SEA0400: A Novel Sodium‐Calcium Exchange Inhibitor with Cardioprotective Properties1

The cardiac sodium‐calcium exchanger (NCX) plays an important role in calcium homeostasis. It is the primary mechanism for removing calcium ions that enter myocytes through L‐type calcium channels on a beat‐to‐beat basis. Its direction of transport is determined by the membrane potential and the ionic concentrations of Na⁺ and Ca²⁺, with the forward (or Ca²⁺‐efflux) mode of transport being the dominant mode under physiological conditions. In contrast, the Ca²⁺‐influx mode (or reverse mode) of NCX becomes important in certain pathophysiological conditions, such as myocardial ischemia and reperfusion. Recent discovery of compounds that inhibit the Ca²⁺‐influx mode (or reverse mode) of NCX has generated intense research interest in the pharmacology of NCX. Among the newer NCX inhibitors described to date, 2‐[4‐[(2,5‐difluorophenyl)methoxy]‐phenoxy]‐5‐ethoxyaniline (SEA0400) appears particularly promising in attenuating cardiac, renal, and cerebral ischemia/reperfusion injuries in various experimental models. Moreover, the mixed results that have emerged from clinical trials evaluating the efficacy and safety of inhibitors of the sodium‐hydrogen exchanger (an upstream target in relation to the sodium‐calcium exchanger) in myocardial protection stimulated interest in evaluating NCX as an alternative therapeutic target. This article reviews the pharmacological profile of SEA0400, as presented in the published literature, and discusses the therapeutic potential of this compound in attenuating myocardial ischemia/reperfusion injury.     

Efonidipine Hydrochloride: A Dual Blocker of L‐ and T‐Type Ca2+ Channels

T‐type Ca²⁺ channels have properties different from those of the L‐type and are involved in cardiac pacemaking and regulation of blood flow, but not in myocardial contraction. Efonidipine is an antihypertensive and antianginal drug with dihydropyridine structure that was recently found to block both L‐ and T‐type Ca²⁺ channels. In isolated myocardial and vascular preparations, efonidipine has potent negative chronotropic and vasodilator effects but only a weak negative inotropic effect. In experimental animals and patients, reduction of blood pressure by the drug was accompanied by no or minimum reflex tachycardia leading to improvement of myocardial oxygen balance and maintenance of cardiac output. Efonidipine increased glomerular filtration rate without increasing intraglomerular pressure. By relaxing both the afferent and efferent arterioles, efonidipine markedly reduced proteinuria. Thus, efonidipine, an L‐ and T‐type dual Ca²⁺ channel blocker, appears to have an ideal profile as an antihypertensive and antianginal drug with organ‐protective effects in the heart and kidney.     

A functional interaction between TRPC/NCKX induced by DAG plays a role in determining calcium influx independently from PKC activation

Ca²⁺influx might occur through K⁺-dependent Na⁺/Ca²⁺ exchanger operating in reverse mode (rNCKX). In a cellular model different from platelets, an interaction between canonical transient receptor potential cation (TRPC) channels and NCX has been found. The aim of this study was to verify whether the TRPC/NCKX interaction operates in human platelets. Our results showed that the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced rNCKX-mediated Ca²⁺ influx through TRPC-mediated Na⁺ influx. DAG-induced activation of TRPC/NCKX occurs independently of protein kinase C (PKC) activation, as PKC inhibitor did not modify OAG-mediated Ca²⁺ influx. Moreover, as both rNCKX and TRPC inhibitors reduced OAG-induced platelet aggregation which, conversely, was increased by flufenamic acid, known to develop TRPC activity, it could be suggested that the TRPC/NCKX interaction has a role in OAG-dependent platelet aggregation.     

