Ion A. Hobai

Decreased Sarcoplasmic Reticulum Calcium Content Is Responsible for Defective Excitation-Contraction Coupling in Canine Heart Failure

BackgroundAltered excitation-contraction (E-C) coupling in canine pacing-induced heart failure involves decreased sarcoplasmic reticulum (SR) Ca uptake and enhanced Na/Ca exchange, which could be expected to decrease SR Ca content (CaSR) and may explain the reduced intracellular Ca (Cai) transient. Studies in other failure models have suggested that the intrinsic coupling between L-type Ca current (ICa,L) and SR Ca release is reduced without a change in SR Ca load. The present study investigates whether CaSR and/or coupling is altered in midmyocardial myocytes from failing canine hearts (F). Methods and ResultsMyocytes were indo-1-loaded via patch pipette (37°C), and Cai transients were elicited with voltage-clamp steps applied at various frequencies. ICa,L density was not significantly decreased in F, but steady-state Cai transients were reduced to 20% to 40% of normal myocytes (N). CaSR, measured by integrating Na/Ca exchange currents during caffeine-induced release, was profoundly decreased in F, to 15% to 25% of N. When CaSR was normalized in F by preloading in 5 mmol/L external Ca before a test pulse at 2 mmol/L Ca, a normal-amplitude Cai transient was elicited. E-C coupling gain was dependent on CaSR but was affected similarly in both groups, indicating that intrinsic coupling is unaltered in F. ConclusionsA decrease in CaSR is sufficient to explain the diminished Cai transients in F, without a change in the effectiveness of coupling. Therefore, therapeutic approaches that increase CaSR may be able to fully correct the Ca handling deficit in heart failure.

Enhanced Ca2+-Activated Na+-Ca2+ Exchange Activity in Canine Pacing-Induced Heart Failure

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca2+ uptake and a greater dependence on transsarcolemmal Na+-Ca2+ exchange (NCX) for Ca2+ removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing–induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca2+ ([Ca2+]i) was buffered to 200 nmol/L. However, when [Ca2+]i was minimally buffered with 50 &mgr;mol/L indo-1, Ca2+ extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca2+ transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca2+ uptake, there is an absolute increase in NCX function that depends on [Ca2+]i in the failing heart.

Role of Sodium-Calcium Exchanger in Modulating the Action Potential of Ventricular Myocytes From Normal and Failing Hearts

Abstract— Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i ≥10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i ≥10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.

Partial Inhibition of Sodium/Calcium Exchange Restores Cellular Calcium Handling in Canine Heart Failure

Sodium/calcium (Na+/Ca2+) exchange (NCX) overexpression is common to human heart failure and heart failure in many animal models, but its specific contribution to the cellular Ca2+ ([Ca2+]i) handling deficit is unclear. Here, we investigate the effects of exchange inhibitory peptide (XIP) on Ca2+ handling in myocytes isolated from canine tachycardic pacing-induced failing hearts. Whole-cell patch-clamped left ventricular myocytes from failing hearts (F) showed a 52% decrease in steady-state sarcoplasmic reticulum (SR) Ca2+ load and a 44% reduction in the amplitude of the [Ca2+]i transient, as compared with myocytes from normal hearts (N). Intracellular application of XIP (30 &mgr;mol/L) normalized the [Ca2+]i transient amplitude in F (3.86-fold increase), concomitant with a similar increase in SR Ca2+ load. The degree of NCX inhibition at this concentration of XIP was ≈27% and was selective for NCX: L-type Ca2+ currents and plasmalemmal Ca2+ pumps were not affected. XIP also indirectly improved the rate of [Ca2+]i removal at steady-state, secondary to Ca2+-dependent activation of SR Ca2+ uptake. The findings indicate that in the failing heart cell, NCX inhibition can improve SR Ca2+ load by shifting the balance of Ca2+ fluxes away from trans-sarcolemmal efflux toward SR accumulation. Hence, inhibition of the Ca2+ efflux mode of the exchanger could potentially be an effective therapeutic strategy for improving contractility in congestive heart failure.

Role of Intracellular Sodium Overload in the Genesis of Cardiac Arrhythmias

Intracellular Na and Arrhythmia. A number of clinical cardiac disorders may be associated with a rise of the intracellular Na concentration (Nai) in heart muscle. A clear example is digitalis toxicity, in which excessive inhibition of the Na/K pump causes the Nai concentration to become raised above the normal level. Especially in digitalis toxicity, but also in many other situations, the rise of Nai may be an important (or contributory) cause of increased cardiac arrhythmias. In this review, we consider the mechanisms by which a raised Nai may cause cardiac arrhythmias. First, we describe the factors that regulate Nai, and we demonstrate that the equilibrium level of Nai is determined by a balance between Na entry into the cell, and Na extrusion from the cell. A numher of mechanisms are responsible for Na entry into the cell, whereas the Na/K pump appears to be the main mechanism for Na extrusion. We then consider the processes by which an increased level of Nai might contrihute to cardiac arrhythmias. A rise of Nai is well known to result in an increase of intracellular Ca, via the important and influential Na/Ca exchange mechanism in the cell membrane of cardiac muscle cells. A rise of intracellular Ca modulates the activity of a numher of sarcolemmal ion channels and aflects release of intracellular Ca from the sarcoplasmic reticulum, all of which might be involved in causing arrhythmia. It is possible that the increase in contractile force that results from the rise of intracellular Ca may initiate or exacerbate arrhythmia, since this will increase wall stress and energy demands in the ventricle, and an increase in wall stress may be arrhythmogenic. In addition, the rise of Nai is anticipated to modulate directly a number of ion channels and to affect the regulation of intracellular pH, which also may be involved in causing arrhythmia. We also present experiments in tbis review, carried out on the working rat heart preparation, which suggest that a rise of Nai causes an increase of wall stress‐induced arrhythmia in this model. In addition, we have investigated the effect on wall stress‐induced arrhythmia of maneuvers that might be anticipated to change intracellular Ca, and this has allowed identification of some of the factors involved in causing arrhythmia in the working rat heart.

