Pharmacological modulation of prostaglandin E2 (PGE2 ) EP receptors improves cardiomyocyte function under hyperglycemic conditions.

Type 2 diabetes (T2D) affects >30 million Americans and nearly 70% of individuals with T2D will die from cardiovascular disease (CVD). Circulating levels of the inflammatory signaling lipid, prostaglandin E2 (PGE2 ), are elevated in the setting of obesity and T2D and are associated with decreased cardiac function. The EP3 and EP4 PGE2 receptors have opposing actions in several tissues, including the heart: overexpression of EP3 in cardiomyocytes impairs function, while EP4 overexpression improves function. Here we performed complementary studies in vitro with isolated cardiomyocytes and in vivo using db/db mice, a model of T2D, to analyze the effects of EP3 inhibition or EP4 activation on cardiac function. Using echocardiography, we found that 2 weeks of systemic treatment of db/db mice with 20 mg/kg of EP3 antagonist, beginning at 6 weeks of age, improves ejection fraction and fractional shortening (with no effect on heart rate). We further show that either EP3 blockade or EP4 activation enhances contractility and calcium cycling in isolated mouse cardiomyocytes cultured in both normal and high glucose. Thus, peak [Ca2+ ]I transient amplitude was increased, while time to peak [Ca2+ ]I and [Ca2+ ]I decay were decreased. These data suggest that modulation of EP3 and EP4 activity has beneficial effects on cardiomyocyte contractility and overall heart function.

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Stimulation of TRPA1 attenuates ischemia-induced cardiomyocyte cell death through an eNOS-mediated mechanism.

The functional expression of transient receptor potential cation channel of the ankyrin-1 subtype (TRPA1) has recently been identified in adult mouse cardiac tissue where stimulation of this ion channel leads to increases in adult mouse ventricular cardiomyocyte (CM) contractile function via a Ca2+-Calmodulin-dependent kinase (CaMKII) pathway. However, the extent to which TRPA1 induces nitric oxide (NO) production in CMs, and whether this signaling cascade mediates physiological or pathophysiological events in cardiac tissue remains elusive. Freshly isolated CMs from wild-type (WT) or TRPA1 knockout (TRPA1-/-) mouse hearts were treated with AITC (100 µM) and prepared for immunoblot, NO detection or ischemia protocols. Our findings demonstrate that TRPA1 stimulation with AITC results in phosphorylation of protein kinase B (Akt) and endothelial NOS (eNOS) concomitantly with NO production in a concentration- and time-dependent manner. Additionally, we found that TRPA1 induced increases in CM [Ca2+]i and contractility occur independently of Akt and eNOS activation mechanisms. Further analysis revealed that the presence and activation of TRPA1 promotes CM survival and viability following ischemic insult via a mechanism partially dependent upon eNOS. Therefore, activation of the TRPA1/Akt/eNOS pathway attenuates ischemia-induced CM cell death.

Modulation of TRPA1 channel activity by Cdk5 in sensory neurons.

Transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is activated by a broad range of noxious stimuli. Cdk5, a member of the Cdk family, has recently been identified as a modulator of pain signaling pathways. In the current study, we investigated the extent to which Cdk5 modulates TRPA1 activity. Cdk5 inhibition was found to attenuate TRPA1 response to agonist in mouse DRG sensory neurons. Additionally, the presence of active Cdk5 was associated with increased TRPA1 phosphorylation in transfected HEK293 cells that was roscovitine-sensitive and absent in the mouse mutant S449A full-length channel. Immunopurified Cdk5 was observed to phosphorylate human TRPA1 peptide substrate at S448A in vitro. Our results point to a role for Cdk5 in modulating TRPA1 activity.

TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway.

RATIONALE: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored. OBJECTIVE: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action. METHODS AND RESULTS: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1-/- mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14-22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1-5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L). CONCLUSIONS: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.

TRPA1 and TRPV1 contribute to propofol-mediated antagonism of U46619-induced constriction in murine coronary arteries.

BACKGROUND: Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. METHODS: Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. RESULTS: Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. CONCLUSIONS: TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.

Chemical composition and phagocyte immunomodulatory activity of Ferula iliensis essential oils.

Essential oil extracts from Ferula iliensis have been used traditionally in Kazakhstan for treatment of inflammation and other illnesses. Because little is known about the biologic activity of these essential oils that contributes to their therapeutic properties, we analyzed their chemical composition and evaluated their phagocyte immunomodulatory activity. The main components of the extracted essential oils were (E)-propenyl sec-butyl disulfide (15.7-39.4%) and (Z)-propenyl sec-butyl disulfide (23.4-45.0%). Ferula essential oils stimulated [Ca2+]i mobilization in human neutrophils and activated ROS production in human neutrophils and murine bone marrow phagocytes. Activation of human neutrophil [Ca2+]i flux by Ferula essential oils was dose-dependently inhibited by capsazepine, a TRPV1 channel antagonist, indicating that TRPV1 channels mediate this response. Furthermore, Ferula essential oils stimulated Ca2+ influx in TRPV1 channel-transfected HEK293 cells and desensitized the capsaicin-induced response in these cells. Additional molecular modeling with known TRPV1 channel agonists suggested that the active component is likely to be (Z)-propenyl sec-butyl disulfide. Our results provide a cellular and molecular basis to explain at least part of the beneficial therapeutic properties of FEOs.

TRPA1 is functionally co-expressed with TRPV1 in cardiac muscle: Co-localization at z-discs, costameres and intercalated discs.

Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) and vanilloid subtype-1 (TRPV1) are structurally related, non-selective cation channels that show a high permeability to calcium. Previous studies indicate that TRP channels play a prominent role in the regulation of cardiovascular dynamics and homeostasis, but also contribute to the pathophysiology of many diseases and disorders within the cardiovascular system. However, no studies to date have identified the functional expression and/or intracellular localization of TRPA1 in primary adult mouse ventricular cardiomyocytes (CMs). Although TRPV1 has been implicated in the regulation of cardiac function, there is a paucity of information regarding functional expression and localization of TRPV1 in adult CMs. Our current studies demonstrate that TRPA1 and TRPV1 ion channels are co-expressed at the protein level in CMs and both channels are expressed throughout the endocardium, myocardium and epicardium. Moreover, immunocytochemical localization demonstrates that both channels predominantly colocalize at the Z-discs, costameres and intercalated discs. Furthermore, specific TRPA1 and TRPV1 agonists elicit dose-dependent, transient rises in intracellular free calcium concentration ([Ca2+]i) that are abolished in CMs obtained from TRPA1-/- and TRPV1-/- mice. Similarly, we observed a dose-dependent attenuation of the TRPA1 and TRPV1 agonist-induced increase in [Ca2+]i when WT CMs were pretreated with increasing concentrations of selective TRPA1 or TRPV1 channel antagonists. In summary, these findings demonstrate functional expression and the precise ultrastructural localization of TRPA1 and TRPV1 ion channels in freshly isolated mouse CMs. Crosstalk between TRPA1 and TRPV1 may be important in mediating cellular signaling events in cardiac muscle.