Dysregulated Smooth Muscle Cell BMPR2-ARRB2 Axis Causes Pulmonary Hypertension.

OBJECTIVE: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (ß-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (ß-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

Selective inhibition of physiological late Na+ current stabilizes ventricular repolarization.

The physiological role of cardiac late Na+ current ( INa) has not been well described. In this study, we tested the hypothesis that selective inhibition of physiological late INa abbreviates the normal action potential (AP) duration (APD) and counteracts the prolongation of APD and arrhythmic activities caused by inhibition of the delayed rectifier K+ current ( IKr). The effects of GS-458967 (GS967) on the physiological late INa and APs in rabbit isolated ventricular myocytes and on the monophasic APs and arrhythmias in rabbit isolated perfused hearts were determined. In ventricular myocytes, GS967 and, for comparison, tetrodotoxin concentration dependently decreased the physiological late INa with IC50 values of 0.5 and 1.9 µM, respectively, and significantly shortened the APD measured at 90% repolarization (APD90). A strong correlation between inhibition of the physiological late INa and shortening of APD by GS967 or tetrodotoxin ( R2 of 0.96 and 0.97, respectively) was observed. Pretreatment of isolated myocytes or hearts with GS967 (1 µM) significantly shortened APD90 and monophasic APD90 and prevented the prolongation and associated arrhythmias caused by the IKr inhibitor E4031 (1 µM). In conclusion, selective inhibition of physiological late INa shortens the APD, stabilizes ventricular repolarization, and decreases the proarrhythmic potential of pharmacological agents that slow ventricular repolarization. Thus, selective inhibition of late INa may constitute a generalizable approach to stabilize ventricular repolarization and suppress arrhythmogenicity associated with conditions whereby AP or QT intervals are prolonged. NEW & NOTEWORTHY The contribution of physiological late Na+ current in action potential duration (APD) of rabbit cardiac myocytes was estimated. The inhibition of this current prevented the prolongation of APD in rabbit cardiac myocytes, the prolongation of monophasic APD, and generation of arrhythmias in rabbit isolated hearts caused by delayed rectifier K+ current inhibition.

A computational model predicts adjunctive pharmacotherapy for cardiac safety via selective inhibition of the late cardiac Na current.

BACKGROUND: The QT interval is a phase of the cardiac cycle that corresponds to action potential duration (APD) including cellular repolarization (T-wave). In both clinical and experimental settings, prolongation of the QT interval of the electrocardiogram (ECG) and related proarrhythmia have been so strongly associated that a prolonged QT interval is largely accepted as surrogate marker for proarrhythmia. Accordingly, drugs that prolong the QT interval are not considered for further preclinical development resulting in removal of many promising drugs from development. While reduction of drug interactions with hERG is an important goal, there are promising means to mitigate hERG block. Here, we examine one possibility and test the hypothesis that selective inhibition of the cardiac late Na current (INaL) by the novel compound GS-458967 can suppress proarrhythmic markers. METHODS AND RESULTS: New experimental data has been used to calibrate INaL in the Soltis-Saucerman computationally based model of the rabbit ventricular action potential to study effects of GS-458967 on INaL during the rabbit ventricular AP. We have also carried out systematic in silico tests to determine if targeted block of INaL would suppress proarrhythmia markers in ventricular myocytes described by TRIaD: Triangulation, Reverse use dependence, beat-to-beat Instability of action potential duration, and temporal and spatial action potential duration Dispersion. CONCLUSIONS: Our computer modeling approach based on experimental data, yields results that suggest that selective inhibition of INaL modifies all TRIaD related parameters arising from acquired Long-QT Syndrome, and thereby reduced arrhythmia risk. This study reveals the potential for adjunctive pharmacotherapy via targeted block of INaL to mitigate proarrhythmia risk for drugs with significant but unintended off-target hERG blocking effects.

Contribution of the late sodium current to intracellular sodium and calcium overload in rabbit ventricular myocytes treated by anemone toxin.