AH‐1058: A Novel Cardioselective Ca2+ Channel Blocker

The pharmacologic profile of a cyproheptadine‐related compound, 4‐(5H‐dibenzo[a,d]cyclohepten‐5‐ylidene)‐1‐[(E)‐3‐(3‐methoxy‐2‐nitro)phenyl‐2‐propenyl]piperidine hydrochloride (AH‐1058), was assessed in various in vivo and in vitro models. In guinea pig cardiomyocytes, AH‐1058 effectively suppressed L‐type Ca²⁺ channel currents without affecting other ion channel or ion exchange currents. In rat cerebral cortical membranes AH‐1058 appears to bind preferentially to L‐type Ca²⁺ channels at phenylalkylamine‐ and benzothiazepine‐binding sites. In canine isolated, blood‐perfused heart preparations, AH‐1058 exerted negative inotropic, dromotropic, and chronotropic and weak coronary vasodilator effects. In halothane‐anesthetized dogs, AH‐1058 suppressed ventricular contractility and decreased blood pressure and cardiac output. Total peripheral vascular resistance was hardly affected by the drug, suggesting that in vivo AH‐1058 can selectively suppress cardiac, as compared to peripheral vascular, function. In conscious dogs, by intravenous administration AH‐1058 reduced systolic blood pressure and maximal upstroke velocity of the left ventricular pressure, while it increased heart rate in a dose‐dependent manner. The drug did not affect diastolic blood pressure, which is quite different from cardiovascular properties of well‐known Ca²⁺ channel blockers, verapamil and diltiazem. This unique cardiovascular profile of AH‐1058 is expected to be useful in the treatment of certain pathological processes such as the obstructive hypertrophic cardiomyopathy, vasovagal syncope, dissecting aortic aneurysm, and ventricular arrhythmias, in which selective inhibition of the ventricular Ca²⁺ channels is essential for drug therapy.     

Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Regulation of ion-transport in the Na⁺/Ca²⁺ exchanger (NCX) occurs via its cytoplasmic Ca²⁺-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca²⁺-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca²⁺-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca²⁺. Striking differences in the electrostatic potential between the Ca²⁺-bound and -free forms strongly suggest that Ca²⁺-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C₂-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca²⁺-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca²⁺ is influenced by the closely located cassette exons. We further propose that Ca²⁺-binding to CBD2 induces a second electrostatic switch, required to alleviate Na⁺-dependent inactivation of Na⁺/Ca²⁺ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.     

Induced Overexpression of Na+/Ca2+ Exchanger Does Not Aggravate Myocardial Dysfunction Induced by Transverse Aortic Constriction

BackgroundAlterations in expression and activity of cardiac Na⁺/Ca²⁺ exchanger (NCX1) have been implicated in the pathogenesis of heart failure.Methods and ResultsUsing transgenic mice in which expression of rat NCX1 was induced at 5 weeks of age, we performed transverse aortic constriction (TAC) at 8 weeks and examined cardiac and myocyte function at 15–18 weeks after TAC (age 23–26 weeks). TAC induced left ventricular (LV) and myocyte hypertrophy and increased myocardial fibrosis in both wild-type (WT) and NCX1-overexpressed mice. NCX1 and phosphorylated ryanodine receptor expression was increased by TAC, whereas sarco(endo)plasmic reticulum Ca²⁺-ATPase levels were decreased by TAC. Action potential duration was prolonged by TAC, but to a greater extent in NCX1 myocytes. Na⁺/Ca²⁺ exchange current was similar between WT-TAC and WT-sham myocytes, but was higher in NCX1-TAC myocytes. Both myocyte contraction and [Ca²⁺]_i transient amplitudes were reduced in WT-TAC myocytes, but restored to WT-sham levels in NCX1-TAC myocytes. Despite improvement in single myocyte contractility and Ca²⁺ dynamics, induced NCX1 overexpression in TAC animals did not ameliorate LV hypertrophy, increase ejection fraction, or enhance inotropic (maximal first derivative of LV pressure rise, +dP/dt) responses to isoproterenol.ConclusionsIn pressure-overload hypertrophy, induced overexpression of NCX1 corrected myocyte contractile and [Ca²⁺]_i transient abnormalities but did not aggravate or improve myocardial dysfunction.     

Reactive oxygen species directly modify sodium–calcium exchanger activity in a splice variant-dependent manner

The sodium–calcium exchanger isoform 1 (NCX1) operating in calcium-efflux mode plays an important role in maintaining calcium homeostasis in the heart. Paradoxically, activity of NCX1 in calcium-influx mode contributes to the pathological intracellular calcium overload during cardiac ischemia–reperfusion injury. Reactive oxygen species (ROS) also contribute to myocardial dysfunction in ischemia–reperfusion and are reported to alter NCX1 activity. However, the molecular mechanism(s) by which ROS modifies NCX1 activity have not been elucidated. Therefore, the effects of the ROS, H₂O₂, on recombinant NCX1 splice variants were studied using the patch-clamp technique. H₂O₂ irreversibly increased calcium-influx mode activity in the cardiac NCX1.1 splice variant, without affecting calcium-efflux mode activity. In direct contrast, H₂O₂ inhibited the calcium-influx mode of the vascular NCX1.3 splice variant indicating that these disparate effects of H₂O₂ may be dependent on the exon complement of the alternative splicing region. Using NCX1 splice variants with various exon compositions, the mutually exclusive exons A and B were found to bestow the differential effects of H₂O₂ on NCX1 function. As NCX1 inhibition is a potential therapeutic strategy for ischemia–reperfusion injury, the effects of the NCX1 inhibitor KB-R7943 were examined. KB-R7943 was ~ 7-fold less potent at inhibiting NCX1 activity after H₂O₂ modification. In summary, this study provides insights into the molecular regulation of NCX1 by ROS and indicates that ROS may elicit differential effects in various tissues depending on the exon composition of the splice variant expressed. These results also highlight that the potency of NCX1 inhibitors may be impaired under conditions of oxidative stress.     

