Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production ...of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine
(Ser
) residue was measured. The contribution of cellular deformation to shear-induced NO production in RBCs was determined by rigidifying RBCs with the thiol-oxidizing agent diamide; rigid RBCs exhibited significantly impaired (up to 80%) capacity to generate NO via RBC-NOS during shear. Standardizing membrane strain of rigid RBCs by applying increased shear did not normalize NO production, or RBC-NOS activation. Calcium imaging with fluo-4 revealed that diamide-treated RBCs exhibited a 42% impairment in Piezo1
mediated calcium movement when compared with untreated RBCs. Pharmacological inhibition of Piezo1 with GsMTx4 during shear inhibited RBC-NOS activation in untreated RBCs, whereas Piezo1 activation with Yoda1 in the absence of shear stimulated RBC-NOS activation. Collectively, a novel, mechanically activated signaling pathway in mature RBCs is described. Opening of Piezo1 and subsequent influx of calcium appear to be required for endogenous production of NO in response to mechanical shear, which is accompanied by phosphorylation of RBC-NOS at Ser
.
The mechano-sensitive ion channel Piezo1 is expressed in enucleated red blood cells and provides a mechanism of shear-induced red cell nitric oxide production via nitric oxide synthase phosphorylation. Thiol oxidation of red cells decreases Piezo1-dependent calcium movement and thus impairs nitric oxide generation in response to mechanical force. The emerging descriptions of exclusively posttranslational signaling networks in circulating red cells as acute regulators of cell function support that these cells play an important role in cardiovascular physiology that extends beyond passive oxygen transport.
Experimental research has recognized the importance of cardiac fibroblast and myofibroblast cells in heart repair and function. In a normal healthy heart, the cardiac fibroblast plays a central role ...in the structural, electrical, and chemical aspects within the heart. Interestingly, the transformation of cardiac fibroblast cells to cardiac myofibroblast cells is suspected to play a vital part in the development of heart failure. The ability to differentiate between the two cells types has been a challenge. Myofibroblast cells are only expressed in the stressed or failing heart, so a better understanding of cell function may identify therapies that aid repair of the damaged heart. This paper will provide an outline of what is currently known about cardiac fibroblasts and myofibroblasts, the physiological and pathological roles within the heart, and causes for the transition of fibroblasts into myoblasts. We also reviewed the potential markers available for characterizing these cells and found that there is no single-cell specific marker that delineates fibroblast or myofibroblast cells. To characterize the cells of fibroblast origin, vimentin is commonly used. Cardiac fibroblasts can be identified using discoidin domain receptor 2 (DDR2) while α-smooth muscle actin is used to distinguish myofibroblasts. A known cytokine TGF-β
1
is well established to cause the transformation of cardiac fibroblasts to myofibroblasts. This review will also discuss clinical treatments that inhibit or reduce the actions of TGF-β
1
and its contribution to cardiac fibrosis and heart failure.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts ...(MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease.
We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression.
Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell ...and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., ‘Gárdos effect’). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.
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•Calcium-induced impairment in erythrocyte deformability explained by cell volume•Decreased cell volume is solely mediated by Gárdos channel activation.•Sensitivity of erythrocytes to mechanical stress is dependent upon cell volume.•Physiologic shear in presence of extracellular calcium causes selective red cell destruction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Intra- and extracellular adenosine levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenge and tissue injury. Extracellular adenosine ...engages members of the G-protein coupled adenosine receptor (AR) family to mediate generally beneficial acute and adaptive responses within all constituent cells of the heart. In this way the four AR sub-types-A1, A2A, A2B, and A3Rs-regulate myocardial contraction, heart rate and conduction, adrenergic control, coronary vascular tone, cardiac and vascular growth, inflammatory-vascular cell interactions, and cellular stress-resistance, injury and death. The AR sub-types exert both distinct and overlapping effects, and may interact in mediating these cardiovascular responses. The roles of the ARs in beneficial modulation of cardiac and vascular function, growth and stress-resistance render them attractive therapeutic targets. However, interactions between ARs and with other receptors, and their ubiquitous distribution throughout the body, can pose a challenge to the implementation of site- and target-specific AR based pharmacotherapy. This review outlines cardiovascular control by adenosine and the AR family in health and disease, including interactions between AR sub-types within the heart and vessels.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
6.
