Contribution of transient receptor potential canonical channels in human and experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is due to progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated store-operated Ca2+ entry (SOCE) contributes to PAH pathogenesis, mediating human PA smooth muscle cell (hPASMC) abnormalities. The transient receptor potential canonical channels (TRPC family) are Ca2+-permeable channels contributing to SOCE in different cell types, including PASMCs. However, the properties, signaling pathways, and contribution to Ca2+ signaling of each TRPC isoform are unclear in human PAH. We studied in vitro the impact of TRPC knockdown on control and PAH-hPASMCs function. In vivo, we analyzed the consequences of pharmacological TRPC inhibition using the experimental model of pulmonary hypertension (PH) induced by monocrotaline (MCT) exposure. Compared with control-hPASMCs cells, in PAH-hPASMCs, we found a decreased TRPC4 expression, overexpression of TRPC3 and TRPC6, and unchanged TRPC1 expression. Using the siRNA strategy, we found that the knockdown of TRPC1-C3-C4-C6 reduced the SOCE and the proliferation rate of PAH-hPASMCs. Only TRPC1 knockdown decreased the migration capacity of PAH-hPASMCs. After PAH-hPASMCs exposure to the apoptosis inducer staurosporine, TRPC1-C3-C4-C6 knockdown increased the percentage of apoptotic cells, suggesting that these channels promote apoptosis resistance. Only TRPC3 function contributed to exacerbated calcineurin activity. In the MCT-PH rat model, only TRPC3 protein expression was increased in lungs compared with control rats, and in vivo "curative" administration of a TRPC3 inhibitor attenuated PH development in rats. These results suggest that TRPC channels contribute to PAH-hPASMCs dysfunctions, including SOCE, proliferation, migration, and apoptosis resistance, and could be considered as therapeutic targets in PAH.NEW & NOTEWORTHY TRPC3 is increased in human and experimental pulmonary arterial hypertension (PAH). In PAH pulmonary arterial smooth muscle cells, TRPC3 participates in the aberrant store-operated Ca2+ entry contributing to their pathological cell phenotypes (exacerbated proliferation, enhanced migration, apoptosis resistance, and vasoconstriction). Pharmacological in vivo inhibition of TRPC3 reduces the development of experimental PAH. Even if other TRPC acts on PAH development, our results prove that TRPC3 inhibition could be considered as an innovative treatment for PAH.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.

T-type Ca2+ channels elicit pro-proliferative and anti-apoptotic responses through impaired PP2A/Akt1 signaling in PASMCs from patients with pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (iPAH) is characterized by obstructive hyperproliferation and apoptosis resistance of distal pulmonary artery smooth muscle cells (PASMCs). T-type Ca2+ channel blockers have been shown to reduce experimental pulmonary hypertension, although the impact of T-type channel inhibition remains unexplored in PASMCs from iPAH patients. Here we show that T-type channels Cav3.1 and Cav3.2 are present in the lung and PASMCs from iPAH patients and control subjects. The blockade of T-type channels by the specific blocker, TTA-A2, prevents cell cycle progression and PASMCs growth. In iPAH cells, T-type channel signaling fails to activate phosphatase PP2A, leading to an increase in ERK1/2, P38 activation. Moreover, T-type channel signaling is redirected towards the activation of the kinase Akt1, leading to increased expression of the anti-apoptotic protein survivin, and a decrease in the pro-apoptotic mediator FoxO3A. Finally, in iPAH cells, Akt1 is no longer able to regulate caspase 9 activation, whereas T-type channel overexpression reverses PP2A defect in iPAH cells but reinforces the deleterious effects of Akt1 activation. Altogether, these data highlight T-type channel signaling as a strong trigger of the pathological phenotype of PASMCs from iPAH patients (hyper-proliferation/cells survival and apoptosis resistance), suggesting that both T-type channels and PP2A may be promising therapeutic targets for pulmonary hypertension.

T-type Ca²âº signalling downregulates MEK1/2 phosphorylation and cross-talk with the RAAS transcriptional response in cardiac myocytes.