Pathological enhancement of late Na(+) current (INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na(+) ([Na(+)]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na(+)]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22-24°C, resting [Na(+)]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na(+)]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM (P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 µM) prevented Na(+) i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na(+)]i (R(2) = 0.88) and between the rise in [Na(+)]i and the increases in cytosolic Ca(2+) (R(2) = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na(+)]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na(+)]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na(+)]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na(+) overload in ventricular myocytes.

Intracellular Na+ overload causes oxidation of CaMKII and leads to Ca2+ mishandling in isolated ventricular myocytes.

An increase of late Na(+) current (INaL) in cardiac myocytes can raise the cytosolic Na(+) concentration and is associated with activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alterations of mitochondrial metabolism and Ca(2+) handling by sarcoplasmic reticulum (SR). We tested the hypothesis that augmentation of INaL can increase mitochondrial reactive oxygen species (ROS) production and oxidation of CaMKII, resulting in spontaneous SR Ca(2+) release and increased diastolic Ca(2+) in myocytes. Increases of INaL and/or of the cytosolic Na(+) concentration led to mitochondrial ROS production and oxidation of CaMKII to cause dysregulation of Ca(2+) handling in rabbit cardiac myocytes.

Inhibition of the late sodium current slows t-tubule disruption during the progression of hypertensive heart disease in the rat.

The treatment of heart failure (HF) is challenging and morbidity and mortality are high. The goal of this study was to determine if inhibition of the late Na(+) current with ranolazine during early hypertensive heart disease might slow or stop disease progression. Spontaneously hypertensive rats (aged 7 mo) were subjected to echocardiographic study and then fed either control chow (CON) or chow containing 0.5% ranolazine (RAN) for 3 mo. Animals were then restudied, and each heart was removed for measurements of t-tubule organization and Ca(2+) transients using confocal microscopy of the intact heart. RAN halted left ventricular hypertrophy as determined from both echocardiographic and cell dimension (length but not width) measurements. RAN reduced the number of myocytes with t-tubule disruption and the proportion of myocytes with defects in intracellular Ca(2+) cycling. RAN also prevented the slowing of the rate of restitution of Ca(2+) release and the increased vulnerability to rate-induced Ca(2+) alternans. Differences between CON- and RAN-treated animals were not a result of different expression levels of voltage-dependent Ca(2+) channel 1.2, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, ryanodine receptor type 2, Na(+)/Ca(2+) exchanger-1, or voltage-gated Na(+) channel 1.5. Furthermore, myocytes with defective Ca(2+) transients in CON rats showed improved Ca(2+) cycling immediately upon acute exposure to RAN. Increased late Na(+) current likely plays a role in the progression of cardiac hypertrophy, a key pathological step in the development of HF. Early, chronic inhibition of this current slows both hypertrophy and development of ultrastructural and physiological defects associated with the progression to HF.

A novel, potent, and selective inhibitor of cardiac late sodium current suppresses experimental arrhythmias.

Inhibition of cardiac late sodium current (late I(Na)) is a strategy to suppress arrhythmias and sodium-dependent calcium overload associated with myocardial ischemia and heart failure. Current inhibitors of late I(Na) are unselective and can be proarrhythmic. This study introduces GS967 (6-[4-(trifluoromethoxy)phenyl]-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine), a potent and selective inhibitor of late I(Na), and demonstrates its effectiveness to suppress ventricular arrhythmias. The effects of GS967 on rabbit ventricular myocyte ion channel currents and action potentials were determined. Anti-arrhythmic actions of GS967 were characterized in ex vivo and in vivo rabbit models of reduced repolarization reserve and ischemia. GS967 inhibited Anemonia sulcata toxin II (ATX-II)-induced late I(Na) in ventricular myocytes and isolated hearts with IC(50) values of 0.13 and 0.21 µM, respectively. Reduction of peak I(Na) by GS967 was minimal at a holding potential of -120 mV but increased at -80 mV. GS967 did not prolong action potential duration or the QRS interval. GS967 prevented and reversed proarrhythmic effects (afterdepolarizations and torsades de pointes) of the late I(Na) enhancer ATX-II and the I(Kr) inhibitor E-4031 in isolated ventricular myocytes and hearts. GS967 significantly attenuated the proarrhythmic effects of methoxamine+clofilium and suppressed ischemia-induced arrhythmias. GS967 was more potent and effective to reduce late I(Na) and arrhythmias than either flecainide or ranolazine. Results of all studies and assays of binding and activity of GS967 at numerous receptors, transporters, and enzymes indicated that GS967 selectively inhibited late I(Na). In summary, GS967 selectively suppressed late I(Na) and prevented and/or reduced the incidence of experimentally induced arrhythmias in rabbit myocytes and hearts.

