SR Ca Content Research Articles

Enhanced Sodium‐Calcium Exchange in the Infarcted Heart

Arrhythmias and contractile dysfunction both contribute to the high morbidity and mortality in patients with congestive heart failure. Contractile dysfunction is generally believed to reflect a decrease in the amplitude of intracellular Ca(2+) transients, whereas tachyarrythmias are often initiated in the setting of cellular Ca(2+) overload. In a rabbit model of left ventricular dysfunction due to myocardial infarction, we found evidence that myocyte sarcoplasmic reticulum Ca(2+) content may be normal or even increased at slow stimulation rates. This may occur because prolonged action potential duration promotes Ca(2+) influx via the Na(+)/Ca(2+) exchanger. Despite preserved SR Ca(2+) content, intracellular Ca(2+) transients and contractions may be reduced in amplitude because of impaired synchronization of Ca(2+) release events throughout the myocyte.

Mechanisms underlying the frequency dependence of contraction and [Ca(2+)](i) transients in mouse ventricular myocytes.

In most mammalian species force of contraction of cardiac muscle increases with increasing rate of stimulation, i.e. a positive force-frequency relationship. In single mouse ventricular cells, both positive and negative relationships have been described and little is known about the underlying mechanisms. We studied enzymatically isolated single ventricular mouse myocytes, at 30 degrees C. During field stimulation, amplitude of unloaded cell shortening increased with increasing frequency of stimulation (0.04 +/- 0.01 Delta L/L(0) at 1 Hz to 0.07 +/- 0.01 Delta L/L(0) at 4 Hz, n = 12, P < 0.05). During whole cell voltage clamp with 50 microM [K5-fluo-3](pip), both peak and baseline [Ca(2+)](i) increased at higher stimulation frequencies, but the net Delta[Ca(2+)](i) increased only modestly from 1.59 +/- 0.08 Delta F/F(0) at 1 Hz, to 1.71 +/- 0.11 Delta F/F(0) at 4 Hz (n = 17, P < 0.05). When a 1 s pause was interposed during stimulation at 2 and 4 Hz, [Ca(2+)](i) transients were significantly larger (at 4 Hz, peak F/F(0) increased by 78 +/- 2 %, n = 5). SR Ca(2+) content assessed during caffeine application, significantly increased from 91 +/- 24 micromol l(-1) at 1 Hz to 173 +/- 20 micromol l(-1) at 4 Hz (n = 5, P < 0.05). Peak I(Ca,L) decreased at higher frequencies (by 28 +/- 6 % at 2 Hz, and 45 +/- 8 % at 4 Hz), due to slow recovery from inactivation. This loss of I(Ca,L) resulted in reduced fractional release. Thus, in mouse ventricular myocytes the [Ca(2+)](i)-frequency response depends on a balance between the increase in SR content and the loss of trigger I(Ca,L). Small changes in this balance may contribute to variability in frequency-dependent behaviour. In addition, there may be a regulation of the contractile response downstream of [Ca(2+)](i).

Overexpression of SERCA2a accelerates repolarisation in rabbit ventricular myocytes

Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation–contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15–25 MΩ resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481±98, n=12; Ad.GFP: 464±85, n=11; Ad.SERCA2a: 285±69, n=13 (mean±S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375±64, n=22; Ad.GFP: 363±47, n=18; Ad.SERCA2a: 231±54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5±4.2, n=8; Ad.SERCA2a: 15.5±4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (μM/l non-mitochondrial volume): non-infected: 25.5±7, n=8; Ad.GFP: 25.7±11, n=6; Ad.SERCA2a: 80.5±19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.

Upregulated Na/Ca exchange is involved in both contractile dysfunction and arrhythmogenesis in heart failure.

Systolic heart failure (HF) is characterized by reduced systolic function and often by arrhythmias. We studied a rabbit model of HF (induced by combined aortic insufficiency and stenosis) which shows both contractile dysfunction and arrhythmogenesis. In this model we find an approximately 100% increase in Na/Ca exchange (NaCaX) expression at the level of mRNA, protein and function, but only a modest decrease in SR Ca-ATPase (approximately 24%, only detectable in cellular function). This combination results in a 40% reduction in SR Ca content in HF, which is sufficient to explain the 40% reduction in twitch Ca transients and 30-38% decrease in contractile function in this HF model. When stimulated by isoproterenol the SR Ca load readily reaches the threshold for spontaneous SR Ca release (this threshold Ca load is unchanged in HF). This SR Ca release activates a transient inward current (I(ti)) carried exclusively by NaCaX. For a given SR Ca release there is greater I(ti) in HF (due to higher NaCaX). We also find a 49% decrease in the inward rectifier potassium current (I(K1)), which allows greater depolarization for a given I(ti). Thus, higher NaCaX and lower I(K1) greatly increase the likelihood that an SR Ca release-induced delayed afterdepolarization (DAD) will trigger an arrhythmogenic action potential. We conclude that NaCaX contributes in major ways to both contractile dysfunction (by reducing SR Ca) and increased propensity for triggered arrhythmias (by increasing I(ti) and DADs).

