Publicaciones CIC

Cardio and neuroprotective consequences of swimming training in ovariectomized rats

Mon, 11 Nov 2024 00:00:00 GMT

Articulo GeroScience; vol. 47, no. 2 Cardiovascular (CV) disease is the major cause of mortality. Estrogens (E) exert multiple CV and neuroprotective effects. During menopause CV and cognitive pathologies increase dramatically. At present, it is known that E exert many of their beneficial effects through the G protein-coupled estrogen receptor (GPER). Exercise reduces the risk of developing CV diseases. Sodium/proton exchanger (NHE-1) is overexpressed in ovariectomized (OVX) rats, probably due by the increase in reactive oxidative species (ROS). Insulin-like growth factor 1 (IGF-1), the main humoral mediator of exercise, inhibits the NHE-1. We aim to explore the subcellular mechanisms involved in the heart and brain impact of physiological exercise in OVX rats. We speculate that physical training, via IGF-1, prevents the increase in ROS; improving heart and brain physiological functions during menopause. Exercise diminished cardiac ROS production and increased catalase (CAT) activity in OVX rats. In concordance, IGF-1 treatment reduces brain ROS, surely contributing to the improvement in brain behavior. Moreover, aerobic routine was able to prevent, and IGF-1 therapy to revert, NHE-1 hyperactivity in OVX rats. Finally, our results confirm the proposed signaling pathway as: IGF-1/PI3K-AKT/NO. Surprisingly, GPER inhibitor (G36) was able to abolish IGF-1 effect, suggesting that directly or indirectly GPER is part of IGF-1 pathway. We propose that IGF-1 is the main responsible for the protective effect of aerobic training both in heart and brain in OVX rats. Moreover, we showed that not only it is possible to prevent, but also to revert the menopause-induced NHE-1 hyperactivity by exercise/IGF-1 cascade.

Cardiovascular risk markers in apparently healthy young adults: Evaluation according to optimal or non-optimal office blood pressure

Sun, 01 Dec 2024 00:00:00 GMT

Preprint Hipertensión y Riesgo Vascular Background: Blood pressure (BP) is linearly related to the incidence of cardiovascular disease from values as low as 115/75 mmHg, even at young ages. A particularly concerning issue is the decrease representation of optimal BP among children and youth. The mechanisms by which minimal elevations in BP increase cardiovascular risk are not defined. The limitations of office BP measurements could be a possible explanation since 24-hour ambulatory measurements (ABPM) better detect the risk of future cardiovascular events. Therefore, we aimed to compare healthy normotensive undergraduate students with optimal vs. non-optimal BP: ABPM, the cardiometabolic risk profile, and echocardiographic characteristics. Methods: medical students from La Plata voluntarily completed a survey to collect personal and family data on cardiovascular risk factors. Subsequently, anthropometric, BP (office and ABPM), and echocardiography determinations were recorded. Cholesterol, triglycerides, and glucose were measured in fasting blood samples. Statistical analyses were performed blinded, using SPSS software. Results: data from 135 students were analyzed (76% female, age 22.5±3.5 years). Mean office BP was 114.5±10.4 and 73.7±7.5 mmHg. Forty percent of students had non-optimal BP (61% females) showing significantly higher BP values in all ABPM periods and higher left ventricular mass index, cardiac wall thicknesses, fasting glucose, TyG index, TG/HDL-c ratio. Seven students met diagnostic criteria for nocturnal hypertension, six of whom were in the non-optimal BP group (11.1% vs 1.2%). Therefore, our study shows that apparently healthy young individuals with non-optimal BP, even if not hypertensive, exhibit differences in several cardiovascular risk markers compared to those with optimal BP.

New advances in the protective mechanism of acidic pH after ischemia: participation of NO

Thu, 01 Aug 2024 00:00:00 GMT

Preprint Archives Biochemistry and Biophysics; vol. 758 Background: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. Objective: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. Methods: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca 2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. Results: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. Conclusions: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.