Modulation of myosin by cardiac myosin binding protein-C peptides improves cardiac contractility in ex-vivo experimental heart failure models.

Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of sarcomeric function. Reduced phosphorylation of cMyBP-C has been linked to compromised contractility in heart failure patients. Here, we used previously published cMyBP-C peptides 302A and 302S, surrogates of the regulatory phosphorylation site serine 302, as a tool to determine the effects of modulating the dephosphorylation state of cMyBP-C on cardiac contraction and relaxation in experimental heart failure (HF) models in vitro. Both peptides increased the contractility of papillary muscle fibers isolated from a mouse model expressing cMyBP-C phospho-ablation (cMyBP-CAAA) constitutively. Peptide 302A, in particular, could also improve the force redevelopment rate (ktr) in papillary muscle fibers from cMyBP-CAAA (nonphosphorylated alanines) mice. Consistent with the above findings, both peptides increased ATPase rates in myofibrils isolated from rats with myocardial infarction (MI), but not from sham rats. Furthermore, in the cMyBP-CAAA mouse model, both peptides improved ATPase hydrolysis rates. These changes were not observed in non-transgenic (NTG) mice or sham rats, indicating the specific effects of these peptides in regulating the dephosphorylation state of cMyBP-C under the pathological conditions of HF. Taken together, these studies demonstrate that modulation of cMyBP-C dephosphorylation state can be a therapeutic approach to improve myosin function, sarcomere contractility and relaxation after an adverse cardiac event. Therefore, targeting cMyBP-C could potentially improve overall cardiac performance as a complement to standard-care drugs in HF patients.

Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling.

Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension, but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of pulmonary arterial hypertension and associated right ventricular (RV) dysfunction, we deleted Bmpr1a in vascular smooth muscle cells and in cardiac myocytes in mice using the SM22alpha;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by Western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in RV systolic pressure and RV hypertrophy following 3 weeks in chronic hypoxia. However, both SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutant mice had fewer muscularized distal pulmonary arteries and attenuated loss of peripheral pulmonary arteries compared with age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by short interference RNA in cultured pulmonary arterial smooth muscle cells, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by short interference RNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RV systolic pressure and RV hypertrophy, despite the attenuated remodeling with chronic hypoxia in the flox/flox mutants versus controls, was not a function of elevated left ventricular end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially influencing vascular stiffness.

Eleclazine exhibits enhanced selectivity for long QT syndrome type 3-associated late Na+ current.

