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.

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 P2YR and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2YR antagonist MRS2578 interrupts heterodimerization between P2YR and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2YR oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases.

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.

Myocardial atrophy, characterized by the decreases in size and contractility of cardiomyocytes, is caused by severe malnutrition and/or mechanical unloading. Extracellular adenosine 5′-triphosphate (ATP), known as a danger signal, is recognized to negatively regulate cell volume. However, it is obscure whether extracellular ATP contributes to cardiomyocyte atrophy. Here, we report that ATP induces atrophy of neonatal rat cardiomyocytes (NRCMs) without cell death through P2Y 2 receptors. ATP led to overproduction of reactive oxygen species (ROS) through increased amount of NADPH oxidase (Nox) 2 proteins, due to increased physical interaction between Nox2 and canonical transient receptor potential 3 (TRPC3). This ATP-mediated formation of TRPC3-Nox2 complex was also pathophysiologically involved in nutritional deficiency-induced NRCM atrophy. Strikingly, knockdown of either TRPC3 or Nox2 suppressed nutritional deficiency-induced ATP release, as well as ROS production and NRCM atrophy. Taken together, we propose that TRPC3-Nox2 axis, activated by extracellular ATP, is the key component that mediates nutritional deficiency-induced cardiomyocyte atrophy.

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Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y 6 receptor (P2Y 6 R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y 6 R prevents or promotes heart failure. We demonstrate that inhibition of P2Y 6 R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y 6 R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y 6 R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y 6 R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y 6 R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y 6 R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y 6 R exacerbates pressure overloadinduced heart failure in mice, although P2Y 6 R in cardiomyocytes contributes to the progression of cardiac fibrosis. Cardiac remodeling is characterized by structural and morphological changes of the heart, including hypertrophy and fibrosis, and is a major clinical outcome of heart failure after cardiac injury 1,2. Structural remodeling is thought to be a plasticity process of the heart to overcome hemodynamic overload, but cardiac resistance (i.e., robustness) to mechanical stress may be reduced by additional environmental factors, such as physical and chemical stresses 3. Purinergic receptors are activated by extracellular nucleotides and play important roles in cardiovascular physiology and pathophysiology 4. Purinergic receptors are divided into two main groups, P1 and P2. P1 receptors are activated by adenosine, and mediate cardiodepressant and cardioprotective effects 4. P2 receptors are subdivided into P2X and P2Y subfamilies, which consist of ligand-gated ion channels and G protein coupled receptors (GPCRs), respectively 4. The P2Y family has eight subtypes (P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 and P2Y 14) that differ in their coupling G protein and ligand selectivity 5. Purinergic signaling must be important for cardiovascular homeostasis because many purinergic receptors are expressed in human and mouse hearts 6,7. The nucleotide, uridine triphosphate (UTP), induces a profibrotic response via P2Y 2 R 8 , while adenosine triphosphate (ATP) induces contraction 9 and negatively regulates hypertrophic growth of cardiomyocytes 10,11 .

Covalent modification of sulfur-containing amino acids in proteins by reactive oxygen species (ROS) has been attracting attention as a major post-translational modification regulating intracellular signal transduction pathways. Angiotensin II Ang II, a major physiologically active substrate in renin-angiotensin (RAS) system, plays a central role in the pathophysiology of cardiovascular systems. Many evidences show that Ang II activates several signaling pathways via an oxidative modification of proteins by Ang II-induced ROS. Ang II induced ROS production is predominantly regulated by three enzymes: NADPH oxidase, mitochondrial respiratory complex, and nitric oxide synthase (NOS), and each enzyme-generating ROS are found to activate appropriate signaling pathways via selective oxidation of specific proteins. These reactions are negatively regulated by ROS-scavenging enzymes or disulfide bridge reducing enzymes, and functional disorders of these enzymes are found to cause cardiovascular dysfunctions. Thus, the spatial and temporal regulation of oxidative modification of signaling proteins by ROS is essential to maintain cardiovascular homeostasis by Ang II. This review brings in the new aspect in understanding ROS-mediated regulation of cardiovascular homeostasis by Ang II, and provides the possible mechanisms underlying metamorphosis of cardiovascular homeostasis by ROS.

This review provided a systematic overview of the questionnaire-related dengue studies conducted in Malaysia and evaluated their reliability and validity used in the questionnaires. An extensive literature search was conducted using various electronic databases, including PubMed, EMBASE, Medline, and ScienceDirect. Systematic reviews and meta-analysis (PRISMA) were selected as the preferred item reporting method. Out of 88 identified dengue-related, 57 published from 2000 to April 2020 met the inclusion criteria and were included. Based on the meta-analysis, a poor mean score was obtained for knowledge (49%), attitude (44%), and preventive practice (55%). The study showed that the level of knowledge on cardinal signs and modes of transmission for dengue virus were highest among health care workers, followed by students (international and local) and lastly community residents. In treatment-seeking behaviours, only half of the respondents (50.8%) would send their child to the nearest health clinics or hospitals when a child became restless or lethargic. The acceptance rate for dengue vaccine, bacteria (Wolbachia), as a vector for dengue control and self-test diagnostic kit for dengue showed considerably high (88.4%, 70%, and 44.8%, respectively). Health belief model (HBM) constructs, such as perceived barriers, perceived severity, perceived susceptibility, self-efficacy, and perceived benefit influence prevention practices. Lastly, only 23 articles (40.3%) had piloted or pretested the questionnaire before surveying, in which three reported Cronbach’s alpha coefficient (0.70–0.90). A need for active participation of communities and healthcare personnel, promotion of awareness, and safe complementary medicines, as well as assessment of psychometric properties of questionnaire use in dengue surveys in Malaysia, in order for assessing dengue reliably and valid.