Apelin/APJ signaling in IGF-1-induced acute mitochondrial and antioxidant effects in spontaneously hypertensive rat myocardium

Fri, 01 Nov 2024 00:00:00 GMT

Articulo Journal of Physiology and Biochemistry; vol. 80, no. 4 IGF-1 and apelin are released in response to exercise training with beneficial effects. Previously we demonstrated that a swimming routine is effective to convert pathological into physiological cardiac hypertrophy, and that IGF-1 improves contractility and the redox state, in spontaneously hypertensive rats (SHR). Now, we hypothesize that the apelinergic pathway is involved in the cardioprotective effects of IGF-1 in the SHR. We assessed the redox state and mitochondrial effects of IGF-1 or apelin in the presence/absence of AG1024 or ML221 [pharmacological antagonists of IGF1 (IGF1R) and apelin (APJ) receptors, respectively] in SHR isolated cardiomyocytes or perfused hearts. Acute IGF-1 (10 nmol/L) significantly: -reduced H2O2 production (IGF-1:62±6; control:100±8.1, %), -increased the activity of superoxide dismutase (IGF-1:193±17, control: 100±13,%), -prevented H2O2-induced m loss (TMREF10min/F0 min: IGF-1:0.93±0.017, control: 0.72±0.029), –reduced mitochondrial permeability transition pore (mPTP) opening estimated by the calcium retention capacity (nmol/mg protein, IGF-1:251±34, control:112±5), and -increased P-AMPK (IGF-1:129±0.9, control: 100±2%) and P-AKT (IGF-1:143±17 control:100±6, %). These effects were suppressed not only by the antagonism of IGF1R but also of APJ. Moreover, IGF-1 significantly increased APJ (IGF-1:198±29 control:100±15,%) and apelin mRNAs (IGF-1:251±48, control:100±6,%). On the other hand, an equipotent dose of exogenous apelin (50 nmol/L) emulated IGF-1 effects being cancelled by the antagonism of APJ however not by AG1024. IGF-1/IGF1R stimulates the apelinergic pathway, improving the redox balance and mitochondria status in the pathologically hypertrophied myocardium of the SHR

A Preclinical Investigation of Estrogenic Bone Protection in a Hypertensive Rat Model Under Gender-Affirming Hormone Therapy

Sun, 01 Jun 2025 00:00:00 GMT

Articulo Biology; vol. 14, no. 6 The goal of gender-affirming hormone therapy (GAHT) is to align an individual’s physical characteristics with their gender identity by suppressing endogenous sex hormones and replacing them with those consistent with their gender. Transgender women undergoing GAHT are at higher risk of cardiovascular complications, and since clinical evidence suggests that hypertension is associated with increased bone loss, we investigated the effects of estrogen treatment on bone health in a hypertensive transgender animal model. Male spontaneously hypertensive rats were orchiectomized (Orch), and half of them received estrogen treatment (Orch + Es), while a third group remained intact as controls. Bone marrow progenitor cells (BMPCs) were isolated to assess osteogenic potential, and femurs were collected for histological and mechanical analysis. BMPCs from Orch + Es rats exhibited enhanced osteogenic potential compared to those from Orch rats. Histological analysis revealed a higher number of osteocytes and fewer adipocytes in the Orch + Es group. Mechanical testing showed reduced bone strength in Orch rats, which was partially preserved in Orch + Es animals. In conclusion, estrogen administration mitigated the deleterious effects of testosterone depletion on BMPCs and provided protective effects on bone structure and strength in this preclinical model of GAHT in hypertensive rats.

Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Sat, 01 Jan 2022 00:00:00 GMT

Articulo Current Research in Physiology; vol. 5 Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca²⁺ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca²⁺ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca²⁺ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca²⁺ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca²⁺ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca²⁺ release propensity or luminal Ca²⁺ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca²⁺ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca²⁺ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.