BACKGROUND: Eleclazine (GS-6615) is a sodium channel blocker designed to improve the selectivity for cardiac late Na+ current (INa) over peak INa. OBJECTIVES: The goals of this study were to investigate the inhibition of late INa by eleclazine using a sample of long QT syndrome type 3 (LQT3) and overlap LQT3/Brugada syndrome mutant channels; to compare the apparent binding rates for eleclazine with those for other class 1 antiarrhythmic agents; and to investigate the binding site. METHODS: Wild-type human cardiac voltage-gated sodium channel (hNaV1.5) and 21 previously reported variants were studied using patch clamp recordings from a heterologous expression system. RESULTS: Eleclazine inhibited anemone toxin II-enhanced late INa from wild-type hNaV1.5 with a drug concentration that causes 50% block of 0.62 ± 0.12 µM (84-fold selectivity over peak INa). The drug concentration that causes 50% block of eleclazine to inhibit the enhanced late INa from LQT3 mutant channels ranged from 0.33 to 1.7 µM. At predicted therapeutic concentrations, eleclazine and ranolazine inhibited peak INa to a similar degree as assessed with 4 overlap LQT3/Brugada syndrome mutations. Eleclazine was found to interact with hNaV1.5 significantly faster than ranolazine and 6 other class 1 antiarrhythmic agents. Engineered mutations (F1760A/Y1767A) located within the local anesthetic binding site decreased the inhibition of late INa and peak INa by eleclazine. CONCLUSION: At predicted therapeutic concentrations, eleclazine elicits potent inhibition of late INa across a cohort of NaV1.5 mutant channels. These properties are consistent with a class 1b antiarrhythmic agent that associates with unusually rapid binding/unbinding rates.

The novel late Na+ current inhibitor, GS-6615 (eleclazine) and its anti-arrhythmic effects in rabbit isolated heart preparations.

BACKGROUND AND PURPOSE: Enhanced late Na+ current (late INa ) in the myocardium is pro-arrhythmic. Inhibition of this current is a promising strategy to stabilize ventricular repolarization and suppress arrhythmias. Here, we describe GS-6615, a selective inhibitor of late INa , already in clinical development for the treatment of long QT syndrome 3 (LQT3). EXPERIMENTAL APPROACH: The effects of GS-6615 to inhibit late INa , versus other ion currents to shorten the ventricular action potential duration (APD), monophasic APD (MAPD) and QT interval, and decrease to the incidence of ventricular arrhythmias was determined in rabbit cardiac preparations. To mimic the electrical phenotype of LQT3, late INa was increased using the sea anemone toxin (ATX-II). KEY RESULTS: GS-6615 inhibited ATX-II enhanced late INa in ventricular myocytes (IC50  = 0.7 µM), shortened the ATX-II induced prolongation of APD, MAPD, QT interval, and decreased spatiotemporal dispersion of repolarization and ventricular arrhythmias. Inhibition by GS-6615 of ATX-II enhanced late INa was strongly correlated with shortening of myocyte APD and isolated heart MAPD (R2  = 0.94 and 0.98 respectively). In contrast to flecainide, GS-6615 had the minimal effects on peak INa . GS-6615 did not decrease the maximal upstroke velocity of the action potential (Vmax) nor widen QRS intervals. CONCLUSIONS AND IMPLICATIONS: GS-6615 was a selective inhibitor of late INa , stabilizes the ventricular repolarization and suppresses arrhythmias in a model of LQT3. The concentrations at which the electrophysiological effects of GS-6615 were observed are comparable to plasma levels associated with QTc shortening in patients with LQT3, indicating that these effects are clinically relevant.

Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium Current Inhibitor (Late INai), a Phase II Agent with Demonstrated Preclinical Anti-Ischemic and Antiarrhythmic Properties.

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Angiotensin II induced increase in frequency of cytosolic and nuclear calcium waves of heart cells via activation of AT1 and AT2 receptors.

