Avoidance of Profound Hypothermia During Initial Reperfusion Improves the Functional Recovery of Hearts Donated After Circulatory Death.

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine-lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p < 0.01). During subsequent EVHP, less myocardial injury (troponin I: 5°C = 91 ± 6, 25°C = 64 ± 16, 35°C = 57 ± 7 pg/mL/g, p = 0.04) and greater preservation of endothelial cell integrity (electron microscopy injury score: 5°C = 3.2 ± 0.5, 25°C = 1.8 ± 0.2, 35°C = 1.7 ± 0.3, p = 0.01) were evident in hearts initially reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of myocardial function (cardiac index: 5°C = 3.9 ± 0.8, 25°C = 6.2 ± 0.4, 35°C = 6.5 ± 0.6 mL/minute/g, p = 0.03) during EVHP. We conclude that the avoidance of profound hypothermia during IR minimizes injury and improves the functional recovery of DCD hearts.

Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation.

Hearts donated following circulatory death (DCD) may represent an additional source of organs for transplantation; however, the impact of donor extubation on the DCD heart has not been well characterized. We sought to describe the physiologic changes that occur following withdrawal of life-sustaining therapy (WLST) in a porcine model of DCD. Physiologic changes were monitored continuously for 20 min following WLST. Ventricular pressure, volume, and function were recorded using a conductance catheter placed into the right (N = 8) and left (N = 8) ventricles, and using magnetic resonance imaging (MRI, N = 3). Hypoxic pulmonary vasoconstriction occurred following WLST, and was associated with distension of the right ventricle (RV) and reduced cardiac output. A 120-fold increase in epinephrine was subsequently observed that produced a transient hyperdynamic phase; however, progressive RV distension developed during this time. Circulatory arrest occurred 7.6±0.3 min following WLST, at which time MRI demonstrated an 18±7% increase in RV volume and a 12±9% decrease in left ventricular volume compared to baseline. We conclude that hypoxic pulmonary vasoconstriction and a profound catecholamine surge occur following WLST that result in distension of the RV. These changes have important implications on the resuscitation, preservation, and evaluation of DCD hearts prior to transplantation.

Biosynthesis and initial processing of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

Based on the deduced amino-acid sequence of the cardiac Na(+)-Ca2+ exchanger, there are six potential N-linked glycosylation sites and a potential cleaved signal sequence. To study the post-translational modifications of the exchanger, in vitro translation was examined in the presence and absence of canine pancreatic microsomes. Glycosylation, detected as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosylation sites by using full and partial length RNA transcripts. In the presence of microsomes, the molecular mass of the full-length clone increased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduction to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleaved during processing. Analysis of molecular-mass shifts obtained with partial transcripts suggested that glycosylation occurs at position N-9. This was confirmed by site-directed mutagenesis studies. A molecular mass of approx. 120 kDa was measured for Western blots of cardiac sarcolemmal membrane or oocytes expressing the wild-type exchanger. The molecular mass was reduced by approx. 10 kDa for the N9Y mutant or from exchanger obtained from a baculovirus-infected insect cell line where glycosylation does not occur. The giant excised patch technique was used to determine the functional consequences of glycosylation. Na(+)-Ca2+ exchange current was examined in patches from oocytes expressing either the wild-type or N9Y mutant. The non-glycosylated mutant exhibited the same properties as the native exchanger with respect to voltage, sodium dependence, and the effects of chymotrypsin. The results indicate that glycosylation does not affect exchanger function in Xenopus oocytes and help to define exchanger topology.

Transport and regulation of the cardiac Na(+)-Ca2+ exchanger, NCX1. Comparison between Ca2+ and Ba2+.

