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Aims Previously we demonstrated that both hypoxia inducible factor-1 (HIF-1) and bone morphogenetic protein-4 (BMP4) up-regulate transient receptor potential canonical (TRPC) 1 and TRPC6, resulting in increased basal intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs), driving development of chronic hypoxia (CH)-induced pulmonary hypertension (CHPH). This study aims to determine whether HIF-1 regulates BMP4, and whether BMP4 mediates TRPC and basal [Ca2+]i increases in hypoxic PASMCs. Methods and results The level of BMP4 mature protein was increased for ∼183% in distal pulmonary arterial smooth muscle (PA) from CH (10% O2 for 21 days; CH) exposed rats, and 143% in PASMCs cultured under prolonged hypoxia (4% O2 for 60 h). In rat PASMCs, HIF-1α overexpression up-regulated, whereas HIF-1α knockdown under hypoxia decreased BMP4 expression; site-mutation identified two functional HIF-1-binding sites in Bmp4 gene promoter; noggin or BMP4 siRNA treatment blocked hypoxia-induced increases of TRPC1 and TRPC6 expression and basal [Ca2+]i. Likewise, in mice, exposure to CH increased BMP4 expression in distal PA for ∼80%, which was absent in HIF-1α heterozygous mutant mice. Comparing with wild-type littermates, BMP4 heterozygous mutant mice exposed to CH displayed lower BMP4 and TRPC levels in PA, decreased basal [Ca2+]i in PASMCs, and attenuated CHPH. In human PASMCs, HIF-1α knockdown attenuated hypoxia-induced BMP4 expression and knockdown of either HIF-1α or BMP4 abolished hypoxia-induced TRPC expression and basal [Ca2+]i. Conclusions BMP4 acts downstream of HIF-1 and mediates hypoxia-induced up-regulation of TRPC, leading to increased basal [Ca2+]i in PASMCs, promoting CHPH pathogenesis.

This editorial refers to ‘Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes’ by A.H.Y. Lin et al ., pp. 94–104. Store-operated Ca2+ entry (SOCE) is an important regulator of vascular tone as it is linked to a variety of functions including contraction, proliferation, and migration.1,2 It is well established that plasma membrane store-operated Ca2+ channel (SOCs) activation induces SOCE. Stimulation of surface receptors G protein and tyrosine kinase by extracellular agonists activates the phospholipase C (PLC)/phosphatidylinositol 4,5-bisphosphate (PIP2)/inositol 1,4,5-triphosphate (IP3) pathway that leads to IP3-mediated depletion of endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+ stores and subsequent activation of SOCs. Stromal interaction molecule (STIM1-2) and Orai (Orai1-3) proteins are identified as critical components in SOCs activation. Of these proteins, structure models for STIM1 and Orai have begun to illustrate the molecular mechanisms controlling activation of SOCs in many cell types including vascular smooth muscle cells (VSMCs).3–5 It has been shown that SOCE is driven by physical contact between STIM1 and Orai1 through the cytosolic CAD domain of STIM1, which binds directly to Orai1 inducing tetrameric channel formation in ER–PM junction.6 Members of the canonical transient receptor potential family (TRPC) have also been proposed to act as SOCs; in particular, TRPC1 is associated with SOCE in VSMCs.7,8 Similar to Orai1, STIM1 is also needed for TRPC1 channel formation in response to ER/SR Ca2+ store depletion. It has been suggested, however, that STIM1-dependent gating of TRPC1 is different from Orai1. Instead of binding directly, an electrostatic model proposed that gating is mediated by an intermolecular electrostatic …

Aims It is well established that dysfunction of voltage-dependent ion channels results in arrhythmias and conduction disturbances in the foetal and adult heart. However, the involvement of voltage-insensitive cationic TRPC (transient receptor potential canonical) channels remains unclear. We assessed the hypothesis that TRPC channels play a crucial role in the spontaneous activity of the developing heart.Methods and results TRPC isoforms were investigated in isolated hearts obtained from 4-day-old chick embryos. Using RT-PCR, western blotting and co-immunoprecipitation, we report for the first time that TRPC1, 3, 4, 5, 6, and 7 isoforms are expressed at the mRNA and protein levels and that they can form a macromolecular complex with the alpha 1C subunit of the L-type voltage-gated calcium channel (Cav1.2) in atria and ventricle. Using ex vivo electrocardiograms, electrograms of isolated atria and ventricle and ventricular mechanograms, we found that inhibition of TRPC channels by SKF-96365 leads to negative chrono-, dromo-, and inotropic effects, prolongs the QT interval, and provokes first-and second-degree atrioventricular blocks. Pyr3, a specific antagonist of TRPC3, affected essentially atrioventricular conduction. On the other hand, specific blockade of the L-type calcium channel with nifedipine rapidly stopped ventricular contractile activity without affecting rhythmic electrical activity.Conclusions These results give new insights into the key role that TRPC channels, via interaction with the Cav1.2 channel, play in regulation of cardiac pacemaking, conduction, ventricular activity, and contractility during cardiogenesis.

