Carmen Delgado

Regional distribution of hyperpolarization‐activated current (If) and hyperpolarization‐activated cyclic nucleotide‐gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts

Hyperpolarization‐activated inward current (If) and changes in the messenger RNA (mRNA) expression levels of hyperpolarization‐activated cyclic nucleotide‐gated channel (HCN)2 and HCN4 encoding If channels of the rat heart were studied in control and hypertrophied myocytes isolated from three ventricular regions: the septum (S), the left ventricular free wall (LV) and the right ventricular free wall (RV). Electrophysiological experiments were conducted by ruptured and perforated‐patch clamp techniques and quantification of mRNA levels was carried out by quantitative reverse transcriptase polymerase chain reaction. The occurrence, density and maximal specific conductance of If were found to be significantly higher in hypertrophied ventricular myocytes isolated from S and LV than in those isolated from RV or sham‐operated rats. Half‐maximal activation potential, the slope of the activation curve and the threshold for activation were similar in ventricular myocytes from sham and aortic stenosed rats in the three regions studied. Isoproterenol 1 µmol l−1 increased current size by shifting current activation to more positive potentials in both sham and hypertrophied myocytes. When we studied the mRNA levels of If channel isoforms present in the ventricle, we found a significant increase of HCN2 and HCN4 mRNA levels in hypertrophied myocytes from S and LV but not in RV. We conclude that the occurrence, density and conductance of If is higher in hypertrophied than in control ventricular myocytes, S being the region where all these changes were most evident. These findings are associated with a higher expression of HCN2 and HCN4 mRNA levels in the two regions that developed hypertrophy.

Electrophysiological effects of propafenone in untreated and propafenone‐pretreated guinea‐pig atrial and ventricular muscle fibres

1 The electrophysiological effects of propafenone (10−7 to 10−4 M) were studied on guinea‐pig isolated atrial and ventricular muscle fibres obtained from untreated animals and animals pretreated with propafenone, 3 and 10 mg kg−1, for 28 days. 2 In untreated atria propafenone produced a dose‐dependent decrease in the rate and maximum following frequency, prolonged the sinus node recovery time and reduced the maximum chronotropic responses to isoprenaline. 3 In untreated atrial and ventricular muscle fibres propafenone depressed action potential amplitude and Vmax, reduced the resting membrane potential and prolonged the action potential duration (APD) and the effective refractory period, lengthening the effective refractory period relative to APD. 4 Propafenone depressed the amplitude and Vmax and shortened the duration of the slow action potentials induced by isoprenaline and caffeine in K‐depolarized papillary muscles. 5 Pretreatment with propafenone reduced atrial rate, but did not modify the action potential characteristics compared to the values obtained in untreated atria. Further addition of propafenone produced similar but more marked changes in untreated atria. 6 In ventricular muscle fibres pretreated with 3 mg kg−1, action potential characteristics before and after further addition of propafenone were similar to those obtained in untreated fibres. However, muscles pretreated with 10 mg kg−1 exhibited a significant prolongation of the APD compared to that in untreated muscles or those pretreated with 3 mg kg−1; further addition of propafenone shortened the APD even when this parameter was of similar value to those observed in the other two series of experiments. 7 It is concluded that even though the effects of propafenone are similar to those of quinidine (class I antiarrhythmic), it also exhibited class II and class IV actions. In pretreated animals a prolongation of the APD (class III action) could also be involved in the antiarrhythmic effects of the drug.

Electrophysiological effects of platelet-activating factor (PAF-acether) in guinea-pig papillary muscles.

