, respectively, indicating that TRPC1-mediated SOCE and TRPC6-mediated ROCE are upregulated by chronic hypoxia (Lin ). TRPC4 is also involved in PAH. In monocrotaline-induced PAH rats, TRPC1 and TRPC4 protein levels were both increased significantly, resulting in enhanced vasoconstriction to endothelin-1 (ET-1) (Liu ). In addition, siRNA specifically targeting TRPC4 reduced increases in TRPC4 expression and capacitative calcium entry (CCE) amplitude and inhibited ATP-induced PASMC proliferation (Zhang ).

The expression and function of TRPCs are variously regulated by molecules in PAH. Wang implied that both bone morphogenetic protein-4 (BMP4) and hypoxia inducible factor-1α (HIF-1α) upregulated TRPC1 and TRPC6, leading to elevated basal [Ca2+]i in PASMCs, driving the development of chronic hypoxia-induced PAH (Wang ). Another study found that TRPC expression was found absent in mice partially deficient for HIF-1α (Wang ). In human PASMCs, siRNA of the HIF-1α reduced hypoxia-induced BMP4 expression and knockout of either HIF-1α or BMP4 abrogated hypoxia-induced basal cytosolic Ca2+ increase and TRPC expression (Zhang ; Wang ). Also, TRPCs have been recognized as reactive oxygen species (ROS)-activated channels and it is suggested that they are critical for hypoxia associated with vascular regulatory procedures in lung tissue.

TRPCs could be regulated by pharmacological intervention during PAH. The treatment of experimental PAH with sildenafil and sodium tanshinone IIA sulfonate suppresses TRPC1/6 expression (Lu ; Wang ). SAR7334, an inhibitor of TRPC6, suppresses native TRPC6 activity in vivo (Maier ) and opens new opportunities for the investigation of TRPC function. In the lung and PASMC from idiopathic PAH patients, the mRNA and protein expression levels of TRPC6 were much higher than that from normotensive or secondary PAH patients. Also, inhibition of TRPC6 expression markedly attenuated idiopathic PAH-PASMC proliferation (Yu ). As a consequence, the participation of TRPC1/4/6 are crucial for PAH.

These results suggest that overexpression of TRPC may partially contribute to the increased PASMC proliferation, hinting at a promising therapeutic strategy for PAH patients.

Role of TRPCs in cardiac hypertrophy

Cardiac hypertrophy serves as a common pathway in cardiovascular diseases. It is the most important pathological foundation resulting in cardiogenic death. Although one study showed that the knockout of some TRPC genes did not result in abnormality in normal mice hearts (Yue ). TRPCs have been demonstrated to play an important role in the pathological progress of cardiac hypertrophy through the mediation of ion channel activities and downstream signaling. Dysregulation of TRPCs may lead to maladaptive cardiac hypertrophy.

Numerous studies have shown that TRPC expression and activity are up-regulated in pathological cardiac hypertrophy (Bush ; Kuwahara ; Ohba ; Seth ). Cardiac hypertrophy induced by transverse aortic constriction (TAC) was improved in Trpc1−/− mice. Meanwhile, downregulation of TRPC1 reduced SOCE and prevented ET-1-, Ang II-, and phenylephrine (PE)-induced cardiac hypertrophy, indicating that deletion of TRPC1 avoided harmful influences in response to increased cardiac stresses in Trpc1−/− mice (Ohba ). Also verified that TRPC1-mediated Ca2+ entry stimulated hypertrophic signaling in cardiomyocytes (Seth ). Similarly, cardiac pathological hypertrophy could be caused by stimulation of pressure overload or overexpression of the TRPC3 gene in cardiomyocytes from TRPC3 transgenic mice, and could be selectively inhibited by Pyr3 (Nakayama ; Kiyonaka ). Also, TRPC6 has been proposed as a critical target of anti-hypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway (Kinoshita ). However, a recent study showed Trpc6−/− mice resulted in an obvious augment in the cardiac mass/tibia length (CM/TL) ratio after Ang II, while the Trpc3−/− mice showed no alteration after Ang II injection. However, the protective effect against hypertrophy of pressure overload was detected in Trpc3−/−/Trpc6−/− mice rather than in Trpc3−/− or Trpc6−/− mice alone (Seo ). Similarly, the newly developed selective TRPC3/6 dual blocker showed an obvious inhibition to myocyte hypertrophy signaling activated by Ang II, ET-1 and PE in a dose-dependent manner in HEK293T cells as well as in neonatal and adult cardiomyocytes (Seo ).

