Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats.

Objective: In spontaneously hypertensive rats (SHR) atrial remodeling has been shown to involve increase in endothelin (ET) signaling. Furthermore, inflammatory processes may further contribute to tissue remodeling. The aimed of this study was to investigate whether an endothelin receptor antagonist, macitentan, could reduce left atrial (LA) remodeling in arterial hypertension. Methods: Molecular characterization of atria was performed in SHR at the age of 8 months and their age-matched normotensive control rats (WKY). SHR were treated with macitentan and, for comparison with a blood pressure reducing drug, with doxazosin. After two months of treatment, molecules involved in endocardial inflammation and atrial calcium handling were assessed. The molecular changes provoked by rapid-pacing (RAP) were analyzed in atrial tissue slices. Results: Doxazosin reduced the systolic blood pressure compared with the untreated SHR (159 ± 26 vs. 176 ± 17; P < 0.05) or macitentan (vs. 189 ± 21; P < 0.05). Macitentan lowered the increased levels of atrial ET-1 and abrogated the pacing-induced upregulation of preproET-1-mRNA in atrial slices from SHR. Macitentan reduced the elevated levels of atrial 8-isoprostanes, the increased expression of pro-inflammatory ICAM-1 and IL-8, the phosphorylation of MAP kinases, ERK and p38, the phosphorylation of NF-κB and the expression of VCAM-mRNA. Major Ca2+-regulating proteins and markers of hypertrophy and fibrosis, however, were not affected. Doxazosin elicited similar changes, except for the alterations in ET-1 levels, NF-κB phosphorylation and VCAM-mRNA. Conclusion: Macitentan reversed pro-inflammatory remodeling in hypertensive atria in a blood pressure-independent manner, which might prevent endocardial dysfunction and thereby, thrombogenesis in arterial hypertension.

JTV519 (K201) reduces sarcoplasmic reticulum Ca²âº leak and improves diastolic function in vitro in murine and human non-failing myocardium.

BACKGROUND AND PURPOSE: Ca²âº leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca²âº leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca²âº leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH: SR Ca²âº leak was induced by ouabain in murine cardiomyocytes. [Ca²âº]-transients, SR Ca²âº load and RyR2-mediated Ca²âº leak (sparks/waves) were quantified, with or without JTV519 (1 µmol·L⁻¹). Contribution of Ca²âº -/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser(2814) phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. KEY RESULTS: Ouabain increased systolic and diastolic cytosolic [Ca²âº](i) , SR [Ca²âº], and SR Ca²âº leak (Ca²âº spark (SparkF) and Ca²âº wave frequency), independently of CaMKII and RyR-Ser(2814) phosphorylation. JTV519 decreased SparkF but also SR Ca²âº load. At matched SR [Ca²âº], Ca²âº leak was significantly reduced by JTV519, but it had no effect on fractional Ca²âº release or Ca²âº wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. CONCLUSIONS AND IMPLICATIONS: JTV519 was effective in reducing SR Ca²âº leak by specifically regulating RyR2 opening at diastolic [Ca²âº](i) in the absence of increased RyR2 phosphorylation at Ser(2814) , extending the potential use of JTV519 to conditions of acute cellular Ca²âº overload.

Activation and propagation of Ca(2+) release during excitation-contraction coupling in atrial myocytes.

Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

Differences in the protein-kinase-A-dependent regulation of CFTR Cl- channels and Na+-K+ pumps in guinea-pig ventricular myocytes.

