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Reduced systolic and diastolic calcium levels in cardiomyocytes from NKA-α2 transgenic mice minimize the desensitization of the β-adrenergic signaling system. These effects result in an improved β-adrenergic reserve and prevent functional deterioration and cardiac remodeling.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP–dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavβ, Cavα2δ, and Cavγ. Cavβ is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cavβ silencing, we demonstrate that Cavβ2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cavβ2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavβ2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cavβ2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavβ2-downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cavβ2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavβ2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways.

In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca2+ levels. We therefore hypothesize that decoding Ca2+ signals for hypertrophic signaling requires intracellular Ca2+ microdomains that are partly independent from cytosolic Ca2+. Furthermore, there is a need for a Ca2+ sensor within these microdomains that translates Ca2+ signals into hypertrophic signaling. Recent evidence suggested that the nucleus of cardiac myocytes might be a Ca2+ microdomain and that calcineurin, once translocated into the nucleus, could act as a nuclear Ca2+ sensor. We demonstrate that nuclear calcineurin was able to act as a nuclear Ca2+ sensor detecting local Ca2+ release from the nuclear envelope via IP3R. Nuclear calcineurin mutants defective for Ca2+ binding failed to activate NFAT-dependent transcription. Under hypertrophic conditions Ca2+ transients in the nuclear microdomain were significantly higher than in the cytosol providing a basis for sustained calcineurin/NFAT-mediated signaling uncoupled from cytosolic Ca2+. Measurements of nuclear and cytosolic Ca2+ transients in IP3 sponge mice showed no increase of Ca2+ levels during diastole as we detected in wild-type mice. Nuclei, isolated from ventricular myocytes of mice after chronic Ang II treatment, showed an elevation of IP3R2 expression which was dependent on calcineurin/NFAT signaling and persisted for 3 weeks after removal of the Ang II stimulus. These data provide an explanation how Ca2+ and calcineurin might regulate transcription in cardiomyocytes in response to neurohumoral signals independently from their role in cardiac contraction control. • Calcineurin acts as an intranuclear Ca2+ sensor to promote NFAT activity. • Nuclear Ca2+ in cardiac myocytes increases via IP3R2 upon Ang II stimulation. • IP3R2 expression is directly dependent on calcineurin/NFAT.

Background: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase–B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell–specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. Methods: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2 ) gene were interbred with Tie2-Cre or PDGF-Rβ-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. Results: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1–induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. Conclusions: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.

Purpose The antiapoptotic B‑cell lymphoma 2 (BCL2) gene is a key player in cancer development and progression. A functional single-nucleotide polymorphism (c.-938C>A, rs2279115) in the inhibitory P2 BCL2 gene promoter has been associated with clinical outcomes in various types of cancer. Aim of the present study was to analyze the role of BCL2-938C>A genotypes in prostate cancer mortality. Methods The association between BCL2-938C>A (rs2279115) genotypes and prostate cancer outcome was studied within the prospective PROCAGENE study comprising 702 prostate cancer patients. Results During a median follow-up time of 92 months, 120 (17.1%) patients died. A univariate Cox regression model showed a significant association of the CC genotype with reduced cancer-specific survival (CSS; hazard ratio, HR, 2.13, 95% confidence interval, CI, 1.10–4.12; p = 0.024) and overall survival (OS; HR 2.34, 95% CI 1.58–3.47; p < 0.001). In a multivariate Cox regression model including age at diagnosis, risk group, and androgen deprivation therapy, the CC genotype remained a significant predictor of poor CSS (HR 2.05, 95% CI 1.05–3.99; p = 0.034) and OS (HR 2.25, 95% CI 1.51–3.36; p < 0.001). Conclusion This study provides evidence that the homozygous BCL2-938 CC genotype is associated with OS and C in prostate cancer patients.