Active modulation of human erythrocyte mechanics Kuck, Lennart; Peart, Jason N.; Simmonds, Michael J.
American Journal of Physiology: Cell Physiology,
08/2020, Volume:
319, Issue:
2
Journal Article
Peer reviewed
Open access
The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the ...absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca 2+ ). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca 2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca 2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.
Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic ...metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function.
We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents.
Analysis of human complex trait genetics indicates that variants in the
genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance.
Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3-5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and ...aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium.
Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip.
Protection via SLP is associated with transcriptional repression of inflammation/immunity, up-regulation of sarcomeric elements and natriuretic peptides, and modulation of cell stress, growth and development, while conventional protective molecules are unaltered.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We critically review potential involvement of trimethylamine N-oxide (TMAO) as a link between diet, the gut microbiota and CVD. Generated primarily from dietary choline and carnitine by gut bacteria ...and hepatic flavin-containing mono-oxygenase (FMO) activity, TMAO could promote cardiometabolic disease when chronically elevated. However, control of circulating TMAO is poorly understood, and diet, age, body mass, sex hormones, renal clearance, FMO3 expression and genetic background may explain as little as 25 % of TMAO variance. The basis of elevations with obesity, diabetes, atherosclerosis or CHD is similarly ill-defined, although gut microbiota profiles/remodelling appear critical. Elevated TMAO could promote CVD via inflammation, oxidative stress, scavenger receptor up-regulation, reverse cholesterol transport (RCT) inhibition, and cardiovascular dysfunction. However, concentrations influencing inflammation, scavenger receptors and RCT (≥100 µm) are only achieved in advanced heart failure or chronic kidney disease (CKD), and greatly exceed pathogenicity of <1–5 µm levels implied in some TMAO–CVD associations. There is also evidence that CVD risk is insensitive to TMAO variance beyond these levels in omnivores and vegetarians, and that major TMAO sources are cardioprotective. Assessing available evidence suggests that modest elevations in TMAO (≤10 µm) are a non-pathogenic consequence of diverse risk factors (ageing, obesity, dyslipidaemia, insulin resistance/diabetes, renal dysfunction), indirectly reflecting CVD risk without participating mechanistically. Nonetheless, TMAO may surpass a pathogenic threshold as a consequence of CVD/CKD, secondarily promoting disease progression. TMAO might thus reflect early CVD risk while providing a prognostic biomarker or secondary target in established disease, although mechanistic contributions to CVD await confirmation.
Department of Pharmacology and Toxicology, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226
Ischemic or myocardial preconditioning (IPC) is a phenomenon whereby brief
periods of ischemia have ...been shown to protect the myocardium against a more
sustained ischemic insult. The result of IPC may be manifest as a marked
reduction in infarct size, myocardial stunning, or incidence of cardiac
arrhythmias. Whereas many endogenous neurotransmitters, peptides, and hormones
have been proposed to play a role in the signal transduction pathways
mediating the cardioprotective effect of IPC, nearly universal evidence
indicates the involvement of the ATP-sensitive potassium (K ATP )
channel. Initial evidence suggested that the surface or sarcolemmal
K ATP (sarcK ATP ) channel triggered or mediated the
cardioprotective effects of IPC; however, more recent findings have suggested
a major role for a mitochondrial site or possibly a mitochondrial
K ATP channel (mitoK ATP ). This review presents evidence
that supports a role for these two channels as a trigger and/or downstream
mediator in the phenomenon of IPC or pharmacologically induced PC as well as
recent evidence that suggests the involvement of a mitochondrial
calcium-activated potassium (mitoK ca ) channel or the electron
transport chain in mediating the beneficial effects of IPC or
pharmacologically induced PC.
ischemia; electron transport chain
Address for reprint requests and other correspondence: G. J. Gross, Dept. of
Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown
Plank Rd., Milwaukee, WI 53226 (E-mail:
ggross{at}mcw.edu ).
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