T cell dysfunction in elderly ARDS patients based on miRNA and mRNA integration analysis.

Background: Acute respiratory distress syndrome (ARDS) is respiratory failure that commonly occurs in critically ill patients, and the molecular mechanisms underlying its pathogenesis and severity are poorly understood. We evaluated mRNA and miRNA in patients with ARDS and elucidated the pathogenesis of ARDS after performing mRNA and miRNA integration analysis. Methods: In this single-center, prospective, observational clinical study of patients with ARDS, peripheral blood of each patient was collected within 24 hours of admission. Sequencing of mRNA and miRNA was performed using whole blood from the ARDS patients and healthy donors. Results: Thirty-four ARDS patients were compared with 15 healthy donors. Compared with the healthy donors, 1233 mRNAs and 6 miRNAs were upregulated and 1580 mRNAs and 13 miRNAs were downregulated in the ARDS patients. For both mRNA and miRNA-targeted mRNA, canonical pathway analysis showed that programmed death-1 (PD-1) and programmed cell death ligand 1 (PD-L1) cancer immunotherapy pathway was most activated and the Th2 pathway was most suppressed. For mRNA, the Th1 pathway was most suppressed. miR-149-3p and several miRNAs were identified as upstream regulators. Conclusion: miRNAs regulated the PD-1 and PD-L1 cancer immunotherapy pathway and Th2 pathway through miRNA interference action of mRNA. Integrated analysis of mRNAs and miRNAs showed that T cells were dysfunctional in ARDS patients.

Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through ß-adrenoceptors.

Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates ß-adrenoceptor (ßAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated ßAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting ß-arrestin-mediated ßAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.

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.

TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.

Vascular smooth muscle cells (VSMCs) play critical roles in the stability and tonic regulation of vascular homeostasis. VSMCs can switch back and forth between highly proliferative synthetic and fully differentiated contractile phenotypes in response to changes in the vessel environment. Although abnormal phenotypic switching of VSMCs is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty, how control of VSMC phenotypic switching is dysregulated in pathologic conditions remains obscure. We found that inhibition of canonical transient receptor potential 6 (TRPC6) channels facilitated contractile differentiation of VSMCs through plasma membrane hyperpolarization. TRPC6-deficient VSMCs exhibited more polarized resting membrane potentials and higher protein kinase B (Akt) activity than wild-type VSMCs in response to TGF-ß1 stimulation. Ischemic stress elicited by oxygen-glucose deprivation suppressed TGF-ß1-induced hyperpolarization and VSMC differentiation, but this effect was abolished by TRPC6 deletion. TRPC6-mediated Ca2+ influx and depolarization coordinately promoted the interaction of TRPC6 with lipid phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of Akt activation. Given the marked up-regulation of TRPC6 observed in vascular disorders, our findings suggest that attenuation of TRPC6 channel activity in pathologic VSMCs could be a rational strategy to maintain vascular quality control by fine-tuning of VSMC phenotypic switching.-Numaga-Tomita, T., Shimauchi, T., Oda, S., Tanaka, T., Nishiyama, K., Nishimura, A., Birnbaumer, L., Mori, Y., Nishida, M. TRPC6 regulates phenotypic switching of vascular smooth muscle cells through plasma membrane potential-dependent coupling with PTEN.

TRPC channels in exercise-mimetic therapy.

Physical exercise yields beneficial effects on all types of muscle cells, which are essential for the maintenance of cardiovascular homeostasis and good blood circulation. Daily moderate exercise increases systemic antioxidative capacity, which can lead to the prevention of the onset and progression of oxidative stress-related diseases. Therefore, exercise is now widely accepted as one of the best therapeutic strategies for the treatment of ischemic (hypoxic) diseases. Canonical transient receptor potential (TRPC) proteins are non-selective cation channels activated by mechanical stress and/or stimulation of phospholipase C-coupled surface receptors. TRPC channels, especially diacylglycerol-activated TRPC channels (TRPC3 and TRPC6; TRPC3/6), play a key role in the development of cardiovascular remodeling. We have recently found that physical interaction between TRPC3 and NADPH oxidase (Nox) 2 under hypoxic stress promotes Nox2-dependent reactive oxygen species (ROS) production and mediates rodent cardiac plasticity, and inhibition of the TRPC3-Nox2 protein complex results in enhancement of myocardial compliance and flexibility similar to that observed in exercise-treated hearts. In this review, we describe current understanding of the roles of TRPC channels in striated muscle (patho)physiology and propose that targeting TRPC-based protein complexes could be a new strategy to imitate exercise therapy.

[New Strategies for Exercise-Mimetic Medication].

　Moderate exercise has been reported to combat several diseases, including cardiovascular diseases and depressants. However, many patients do not have ability to undergo exercise therapy due to aging and severity of the symptoms. Therefore development of new drugs that can imitate exercise therapy is desired and actually studied worldwide. The heart is one of the physical load-responsive target organs such as skeletal muscles and vascular smooth muscles. The heart can adapt from environmental stress by changing its structure and morphology (i.e., remodeling). Physiological remodeling, caused by exercise or pregnancy, can be defined by compensative and reversible changes to the heart, whereas pathological remodeling can be defined by irreversible changes of the heart, through aberrant calcium ion (Ca2+) signaling as well as production of reactive oxygen species (ROS). However, crosstalk between Ca2+ and ROS remains obscure. In this review we will introduce our recent findings on the functional crosstalk between transient receptor potential canonical (TRPC) 3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) 2 as a novel molecular target to mimic exercise therapy.

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.

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.

TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice.

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.

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.

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.

Brazilian Green Propolis Promotes Weight Loss and Reduces Fat Accumulation in C57BL/6 Mice Fed A High-Fat Diet.

Propolis is a bee product with various biological properties. C57BL/6 mice were fed a high-fat diet and treated with propolis for 14 weeks. Body weight in mice treated with 2% propolis was less than that in control mice from 3 weeks after the start of treatment until 14 weeks except for the 7th week. Mice treated with propolis showed significantly lower epididymal fat weight and subcutaneous fat weight. Infiltration of epididymal fat by macrophages and T cells was reduced in the propolis group. Supplementation of propolis increased feces weight and fat content in feces, suggesting that mechanisms of weight reduction by propolis partly include a laxative effect and inhibition of fat absorption.