Calcitonin Gene-Related Peptide Regulates Cardiomyocyte Survival through Regulation of Oxidative Stress by PI3K/Akt and MAPK Signaling Pathways.

CGRP and specific CGRP receptors are found in the heart where they produce positive-inotropic and anti-apoptotic effects, key adaptations to exercise and cardiovascular disease. PI3K/Akt and MAPK signaling imbalances are associated with cardiomyocyte pathologies; however, the effects of CGRP on these pathways are unclear. Therefore, we hypothesized that CGRP modulates inotropic and apoptotic adaptations of cardiomyocytes by regulating PI3K/Akt and MAPK/ERK signaling balances. We treated cardiomyocytes with combinations of CGRP, PI3K/Akt and MAPK signaling agonists and antagonists. We evaluated expression of the mRNA and proteins levels of survival signaling molecules related to the PI3K/Akt and MAPK and measured apoptosis by caspase 3/7 activity. CGRP1-37 decreased Akt, NFκB, SOD-3 and increased ERK1/2 and p38 MAPK expressions, which was antagonized by CGRP8-37. Akt-negative construct transfection, Ad.Akt(K179M), inhibited the CGRP1-37-induced increment in MAPK expressions. A PI3K-antagonist treatment with LY294002 or CGRP1-37/Ad.Akt(K179M) co-treatment alleviated the CGRP-increased caspase activity and -decrements in SOD-3. These findings demonstrate a CGRP negative effect on the PI3K/Akt signaling pathway and CGRP receptor-induced crosstalk between PI3K/Akt and MAPK in normal cardiomyocytes. Future studies to differentiate CGRP effects on intracellular signal transduction mechanisms in pathological conditions will elucidate the significance of CGRP in, and provide novel therapeutic targets for, heart failure.

Acute alcohol modulates cardiac function as PI3K/Akt regulates oxidative stress.

BACKGROUND: Clinical manifestations of alcohol abuse on the cardiac muscle include defective contractility with the development of heart failure. Interestingly, low alcohol consumption has been associated with reduced risk of cardiovascular disease. Although several hypotheses have been postulated for alcoholic cardiomyopathy and for the low-dose beneficial cardiovascular effects, the precise mechanisms and mediators remain largely undefined. We hypothesize that modulation of oxidative stress by PI3K/Akt plays a key role in the cardiac functional outcome to acute alcohol exposure. METHODS: Thus, acutely exposed rat cardiac tissue and cardiocytes to low (LA: 5 mM), moderate (MA: 25 mM), and high (HA: 100 mM) alcohol were assessed for markers of oxidative stress in the presence and absence of PI3K/Akt activators (IGF-1 0.1 µM or constitutively active PI3K: Ad.BD110 transfection) or inhibitor (LY294002 1 µM or Akt-negative construct Ad.Akt(K179M) transfection). RESULTS: Acute LA reduced Akt, superoxide dismutase (SOD-3) and NFκB, ERK1, and p38 MAPK gene expression. Acute HA only increased that of SOD-3 and NFκB. These effects were generally inhibited by Ad.Akt(K179M) and enhanced with Ad.BD110 transfection. In parallel, LA reduced but HA enhanced Akt activity, which was reversed by IGF-1 and inhibited by Ad.Akt(K179M), respectively. Also, LA reduced caspase 3/7 activity and oxidative stress, while HA increased both. The former was blocked, while the latter effect was enhanced by Ad.Akt(K179M). The reverse was true with PI3K/Akt activation. This translated into reduced viability with HA, with no effect with LA. On the functional level, acute LA improved cardiac output and ejection fraction, mainly through increased stroke volume. This was accompanied with enhanced end-systolic pressure-volume relationship and preload recruitable stroke work. Opposite effect was recorded for HA. LA and HA in vivo functional effects were alleviated by LY and enhanced by IGF-1 treatment. CONCLUSIONS: Acute LA and HA seem to oppositely affect cardiac function through modulation of oxidative stress where PI3K/Akt plays a pivotal role.

Molecular steps in the immune signaling pathway evoked by plant elicitor peptides: Ca2+-dependent protein kinases, nitric oxide, and reactive oxygen species are downstream from the early Ca2+ signal.

Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca(2+) elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca(2+) signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca(2+)-dependent protein kinases (CPKs) decode the Ca(2+) signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca(2+) signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca(2+)-conducting channel in the Pep immune signaling pathway.

Detection of reactive oxygen species downstream of cyclic nucleotide signals in plants.