The transient receptor potential (TRP; C-classical, TRPC) channel TRPC3 allows a cation (Na

In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca

Rationale: The Na + /K + ATPase (NKA) directly regulates intracellular Na + levels, which in turn indirectly regulates Ca 2+ levels by proximally controlling flux through the Na + /Ca 2+ exchanger (NCX1). Elevated Na + levels have been reported during heart failure, which permits some degree of reverse-mode Ca 2+ entry through NCX1, as well as less efficient Ca 2+ clearance. Objective: To determine whether maintaining lower intracellular Na + levels by NKA overexpression in the heart would enhance forward-mode Ca 2+ clearance and prevent reverse-mode Ca 2+ entry through NCX1 to protect the heart. Methods and Results: Cardiac-specific transgenic mice overexpressing either NKA-α1 or NKA-α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-α1 had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-α2 overexpression but not NKA-α1 led to significantly faster removal of bulk Ca 2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-α2 showed greater affinity for Na + compared with NKA-α1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-α2 but not NKA-α1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca 2+ removal. Conclusions: Our results suggest that the protective effect produced by increased expression of NKA-α2 on the heart after pressure overload is due to more efficient Ca 2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-α1.

Transient receptor potential (TRP) channels of multiple subclasses are expressed in the heart, although their functions are only now beginning to emerge, especially for the TRPC subclass that appears to regulate the cardiac hypertrophic response. Although TRP channels permeate many different cations, they are most often ascribed a specific biological function because of Ca 2+ influx, either for microdomain signaling or to reload internal Ca 2+ stores in the endoplasmic reticulum through a store-operated mechanism. However, adult cardiac myocytes arguably do not require store-operated Ca 2+ entry to regulate sarcoplasmic reticulum Ca 2+ levels and excitation–contraction coupling; hence, TRP channels expressed in the heart most likely coordinate signaling within local domains or through direct interaction with Ca 2+ -dependent regulatory proteins. Here, we review the emerging evidence that TRP channels, especially TRPCs, are critical regulators of microdomain signaling in the heart to control pathological hypertrophy in coordination with signaling through effectors such as calcineurin and NFAT (nuclear factor of activated T cells).

VEGF-induced Ca2+ signalling was investigated in CD133+/VEGFR-2+ progenitor cells isolated from human adipose stroma. Colonies derived from CD133+ immunoselected cells displayed inhomogenous Ca2+ signals, with variable magnitude of VEGF-induced Ca2+ entry, which positively correlated with expression of the Ca2+ channel protein TRPC3. High levels of VEGF-induced Ca2+ entry and TRPC3 expression were preferentially detected in rim areas of expanding colonies. Dominant negative suppression of TRPC3 inhibited VEGF-induced Ca2+entry into CD133+ cells. Our results identify TRPC3 as a key Ca2+ entry channel in a subset of CD133+ stem cells. We suggest TRPC3 as an essential determinant of cell fate in CD133+ progenitor-derived colonies.

Nonselective and lipid-regulated cation channels formed by TRPC3, TRPC6 and TRPC7 have recently obtained attention in view their potential pathophysiological impact. It appears as a particular challenge to understand the molecular basis of TRPC3/6/7-related diseases in order to further delineate their value as therapeutic targets. The multifunctional nature of these channel proteins, based on a complex, versatile heteromerization potential along with highly promiscuous gating and mixed cation permeation properties, make these channels pivotal players in cellular signaling networks. Here we summarize newsworthy aspects of TRPC3/6/7 physiology and pathophysiology that open the view on novel therapeutic strategies based on these TRPCs as target structures.

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.

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca(2+) homeostasis. As permeation of Ca(2+) through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca(2+) entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na(+) permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na(+) entry may be converted into a distinct pattern of cellular Ca(2+) signals by interaction with Na(+)/Ca(2+) exchanger proteins. This review discusses current concepts regarding the link between Na(+) entry through TRPC channels and cellular Ca(2+) signaling.

Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

Impaired cardiomyocyte Ca2+ handling is considered as crucial factor programming cardiac hypertrophy and heart failure. It has, however, not been entirely resolved what type of Ca2+ signals is involved in mechanisms inducing hypertrophic remodelling. Ca2+ microdomains, which are restricted from the fluctuating Ca2+ during excitation–contraction coupling, could generate local Ca2+ signals and activate downstream Ca2+ dependent signalling pathways. With the non-selective ion channels of the TRPC family, attractive entities in microdomain Ca2+ signalling have been found. Within a short period of time important aspects of TRPC dependent signalling have been revealed. TRPC channels contribute to a store-operated Ca2+ entry, are functionally cross-linked with L-type Ca2+ channels and can indirectly sense stress stimuli during cardiac hypertrophy. Pressure-overload or agonist stimulation of the heart results in an increased TRPC channel activity which is mechanistically coupled to Ca2+ dependent signalling pathways. In particular, the calcineurin/nuclear factor of activated T-cells signalling pathway has been elucidated as central mediator translating TRPC dependent Ca2+ signals to hypertrophic cardiac growth. Interference with the TRPC/calcineurin/nuclear factor of activated T-cells circuit or TRPC blockade has been found beneficial in opposing cardiac remodelling. TRPC channels can therefore be regarded as promising targets in the therapy of heart failure