Changes in activity and regulation of the cardiac Ca2+ channel (L-type) by protein kinase C in chronic alcohol-exposed rats

Background: It has been reported recently that long‐term alcohol exposure in rats increases the number of dihydropyridine binding sites in cardiac membrane preparations. We fed Sprague Dawley® rats a liquid diet that contained ethanol as 36% of total calories for 4 to 6 months and studied how alcohol exposure affected the activity and regulation of the cardiac Ca²⁺ channel. Methods: Dihydropyridine‐sensitive cardiac Ca²⁺ channel activity was measured as the rate of Mn²⁺ quench of the cytosolic fura‐2 signal in electrically stimulated myocytes. Results: In control rat myocytes, pretreatment with phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), reduced the rate of Mn²⁺ quench to 68% of the untreated cell response. Pretreatment with GF109203X, a protein kinase C inhibitor, enhanced the rate of influx by 56%, whereas Gö6976, an inhibitor of PKC α, β, and γ, did not affect the rate of influx. By contrast, PMA did not affect the rate of Mn²⁺ quench in alcoholic myocytes; however, the PKC inhibitor GF109203X still enhanced the rate of Mn²⁺ quench by 33%. Similar to control myocytes, no effect was observed after pretreatment with Gö6976 in the alcoholic cells. In both Western blot and immunoprecipitation experiments, PKC ? expression in alcohol‐exposed myocytes was reduced to 68% of the control. However, the ratio of membrane/cytosolic distribution of PKC ? in alcoholic myocytes was increased from 1.6 to 2.6. No change was detected in the expression of PKC α and PKC δ. PKC activity, measured in the presence of Gö6976, which inhibits PKC α, β, and γ, was reduced in alcoholic myocytes to 57% of the control, but the proportion of PKC activity in the particulate fraction was increased from 26% in the control myocytes to 36% in the alcoholic myocytes. Conclusions: Altered expression and activity of PKC may be associated with changes in the regulation of the cardiac Ca²⁺ channel found in the hearts of rats chronically exposed to alcohol. Specifically, we found that the novel class of PKC isozymes is responsible for regulating the cardiac Ca²⁺ channel in control cardiomyocytes, and that the loss of PMA modulation found in the alcoholic cells may be due, in part, to reduced expression and altered distribution of PKC ?.     

Hepatic stellate cells express Ca2+ pump-ATPase and Ca2+-Mg2+-ATPase in plasma membrane of caveolae

Intracytoplasmic free calcium ions (Ca²⁺) are maintained at a very low concentration in mammalian tissue by the extrusion of Ca²⁺ across a steep extracellular Ca²⁺ gradient, mainly through the activity of plasma membrane Ca²⁺ pump-ATPase. The present study aimed to identify, by electron cytochemical and electron immunogold methods, the ultrastructural localizations of two types of plasma membrane Ca²⁺-ATPase; Ca²⁺-Mg²⁺-ATPase and Ca²⁺ pump-ATPase, in hepatic stellate cells. Liver tissues and isolated hepatic stellate cells (HSCs) were studied. The ultrastructural localization of Ca²⁺-Mg²⁺-ATPase activity was examined by the electron cytochemical method of Ando. The localization of Ca²⁺ pump-ATPase was identified by immunofluorescence. The ultrastructural localization of Ca²⁺ pump-ATPase was identified by the electron immunogold method. The cytochemical reaction products of Ca²⁺-Mg²⁺-ATPase activity were localized on the outer (cavity) side of the plasma membrane of caveolae. Immunofluorescence of Ca²⁺ pump-ATPase was seen as small dots along the cell edge in HSCs. Immunogold particles indicating the presence of Ca²⁺ pump-ATPase were identified on the inner (cytoplasmic) side of the plasma membrane of caveolae. We localized Ca²⁺ pump-ATPase on the inner side of the plasma membrane caveolae and Ca²⁺-Mg²⁺-ATPase on the outer leaflet of the caveolar plasma membrane in stellate cells, suggesting that Ca²⁺ pump-ATPase may play a key role in the Ca²⁺ reflux. Received: March 7, 2000 / Accepted: July 7, 2000     