Sympathetic regulation of cardiac contractility is mediated in part by α(1)-adrenergic receptors (ARs), and the α(1A)-subtype has been implicated in the pathogenesis of cardiac hypertrophy. However, little is known about α(1A)-AR signalling pathways in ventricular myocardium. The aim of this study was to determine the signalling pathway that mediates α(1A)-AR-coupled cardiac contractility.Using a transgenic model of enhanced cardiac α(1A)-AR expression and signalling (α(1A)-H mice), we identified a receptor-coupled signalling pathway that enhances Ca(2+) entry and increases contractility. This pathway involves α(1A)-AR-activated translocation of Snapin and the transient receptor potential canonical 6 (TRPC6) channel to the plasma membrane. In ventricular cardiomyocytes from α(1A)-H and their non-transgenic littermates (or WTs), stimulation with α(1A)-AR-specific agonists resulted in increased [Ca(2+)](i), which was dose-related and proportional to the level of α(1A)-AR expression. Blockade of TRPC6 inhibited the α(1A)-AR-mediated increase in [Ca(2+)](i) and contractility. External Ca(2+) entry, underlying the [Ca(2+)](i) increase, was not due to store-operated Ca(2+) entry but to a receptor-operated mechanism of Ca(2+) entry resulting from α(1A)-AR activation.These findings indicate that Ca(2+) entry via the α(1A)-AR-Snapin-TRPC6-pathway plays an important role in physiological regulation of cardiac contractility and may be an important target for augmenting cardiac performance.

Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca 2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na + /Ca 2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. Methods: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein–protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. Results: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Coimmunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GSTpulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca 2+ entry, which was dependent on TRPC3-mediated Na + loading of myocytes. This NCX-mediated Ca 2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. Conclusion: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca 2+ homeostasis.

Aims Macrophage apoptosis plays a determinant role in progression of atherosclerotic lesions. An important goal in atherosclerosis research is to identify new components of macrophage apoptosis that can eventually be exploited as molecular targets in strategies aimed at manipulating macrophage function in the lesion. In the previous work from our laboratory, we have shown that transient receptor potential canonical 3 (TRPC3) channel is an obligatory component of survival mechanisms in human and murine macrophages and that TRPC3-deficient non-polarized bone marrow-derived macrophages exhibit increased apoptosis, suggesting that in vivo TRPC3 might influence lesion development. In the present work, we used a bone marrow transplantation strategy as a first approach to examine the impact of macrophage deficiency of TRPC3 on early and advanced atherosclerotic lesions of Apoe−/− mice. Methods and results After 3 weeks of high-fat diet, lesions in mice transplanted with bone marrow from Trpc3−/− donors were smaller and with reduced cellularity than controls. Advanced lesions from these mice exhibited reduced necrotic core, less apoptotic macrophages, and increased collagen content and cap thickness. In vitro , TRPC3-deficient macrophages polarized to the M1 phenotype showed reduced apoptosis, whereas both M1 and M2 macrophages had increased efferocytic capacity. Conclusions Bone marrow deficiency of TRPC3 has a dual beneficial effect on lesion progression by reducing cellularity at early stages and necrosis in the advanced plaques. Our findings represent the first evidence for a role of a member of the TRPC family of cation channels in mechanisms associated with atherosclerosis.

This editorial refers to ‘A key role of TRPC channels in the regulation of electromechanical activity of the developing heart’ by J. Sabourin et al. , pp. 226–236, this issue . It is well established that in the adult mammalian heart, small amounts of Ca2+ entry via voltage-dependent sarcolemmal Cav1.2 L-type Ca2+ channels can trigger the release of a significant amount of Ca2+ from the sarcoplasmic reticulum (SR) that is followed by muscle contraction, a process known as excitation–contraction (EC) coupling.1 However, for the developing mammalian and avian hearts, earlier studies proposed that EC coupling relies essentially on transsarcolemmal influx of Ca2+ through L-type Ca2+ channels, mainly Cav1.2, and that the contribution of the SR is minimal.2 In the embryonic chick heart, Ca2+ channel density is maximal at 3 days and decreases at 17 days,3 whereas Ca2+ channel density is low in the foetal heart and increases with development in guinea-pig,4 rabbit,5 and human,6 consistent with the need for additional Ca2+ entry through other sarcolemmal voltage-dependent proteins such as the T-type Ca2+ channel, the Cav1.3 L-type Ca2+ channel, and the Na/Ca2+ exchanger (NCX).2,6–8 Other candidate channels …