Cyclic nucleotides act in plant cell signal transduction cascades by activating cyclic nucleotide gated cation-conducting ion channels (CNGCs). Activation of CNGCs results in inward cation (including Ca(2+)) conductance across the plasma membrane. Elevation of cytosolic Ca(2+) is an early step in numerous plant cell signal transduction cascades, including plant immune responses to pathogens. CNGC involvement, along with cyclic nucleotides cAMP and cGMP, in pathogen defense programs is one relatively well-studied area of cyclic nucleotide signaling in plants. During plant immune responses, CNGC-dependent Ca(2+) elevations lead to a signaling cascade that results in the generation of defense molecules such as hydrogen peroxide and nitric oxide, and induction of defense gene expression. This pathogen defense response is discussed, and methods to detect some of the downstream signaling steps in the pathway are presented.

Inotropic and lusitropic effects of calcitonin gene-related peptide in the heart.

Previous studies have demonstrated positive-inotropic effects of calcitonin gene-related peptide (CGRP), but the mechanisms remain unclear. Therefore, two experiments were performed to determine the physiological correlates of the positive-inotropic effects of CGRP. Treatments designed to antagonize the effects of physiologically active CGRP1₋37 included posttreatment with CGRP8₋37 and pretreatment with LY-294002 (LY, an inhibitor of phosphatidylinositol 3-kinase), 17ß-estradiol (E), and progesterone (P) were also used to modulate the effects of CGRP1₋37. Experiment 1 was in vitro studies on sarcomeres and cells of isolated adult rat cardiac myocytes. CGRP1₋37, alone and in combination with E and P, decreased sarcomere shortening velocities and increased shortening percentages, effects that were antagonized by CGRP8₋37, but not by LY. CGRP1₋37 increased resting intracellular calcium ion concentrations and Ca(2+) influxes, effects that were also antagonized by both CGRP8₋37 and LY. Experiment 2 was in vivo studies on left ventricular pressure-volume (PV) loops. CGRP1₋37 increased end-systolic pressure, ejection fraction, and velocities of contraction and relaxation while decreasing stroke volume, cardiac output, stroke work, PV area, and compliance. After partial occlusion of the vena cava, CGRP1₋37 increased the slope of the end-systolic PV relationship. CGRP8₋37 and LY attenuated most of the CGRP-induced changes. These findings suggest that CGRP-induced positive-inotropic effects may be increased by treatments with estradiol and progesterone and inhibited by LY. The physiological correlates of CGRP-induced positive inotropy observed in rat sarcomeres, cells, and intact hearts are likely to reveal novel mechanisms of heart failure in humans.

The good, the bad, and the ugly with alcohol use and abuse on the heart.

Since its advent, alcohol has been utilized throughout history socially, for rituals, worship, and for its therapeutic, antibacterial, and analgesic properties. In moderation, alcohol consumption and its use are generally viewed as clinically beneficial. Excessive alcohol consumption on the other hand has been recognized as having several adverse implications. Excessive use increases the risk of liver and heart disease, metabolic disturbances, nutritional deficiencies, certain cancers, brain damage, dementia, neuropathy, as well as other facets of morbidity and mortality. This review targets the sequelae of alcohol consumption on the heart, specifically on myocardial contractility, calcium channel signaling, and intracellular signaling pathways. With the incidence of alcohol-induced cardiac abnormalities being higher than previously thought, it is of increasing importance to elucidate the mechanisms behind them. Here, the cardiac effects of alcohol were not discussed in isolation but in conjunction with other important factors, such as high- and low-density lipoprotein levels and vascular dilatory influences. We explore these mechanisms, in particular, the oxidative stress as the major contributor, as well as pathways that may prove to be cardioprotective. As such, we demonstrate the involvement of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) as well as AKT that act as regulators of oxidative balance during oxidative stress responses. Thus, alcohol consumption may confer a cardioprotective effect when used in moderation through an AKT/NRF2-dependent mechanism.

Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants.

Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca(2+) elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca(2+) elevation is attributable to cGMP activation of a Ca(2+) channel. Some differences were identified between Pep/PEPR signaling and the Ca(2+)-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca(2+) stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca(2+). Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca(2+) elevation, H(2)O(2) generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen.

Leaf senescence signaling: the Ca2+-conducting Arabidopsis cyclic nucleotide gated channel2 acts through nitric oxide to repress senescence programming.

Ca(2+) and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca(2+). The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca(2+) modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca(2+) and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca(2+) uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca(2+) accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H(2)O(2) generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca(2+) channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca(2+) signaling provides evidence consistent with genetic studies of the relationship between Ca(2+) signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca(2+) uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.

Ca2+, cAMP, and transduction of non-self perception during plant immune responses.

Ca(2+) influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca(2+) influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca(2+) flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca(2+) elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca(2+) channel-dependent cytosolic Ca(2+) elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca(2+) rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca(2+) channels and immune signaling downstream from cytosolic Ca(2+) elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.