Protein-kinase-A- (PKA-) dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current (I(CFTR)) and Na+-K+ pump current (Ip) was studied in single guinea-pig ventricular myocytes. Both currents were measured simultaneously by means of whole-cell recording at 30 degrees C. The adenylyl cyclase activator forskolin was used to stimulate PKA activity. At -20 mV, forskolin (4 microM) induced a fast activation of I(CFTR) and a delayed stimulation of Ip. Despite the strikingly different time courses, however, the potency of the drug to regulate both currents was identical. Half-maximal activation of I(CFTR) and stimulation of Ip, respectively, were observed at 9.6 x 10(-8) M and 9.9 x 10(-8) M forskolin. Inclusion of a specific peptide inhibitor of PKA in the pipette solution (PKI, 20 microM) blocked forskolin's effect on Ip. However, regardless of the time allowed for cell dialysis, there still was a marked, transient activation of I(CFTR), which could be prevented by: (1) a short pre-activation of I(CFTR) with forskolin or (2) the additional inclusion in the pipette solution of a synthetic peptide (Ht31 peptide, 60 microM) that interferes with PKA binding to its anchoring proteins. Thus, there is a tight functional coupling between PKA and CFTR Cl- channels in guinea-pig ventricular myocytes. The coupling is probably due to the close physical proximity of channels and kinases mediated by PKA anchoring proteins. Na+-K+ pumps, on the other hand, though also regulated by PKA, appear to be loosely coupled to the kinases.

Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+-K+ pump in guinea-pig ventricular myocytes.

1. The effects of the adenylyl cyclase activator forskolin on steady-state and transient currents generated by the Na+-K+ pump were studied in guinea-pig ventricular myocytes by means of whole-cell voltage clamp at 30 C. 2. In external solution containing 144 mM Na+ (Na+o) and 10 mM K+ (K+o), steady-state Na+-K+ pump current (Ip) activated by 5 mM pipette Na+ (Na+pip) at -20 mV was reversibly augmented by forskolin (4 microM) to 133 +/- 4 % of the control current (n = 15). The forskolin analogue 1, 9-dideoxyforskolin (10 microM), which does not activate adenylyl cyclases, did not increase Ip (n = 2). Application of the protein kinase A (PKA) inhibitor H-89 (10 microM) in the continued presence of forskolin reversed the forskolin-induced elevation of Ip (n = 3). 3. The forskolin effect on Ip persisted in the presence of 50 mM Na+pip which ensured that the internal Na+-binding sites of the Na+-K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 +/- 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0.013 nM (n = 5) and to 138 +/- 4 % of the control Ip when free [Ca2+]pip was 15 nM (n = 9). 4. In Na+-free external solution, Ip activated by 50 mM Na+pip and 1.5 mM K+o was likewise increased by forskolin but to a lesser extent than in Na+-containing medium (116 +/- 3 % of control, n = 10). 5. In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+-Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q-V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0.5) and the maximum amount of movable charge (DeltaQmax). Forskolin (2-10 microM) shifted V0.5 to more negative values while DeltaQmax was not affected (n = 11). The effects of forskolin on transient pump currents were mimicked by 8-bromo-cAMP (500 microM; n = 2) and abolished by a peptide inhibitor of PKA (PKI, 10 microM; n = 5). 6. We conclude that activation of the cAMP-PKA pathway in guinea-pig ventricular myocytes increases Na+-K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+-K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart.

Depolarization increases the apparent affinity of the Na+-K+ pump to cytoplasmic Na+ in isolated guinea-pig ventricular myocytes.

1. In order to investigate the possible effect of membrane potential on cytoplasmic Na+ binding to the Na+-K+ pump, we studied Na+-K+ pump current-voltage relationships in single guinea-pig ventricular myocytes whole-cell voltage clamped with pipette solutions containing various concentrations of Na+ ([Na+]pip) and either tetraethylammonium (TEA+) or N-methyl-D-glucamine (NMDG+) as the main cation. The experiments were conducted at 30 C under conditions designed to abolish the known voltage dependence of other steps in the pump cycle, i.e. in Na+-free external media containing 20 mM Cs+. 2. Na+-K+ pump current (Ip) was absent in cells dialysed with Na+-free pipette solutions and was almost voltage independent at 50 mM Na+pip (potential range: -100 to +40 mV). By contrast, the activation of Ip by 0.5-5 mM Na+pip was clearly voltage sensitive and increased with depolarization, independently of the main intracellular cation species. 3. The apparent affinity of the Na+-K+ pump for cytoplasmic Na+ increased monotonically with depolarization. The [Na+]pip required for half-maximal Ip activation (K0.5 value) amounted to 5.6 mM at -100 mV and to 2.2 mM at +40 mV. 4. The results suggest that cytoplasmic Na+ binding and/or a subsequent partial reaction in the pump cycle prior to Na+ release is voltage dependent. From the voltage dependence of the K0.5 values the dielectric coefficient for intracellular Na+ binding/translocation was calculated to be approximately 0.08. The voltage-dependent mechanism might add to the activation of the cardiac Na+-K+ pump during cardiac excitation.