Although the TRPCs role in myocardial hypertrophy is controversial, it is generally believed that calcineurin-nuclear factor of activated T-cells (Cn/NFAT) is a critical factor of microdomain signaling in the heart to control pathological hypertrophy. Studies found that transgenic mice that express dominantnegative myocyte-specific TRPC3, TRPC6 or TRPC4 attenuated the reactivity following either neuroendocrine-like or pressure overload-induced pathologic cardiac hypertrophy through Cn/NFAT stimulation in vivo, demonstrating that blockades of TRPCs are necessary adjusters of hypertrophy (Dietrich ; Wu ; Eder and Molkentin, 2011).

Undoubtedly, TRPCs play an important role in cardiac hypertrophy and can be regarded as new therapeutic target in the development of new drugs.

Role of TRPCs in atherosclerosis

Atherosclerosis is commonly considered a chronic disease with dominant accumulation of lipids and inflammatory cells of the arterial wall throughout all stages of the disease (Tabas ). Several types of cells such as VSMCs, ECs, monocytes/macrophages, and platelets are involved in the pathological mechanisms of atherosclerosis.

It has been reported that the participation of proliferative phenotype of VSMCs is a consequential part in atherosclerosis. Cytoplasmic Ca2+ dysregulation via TRPC1 can mediate VSMC proliferation (Edwards ). Studies have established that TRPC1 is implicated in coronary artery disease (CAD), during which the expression of TRPC1 mRNA and protein are elevated (Cheng ; Edwards ). Kumar showed the upregulated TRPC1 in hyperplastic VSMCs was related to cell cycle activity and enhanced Ca2+ entry using a model of vascular injury in pigs and rats. In addition, the inhibition of TRPC1 effectively attenuates neointimal growth in veins (Kumar ). These results indicate that upregulation of TRPC1 in VSMCs is a general feature of atherosclerosis.

The vascular endothelium is a polyfunctional organ, and ECs can generate extensive factors to mediate cellular adhesion, smooth muscle cell proliferation, thromboresistance, and vessel wall inflammation. Vascular endothelial dysfunction is the earliest detectable manifestation of atherosclerosis, which is associated with the malfunction of multiple TRPCs (Poteser ). Tauseef showed that TRPC1 maintained adherens junction plasticity and enabled EC-barrier destabilization by suppressing sphingosine kinase 1 (SPHK1) expression to induce endothelial hyperpermeability. Also, Poteser demonstrated that porcine aorta endothelial cells, which co-expressed a redox-sensitive TRPC3 and TRPC4 complex, could give rise to cation channel activity. Furthermore, mice transfected with TRPC3 showed increased size and cellularity of advanced atherosclerotic lesions (Smedlund ). In addition, studies further supported the relevance of EC migration to the healing of arterial injuries, suggesting TRPC5 and TRPC6 were activated by hypercholesterolemia, which impairs endothelial healing in vitro and in vivo (Rosenbaum ; Chaudhuri ).

Monocyte activation, adhesion to the endothelium, and transmigration into the sub-endothelial space are critical for early pathogenesis of atherosclerosis. The roles of TRPCs have been identified in the macrophage efferocytosis and survival, two crucial events in atherosclerosis lesion development (Tano ). It has been shown that high D-glucose or peroxynitrite-induced oxidative stress significantly increased the expression of TRPCsin human monocytes (Wuensch ). Vascular cell adhesion molecule-1 (VCAM-1) is important in monocyte recruitment to the endothelium as a critical factor in the development of atherosclerotic lesions. Smedlund et al. suggested that inhibition of TRPC3 expression could significantly attenuate ATP-induced VCAM-1 and monocyte adhesion (Smedlund and Vazquez, 2008; Smedlund ), indicating TRPC3 is involved in atherosclerosis lesion development. The platelet also plays important roles in cardiovascular diseases, especially in atherosclerosis, by participating in the formation of thrombosis and the induction of inflammation (Wang ). Liu investigated platelets in type II diabetes mellitus (DM) patients and found a time-dependent and concentration-dependent amplification of TRPC6 expression on the platelet membrane after challenge with high glucose. These results indicate that the incremental expression and activation of TRPC6 in platelets of DM patients may result in the risk of increasing atherosclerosis.