Sodium Pump in T-Tubules of Frog Muscle Fibers

Mon, 01 Jan 1990 00:00:00 GMT

Capitulo de libro Transduction in Biological Systems Hidalgo, Cecilia; Bacigalupo, Juan; Jaimovich, Enrique; Vergara, Julio It is well known that in the cytosol of most cells the high concentration of K+ ([K +] i ) and the relatively low concentration of Na+ ([Na+] i ) are kept constant in spite of their electrochemical gradients, which promotes the loss of K+ and the gain of Na+. The steadiness of [K +], and [Na+] i , is maintained by a metabolic energy-dependent active transport process first proposed by Dean(1) in skeletal muscle and known as the Na+ pump (Dean coined the name) or more properly as the Na + /K + pump. Springer

Bay K 8644 enhances the outflow of [³H]-noradrenaline and [³H]-DOPEG from isolated rat atria

Sat, 01 Jun 1991 00:00:00 GMT

Articulo Naunyn-Schmiedeberg's Archives of Pharmacology; vol. 343, no. 6 The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3μmol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min⁻¹ (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min⁻¹ × 10³) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.

Lusitropic effects of α- and β-adrenergic stimulation in amphibian heart

Wed, 21 Dec 1994 00:00:00 GMT

Articulo Molecular and Cellular Biochemistry; vol. 141, no. 2 The effects ofl 3 and ct-adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3 • 10 .8 M isoproterenol produced a positive inotropic effect, as detected by a 92 + 24% increase in the maximal rate of contraction (+T) and a positive lusitropic effect characterized by a decrease in both the ratio +'F/-i" (23 + 5%) and the half relaxation time (tt/2) (19 + 4%). The mechanical behavior induced by the [3-agonist was associated with an increase in the intracellular cAMP levels from control values of 173 + 19 to 329 + 28 nmol/mg wet tissue. Hearts superfused with 32p in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151 + 13 to 240 + 44 pmo132p/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by [3-adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [y 32p] ATE The effect ofct-adrenergic stimulation on ventricular strips was studied at 30 and 22~ At 30~ the effects of 10 5 to 104M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22~ blockage with propranolol left a remanent positive inotropic effect (10% of the total effect ofphenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action. These propranolol-resistant effects were abolished by prazosin. Our results suggest that in amphibian heart, both the inotropic and lusitropic responses to catecholamines are mainly due to a [3-adrenergic stimulation which predominates over the ct-adrenergic response. Phospholamban phosphorylation seems not to be involved in mediating the positive lusitropic effect of [3-adrenergic agents whereas phosphorylation oftroponin I may play a critical role. (Mol Cell Biochem 141: 87-95, 1994).

Angiotensin II Activates Na+-Independent Cl−-HCO3− Exchange in Ventricular Myocardium

Mon, 09 Mar 1998 00:00:00 GMT

Articulo Circulation research; vol. 82, no. 4 The effect of angiotensin II (Ang II) on the activity of the cardiac Na+-independent Cl−-HCO3− exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pHi was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-( N -ethyl- N -isopropyl)amiloride–sensitive increase in pHi in the absence of external HCO3− (HEPES buffer), consistent with its stimulatory action on Na+-H+ exchange (NHE). This alkalinizing effect was not detected in the presence of a CO2-HCO3− buffer (pHi 7.07±0.02 and 7.08±0.02 before and after Ang II, respectively; n=17). Moreover, in Na+-free HCO3−–buffered medium, in which neither NHE nor Na+-HCO3− cotransport are acting, Ang II decreased pHi, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II–induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO3−-dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO3− buffer that were first exposed to 1 μmol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pHi from 7.05±0.05 to 7.22±0.05 ( P <.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pHi recovery after the imposition of an intracellular alkali load in a HCO3−-containing solution either with or without Ang II. The rate of myocardial pHi recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl− in a Na+-free solution. Under this condition, the rate of intracellular alkalinization increased from 0.053±0.016 to 0.108±0.026 pH unit/min (n=6, P <.05) in the presence of Ang II. This effect was canceled either by the presence of the AT1 receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C–dependent regulatory pathway linked to the AT1 receptors.