Focal agonist stimulation results in spatially restricted Ca2+ release and capacitative Ca2+ entry in bovine vascular endothelial cells.

1. Intracellular Ca2+ ([Ca2+]i) signals were studied with spatial resolution in bovine vascular endothelial cells using the fluorescent Ca2+ indicator fluo-3 and confocal laser scanning microscopy. Single cells were stimulated with the purinergic receptor agonist ATP resulting in an increase of [Ca2+]i due to intracellular Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores. ATP-induced Ca2+ release was quantal, i.e. submaximal concentrations mobilized only a fraction of the intracellularly stored Ca2+. 2. Focal receptor stimulation in Ca2+-free solution by pressure application of agonist-containing solution through a fine glass micropipette resulted in a spatially restricted increase in [Ca2+]i. Ca2+ release was initiated at the site of stimulation and frequently propagated some tens of micrometres into non-stimulated regions. 3. Local Ca2+ release caused activation of capacitative Ca2+ entry (CCE). CCE was initially colocalized with Ca2+ release. Following repetitive focal stimulation, however, CCE became detectable at remote sites where no Ca2+ release had been observed. In addition, the rate of Ca2+ store depletion with repetitive local activation of release in Ca2+-free solution was markedly slower than that elicited by ATP stimulation of the entire cell. 4. From these experiments it is concluded that both intracellular IP3-dependent Ca2+ release and activation of CCE are controlled locally at the subcellular level. Moreover, redistribution of intracellular Ca2+ stored within the endoplasmic reticulum efficiently counteracts local store depletion and accounts for the spatial spread of CCE activation.

High affinity regulation of cardiac Cl- and Ca2+ conductances by (13R)-spiroforskolin.

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I(Cl(cAMP))) and the atrial L-type calcium current (I(Ca,L)) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30 degrees C and 20-22 degrees C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I(Cl(CAMP)) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC50 value) amounted to 9.6x10(-11) M and was lower than the EC50 value for forskolin (2.4x10(-8) M). (13R)-spiroforskolin evoked a smaller maximal I(Cl(cAMP)) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I(Cl(cAMP)) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I(Cl(cAMP)) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I(Ca,L) (13R)-spiroforskolin displayed a smaller activation and a lower EC50 value (5.8x10(-10) M) than forskolin (EC50 value: 3.7x10(-7) M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase.

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells.

The Na/K pump current I(p) of single HEK293 cells either untransfected (endogenous I(p)) or transfected with the alpha1 subunit of the rat Na/K pump (exogenous I(p)) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I(p) was irreversibly blocked by 10(-4) M ouabain or 2 x 10(-4) M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K(o). It was half maximally activated at 1.5 mM K(o) and increased linearly with depolarization over the entire voltage range studied (-80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant alpha1 subunit of the rat Na/K pump showed an I(p) in the presence of 10(-4) M ouabain and 2 x 10(-4) M DHO, respectively. This exogenous I(p) was reversibly blocked by 10(-2) M ouabain. Half maximal activation of the exogenous I(p) occurred at 1.7 mM K(o). Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I(p) of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I(p) in HEK293 cells is generated by the alpha1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.