The aim of this work is to verify if Angiotensin II (Ang II) affects the frequency of spontaneous cytosolic and nuclear Ca2+ waves in chick embryonic cardiomyocytes and if this effect is mediated via the activation of AT1 and/or AT2 receptors. Using the rapid scan technique of confocal microscopy, we observed that Ang II (10(-8)M) increases the frequency of cytosolic and nuclear Ca2+ waves. This effect was accompanied by a decrease in the amplitude of nuclear Ca2+ waves and an absence of effect on the amplitude of cytosolic Ca2+ waves. The effect of the octapeptide on both frequency and amplitude of the nuclear waves was prevented by the AT1 receptor antagonist L158809. However, blockade of the AT2 receptor using the antagonist PD123319 (10(-7)M) only prevented the effect of Ang II on the frequency of Ca2+ waves. Furthermore, the effect was prevented by both a PKC inhibitor (bisindolylmaleimide) and a PKC activator (phorbol 12,13-dibutyrate). In addition, the Ang II effect was not prevented by the blocker of the pacemaker current If. These results demonstrate that Ang II, via the activation of its receptors AT1 and AT2, affects the frequency of spontaneous Ca2+ waves and this effect seems to be mediated by the PKC pathway.

Patchy deletion of Bmpr1a potentiates proximal pulmonary artery remodeling in mice exposed to chronic hypoxia.

Reduced vascular expression of bone morphogenetic protein type IA receptor (Bmpr1a) has been found in patients with pulmonary arterial hypertension. Our previous studies in mice with patchy deletion of Bmpr1a in vascular smooth muscle cells and cardiac myocytes showed decreased distal vascular remodeling despite a similar severity of hypoxic pulmonary hypertension (HPH). We speculate increased stiffness from ectopic deposition of collagen in proximal pulmonary arteries might account for HPH. Pulsatile pressure-flow relationships were measured in isolated, ventilated, perfused lungs of SM22α;TRE-Cre;R26R;Bmpr1a(flox/flox) (KO) mice and wild-type littermates, following 21 days (hypoxia) and 0 days (control) of chronic hypoxia. Pulmonary vascular impedance, which yields insight into proximal and distal arterial remodeling, was calculated. Reduced Bmpr1a expression had no effect on input impedance Z(0) (P = 0.52) or characteristic impedance Z(C) (P = 0.18) under control conditions; it also had no effect on the decrease in Z(0) via acute rho kinase inhibition. However, following chronic hypoxia, reduced Bmpr1a expression increased Z(C) (P < 0.001) without affecting Z(0) (P = 0.72). These results demonstrate that Bmpr1a deficiency does not significantly alter the hemodynamic function of the distal vasculature or its response to chronic hypoxia but larger, more proximal arteries are affected. In particular, reduced Bmpr1a expression likely decreased dilatation and increased stiffening in response to hypoxia, probably by collagen accumulation. Increased PA stiffness can have a significant impact on right ventricular function. This study illustrates for the first time how proximal pulmonary artery changes in the absence of distal pulmonary artery changes contribute to pulmonary arterial hypertension.

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension.

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Block of Na+ currents and suppression of action potentials in embryonic rat dorsal root ganglion neurons by ranolazine.

Ranolazine, an anti-anginal drug, reduces neuropathic and inflammatory-induced allodynia in rats. However, the mechanism of ranolazin's anti-allodynic effect is not known. We hypothesized that ranolazine would reduce dorsal root ganglion (DRG) Na(+) current (I(Na)) and neuronal firing by stabilizing Na(+) channels in inactivated states to cause voltage- and frequency-dependent block. Therefore, we investigated the effects of ranolazine on tetrodotoxin-sensitive (TTXs) and tetrodotoxin-resistant (TTXr) I(Na) and action potential parameters of small diameter DRG neurons from embryonic rats. Ranolazine (10 and 30 µM) significantly reduced the firing frequency of evoked action potentials in DRG neurons from 19.2 ± 1.4 to 9.8 ± 2.7 (10 µM) and 5.7 ± 1.3 (30 µM) Hz at a resting membrane potential of -40 mV. Ranolazine blocked TTXs and TTXr in a voltage- and frequency-dependent manner. Furthermore, ranolazine (10 µM) blocked hNa(v)1.3 (expressed in HEK293 cells) and caused a hyperpolarizing shift in the voltage dependence of steady-state intermediate and slow inactivation Na(v)1.3 current. Taken together, the results suggest that ranolazine suppresses the hyperexcitability of DRG neurons by interacting with the inactivated states of Na(+) channels and these actions may contribute to its anti-allodynic effect in animal models of neuropathic pain.