Role of a Ca²⁺-activated K⁺ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells

Mon, 18 Jan 1999 00:00:00 GMT

Articulo Pflügers Archiv; vol. 437, no. 3 Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (Vm) was -41+/-2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) Vm became significantly less negative: -13+/-2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of Vm. Depolarizing voltage steps up to +60 mV from holding potentials of -60 mV resulted in large (1-10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above -20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca²⁺-activated potassium channels (BKCa), diminished the total current recorded at +60 mV by 45+/-14% (P<0.05, n=5) and 50+/-6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BKCa channels present in HSV smooth muscle cells contribute to the maintenance of the Vm and sustain a significant portion of the total voltage-activated, outward current. Finally, BKCa channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity.

Calcium-induced calcium release in EGTA-skinned aortic strips from genetically hypertensive and normotensive rats

Sun, 01 Jan 1995 00:00:00 GMT

Articulo Japanese Heart Journal; vol. 36, no. 2 The release of 45Ca induced by Ca2+ was studied in genetically hypertensive (SHR) and normotensive (WKY) rats using EGTA-skinned aortic strips. Strips preloaded with 45Ca (pCa 6.6) were desaturated at 5mM of EGTA. A slow component of the washout in aorta from SHRs exhibited higher Ca content and a lower rate of Ca leak than that in aorta from WKY rats. This slow component was stimulated during washing with 0.03, 0.3, 1 or 10μM free Ca2+. The release of 45Ca induced by Ca2+ proceeded at similar rates in preparations of the two strains. Compared to WKY aortic strips the stimulated efflux of 45Ca was greater in SHR aortic strips due to the higher Ca content. About half of the release of 45Ca induced by 1μM free Ca2+ during the first 6 minutes of stimulation was blocked by 0.6mM of ryanodine or 50μM of ruthenium red, thus identifying the sarcoplasmic reticulum as a source of Ca release. The results suggest that this intracellular storage of Ca in aorta from genetically hypertensive rats is relevant for the generation of high levels of cytosolic Ca2+.

Effect of caffeine on K+ efflux in frog skeletal muscle

Mon, 18 Jan 1999 00:00:00 GMT

Articulo Pflugers Archiv European Journal of Physiology; vol. 437, no. 3 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (k; K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 µM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 µM tetracaine significantly reduced the increase in k; K,o (Δk; K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced Δk; K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect Δk; K,o. Fifth, tolbutamide (800 µM), an inhibitor of KATP channels, reduced Δk; K,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced Δk; K,o. Seventh, omitting Na+ from the external medium reduced Δk; K,o by about 40%. Eight, amiloride (5 mM) decreased Δk; K,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.

Antithrombotic Effects of Amaranthus hypochondriacus Proteins in Rats

Tue, 01 Mar 2016 00:00:00 GMT

Articulo Plant Foods for Human Nutrition; vol. 71, no. 1 Cardiovascular disease (CVD) is a major cause of disability and premature death throughout the world. Diets with antithrombotic components offer a convenient and effective way of preventing and reducing CVD incidence. The aim of the present work was to assess in vivo and ex vivo effects of Amaranthus hypochondriacus proteins on platelet plug formation and coagulation cascade. Amaranth proteins were orally administrated to rats (AG, 8 animals) and bleeding time was determined showing no significant difference compared with control rats (CG, 7 animals). However, results show a strong tendency, suggesting that amaranth proteins are involved in the inhibition of thrombus formation. Non-anticoagulated blood extracted from animals was analyzed with the hemostatometer, where AG parameters obtained were twice the values showed by CG. The clotting tests, thrombin time (TT) and activated partial thromboplastin time (APTT), presented a 17 and 14% clotting formation increase respectively when comparing AG with CG. The ex-vivo assays confirm the hypothesis inferring that amaranth proteins are a potential antithrombotic agent.

