The Pleiotropic Role of Extracellular ATP in Myocardial Remodelling.

Myocardial remodelling is a molecular, cellular, and interstitial adaptation of the heart in response to altered environmental demands. The heart undergoes reversible physiological remodelling in response to changes in mechanical loading or irreversible pathological remodelling induced by neurohumoral factors and chronic stress, leading to heart failure. Adenosine triphosphate (ATP) is one of the potent mediators in cardiovascular signalling that act on the ligand-gated (P2X) and G-protein-coupled (P2Y) purinoceptors via the autocrine or paracrine manners. These activations mediate numerous intracellular communications by modulating the production of other messengers, including calcium, growth factors, cytokines, and nitric oxide. ATP is known to play a pleiotropic role in cardiovascular pathophysiology, making it a reliable biomarker for cardiac protection. This review outlines the sources of ATP released under physiological and pathological stress and its cell-specific mechanism of action. We further highlight a series of cardiovascular cell-to-cell communications of extracellular ATP signalling cascades in cardiac remodelling, which can be seen in hypertension, ischemia/reperfusion injury, fibrosis, hypertrophy, and atrophy. Finally, we summarize current pharmacological intervention using the ATP network as a target for cardiac protection. A better understanding of ATP communication in myocardial remodelling could be worthwhile for future drug development and repurposing and the management of cardiovascular diseases.

Modulation of POPDC1 Expression by Phenothiazine and Trifluoperazine Suppress Colon Cancer Growth and Migration.

OBJECTIVE: The aim of this study was to investigate the effects of CaM antagonist, PTZ, and TFP on cell proliferation and migration of colon cancer cells and its impact on POPDC protein expression. METHODS: The 50% inhibitory concentration (IC50) of PTZ and TFP in SW1116, SW480, HCT-15, and COLO205 colon cancer cell lines are measured using MTT. Western blot and immunocytochemistry were used to determine the expression of PCNA, cyclin D1 (CD1), and POPDC proteins. Cell migration was observed using a scratch wound-healing assay. RESULTS: Treatment with PTZ and TFP inhibited colon cancer cells growth in a dose-dependent manner. PTZ and TFP significantly inhibited the activation of proliferation markers, PCNA and CD1, and the migration of colon cancer cells. Furthermore, POPDC protein was significantly suppressed in all cell types of colon cancer, particularly in SW480. Finally, the CaM antagonist upregulates the POPDC1 expression in colon cancer cells. CONCLUSION: These findings suggest that CaM antagonists suppress colon cancer cells proliferation via downregulation of CD1 and PCNA. In addition, POPDC protein could be used as a biomarker in colon cancer, and CaM antagonist could be used to regulate POPDC1 expression. This study suggests that targeting POPDC1 with CaM inhibition could be a potential therapeutic strategy for colon cancer treatment. 
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Carpaine Promotes Proliferation and Repair of H9c2 Cardiomyocytes after Oxidative Insults.

Carpaine has long been identified as the major alkaloid in Carica papaya leaves that possess muscle relaxant properties. Limited study on the molecular signaling properties of carpaine urges us to conduct this study that aims to elucidate the mechanism underlying the cardioprotective effect of carpaine in embryonic cardiomyocytes of the H9c2 cell line. The 50% inhibitory concentration (IC50) of carpaine was first determined using a colorimetric MTT assay to establish the minimum inhibitory concentration for the subsequent test. Using a 1 µM carpaine treatment, a significant increase in the H9c2 proliferation rate was observed following 24 and 48 h of incubation. A Western blot analysis also revealed that carpaine promotes the upregulation of the cell cycle marker proteins cyclin D1 and PCNA. Carpaine-induced H9c2 cell proliferation is mediated by the activation of the FAK-ERK1/2 and FAK-AKT signaling pathways. In the setting of ischemia-reperfusion injury (IRI), carpaine provided a significant protective role to recover the wounded area affected by the hydrogen peroxide (H2O2) treatment. Furthermore, the oxidative-stress-induced reduction in mitochondrial membrane potential (MMP) and overproduction of reactive oxygen species (ROS) were attenuated by carpaine treatment. The current study revealed a novel therapeutic potential of carpaine in promoting in vitro cardiomyocyte proliferation and repair following injury.