Ranolazine block of human Na v 1.4 sodium channels and paramyotonia congenita mutants.

The antianginal drug ranolazine exerts voltage- and use-dependent block (UDB) of several Na+ channel isoforms, including Na(v) 1.4. We hypothesized that ranolazine will similarly inhibit the paramyotonia congenita Na(v) 1.4 gain-of-function mutations, R1448C, R1448H, and R1448P that are associated with repetitive action potential firing. Whole-cell Na+ current (I(Na)) was recorded from HEK293 cells expressing the hNa(v) 1.4 WT or R1448 mutations. At a holding potential (HP) of -140 mV, ranolazine exerted UDB (10 Hz) of WT and R1448 mutations (IC 50 = 59 - 71 µM). The potency for ranolazine UDB increased when the frequency of stimulation was raised to 30 Hz (IC 50 = 20 - 27 uM). When the HP was changed to -70 mV to mimic the resting potential of an injured skeletal muscle fibre, the potency of ranolazine to block I(Na) further increased; values of ranolazine IC 50 for block of WT, R1448C, R1448H, and R1448P were 3.8, 0.9, 6.3, and 0.9 uM, respectively. Ranolazine (30 uM) also caused a hyperpolarizing shift in the voltage-dependence of inactivation of WT and R1448 mutations. The effects of ranolazine (30 uM) to reduce I(Na) were similar (~35% I(Na) inhibition) when different conditioning pulse durations (2-20 msec) were used. Ranolazine (10 µM) suppressed the abnormal I(Na) induced by slow voltage ramps for R1448C channels. In computer simulations, 3 µM ranolazine inhibited the sustained and excessive firing of skeletal muscle action potentials that are characteristic of myotonia. Taken together, the data indicate that ranolazine interacts with the open state and stabilizes the inactivated state(s) of Na(v)1.4 channels, causes voltage- and use-dependent block of I(Na) and suppresses persistent I(Na). These data further suggest that ranolazine might be useful to reduce the sustained action potential firing seen in paramyotonia congenita.

Use-dependent block of cardiac late Na(+) current by ranolazine.

BACKGROUND: Ranolazine is an antianginal drug that inhibits the cardiac late Na+ current (INa). The selectivity of ranolazine to block late INa relative to peak INa at rapid heart rates has not been determined, but is potentially important to drug efficacy and safety. OBJECTIVE: This study sought to quantify use-dependent block (UDB) of cardiac peak and late INa by ranolazine. METHODS: Wild-type (WT) and long QT3 mutation R1623Q channels were expressed in HEK293 cells and studied using whole-cell patch-clamp technique. RESULTS: Ranolazine (1 to 300 microM caused tonic (0.1 Hz) and UDB (1, 2, and 5 Hz) of WT and R1623Q peak INa. The IC50 values for block of WT and R1623Q peak INa at 0.1, 1, 2, and 5 Hz were 430, 260, 157, and 154 microM, and 95, 77, 37, and 25 microM, respectively. The IC50 values for block of R1623Q late INa at 0.1, 1, 2, and 5 Hz were 7.5, 7.3, 2.2, and 1.9 microM, respectively. Ranolazine (10 microM) caused a hyperpolarizing shift of WT and R1623Q peak INa steady-state inactivation without affecting steady-state activation, suggesting that ranolazine interacts with inactivated states of the channels. Ranolazine (30 microM) significantly slowed the recovery from inactivation of peak INa of both WT and R1623Q and late INa of R1623Q. CONCLUSION: Ranolazine slowed recovery of late INa from inactivation and thus caused UDB of late INa. These data suggest that the effect of ranolazine to block late INa may be increased and the selectivity to block late INa relative to peak INa may be retained during tachycardia.

SM22alpha-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis.

Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain.

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.