The potential of Na+/Ca2+ exchange blockers in the treatment of cardiac disease

The Na+/Ca2+ exchanger (NCX), a surface membrane antiporter, is the primary pathway for Ca2+ efflux from the cardiac cell and a determinant of both the electrical and contractile state of the heart. Enhanced expression of NCX has recently been recognised as one of the molecular mechanisms that contributes to reduced Ca2+ release, impaired contractility and an increased risk of arrhythmias during the development of cardiac hypertrophy and failure. The NCX has also been implicated in the mechanism of arrhythmias and cellular injury associated with ischaemia and reperfusion. Hence, NCX blockade represents a potential therapeutic strategy for treating cardiac disease, however, its reversibility and electrogenic properties must be taken into consideration when predicting the outcome. NCX inhibition has been demonstrated to be protective against ischaemic injury and to have a positive inotropic and antiarrhythmic effect in failing heart cells. However, progress has been impaired by the absence of clinically useful agents. Two drugs, KB-R7943 and SEA-0400, have been developed as NCX blockers but both lack specificity. Selective peptide inhibitors have been well characterised but are active only when delivered to the intracellular space. Gene therapy strategies may circumvent the latter problem in the future. This review discusses the effects of NCX blockade, supporting its potential as a new cardiovascular therapeutic strategy.

The peptide ”FRCRCFa”, dialysed intracellularly, inhibits the Na/Ca exchange in rabbit ventricular myocytes with high affinity

Abstract We investigated the effect in rabbit ventricular myocytes of ”FRCRCFa”, a newly developed peptide inhibitor of the Na/Ca exchange. Myocytes were whole-cell patch clamped and experiments were carried out at 36°C. The Na/Ca exchange was measured selectively, by blocking interfering ion channel currents and the Na/K pump, as the membrane current which could be inhibited by 5 mM nickel (Ni; a known blocker of the Na/Ca exchange). Increasing concentrations of FRCRCFa dialysed into the cell from the patch-pipette inhibited the Na/Ca exchange current. The dose/response curve could be fitted by a function for co-operative ligand binding, which predicted a KD for FRCRCFa-mediated inhibition of 22.7 ± 3.7 nM, with a Hill coefficient of 0.61 ± 0.06. Pipette FRCRCFa concentrations of 1 μM and above were sufficient to cause complete inhibition of Na/Ca exchange current. The inhibitory effect of FRCRCFa was independent of membrane potential and relatively selective: 10 μM FRCRCFa dialysed into the cell had no effect on the L-type Ca current and delayed rectifier and inward rectifier K currents. Thus FRCRCFa appears to be a potent and relatively selective inhibitor of the Na/Ca exchange in intact cardiac myocytes, and may be of value for studies of the Na/Ca exchange.

Dysregulation of intracellular calcium transporters in animal models of sepsis-induced cardiomyopathy.

ABSTRACT Sepsis-induced cardiomyopathy (SIC) develops as the result of myocardial calcium (Ca2+) dysregulation. Here we reviewed all published studies that quantified the dysfunction of intracellular Ca2+ transporters and the myofilaments in animal models of SIC. Cardiomyocytes isolated from septic animals showed, invariably, a decreased twitch amplitude, which is frequently caused by a decrease in the amplitude of cellular Ca2+ transients (&Dgr;Cai) and sarcoplasmic reticulum (SR) Ca2+ load (CaSR). Underlying these deficits, the L-type Ca2+ channel is downregulated, through mechanisms that may involve adrenomedullin-mediated redox signaling. The SR Ca2+ pump is also inhibited, through oxidative modifications (sulfonylation) of one reactive thiol group (on Cys674) and/or modulation of phospholamban. Diastolic Ca2+ leak of ryanodine receptors is frequently increased. In contrast, Na+/Ca2+ exchange inhibition may play a partially compensatory role by increasing CaSR and &Dgr;Cai. The action potential is usually shortened. Myofilaments show a bidirectional regulation, with decreased Ca2+ sensitivity in milder forms of disease (due to troponin I hyperphosphorylation) and an increase (redox mediated) in more severe forms. Most deficits occurred similarly in two different disease models, induced by either intraperitoneal administration of bacterial lipopolysaccharide or cecal ligation and puncture. In conclusion, substantial cumulative evidence implicates various Ca2+ transporters and the myofilaments in SIC pathology. What is less clear, however, are the identity and interplay of the signaling pathways that are responsible for Ca2+ transporters dysfunction. With few exceptions, all studies we found used solely male animals. Identifying sex differences in Ca2+ dysregulation in SIC becomes, therefore, another priority.

Perioperative spinal cord infarction in nonaortic surgery: report of three cases and review of the literature

Paraplegia caused by a spinal cord infarction (SCI) is a devastating perioperative complication, most often associated with aortic and spine surgery. We present two other clinical scenarios in which perioperative SCI may occur. They happened during surgical procedures performed with epidural anesthesia, in the presence of several specific risk factors such as spinal stenosis, vascular disease, intraoperative hypotension, or the use of epinephrine in the local anesthetic solution. Second, SCI may occur during episodes of postoperative hypotension in patients with a history of aortic aneurysms.

SERCA Cys674 sulphonylation and inhibition of L-type Ca2+ influx contribute to cardiac dysfunction in endotoxemic mice, independent of cGMP synthesis

The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 μg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.