The Na+/K+ ATPase (NKA) directly regulates intracellular Na+ levels, which indirectly regulate Ca2+ levels by controlling flux through the Na+/Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse mode Ca2+ entry through NCX1 and less efficient Ca2+ clearance. To determine if lower intracellular Na+ levels would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 as a protective measure, we generated cardiac-specific transgenic mice overexpressing either the NKA-α1 or α2 isoform and subjected them to pressure overload hypertrophic stimulation. We found that while increased expression of the NKA-α1 isoform had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload at 2, 10 and 16 weeks of stimulation. Remarkably, total NKA protein expression was not altered in either of these 2 transgenic models, as increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. While total NKA ATPase activity and intracellular Na+ levels were unchanged in either overexpression model, and both showed reduced Ca2+ transient amplitudes and sarcoplasmic reticulum Ca2+ load, only NKA-α2 overexpression led to faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. This increased NCX1 activity, though correlated with improved outcome after pressure overload, did not affect signaling through Ca2+-sensitive signaling pathways such as calcineurin/nuclear factor of activated T-cells, Ca2+/calmodulin-dependent kinase II, or protein kinase Cα. Overexpression of NKA-α2 did, however, result in reduced expression of phospholemman (PLM), an inhibitor of NKA activity (when dephosphorylated) and NCX1 activity (when phosphorylated). Our results suggest that the protective effect produced by increased expression of NKA-α2 after pressure overload is likely due to: 1) Na+ regulation in a unique signaling microdomain distinct from NKA-α1, and 2) downregulation of PLM expression that removes a negative regulator of NCX1 activity, both leading to preservation of forward-mode NCX1 activity during disease, in association with optimized cardiac function.

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca\(^{2+}\) component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na\(^{+}\) and Ca\(^{2+}\) influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca\(^{2+}\) influx which has been regarded as ideal Ca\(^{2+}\) source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca\(^{2+}\) signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca\(^{2+}\) transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca\(^{2+}\) cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca\(^{2+}\) transient amplitude at baseline and TRPC4β increasing the Ca\(^{2+}\) peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca\(^{2+}\) influx when treated with Ang II under non-pacing conditions. Consistent with the Ca\(^{2+}\) data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca\(^{2+}\) signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.

Pathologic hypertrophy of the heart is regulated through membrane-bound receptors and intracellular signaling pathways that function, in part, by altering Ca 2+ handling and Ca 2+ -dependent signaling effectors. Transient receptor potential canonical (TRPC) channels are important mediators of Ca 2+ -dependent signal transduction that can sense stretch or activation of membrane-bound receptors. Here we generated cardiac-specific transgenic mice that express dominant-negative (dn) TRPC3, dnTRPC6, or dnTRPC4 toward blocking the activity of the TRPC3/6/7 or TRPC1/4/5 subfamily of channels in the heart. Remarkably, all three dn transgenic strategies attenuated the cardiac hypertrophic response following either neuroendocrine agonist infusion or pressure-overload stimulation. dnTRPC transgenic mice also were partially protected from loss of cardiac functional performance following long-term pressure-overload stimulation. Importantly, adult myocytes isolated from hypertrophic WT hearts showed a unique Ca 2+ influx activity under store-depleted conditions that was not observed in myocytes from hypertrophied dnTRPC3, dnTRPC6, or dnTRPC4 hearts. Moreover, dnTRPC4 inhibited the activity of the TRPC3/6/7 subfamily in the heart, suggesting that these two subfamilies function in coordinated complexes. Mechanistically, inhibition of TRPC channels in transgenic mice or in cultured neonatal myocytes significantly reduced activity in the calcineurin–nuclear factor of activated T cells (NFAT), a known Ca 2+ -dependent hypertrophy-inducing pathway. Thus, TRPC channels are necessary mediators of pathologic cardiac hypertrophy, in part through a calcineurin–NFAT signaling pathway.