The effects of PAF-acether (10(-11) to 10(-7) M) were studied on the electrical and mechanical activity of guinea-pig papillary muscles. At 10(-11) M PAF-acether did not modify the amplitude and Vmax of the upstroke or the resting membrane potential. At higher concentrations PAF-acether produced a dose-dependent increase in the amplitude and Vmax of the upstroke, shortened the action potential duration and hyperpolarized the resting membrane potential. These effects were accompanied by a biphasic effect on ventricular contractile force. The shortening of the APD was inhibited in muscles pretreated with tetraethylammonium or verapamil. In papillary muscles depolarized by 27 mM K Tyrode solution PAF-acether induced slow action potentials which were blocked by verapamil. PAF-acether produced a dose-dependent increase in amplitude and Vmax of the upstroke on the slow action potentials elicited by isoproterenol, prolonged the action potential duration and hyperpolarized the resting membrane potential. These results suggest that in guinea-pig papillary muscles PAF-acether increased Ca influx via the slow inward current.

LA419, a Novel Nitric Oxide Donor, Prevents Pathological Cardiac Remodeling in Pressure-Overloaded Rats Via Endothelial Nitric Oxide Synthase Pathway Regulation

Reduced endogenous NO production has been described in cardiovascular disorders as cardiac hypertrophy and heart failure. The therapy with conventional nitrates is limited by their adverse hemodynamic effects and drug tolerance. The novel NO donor LA419 has demonstrated important antithrombotic and anti-ischemic properties without those adverse effects. The aim of this study was to evaluate the effect of LA419 chronic treatment on cardiac hypertrophy development in a progressive model of left ventricular hypertrophy. Rats were randomly divided into 6 groups: sham and clip (euthanized 7 weeks after aortic stenosis), sham+vehicle, sham+LA419, clip+vehicle, and clip+LA419 (euthanized 14 weeks after the surgery and treated with vehicle or 30 mg/kg of LA419 once left ventricular hypertrophy was established). LA419 treatment for 7 weeks induced a marked reduction in the heart:body weight ratio (4.10±0.28 and 3.38±0.06 mg/g in clip+vehicle versus clip+LA419; P<0.001) and left ventricular diameter (11.96±0.25 and 9.90±0.20 mm in clip+vehicle versus clip+LA419; P<0.001) without modifying the high blood pressure observed in stenosed rats. Histological analysis revealed that LA419 attenuated myocardial and perivascular fibrosis observed in rats with pressure overload for 14 weeks. In addition, LA419 treatment restored endothelial NO synthase and caveolin-3 expression levels, enhanced the interaction between endothelial NO synthase and its positive regulator the heat shock protein 90, and re-established the normal cardiac content of cGMP in stenosed rats. Thus, LA419 prevented the progression to maladaptative cardiac hypertrophy in response to prolonged pressure overload through a mechanism that involved the re-establishment of the endothelial NO synthase signaling pathway.

NOD1 activation induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.

Urocortin induces positive inotropic effect in rat heart.

AIMS The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways. METHODS AND RESULTS Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum. CONCLUSION Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

Slow inward current in single cells isolated from adult human ventricles

Characteristics of the slow inward current (Isi) in human ventricular myocytes isolated from septal specimens obtained in patients undergoing corrective cardiac surgery were studied using the whole-cell clamp method. A first series of experiments was performed under normal standard superfusion. Clamping from −60 mV evoked an inward current with a threshold at about −35 mV, a maximum around +10 mV and an apparent reversal potential at about +55 mV. No overlapping transient or background outward currents were detected in the −60 to +30 mV potential range, but time-dependent and steady-state outward currents were elicited at potentials above +30 mV. An overlap of steady-state activation and inactivation curves was present between −30 and +10 mV and a slight relief from inactivation was observed for voltages positive to +10mV. The time course of inactivation consisted of fast and slow phases with time constants differing by a factor of eight. Slow time constants of inactivation were shorter at potentials that elicited larger Isi, and longer at potentials inducing smaller Isi. Recovery from inactivation evolved slowly with 100% reactivation occurring in about 4000 ms. Switching the holding potential from −60 to −40 mV led to a reversible decline of Isi without any change of the decay time constants. Isi was significantly increased by 0.1 μM isoproterenol. Total or partial inhibition by inorganic (2 mM Mn2+, 3 mM Co2+, 1 mM Cd2+) and organic (1 μM methoxyverapamil, 5 μM diltiazem) calcium antagonists did not unmask any transient outward current. However, a consistent increase of Isi was reversibly observed with 3 mM 4-aminopyridine while using standard solutions. A second series of experiments carried out with K+- and Na+-free solutions did not demonstrate any significant change from data observed with standard solutions except a reduction of outward currents at steps above +30 mV and alteration of inactivation kinetics. In this experimental setting, 4-aminopyridine also increased Isi but to a lesser degree. We conclude that Isi, as compared to the outward currents, is dominant in the diseased human ventricular cells we have studied.