Retraction Note to: Compensatory role of the NBCn1 sodium/bicarbonate cotransporter on Ca2+-induced mitochondrial swelling in hypertrophic hearts

Wed, 18 Apr 2018 00:00:00 GMT

Otros Basic Research in Cardiology; vol. 113, no. 3; https://sedici.unlp.edu.ar/handle/10915/102178 The authors have retracted this article because of modifications in the control lanes of Figs. 2a and 8a of the COX1 blot obtained for 18-week-old rats (rotation, horizontal flipping and re-use of the control lanes for the 35-week-old rats blot). In light of the concerns raised, the conclusions drawn in this article cannot be relied upon. Este documento es una retractación del artículo citado en el título (ver "Documentos relacionados").

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase

Fri, 01 Dec 2017 00:00:00 GMT

Articulo Pflügers Archiv - European Journal of Physiology; vol. 469, no. 12 The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca²⁺ transients and intracellular pHᵢ were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHᵢ. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca²⁺ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca²⁺.

The relative relevance of phosphorylation of the Thr¹⁷ residue of phospholamban is different at different levels of β-adrenergic stimulation

Thu, 01 Aug 2002 00:00:00 GMT

Articulo Pflügers Archiv - European Journal of Physiology; vol. 444, no. 6 Contractility and relaxation measurements were combined with the determination of total phospholamban (PLB) phosphorylation and the immunodetection of PLB-phosphorylation sites in the intact, beating rat heart to identify the contributions of PLB phosphorylation at the Thr¹⁷ and Ser¹⁶ residues at different levels of β-adrenoceptor stimulation. Whereas with 30-300 nM isoproterenol, phosphorylation of Thr¹⁷, the Ca²⁺-calmodulin-dependent protein kinase-II (CaMKII) site and Ser¹⁶, the protein kinase A (PKA) site, contributed approximately 50% each to PLB phosphorylation, and both participated in the relaxant action of isoproterenol, at lower a level of β-adrenoceptor stimulation (isoproterenol 0.3-3 nM), both effects were exclusively due to Ser¹⁶ phosphorylation. Increasing [Ca]_o at 3 nM isoproterenol, to obtain an increase in contractility comparable to that produced by 30 nM isoproterenol, significantly increased Thr¹⁷ phosphorylation and the relaxant effect produced by 3 nM isoproterenol. An increase in Thr¹⁷ phosphorylation and in the relaxant effect of 3 nM isoproterenol was also obtained by phosphatase inhibition (okadaic acid). In this case, Ser¹⁶ phosphorylation was also increased. Moreover, perfusion with 30 nM isoproterenol in the presence of the PKA inhibitor H-89 decreased phosphorylation at both PLB residues and diminished the inotropic and relaxant responses to the β-agonist. The relative contribution of Thr¹⁷ phosphorylation to the isoproterenol-induced phosphorylation of PLB and relaxation thus increased with the level of β-adrenoceptor stimulation and the consequent increase in PKA activity. The lack of Thr¹⁷ phosphorylation at low isoproterenol concentrations might therefore be attributed to a level of PKA activity insufficient to increase [Ca]_i to activate the CaMKII system and/or to inhibit the phosphatase that dephosphorylates PLB.

Positive chronotropic effect of Bay K 8644: participation of endogenous norepinephrine

Sun, 01 Jan 1989 00:00:00 GMT

Articulo Naunyn-Schmiedeberg's Archives of Pharmacology; vol. 340, no. 4 The chronotropic effect of Bay K 8644, a dihydropyridine known to increase the slow inward current, was studied in spontaneously beating rat atria. Increases in atrial rate were concentration-dependent and the maximal increase (106 ± 10 beats/min) was obtained at 3 × 10⁻⁶ mol/l. Reserpine pretreatment, or propranolol 3 × 10⁻⁷ mol/l, or propranolol plus prazosin 10⁻⁶ mol/l decreased the maximum chronotropic effect of Bay K 8644 by about 60%. Blockade of the removal mechanisms of catecholamines (hydrocortisone 3 × 10⁻⁵ mol/l plus cocaine 10⁻⁵ mol/1) did not prevent the chronotropic effect of the compound. Exposure to Bay K 8644 increased the spontaneous outflow of tritium from atria preloaded with [3H]-norepinephrine by 30%. The results indicate that Bay K 8644 produces positive chronotropic effects through two mechanisms: a direct one and an indirect mechanism that involves the participation of norepinephrine released from sympathetic nerve endings.