Cardiac dysfunction is often associated with an increase in the activity of the renin-angiotensin II-aldosterone system (RAAS). Here, we highlight the cross-talk between the Ca(2+) signalling generated by cardiac T-type current (I(CaT)) and RAAS signalling. Neonatal rat cardiomyocytes exposed to aldosterone, angiotensin II or aldosterone plus angiotensin II co-treatment (AA) show an increase in I(CaT) density, with no cumulative effect of the AA co-treatment. AA increases the amount of T-type channel Ca(v)3.1 mRNA in a time-dependent manner. Angiotensin II increases Ca(v)3.1 mRNA stability, whereas aldosterone increases the transcriptional activity of the Ca(v)3.1 gene promoter. However, in AA-treated cells, angiotensin II decreases aldosterone-induced promoter activity, and aldosterone decreases angiotensin II-induced mRNA stability. The mitogen-activated protein kinase kinase (MEK1/2), which is synergically phosphorylated in AA-treated cells, alters the translocation of glucocorticoid receptors (GR) into the nucleus and attenuates aldosterone-induced promoter activity. In contrast, MEK1/2 has no effect on the NFkB-induced increase in Ca(v)3.1 mRNA and MEK1/2 promoted CREB-target gene transcription. Aldosterone and AA-induced I(CaT) signalling result in a time-dependent activation of the phosphatase PP2A, which dephosphorylates MEK1/2 and CREB. Finally, angiotensin II alone also activates PP2A, which targets MEK1/2, but this activation is independent of I(CaT) calcium signalling and has no effect on CREB phosphorylation. In conclusion, our data demonstrate the cross-talk between a GR-mediated aldosterone response, angiotensin II and the I(CaT) signalling pathways and identify MEK1/2 as a point of connection. This cross-talk results in the fine control of GR- and/or CREB-target gene expression.

T-type Ca²+ signalling regulates aldosterone-induced CREB activation and cell death through PP2A activation in neonatal cardiomyocytes.

AIMS: We have investigated Ca²(+) signalling generated by aldosterone-induced T-type current (I(CaT)), the effects of I(CaT) in neonatal cardiomyocytes, and a putative role for I(CaT) in cardiomyocytes during cardiac pathology induced by stenosis in an adult rat. METHODS AND RESULTS: Neonatal rat cardiomyocytes treated with aldosterone showed an increase in I(CaT) density, principally due to the upregulation of the T-type channel Ca(v)3.1 (by 80%). Aldosterone activated cAMP-response element-binding protein (CREB), and this activation was enhanced by blocking I(CaT) or by inhibiting protein phosphatase 2A (PP2A) activity. Aldosterone induced PP2A activity, an induction that was prevented upon I(CaT) blockade. I(CaT) exerted a negative feedback regulation on the transcription of the Ca(v)3.1 gene, and the activation of PP2A by I(CaT) led to increased levels of the pro-apoptotic markers caspase 9 and Bcl-x(S) and decreased levels of the anti-apoptotic marker Bcl-2. These findings were corroborated by flow cytometry analysis for apoptosis and necrosis. Similarly, in a rat model of cardiac disease, I(CaT) re-emergence was associated with a decrease in CREB activation and was correlated with increases in caspase 9 and Bcl-x(S) and a decrease in Bcl-2 levels. CONCLUSION: Our findings establish PP2A/CREB as targets of I(CaT)-generated Ca²(+) signalling and identify an important role for I(CaT) in cardiomyocyte cell death.

Identification of functional corticosteroid response elements involved in regulation of Cacna1g expression in cardiac myocytes.