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel.

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na–Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na–Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.

Sarcoplasmic Reticulum Ca Content, Sarcolemmal Ca Influx and the Genesis of Arrhythmias in Isolated Guinea-pig Cardiomyocytes

Early afterdepolarizations are seen during the repolarization phases of the action potential and delayed afterdepolarizations appear later, usually following complete repolarization of the cell membrane potential. Both forms of afterdepolarization are linked to the occurrence of aftercontractions, seem to play a role in the generation of ventricular arrhythmias and are believed to be the result of abnormalities of intracellular Ca handling. Suggestions for the mechanisms responsible vary from both types of afterpotential being mediated by Ca release from the sarcoplasmic reticulum, to early afterdepolarization formation being due to reactivation of the L-type sarcolemmal Ca channels during the action potential. We tried to assess the functional importance of the sarcoplasmic reticulum or Ca influx in the development of afterpotentials and abnormal contractile activity in guinea-pig cardiac myocytes. Ca influx was increased using isoproterenol, Bay K8644 or increasing extracellular [Ca]. Sarcoplasmic reticulum Ca content was measured using rapid cooling contractures or caffeine-induced Na/Ca exchange current and the sarcoplasmic reticulum was inhibited using caffeine or thapsigargin. Aftercontractions associated with either early or delayed afterdepolarizations could be induced by increasing Ca influx. The increased Ca influx produced increases in sarcoplasmic reticulum Ca content and aftercontractions were associated with a larger SR Ca content. However, the sarcoplasmic reticulum was no more loaded with Ca when aftercontractions occurred than when aftercontractions did not occur. Preventing Ca sequestration by the sarcoplasmic reticulum inhibited the formation of aftercontractions. The results suggest that alterations to both Ca influx and sarcoplasmic reticulum Ca content are required to produce aftercontractions.

Evidence that reverse Na-Ca exchange can trigger SR calcium release.

Several results suggest that the Na-Ca exchange can function as a trigger promoting SR Ca release and ensuing contractions. First, if the Ca current was the sole trigger for contraction we would expect the relationship between triggered contractions and voltage to be similar to the relationship between Ca current and contraction. When Na is present in the pipette this is not observed. Between -40 and +10 mV the relationships between contractions and voltage and current and voltage are similar. At potentials positive to 10 mV the Ca current declines as expected but contractions either decline much more slowly or continue to increase depending upon the concentration of intracellular Na. In addition, we have observed that contractions can be activated when Ca current is largely or completely blocked. Since these contractions are sensitive to the presence of ryanodine and thapsigargin they appear to be triggered by Na-Ca exchange. Also, contractions that are activated in the presence of nifedipine are sensitive to the Na-Ca exchange inhibitor XIP. Finally, rapid removal of extracellular Na apparently stimulates enough reverse exchange triggering of SR Ca release without affecting the SR content. It is clear that the shape of the shortening voltage relationship depends upon the concentration of dialyzing Na. This is likely to occur for two reasons. Either the shape of the shortening voltage relationship depends upon the extent to which Na-Ca exchange contributes a trigger for SR Ca release or alternatively the shape of the shortening voltage relationship depends upon SR Ca content. The latter is known to depend upon the Na concentration. In addition it is now established that the gain of SR Ca release is influenced by SR content. However, we studied triggered contractions in the absence of a Na gradient when the only available trigger is the Ca current. We measured triggered contractions over a range of voltages between -30 and +60 mV. Between each measurement we reestablished the Na gradient and activated a series of conditioning pulses to standardize the SR Ca content. Just before a test pulse we removed extracellular Na and activated either 3 or 6 pulses to produce two different SR Ca loads (in the absence of a Na gradient entering Ca cannot be extruded and therefore changes the SR Ca content). Regardless of the number of prepulses in the absence of a Na gradient the shortening voltage relationship was similar and bell shaped. From this we conclude that the shape of the relationship between shortening and voltage does not depend upon SR Ca content. Therefore, we conclude that the asymmetry in the shortening voltage relationship that depends upon intracellular Na is due to a contribution of reverse Na-Ca exchange. It is too early to say what the physiological significance (if any) of triggering by reverse exchange actually is. However, it does seem likely that it might provide a powerful inotropic mechanism. For example intracellular Na might be expected to change with heart rate and to be elevated at higher heart rates. Presumably this increased intracellular Na would tend to favor triggering by reverse exchange and would therefore enhance contractility at a time when it would be most required.