Prevention of Ventricular Arrhythmia and Calcium Dysregulation in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin‐2 Mutation

Arrhythmia Prevention in CPVT . Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium‐regulation proteins cardiac ryanodine receptor (RyR2) or calsequestrin‐2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca²⁺ overload and increased Ca²⁺ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT. Method and results: CPVT mouse models that lack CASQ2 were treated with Ca²⁺‐channel inhibitors, β‐adrenergic inhibitors, or Mg²⁺. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca²⁺ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress‐induced ventricular arrhythmia in mutant mice. The Ca²⁺ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca²⁺ content in mutant myocytes, diminished diastolic Ca²⁺ overload, increased systolic Ca²⁺ amplitude, and prevented Ca²⁺ oscillations in stressed mutant myocytes. Conclusions: Ca²⁺ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil‐induced partial normalization of SR Ca²⁺ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca²⁺ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT . (J Cardiovasc Electrophysiol, Vol. 22, pp. 316‐324, March 2011)     

Blockade of the Na+-Ca2+ exchanger is more efficient than blockade of the Na+-H+ exchanger for protection of the myocardium from lethal reperfusion injury

Since the Na⁺-H⁺ exchanger (NHE) is not the only pathway of Na⁺ influx into cardiomyocytes during ischemia/reperfusion, we hypothesized that blockade of Na⁺-Ca²⁺ exchanger (NCX) may be a more efficient strategy than is NHE inhibition for protecting the myocardium from infarction. To test this hypothesis, we compared KB-R7943 (KBR), a novel selective NCX blocker, with cariporide, a selective NHE blocker, with regard to their protective effects against infarction. In isolated rabbit hearts, infarction was induced by 30-min global ischemia/2-h reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Hearts received no drugs, or were infused with cariporide (1 M) for 10 min or KBR (0.3 or 10 M) for 5 min before ischemia or after the onset of reperfusion. Protein level of NCX was assessed by Western blotting. Cariporide infusion before ischemia significantly reduced %IS/AR from 63.9 ± 2.9% to 20.2 ± 3.0%, but its infusion upon reperfusion failed to achieve a significant protection (%IS/AR = 53.8 ± 4.6%). In contrast, KBR infusion similarly reduced infarct size both when infused before ischemia (%IS/AR = 33.3 ± 6.3% and 21.9 ± 4.7% by 0.3 and 10 M KBR, respectively) and when infused for only 5 min after reperfusion (%IS/AR = 35.3 ± 7.1% and 31.5 ± 2.1% by 0.3 and 10 M KBR, respectively). Protein levels of NCX after 30-min ischemia and 30-min ischemia/30-min reperfusion were similar to baseline values in both untreated controls and hearts treated with 0.3 M KBR upon reperfusion. These results suggest that lethal reperfusion injury is more efficiently suppressed by blockade of the NCX than by blockade of the NHE.     

Activation of the cardiac Na-Ca exchanger by sorcin via the interaction of the respective Ca-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca²⁺ binding. The sarcolemmal Na⁺/Ca²⁺ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca²⁺ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca²⁺ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca²⁺, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca²⁺-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.     

Clinical Potential of Lomerizine,a Ca2+ Channel Blocker as an Anti‐Glaucoma Drug: Effects on Ocular Circulation and Retinal Neuronal Damage

Glaucoma is defined as an optic neuropathy with characteristic changes in the optic nerve head and ultimate loss of visual field. Previous studies have suggested that (a) mechanical damage due to raised intraocular pressure and (b) a compromised tissue circulation in the optic nerve head play significant roles in the development of glaucomatous damage in the optic nerve head. Recently, we found that lomerizine, a new Ca²⁺ channel blocker, increased ocular circulation and protected neuronal cells against retinal neurotoxicity both in vitro and in vivo with minimal cardiovascular side effects. We examined the effect of lomerizine on the ocular circulation and compared it with those of other Ca²⁺ channel blockers in normal rabbits and in rabbits with an endothelin‐1‐disturbed circulation in the optic nerve head. In anesthetized rabbits, lomerizine and the other Ca²⁺ channel blockers increased the ocular circulation and also inhibited the hypoperfusion induced in optic nerve head tissue by an intravitreous injection of endothelin‐1. Whereas the other Ca²⁺ channel blockers produced changes in blood pressure and heart rate, the effects of lomerizine on these parameters were slight. In healthy humans, lomerizine increased blood velocity in the optic nerve head, without significantly altering blood pressure or heart rate. Moreover, lomerizine reduced retinal damage in rats both in vitro and in vivo, presumably through a Ca²⁺ channel blocking effect via an action that may involve a direct protection of retinal neurons as well as an improvement in the ocular circulation. These results indicate that lomerizine may be useful as a therapeutic drug against ischemic retinal diseases (such as glaucoma and retinal vascular occlusive diseases) that involve a disturbance of the ocular circulation.     