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

TRPC3 represents one of the first identified mammalian relatives of the Drosophila trp gene product. Despite intensive biochemical and biophysical characterization as well as numerous attempts to uncover its physiological role in native cell systems, this channel protein still represents one of the most enigmatic members of the transient receptor potential (TRP) superfamily. TRPC3 is significantly expressed in brain and heart and likely to play a role in both non-excitable as well as excitable cells, being potentially involved in a wide spectrum of Ca2+ signalling mechanisms. Its ability to associate with a variety of partner proteins apparently enables TRPC3 to form different cation channels in native cells. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators and cellular Ca2+ gradients as well as redox signals. The physiological/pathophysiological functions of this highly versatile cation channel protein are as yet barely delineated. Here we summarize current knowledge on properties and possible signalling functions of TRPC3 and discuss the potential biological relevance of this signalling molecule.

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.

TRPC channels have been identified as components of Ca2+ signaling pathways that promote maladaptive growth of the myocardium. Transgenic mice over-expressing TRPC3 or 6 exhibited an increased propensity towards hypertrophic progression in response to pressure overload, in part through a Ca2+-dependent calcineurin-NFAT signaling pathway. Moreover, different TRPC subunits are known to be up-regulated in heart failure and in hypertrophic hearts after pressure overload. In this study we inhibited TRPC channel function with dominant negative (dn) TRPC mutants and examined the ensuing effect on cardiac hypertrophy and Ca2+ influx dynamics. One of these mutants comprises an N-terminal TRPC4 fragment that we used to generate cardiac restricted transgenic mice. Intracellular Ca2+ signals were measured in adult cardiac myocytes of wildtype (WT) and dn-TRPC4 mice that underwent TAC (transverse aortic banding) or a sham procedure. Isolated myocytes maintained in Ca2+-free buffer were store-depleted with CPA while being treated with Ang II, followed by perfusion with Ca2+ containing buffer to analyze Ca2+ influx with Fura-2 fluorescence. WT myocytes showed essentially no Ca2+ entry, however, hypertrophic myocytes that underwent TAC showed abundant Ca2+ influx. SKF96365 inhibited this Ca2+ influx, suggesting a role for TRPC channels. Indeed, isolated myocytes from dnTRPC4 transgenic mice subjected to TAC lacked this Ca2+ influx, confirming that TRPC channels mediate this previously unrecognized influx activity associated with hypertrophy in cardiac myocytes. Interestingly, Ca2+ transients from myocytes of dn-TRPC4 mice showed increased peak amplitudes, which might indicate an increased cardiac contractility and cardiac performance when dn-TRPC4 is expressed. More importantly, dnTRPC4 mice showed significant attenuation of cardiac hypertrophy following TAC stimulation. These data show that inhibition of TRPC channels exerts ameliorative effects on the development of cardiac hypertrophy by decreasing Ca2+ signals that likely regulate pro-hypertrophic pathways.

Recent evidence suggests involvement of transient receptor potential (TRP)-related cation channels in cardiac physiology and pathophysiology, with TRPC3 as one potential key player in cardiac hypertrophy. It has been suggested that TRPC3 is upregulated in hypertrophy development and contributes to Ca2+ signals that govern pathological remodelling. As TRPC proteins form nonselective cation channels, we hypothesized that these molecules determine not only Ca2+-mediated gene expression but also excitability and basic electrical properties of the myocardium in pathological states. We utilized the patch clamp technique to characterize the electrical remodelling associated with enhanced TRPC3 expression in cardiac myocytes. The murine HL-1 atrial cell model, which was found to express both TRPC3 and TRPC6 at low levels along with typical signalling partners of TRPC proteins such as Na+/Ca2+-exchanger-1 and CaV1.2, was modified to overexpress a functional YFP-TRPC3 fusion protein. TRPC3 over-expressing cells displayed substantially altered electrical properties even in the absence of TRPC-activating stimuli, as evident from a significant shortening of basal action potential duration and impaired frequency-dependent adaptation of action potential shape. Action potential-clamp experiments as well as mathematical modelling attempts were performed to delineate the consequences of TRPC3 overexpression and to analyze the mechanisms of TRPC3-mediated electrical remodelling in the heart. Our results suggest a pivotal role of TRPC channels in cardiac electrical remodelling associated with maladaptive hypertrophy.