In summary, the pathophysiological relevance of TRPCs in several critical progresses has been linked to atherosclerosis.

Role of TRPCs in arrhythmia

Arrhythmia is a group of conditions in which the electrical activity of the heart is irregular, either too fast (above 100 beats per minute, called tachycardia) or too slow (below 60 beats per minute, called bradycardia). Several experiments have shed light on TRPC-regulated Ca2+ entry in arrhythmia. Sabourin et al. (2011) found that the existence of TRPC1,3,4,5,6 and 7 in the atria and ventricle, via association with the L-type voltagegated calcium channel (LTCC), plays a role in the modulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis. Mechanical stretch is one of the causes of cardiac arrhythmia. It has been demonstrated that mechanical transformation of ventricular myocytes can modulate TRPC6. The process can be inhibited by GsMTx-4, which is a peptide isolated from tarantula venom and a specific inhibitor of stretch-activated channels (SAC) (Dyachenko ; Anderson ; Gopal ).

One of the most common arrhythmias is atrial fibrillation (AF) (Nattel, 2011; Wakili ). By researching fibroblast regulation by Ca2+-permeable TRPC3, Harada found that AF increased expression of TRPC3 by activating NFAT-mediated downregulation of microRNA-26. Further, they found that AF induced TRPC3-dependent increase of fibroblast proliferation and differentiation, likely by mediating the Ca2+ entry that stimulates extracellular signal-regulated kinase signaling. TRPC3 blockade prevented AF substrate development in a dog model of electrically maintained AF in vivo (Harada ). In conclusion, by promoting fibroblast pathophysiology, TRPC3 is likely to play an important role in AF.

Role of TRPCs in ischemia reperfusion injury

Tissue injury led by ischemia reperfusion is the main cause of cell apoptosis and necrosis leading to myocardial infarction, stroke, and other deadly diseases. After focal cerebral ischemia, brain injury results from a suite of pathological progresses, including inflammation, excitotoxicity, and apoptosis. Researchers have indicated that an increase in cytosolic Ca2+ is a critical step in initiating myocardial cell apoptosis and necrosis responding to ischemia reperfusion (Carafoli, 2002; Brookes ). Several Ca2+ entry pathways, including the CCE and the Na+/Ca2+ exchanger channel, have been implicated in mediating myocardial cell Ca2+ overload (Carafoli, 2002; Brookes ; Piper ). An increasing number of studies show that members of the TRPC proteins are involved in regulating CCE. Given this growing evidence linking TRPC proteins to CCE in myocardial cells subjected to ischemia reperfusion injury, Liu tested the assumption that increased expression of TRPC3 in myocardial cells results in increased sensitivity to the injury after ischemia reperfusion, and found that the treatment of CCE inhibitor SKF96365 markedly improved cardiomyocytes viability in response to overexpressed TRPC3. In contrast, the LTCC inhibitor verapamil had no effect (Shan ; Liu ). These data strongly indicate that CCE mediated through TRPCs may lead to Ca2+-induced cardiomyocyte apoptosis caused by ischemia reperfusion injury.

Intracellular Ca2+ overload is also the major reason of neuronal death after cerebral ischemia. TRPC6 protein is hydrolyzed by the activation of calpain induced by intracellular Ca2+ overload in the neurons after ischemia, which precedes ischemic neuronal cell death. The inhibition of proteolytic degeneration of TRPC6 protein by blocking calpain prevented ischemic neuronal death in an animal model of stroke (Du ). Studies found that the upregulated TRPC6 could activate downstream effectors cAMP/Ca2+-response elementbinding (CREB) proteins, which are activated in neurons linked to a number of stimuli including growth factors, hormones, and neuronal activity through the Ras/MEK/ERK and CaM/CaMKIV pathways (Shaywitz and Greenberg, 1999; Tai ; Du ). It was also demonstrated that enhanced CREB activation activated neurogenesis, avoided myocardial infarct expansion, and reduced the penumbra region of cerebral ischemia and infarct volumes (Zhu ). Thus, TRPC6 neuroprotection relied on CREB activation. Similarly, Lin demonstrated that resveratrol prevented cerebral ischemia/reperfusion injury through the TRPC6-MEK-CREB and TRPC6-CaMKIV-CREB pathway.