Carbonic anhydrase IX and hypoxia-inducible factor 1 attenuate cardiac dysfunction after myocardial infarction

Tue, 06 Jul 2021 00:00:00 GMT

Articulo Pflügers Archiv: European Journal of Physiology; vol. 473, no. 8 Myocardial infarction (MI) is one of the leading causes of death worldwide. Prognosis and mortality rate are directly related to infarct size and post-infarction pathological heart remodeling, which can lead to heart failure. Hypoxic MI-affected areas increase the expression of hypoxia-inducible factor (HIF-1), inducing infarct size reduction and improving cardiac function. Hypoxia translocates HIF-1 to the nucleus, activating carbonic anhydrase IX (CAIX) transcription. CAIX regulates myocardial intracellular pH, critical for heart performance. Our objective was to investigate CAIX participation and relation with sodium bicarbonate transporters 1 (NBC1) and HIF-1 in cardiac remodeling after MI. We analyzed this pathway in an "in vivo" rat coronary artery ligation model and isolated cardiomyocytes maintained under hypoxia. Immunohistochemical studies revealed an increase in HIF-1 levels after 2 h of infarction. Similar results were observed in 2-h infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes with a nuclear distribution (confocal microscopy). Immunohistochemical studies showed an increase CAIX in the infarcted area at 2 h, mainly distributed throughout the cell and localized in the plasma membrane at 24 h. Similar results were observed in 2 h in infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes (confocal microscopy). NBC1 expression increased in cardiac tissue after 2 h of infarction (immunoblotting). CAIX and NBC1 interaction increases in cardiac tissue subjected to MI for 2h when CAIX is present (immunoprecipitation). These results suggest that CAIX interacts with NBC1 in our infarct model as a mechanism to prevent acidic damage in hypoxic tissue, making it a promising therapeutic target.

Dissociation between myocardial relaxation and diastolic stiffness in the stunned heart: its prevention by ischemic preconditioning

Wed, 22 Dec 1993 00:00:00 GMT

Articulo Molecular and Cellular Biochemistry; vol. 129, no. 2 The effects of myocardial stunning and ischemic preconditioning on left-ventricular developed pressure and end-diastolic pressure (diastolic stiffness) as well as on coronary-perfusion pressure were examined in isolated isovolumic rabbit hearts. The isovolumic relaxation was evaluated, and the time constant of pressure decay during the isovolumic period was calculated. Our experimental protocol comprised: 1) myocardial stunning-global ischemia (15 min) followed by reperfusion (30 min); 2) myocardial stunning-global ischemia (20 min) followed by reperfusion (30 min); and 3) ischemic preconditioning — a single cycle of brief global ischemia and reperfusion (5 min each), before a second ischemic period, of 20-min duration. There was no effect upon systolic and diastolic parameters when 15 and 20 minutes of ischemia were evaluated. In both stunned groups the left ventricular developed pressure first recovered to near control values, but then stabilized at only 60% of the control values. Whereas the isovolumic relaxation time constant was increased after 5 min of reperfusion, and return to control values at late reperfusion, the end diastolic pressure remained elevated during the entire period. Values of dP/dV calculated at common pressure levels, were used as a second index of diastolic stiffness. They were increased after stunning, as also was the coronary perfusion pressure. When the heart was preconditioned with a single episode of ischemia, the systolic and diastolic alterations were completely abolished. We thus concluded that diastolic abnormalities incurred by myocardial stunning consist in both an increase in diastolic stiffness and an early impairment of isovolumic relaxation. The increase in stiffness cannot result from incomplete relaxation since these two parameters become temporally dissociated during the reperfusion period.