We recently reported that corticosteroids increase the expression of the T-type channel Ca(v)3.1 through a transcriptional up-regulation of the Ca(v)3.1 encoding gene cacna1g. The nucleotide sequence analysis of cacna1g promoter revealed putative glucocorticoid response elements (GREs). However, the functional GREs involved in the regulation of cacna1g expression in neonatal cardiac myocytes are unknown. In the present study we have investigated the nuclear targets responsible for the transcriptional regulation of cacna1g. We identified five GREs from the nucleotide sequence of cacna1g promoter. Additionally, using punctual mutagenesis approach, three functional categories of GREs have been identified: (i) GRE-1 involved in promoter activity induced by aldosterone (Aldo, 1 microM); (ii) GRE-4 and GRE-5 involved in promoter activity induced by dexamethasone (Dex, 1 microM); and (iii) GRE-2 and GRE-3 involved in the basal level of neonatal promoter activity. The data presented here lead to better understanding of the molecular mechanisms underlying the regulation of Ca(v)3.1 channel expression by corticosteroids. These new findings have attractive physiological features during cardiac development and pathology such as arrhythmias.

Regulation of T-type Cav3.1 channels expression by synthetic glucocorticoid dexamethasone in neonatal cardiac myocytes.

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.

Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels.

Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40+/-0.05 pA/pF (n=26) to 0.20+/-0.03 pA/pF (n=27, P<0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 micromol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II-increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II-increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II-increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II-increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.

Expression of heart K+ channels in adrenalectomized and catecholamine-depleted reserpine-treated rats.

We studied cardiac outward K currents (transient and sustained) by the whole-cell patch-clamp technique and the Kv4.2, Kv4.3, Kv1.4, Kv1.5, Kv1.2 and Kv2.1 expression of voltage-gated K channel by RT-PCR, in ventricular myocytes from two models of catecholamine-depleted adult rats. We induced endogenous catecholamine depletion by reserpine treatment and used adrenalectomized rats as a model of plasma catecholamine depletion. In reserpine-treated rats (97% decrease in endogenous norepinephrine content of the heart), the amplitude of the transient outward current was decreased by 48% and Kv4.2 and Kv4.3 mRNA levels were decreased by 57% and 34%, respectively. The amount of Kv1.5 mRNA tripled, with no change in sustained current density. This increase was not confirmed by immunostaining for the Kv1.5 protein. The amplitude of K currents and their corresponding mRNA levels returned to control values following recovery from reserpine treatment. In contrast, in adrenalectomized rats (98% decrease in plasma epinephrine concentration), we observed no change in the amplitude of outward K currents or in Kv mRNA levels. These results suggested a role for sympathetic innervation and endogenous norepinephrine in the regulation of transcription of cardiac outward K currents in physiological and pathological situations.

Ventricular hypertrophy induced by mineralocorticoid treatment or aortic stenosis differentially regulates the expression of cardiac K+ channels in the rat.

Rats treated with DOCA salts and subjected to abdominal aortic stenosis display left ventricle hypertrophy associated with a decrease in cardiac I(to) current density and prolongation of the action potential duration. We investigated the molecular basis of these electrophysiological defects by analyzing the amount of mRNA corresponding to the genes encoding the a subunits of the left ventricle K+ channel at the steady state. The mRNAs corresponding to the a subunits of the K+ channel (Kv1.2, Kv1.4, Kv1.5, Kv2. 1, Kv4.2 and Kv4.3) were measured by quantitative RT-PCR using a specific Kv internal standard. In control rats, the Kvl.5 gene was only expressed at a low level, whereas the Kv4.2 and Kv4.3 genes were expressed at a high level. Regardless of the etiology of the hypertrophy, the amounts of Kv1.4 and Kv1.5 mRNAwere similar in treated, sham and control rats. The amounts of Kv1.2 and Kv2.1 mRNA were markedly lower in DOCA-salt treated rats (66%) than in sham-DOCA rats, but no effect was observed after stenosis. The very conservative Kv4.2 and Kv4.3 genes were found to be downregulated simultaneously in both type of hypertrophy. However, the steady-state amount of Kv4 mRNA was even lower in rats with DOCA-salt-induced hypertrophy than in those with stenosis-induced ventricular hypertrophy. Therefore, the decrease in I(to) density, consecutively to pressure- and volume-overload, is due to a large decrease in the amount of Kv4.2 and Kv4.3 mRNA. In addition, DOCA-salt treatment alters the amounts of Kv transcripts independently to cardiac hypertrophy, suggesting that the mineralocorticoid may be involved in Kv gene expression.