A Review of HNS‐32: A Novel Azulene‐l‐Carboxamidine Derivative with Multiple Cardiovascular Protective Actions

HNS‐32 [N¹,N¹‐dimethyl‐N²‐(2‐pyridylmethyl)‐5‐isopropyl‐3,8‐dimethylazulene‐1‐carboxamidine] (CAS Registry Number: 186086‐10‐2) is a newly synthesized azulene derivative. Computer simulation showed that its three dimensional structure is similar to that of the class Ib antiarrhythmic drugs, e.g., lidocaine or mexiletine. HNS‐32 potently suppressed ventricular arrhythmias induced by ischemia due to coronary ligation and/or ischemia‐reperfusion in dogs and rats. In the isolated dog and guinea pig cardiac tissues, HNS‐32 had negative inotropic and chronotropic actions, prolonged atrial‐His and His‐ventricular conduction time and increased coronary blood flow. In the isolated guinea pig ventricular papillary muscle, HNS‐32 decreased maximal rate of action potential upstroke (V?_max) and shortened action potential duration (APD). These findings suggest that HNS‐32 inhibits inward Na⁺ and Ca²⁺ channel currents. In the isolated pig coronary and rabbit conduit arteries, HNS‐32 inhibited both Ca²⁺ channel‐dependent and ‐independent contractions induced by a wide variety of chemical stimuli. HNS‐32 is a potent inhibitor of protein kinase C (PKC)‐mediated constriction of cerebral arteries. It is likely to block both, Na⁺ and Ca²⁺ channels expressed in cardiac and vascular smooth muscles. These multiple ion channel blocking effects are largely responsible for the antiarrhythmic and vasorelaxant actions of HNS‐32. This drug may represent a novel approach to the treatment of arrhythmias.     

The signalling pathway of CaMKII-mediated apoptosis and necrosis in the ischemia/reperfusion injury

Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) plays an important role mediating apoptosis/necrosis during ischemia-reperfusion (IR). We explored the mechanisms of this deleterious effect. Langendorff perfused rat and transgenic mice hearts with CaMKII inhibition targeted to sarcoplasmic reticulum (SR-AIP) were subjected to global IR. The onset of reperfusion increased the phosphorylation of Thr¹⁷ site of phospholamban, without changes in total protein, consistent with an increase in CaMKII activity. Instead, there was a proportional decrease in the phosphorylation of Ser2815 site of ryanodine receptors (RyR2) and the amount of RyR2 at the onset of reperfusion, i.e. the ratio Ser2815/RyR2 did not change. Inhibition of the reverse Na⁺/Ca²⁺exchanger (NCX) mode (KBR7943) diminished phospholamban phosphorylation, reduced apoptosis/necrosis and enhanced mechanical recovery. CaMKII-inhibition (KN-93), significantly decreased phospholamban phosphorylation, infarct area, lactate dehydrogenase release (LDH) (necrosis), TUNEL positive nuclei, caspase-3 activity, Bax/Bcl-2 ratio and Ca²⁺-induced mitochondrial swelling (apoptosis), and increased contractile recovery when compared with non-treated IR hearts or IR hearts pretreated with the inactive analog, KN-92. Blocking SR Ca²⁺ loading and release (thapsigargin/dantrolene), mitochondrial Ca²⁺ uniporter (ruthenium red/RU360), or mitochondrial permeability transition pore (cyclosporine A), significantly decreased infarct size, LDH release and apoptosis. SR-AIP hearts failed to show an increase in the phosphorylation of Thr¹⁷ of phospholamban at the onset of reflow and exhibited a significant decrease in infarct size, apoptosis and necrosis respect to controls. The results reveal an apoptotic-necrotic pathway mediated by CaMKII-dependent phosphorylations at the SR, which involves the reverse NCX mode and the mitochondria as trigger and end effectors, respectively, of the cascade.     

Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes

The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

ATP-Dependent Calcium Uptake in Myocardial Sarcoplasmic Reticulum from Spontaneously Hypertensive Rats: Effect of Modification of Dietary Protein

Membrane fractions enriched in sarcoplasmic reticulum (SR) were isolated from the cardiac ventricles of 10-month-old, stroke-prone spontaneously hypertensive rats (SHRSP) which had been maintained for nine months on one of four experimental diets: low protein (LP) (19% protein), standard (STD) (24% protein), high protein (HP) (32% protein), or high methionine (1.9% methionine) (MET). ATPase activities, as well as ATP-dependent Ca²⁺ binding and Ca²⁺ uptake activities, of the isolated SR were determined to examine the influence of diet on myocardial Ca²⁺ -pump activity. SR from all four groups exhibited similar Mg²⁺ ATPase activity. However, the (Ca²⁺ Mg²⁺)-ATPase activity was significantly elevated in SR from rats on the MET diet while the activity in the other groups showed no significant differences. After 15 sec of incubation. Ca²⁺ -uptake (presence of oxalate) in SR from the LP group was significantly less than Ca²⁺ -uptake in SR from each of the three other diet groups. Ca²⁺ binding (absence of oxalate) in the SR from the LP group was also significantly less than that from each of the three other diet groups. Kinetic analysis of SR Ca²⁺ -uptake over 60 sec revealed that the B_max of the MET group was significantly higher than B_max of the STD diet group. In addition, the B_max of the LP group was significantly lower than B_max of the HP and MET groups. There was no significant difference in affinity of the SR Ca²⁺ -uptake system among the four diet groups. These results indicate that modification of dietary protein can influence myocardial SR Ca²⁺ -pump function.     

SR-targeted CaMKII inhibition improves SR Ca²+ handling, but accelerates cardiac remodeling in mice overexpressing CaMKIIδC

Cardiac myocyte overexpression of CaMKIIδ_C leads to cardiac hypertrophy and heart failure (HF) possibly caused by altered myocyte Ca²⁺ handling. A central defect might be the marked CaMKII-induced increase in diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak which decreases SR Ca²⁺ load and Ca²⁺ transient amplitude. We hypothesized that inhibition of CaMKII near the SR membrane would decrease the leak, improve Ca²⁺ handling and prevent the development of contractile dysfunction and HF. To test this hypothesis we crossbred CaMKIIδ_C overexpressing mice (CaMK) with mice expressing the CaMKII-inhibitor AIP targeted to the SR via a modified phospholamban (PLB)-transmembrane-domain (SR-AIP). There was a selective decrease in the amount of activated CaMKII in the microsomal (SR/membrane) fraction prepared from these double-transgenic mice (CaMK/SR-AIP) mice. In ventricular cardiomyocytes from CaMK/SR-AIP mice, SR Ca²⁺ leak, assessed both as diastolic Ca²⁺ shift into SR upon tetracaine in intact myocytes or integrated Ca²⁺ spark release in permeabilized myocytes, was significantly reduced. The reduced leak was accompanied by enhanced SR Ca²⁺ load and twitch amplitude in double-transgenic mice (vs. CaMK), without changes in SERCA expression or NCX function. However, despite the improved myocyte Ca²⁺ handling, cardiac hypertrophy and remodeling was accelerated in CaMK/SR-AIP and cardiac function worsened. We conclude that while inhibition of SR localized CaMKII in CaMK mice improves Ca²⁺ handling, it does not necessarily rescue the HF phenotype. This implies that a non-SR CaMKIIδ_C exerts SR-independent effects that contribute to hypertrophy and HF, and this CaMKII pathway may be exacerbated by the global enhancement of Ca transients.     

Therapeutic potential of novel Na+-Ca2+ exchange inhibitors in attenuating ischemia-reperfusion injury

The cardiac Na+-Ca2+ exchanger (NCX) plays an essential role in regulating Ca2+ under physiological and pathophysiological conditions. In its forward mode of operation, which predominates under physiological conditions, it extrudes the Ca2+ that enters the cardiac myocyte on a beat-to-beat basis. During ischemia and reperfusion, increased intracellular Na+ leads to a decrease in Ca2+ efflux and enhanced Ca2+ influx via the NCX, potentially leading to Ca2+ overload, which is one of the major pathophysiological mechanisms for ischemia-reperfusion injury. Novel NCX inhibitors discovered in recent years have shown great promise in attenuating ischemia-reperfusion injury.