Localization of the Na/Ca exchange-dependent Ca compartment in cultured neonatal rat heart cells.

It has been previously established, in both adult and cultured neonatal cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarcoplasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemmal leaflet represent together the subcellular locus of the compartment. The present study examines this proposal. The amount of Ca in the total compartment is measured isotopically in intact functional cells (using the on-line "scintillation disk" technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated from another set of intact cells by "gas dissection," and the amount of Ca bound to the membranes is measured. Probes that specifically decrease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrease total cell content and sarcolemmal binding proportionally. High-dose ryanodine (producing closure of SR channels) markedly reduces sarcolemmal binding relative to total content of the compartment. The sarcolemmal sites saturate between 1 and 2 mM extracellular Ca ([Ca]o), whereas the total compartment saturates between 4 and 6 mM [Ca]o. Below 1 mM [Ca]o, sarcolemmal binding is maintained relative to total compartment content. Finally, the total compartment increases after reversal of the intracellular Na to extracellular Na ([Na]i/[Na]o) gradient with sarcolemmal content-to-total content ratio dependent on the method used to reverse the [Na]i/[Na]o ratio. The results are consistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region ("cleft") where JSR Ca is in equilibrium with anionic inner sarcolemmal leaflet Ca binding sites.

Na-Ca Exchange is Required for Rest-decay but not for Rest-potential of Twitches in Rabbit and Rat Ventricular Myocytes

The influence of the Na-Ca exchange (NaCaX) on the effects of rest (30-300s) on twitch amplitude and SR Ca content (assessed by caffeine contractures) was studied in ventricular myocytes isolated from rat and rabbit. In control conditions, rabbit cells showed monotonic rest-decay of the amplitudes of both twitch and caffeine contractures, while rat myocytes developed rest-potential of twitches change in SR Ca content. Inhibition of the Na-Ca exchange during rest by perfusion with ONa.OCa solution did not affect the responses in rat cells but abolished rest-dependent SR Ca loss in rabbit cells. Indeed, when NaCaX was blocked during rest, then rabbit cells, like rat, displayed rest-potential of twitches. Stimulation of net Ca extrusion via NaCaX during rest by perfusion with OCa solution induced rest-decay of twitches and contractures in rat cells similar to that observed in rabbit cells. This maneuver also accelerated decline in SR Ca during rest and amplitude of the first post-rest twitch in rabbit myocytes. These effects were only slightly enhanced by preperfusion with ONa,OCa solution to deplete Na i. We were thus able to interconvert the contractile responses to rest between these cell types solely by modifying the driving force for Ca transport by the exchange. Our results indicate that SR Ca is lost during quiescence in both species, but only if the NaCaX is able to promote diastolic Ca extrusion will net decline of SR Ca (and twitch amplitude) occur. On the other hand, post-rest twitch potentiation in both rat and rabbit cells can occur without a change in SR Ca content. This effects might be attributable, at least in part, to a slow phase of recovery of excitation-contraction coupling.

Paradoxical Twitch Potentiation After Rest in Cardiac Muscle: Increased Fractional Release of SR Calcium

Rest interval dependent changes in contractile force (rest decay and rest potentiation) were studied in rabbit, rat and ferret ventricular muscle and myocytes. The SR Ca content was assessed by rapid cooling contractures or caffeine induced contractures. Intracellular Ca transients, action potentials and Ca current were also recorded. Rest decay of twitches ventricle are roughly paralleled by a decline in SR Ca content. Rat ventricle exhibits primarily rest potentiation, which is not necessarily paralleled by an increased SR Ca content. Ferret ventricle exhibits both rest potentiation and rest decay. However, the SR Ca content in ferret appears to decline monotonically throughout the rest. It is demonstrated that the rest potentiation is not due to an increase in Ca current or in action potential duration. We conclude that there is increase in the fraction of SR Ca content which is released the time that rest potentiation develops. The differences in post-rest contractile function among different cardiac preparations can be described by a simple unifying mechanistic model. In this model there is an exponential time dependent recovery of the ability of the SR to release Ca (e.g. recovery from inactivation) which can be considered to increase the fraction os SR Ca release in response to activation. This fractional SR Ca release is multiplied by the SR Ca content (which may decline exponentially) to provide a measure of the Ca available for activation of contractile force.

Modulation of SR-Ca content by Na/Ca exchange in atrial ferret heart: C. Huchet, J. Noireaud, C. Léoty. URA CNRS 1340, Laboratory of General Physiology, E.N.V., F-44087 Nantes Cedex 03 France

Modulation of SR-Ca content by Na/Ca exchange in atrial ferret heart: C. Huchet, J. Noireaud, C. Léoty. URA CNRS 1340, Laboratory of General Physiology, E.N.V., F-44087 Nantes Cedex 03 France