Genotype-Phenotype Study of ß-Thalassemia Patients in Sabah.

ß-thalassemia is a serious public health problem in Sabah due to its high prevalence. This study aimed to investigate the effects of different types of ß-globin gene mutations, coinheritance with α-globin gene mutations, XmnI-Gγ, and rs368698783 polymorphisms on the ß-thalassemia phenotypes in Sabahan patients. A total of 111 patients were included in this study. The sociodemographic profile of the patients was collected using a semi-structured questionnaire, while clinical data were obtained from their medical records. Gap-PCR, ARMS-PCR, RFLP-PCR, and multiplex PCR were performed to detect ß- and α-globin gene mutations, as well as XmnI-Gγ and rs368698783 polymorphisms. Our data show that the high prevalence of ß-thalassemia in Sabah is not due to consanguineous marriages (5.4%). A total of six different ß-globin gene mutations were detected, with Filipino ß°-deletion being the most dominant (87.4%). There were 77.5% homozygous ß-thalassemia patients, 16.2% compound heterozygous ß-thalassemia patients, and 6.3% ß-thalassemia/Hb E patients. Further evaluation on compound heterozygous ß-thalassemia and ß-thalassemia/Hb E patients found no concomitant α-globin gene mutations and the rs368698783 polymorphism. Furthermore, the XmnI-Gγ (-/+) genotype did not demonstrate a strong impact on the disease phenotype, as only two of five patients in the compound heterozygous ß-thalassemia group and two of three patients in the ß-thalassemia/Hb E group had a moderate phenotype. Our findings indicate that the severity of the ß-thalassemia phenotypes is closely related to the type of ß-globin gene mutations but not to the XmnI-Gγ and rs368698783 polymorphisms.

Different Domains of Dengue Research in Malaysia: A Systematic Review and Meta-Analysis of Questionnaire-Based Studies.

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.

Modulation of P2Y6R expression exacerbates pressure overload-induced cardiac remodeling in mice.

Cardiac tissue remodeling caused by hemodynamic overload is a major clinical outcome of heart failure. Uridine-responsive purinergic P2Y6 receptor (P2Y6R) contributes to the progression of cardiovascular remodeling in rodents, but it is not known whether inhibition of P2Y6R prevents or promotes heart failure. We demonstrate that inhibition of P2Y6R promotes pressure overload-induced sudden death and heart failure in mice. In neonatal cardiomyocytes, knockdown of P2Y6R significantly attenuated hypertrophic growth and cell death caused by hypotonic stimulation, indicating the involvement of P2Y6R in mechanical stress-induced myocardial dysfunction. Unexpectedly, compared with wild-type mice, deletion of P2Y6R promoted pressure overload-induced sudden death, as well as cardiac remodeling and dysfunction. Mice with cardiomyocyte-specific overexpression of P2Y6R also exhibited cardiac dysfunction and severe fibrosis. In contrast, P2Y6R deletion had little impact on oxidative stress-mediated cardiac dysfunction induced by doxorubicin treatment. These findings provide overwhelming evidence that systemic inhibition of P2Y6R exacerbates pressure overload-induced heart failure in mice, although P2Y6R in cardiomyocytes contributes to the progression of cardiac fibrosis.

TRPC3-Nox2 axis mediates nutritional deficiency-induced cardiomyocyte atrophy.

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 P2Y2 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.

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.

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

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.

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.

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

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 ß-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 ß-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.

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice.

N-type voltage-dependent Ca(2+)channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca(2+) influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice.

Redox control of cardiovascular homeostasis by angiotensin II.

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.