Effects of propafenone on calcium current in guinea-pig ventricular myocytes.

1 The modulation of L‐type voltage‐sensitive calcium channels in guinea‐pig isolated ventricular myocytes by propafenone was examined by the whole cell voltage‐clamp technique. 2 Propafenone, 10−7 −5 × 10−5 m, produced a concentration‐dependent inhibition of Ca current (ICa) without any significant change in the current‐voltage relation. Half‐blocking concentration (IC50) of propafenone for the peak ICa at +10 mV was 5 × 10−6 m. 3 The voltage‐dependence of ICa inactivation was shifted in the hyperpolarizing direction in the presence of 5 × 10−6 m propafenone. 4 A frequency‐dependent relative block by propafenone was observed after repetitive depolarizing test pulses at a frequency of 0.5 and 1 Hz. The recovery of ICa from inactivation was prolonged by propafenone and the reactivation exhibited an additional exponential component attributed to the slow release from drug block of Ca channels. 5 These results suggest that propafenone, at therapeutic concentrations exhibits Ca channel blocking properties that may be involved in its antiarrhythmic mechanism.

Effect of milrinone on contractility and 45Ca movements in the isolated rabbit aorta

The effects of milrinone on 45Ca movements and on contractile responses were studied in rabbit aortic smooth muscle. Milrinone, 10(-7)-5 x 10(-4) M, inhibited the responses induced by high K and noradrenaline (NA). These effects were observed when milrinone was added before or after the induced contractions and were similar in aortas with or without endothelium. The Ca-induced contractions of K-depolarized aortas as well as the contractions induced by caffeine in normal and in Ca-free solution were also inhibited dose dependently by milrinone. Milrinone reduced the 45Ca influx in resting or non-stimulated aortic rings as well as in aortic rings stimulated by NA. On the other hand, neither the 45Ca influx stimulated by high K or the 45Ca efflux stimulated by NA were altered by pretreatment with milrinone. It is concluded that milrinone inhibits vascular smooth muscle contractility by probably acting at multiple sites to decrease the availability of intracellular free Ca required for activation.

Effects of chlorbutol on 45 Ca movements and contractile responses of rat aorta and its relevance to the actions of Syntocinon

The effects of chlorbutol (0.7, 1.4 and 2.8 mM) on the contractile responses induced by KCl and noradrenaline (NA) and on 45Ca movements have been studied on rat isolated thoracic aorta. Chlorbutol decreased, in a dose-dependent manner, contractions induced by KCl and NA and this effect was observed whether it was added before or after the induced contractions. Preincubation with chlorbutol inhibited the contractile responses elicited by addition of Ca (1-5 mM) to Ca-free high-potassium solution. It also inhibited in a dose-dependent manner the 45Ca influx but increased 45Ca efflux in rat aortic strips. These results suggest that chlorbutol decreases peripheral resistance by reducing the availability of intracellular Ca to the contractile machinery in vascular smooth muscle cells. The effects of synthetic oxytocin (Syntocinon) at concentrations containing the same chlorbutol concentration were quantitatively similar from those produced by chlorbutol alone. Therefore, the inhibitory cardiovascular effects ascribed previously to synthetic oxytocin may be attributed to its preservative, chlorbutol, and not to oxytocin itself.