Unraveling the Cardiac Effects Induced by Carvacrol in Spontaneously Hypertensive Rats: Involvement of Transient Receptor Potential Melastatin Subfamily 4 and 7 Channels.

ABSTRACT: Accumulating evidence indicates that transient receptor potential (TRP) channels are involved in the pathophysiological process in the heart, and monoterpenes, such as carvacrol, are able to modulate these channels activity. In this article, our purpose was to evaluate the direct cardiac effect of carvacrol on the contractility of cardiomyocytes and isolated right atria from spontaneously hypertensive and Wistar Kyoto rats. In this way, in vitro experiments were used to evaluate the ventricular cardiomyocytes contractility and the Ca2+ transient measuring, in addition to heart rhythm in the right atria. The role of TRPM channels in carvacrol-mediated cardiac activities was also investigated. The results demonstrated that carvacrol induced a significant reduction in ventricular cell contractility, without changes in transient Ca2+. In addition, carvacrol promoted a significant negative chronotropic response in spontaneously hypertensive and Wistar Kyoto rats' atria. Selective blockage of TRPM channels suggests the involvement of TRP melastatin subfamily 2 (TRPM2), TRPM4, and TRPM7 in the carvacrol-mediated cardiac effects. In silico studies were conducted to further investigate the putative role of TRPM4 in carvacrol-mediated cardiac action. FTMap underscores a conserved pocket in both TRPM4 and TRPM7, revealing a potential carvacrol binding site, and morphological similarity analysis demonstrated that carvacrol shares a more than 85% similarity to 9-phenanthrol. Taken together, these results suggest that carvacrol has direct cardiac actions, leading to reduced cellular contractility and inducing a negative chronotropic effect, which may be related to TRPM7 and TRPM4 modulation.

Larvicide Activity on Aedes aegypti of Essential Oil Nanoemulsion from the Protium heptaphyllum Resin.

The aim of this work was to prepare a nanoemulsion containing the essential oil of Protium heptaphyllum resin and to evaluate the larvicidal activity and the residual larvicidal effect against Aedes aegypti. The essential oil was identified by gas chromatography coupled to a mass spectrometer, and the nanoemulsions were prepared using a low-energy method and characterized by photon correlation spectroscopy. The results indicated the major constituents as p-cimene (27.70%) and α-Pinene (22.31%). Nanoemulsions had kinetic stability and a monomodal distribution in a hydrophilic-lipophilic balance of 14 with particle diameters of 115.56 ± 1.68 nn and zeta potential of -29.63 ± 3.46 mV. The nanoemulsion showed larvicidal action with LC50 = 2.91 µg∙mL-1 and residual larvicidal effect for 72 h after application to A. aegypti larvae. Consequently, the nanobiotechnological product derived from the essential oil of P. heptaphyllum resin could be used against infectious disease vectors.

Absence of suppressor of cytokine signaling 2 turns cardiomyocytes unresponsive to LIF-dependent increases in Ca2+ levels.

Little is known regarding the role of suppressor of cytokine signaling (SOCS) in the control of cytokine signaling in cardiomyocytes. We investigated the consequences of SOCS2 ablation for leukemia inhibitory factor (LIF)-induced enhancement of intracellular Ca2+ ([Ca2+]i) transient by performing experiments with cardiomyocytes from SOCS2-knockout (ko) mice. Similar levels of SOCS3 transcripts were seen in cardiomyocytes from wild-type and SOCS2-ko mice, while SOCS1 mRNA was reduced in SOCS2-ko. Immunoprecipitation experiments showed increased SOCS3 association with gp130 receptor in SOCS2-ko myocytes. Measurements of Ca2+ in wild-type myocytes exposed to LIF showed a significant increase in the magnitude of the Ca2+ transient. This change was absent in LIF-treated SOCS2-ko cells. LIF activation of ERK and STAT3 was observed in both wild-type and SOCS2-ko cells, indicating that in SOCS2-ko, LIF receptors were functional, despite the lack of effect in the Ca2+ transient. In wild-type cells, LIF-induced increase in [Ca2+]i and phospholamban Thr17 [PLN(Thr17)] phosphorylation was inhibited by KN-93, indicating a role for CaMKII in LIF-induced Ca2+ raise. LIF-induced phosphorylation of PLN(Thr17) was abrogated in SOCS2-ko myocytes. In wild-type cardiomyocytes, LIF treatment increased L-type Ca2+ current (ICa,L), a key activator of CaMKII in response to LIF. Conversely, SOCS2-ko myocytes failed to activate ICa,L in response to LIF, providing a rationale for the lack of LIF effect on Ca2+ transient. Our data show that absence of SOCS2 turns cardiomyocytes unresponsive to LIF-induced [Ca2+] raise, indicating that endogenous levels of SOCS2 are crucial for full activation of LIF signaling in the heart.

Blocking the L-type Ca2+ channel (Cav 1.2) is the key mechanism for the vascular relaxing effect of Pterodon spp. and its isolated diterpene methyl-6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate.

Pterodon spp. Vogel (Fabaceae), popularly known as "sucupira", has ethnopharmacological application which is described as having antispasmodic and relaxant effects. Hence, it was hypothesized that sucupira oil-resin (SOR) could induce smooth muscle relaxation. So, this study investigated the mechanisms involved in the vasorelaxant effect of SOR and its isolated diterpene (methyl-6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate). Vascular reactivity experiments were performed using rat aortic rings (n=5-8) with (E+) or without endothelium (E-) in an isolated bath organ. The SOR (0-56 µg/mL) relaxed phenylephrine (E+: 86.7±7.1%; E-: 92.3±4.7%) and KCl contracted rings (E-: 97.1±2.8%). In the same way, diterpene (0-48 µg/mL) also relaxed phenylephrine (E+: 94.5±3.6%; E-: 92.2±3.4%) and KCl contracted rings (E-: 99.7±0.2%). The pre-incubation of arterial rings with cyclopiazonic acid (reticular Ca2+-ATPase inhibitor), tetraethylammonium (K+ channels blocker) or MDL-12,330A (adenylyl cyclesinhibitor) did not modify either SOR- or diterpeneinduced vasorelaxation. However, ODQ (guanylyl cyclase inhibitor) impaired only diterpene-induced vasorelaxation. SOR and diterpene significantly reduced CaCl2-induced contraction stimulated by Bay K8644 (1 µM), phenylephrine (0.1 µM) or KCl solution (40 mM). Computational molecular docking studies demonstrated that the vasodilator effect of diterpene relies on blocking the Cav 1.2 channel, and patch clamp results showed that diterpene substantially decreased the ionic current through Cav 1.2 in freshly dissociated vascular smooth muscle cells. These findings suggest that SOR and its isolated diterpene induce endothelium-independent vascular relaxation by blocking the L-type Ca2+ channel (Cav 1.2).

Functional cross-talk between aldosterone and angiotensin-(1-7) in ventricular myocytes.

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca(2+)) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca(2+) transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca(2+) transient parameters, whereas aldosterone increased the magnitude of the Ca(2+) transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca(2+) transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca(2+) transient amplitude was mediated by protein kinase A, and was related to an increase in Ca(2+) current (I(Ca)) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca(2+) spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca(2+) spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca(2+) transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca(2+) signals in cardiomyocytes.

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart.

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release.

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.