Takuro Numaga-Tomita

Purinergic P2Y6 receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension

Aging changes the response of blood vessels to angiotensin II, contributing to hypertension. Raising blood pressure with age Activation of angiotensin AT1 receptors in vascular smooth muscle cells by angiotensin II can trigger either proliferation or hypertrophy (the latter of which increases blood pressure), depending on age. Nishimura et al. discovered that an age-related increase in the heterodimerization of AT1 receptors with purinergic P2Y6 receptors explained the different responses to angiotensin II. Blood pressure increased to a lesser extent in response to angiotensin II in mice lacking P2Y6 receptors. Vascular smooth muscle cells from young mice had less P2Y6 receptor than those from older mice. Angiotensin II increased the proliferation of vascular smooth muscle cells from young mice but increased cell size in those from older mice by activating different signaling pathways. Thus, targeting the interaction between AT1 and P2Y6 receptors may decrease age-associated high blood pressure. The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR), promoted Ang II–induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II–induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein–dependent vascular hypertrophy but reduced β-arrestin–dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II–induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascular smooth muscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin–dependent proliferation to G protein–dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II.

TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.

TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis

Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.

TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca2+ concentration in mouse cardiomyocytes

When a cardiac muscle is held in a stretched position, its [Ca2+] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca2+ concentration ([Ca2+]i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca2+]i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC.

Purinergic P2Y receptors: Molecular diversity and implications for treatment of cardiovascular diseases

&NA; Purinergic signaling, mediated mainly by G protein‐coupled P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR‐mediated biological functions are predominantly activated by mononucleotides released from non‐adrenergic, non‐cholinergic nerve endings or non‐secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand‐independent P2YR function through receptor‐receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein‐coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide‐responsive P2Y6R and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2Y6R antagonist MRS2578 interrupts heterodimerization between P2Y6R and AT1R, thereby reducing the risk of AT1R‐stimulated hypertension in mice. These results strongly suggest that targeting P2Y6R oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases.

Purinergic P2Y6 receptors: A new therapeutic target of age-dependent hypertension

&NA; Aging has a remarkable effect on cardiovascular homeostasis and it is known as the major non‐modifiable risk factor in the development of hypertension. Medications targeting sympathetic nerve system and/or renin‐angiotensin‐aldosterone system are widely accepted as a powerful therapeutic strategy to improve hypertension, although the control rates remain unsatisfactory especially in the elder patients with hypertension. Purinergic receptors, activated by adenine, uridine nucleotides and nucleotide sugars, play pivotal roles in many biological processes, including platelet aggregation, neurotransmission and hormone release, and regulation of cardiovascular contractility. Since clopidogrel, a selective inhibitor of G protein‐coupled purinergic P2Y12 receptor (P2Y12R), achieved clinical success as an anti‐platelet drug, P2YRs has been attracted more attention as new therapeutic targets of cardiovascular diseases. We have revealed that UDP‐responsive P2Y6R promoted angiotensin type 1 receptor (AT1R)‐stimulated vascular remodeling in mice, in an age‐dependent manner. Moreover, the age‐related formation of heterodimer between AT1R and P2Y6R was disrupted by MRS2578, a P2Y6R‐selective inhibitor. These findings suggest that P2Y6R is a therapeutic target to prevent age‐related hypertension. Graphical abstract Figure. No caption available.

TRPC3 Channels in Cardiac Fibrosis

Cardiac stiffness, caused by interstitial fibrosis due to deposition of extracellular matrix proteins, is thought as a major clinical outcome of heart failure with preserved ejection fraction (HFpEF). Canonical transient receptor potential (TRPC) subfamily proteins are components of Ca2+-permeable non-selective cation channels activated by receptor stimulation and mechanical stress, and have been attracted attention as a key mediator of maladaptive cardiac remodeling. How TRPC-mediated local Ca2+ influx encodes a specific signal to induce maladaptive cardiac remodeling has been long obscure, but our recent studies suggest a pathophysiological significance of channel activity-independent function of TRPC proteins for amplifying redox signaling in heart. This review introduces the current understanding of the physiological and pathophysiological roles of TRPCs, especially focuses on the role of TRPC3 as a positive regulator of reactive oxygen species (PRROS) in heart. We have revealed that TRPC3 stabilizes NADPH oxidase 2 (Nox2), a membrane-bound reactive oxygen species (ROS)-generating enzyme, by forming stable protein complex with Nox2, which leads to amplification of mechanical stress-induced ROS signaling in cardiomyocytes, resulting in induction of fibrotic responses in cardiomyocytes and cardiac fibroblasts. Thus, the TRPC3 function as PRROS will offer a new therapeutic strategy for the prevention or treatment of HFpEF.

TRPC3-Nox2 complex mediates doxorubicin-induced myocardial atrophy

Myocardial atrophy is a wasting of cardiac muscle due to hemodynamic unloading. Doxorubicin is a highly effective anticancer agent but also induces myocardial atrophy through a largely unknown mechanism. Here, we demonstrate that inhibiting transient receptor potential canonical 3 (TRPC3) channels abolishes doxorubicin-induced myocardial atrophy in mice. Doxorubicin increased production of ROS in rodent cardiomyocytes through hypoxic stress-mediated upregulation of NADPH oxidase 2 (Nox2), which formed a stable complex with TRPC3. Cardiomyocyte-specific expression of TRPC3 C-terminal minipeptide inhibited TRPC3-Nox2 coupling and suppressed doxorubicin-induced reduction of myocardial cell size and left ventricular (LV) dysfunction, along with its upregulation of Nox2 and oxidative stress, without reducing hypoxic stress. Voluntary exercise, an effective treatment to prevent doxorubicin-induced cardiotoxicity, also downregulated the TRPC3-Nox2 complex and promoted volume load-induced LV compliance, as demonstrated in TRPC3-deficient hearts. These results illustrate the impact of TRPC3 on LV compliance and flexibility and, focusing on the TRPC3-Nox2 complex, provide a strategy for prevention of doxorubicin-induced cardiomyopathy.

TRPC3 amplifies B-cell receptor-induced ERK signalling via protein kinase D-dependent Rap1 activation

Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca(2+) influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

TRPC5-eNOS axis negatively regulates ATP-induced cardiomyocyte hypertrophy

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca2+-dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5′-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.