The aforementioned results provide further evidence that TRPC3 and TRPC6 play roles in the mediation of cardiomyocyte function and suggest that TRPC3 and TRPC6 may contribute to increased tolerance to ischemia reperfusion injury.

DISCUSSION

Mechanisms including elevated activation or expression of TRPCs that partake in mediating Ca2+ influx activated by GPCRs offer the chance to interfere with Ca2+-dependent signaling processes, thus playing a significant role in cardio/cerebro-vascular diseases. The primary regulatory paradigm for most of these activities takes charge of total cytosolic Ca2+ or the propagation of intracellular Ca2+ signaling events that regulate cellular activity. Strong evidence indicates that TRPCs conduce to mechanical and agonist-induced SMC or fibroblast proliferation, cardiomyocytes apoptosis, and endothelium dysfunction. TRPCs were also present in Ang II-induced endothelium-dependent vasodilation and elevated contractility, regulation of vascular angiogenesis to participate in hypertension, pulmonary arterial hypertension, cardiac hypertrophy, atherosclerosis, arrhythmia, and ischemia reperfusion injury. These new findings permit a more comprehensive assessment of the molecular and cellular importance of TRPCs in physiology and pathophysiology.

Many questions remain to be elucidated. Therefore, researchers should keep a watchful eye on how the novel effects of TRPCs can be committed to human cardio/cerebro-vascular diseases and clarify the clinical relevance of TRPC activities. An improved understanding of the underlying mechanisms of cardiovascular and cerebrovascular diseases may assist in the design of new therapies and the identification of more selective pharmacological agonists and antagonists (Table 3) for TRPCs or interdependent channels as well as promote exciting chances to develop new therapies that prevent or treat cardio/cerebro-vascular diseases.

Table 3.

The essential information about inhibitors of TRPC channels or interdependent channels.

Inhibitor	Chemical structure	Targeting channels	Predicted effects	Action mechanism	Reference
SKF96365		TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7	Selectively decrease receptor-mediated calcium entry (RMCE) in human platelets, neutrophils and endothelial cells	Inhibit receptor-mediated Ca2+ entry and voltage-gated Ca2+ entry	Merritt et al., 1990; Farooqi et al., 2013
Pyrazole-3 (Pyr3)		TRPC3	Prevent stent-induced arterial remodeling and inhibit SMC proliferation	Inhibit TRPC3 by binding to the extracellular side of the receptor	Rowell et al., 2010; Christian and Maik, 2011; Koenig et al., 2013
SAR7334		TRPC3, TRPC6, TRPC7	Effect on acute hypoxic pulmonary vasoconstriction and systemic blood pressure	Inhibit TRPC3, TRPC6, TRPC7-mediated Ca2+ influx into cells	Maier et al., 2015
GsMTx-4	Gly-Cys-Leu-Glu-Phe-Trp-Trp-Lys-Cys-Asn-Pro-Asn-Asp-Asp-Lys-Cys-Cys-Arg-Pro-Lys-Leu-Lys-Cys-Ser-Lys-Leu-Phe-Lys-Leu-Cys-Asn-Phe-Ser-Phe-NH2	Stretch-activated ion channels and NaV1.7 Na + channels and TRPC1, TRPC6	Potential therapeutic targets for cardiac arrhythmias	Inhibit Na+ voltage-gated channels and cation-selective mechanosensitive channels	Franz and Bode, 2003; Bowman et al.,2007; Rowell et al., 2010
BTP2		TRPCs and Store-operated Ca2+ influx and Ca2+ release-activat ed Ca2+ channels	Suppresses cytokine production (IL-2, IL-4, IL-5, IFN-γ, etc.) and proliferation in T cells in vitro	Inhibit anti-CD3 antibody-induced sustained Ca2+ influx	Ohga et al., 2008; Rowell et al., 2010