Cardiac muscle fails to relax upon replacement of extracellular Ca2+ with Ba2+. Among the manifold consequences of this intervention, one major possibility is that Na(+)-Ba2+ exchange is inadequate to support normal relaxation. This could occur due to reduced transport rates of Na(+)-Ba2+ exchange and/or by failure of Ba2+ to activate the exchanger molecule at the high affinity regulatory Ca2+ binding site. In this study, we examined transport and regulatory properties for Na(+)-Ca2+ and Na(+)-Ba2+ exchange. Inward and outward Na(+)-Ca2+ or Na(+)-Ba2+ exchange currents were examined at 30 degrees C in giant membrane patches excised from Xenopus oocytes expressing the cloned cardiac Na(+)-Ca2+ exchanger, NCX1. When excised patches were exposed to either cytoplasmic Ca2+ or Ba2+, robust inward Na(+)-Ca2+ exchange currents were observed, whereas Na(+)-Ba2+ currents were absent or barely detectable. Similarly, outward currents were greatly reduced when pipette solutions contained Ba2+ rather than Ca2+. However, when solution temperature was elevated from 30 degrees C to 37 degrees C, a substantial increase in outward Na(+)-Ba2+ exchange currents was observed, but not so for inward currents. We also compared the relative abilities of Ca2+ and Ba2+ to activate outward Na(+)-Ca2+ exchange currents at the high affinity regulatory Ca2+ binding site. While Ba2+ was capable of activating the exchanger, it did so with a much lower affinity (KD approximately 10 microM) compared with Ca2+ (KD approximately 0.3 microM). Moreover, the efficiency of Ba2+ regulation of Na(+)-Ca2+ exchange is also diminished relative to Ca2+, supporting approximately 60% of maximal currents obtainable with Ca2+. Ba2+ is also much less effective at alleviating Na+i-induced inactivation of NCX1. These results indicate that the reduced ability of NCX1 to adequately exchange Na+ and Ba2+ contributes to failure of the relaxation process in the cardiac muscle.

Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain.

The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.

Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current-voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being approximately 80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced ( approximately 20-40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+.

The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1-10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.

Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.

Impairment of Ca release from mammalian ventricular sarcoplasmic reticulum by the calcium channel agonist Bay K 8644.

Positive inotropic concentration of the Ca-channel agonist, Bay K 8644, depressed contraction of canine right ventricular trabecula immediately after a rest period of 8 min, without decreasing action potential plateau amplitude. In contrast, high external Ca and ouabagenin caused only a slight decrease in post-rest contraction. Bay K 8644-induced post-rest depression was inversely related to the extracellular Ca concentration. Hence it could not be due to cellular Ca overload. Since post-rest potentiation is due to increased contribution of Ca from the sarcoplasmic reticulum, these results suggest that Bay K 8644 decrease the amount of releasable Ca from this structure during rest.

Effects of caffeine and ryanodine on depression of post-rest tension development produced by Bay K 8644 in canine ventricular muscle.

1. Post-rest inotropy in canine ventricular myocardium has proved a useful indicator of sarcoplasmic reticular calcium release. This phenomenon is converted to rest depression by the calcium channel activator (agonist), Bay K 8644 as well as other chemically diverse agents such as caffeine and ryanodine. 2. Rapid cooling contractures and post-rest contraction amplitude were used as independent measures of sarcoplasmic reticular calcium content and release. Simultaneous recordings of transmembrane action potentials and their accompanying contractions were obtained to determine the association between electrophysiological and mechanical events. The present study was designed to elucidate the mechanism by which Bay K 8644, caffeine and ryanodine alter force production after variable periods of rest. 3. Bay K 8644 (1 microM) increased steady state contraction in response to a constant train of stimulation, caused rest-depression after 2 and 8 min rest, prolonged action potential duration and increased action potential plateau amplitude. Augmented steady state tension was not accompanied by any change in time to peak tension or rapid cooling contracture amplitude. However, the post-rest rapid cooling contracture was greatly diminished compared to that observed prior to Bay K 8644 treatment. 4. Caffeine (3 and 5 mM) caused rest-depression with an increase in steady state contraction amplitude. Along with this there was a slight decrease in action potential duration and plateau amplitude and an increase in time to peak tension. The rapid cooling contractures were virtually abolished at all conditioning intervals. The effect of caffeine on twitch tension and cooling contracture is consistent with the ability of this compound to inhibit calcium sequestration by the sarcoplasmic reticulum. 5. A combination of Bay K 8644 and caffeine caused significantly less rest-depression than that seen with Bay K 8644 alone. The augmented twitch tension was accompanied by a long time to peak tension and action potential duration. However, there was no increase in the amplitude of the rapid cooling contracture, either after a regular train of stimulation or after rest, compared to that seen after Bay K 8644. 6. Ryanodine (10 nM), produced rest-depression, reduced steady state twitch tension and augmented the rest-depression produced by Bay K 8644. The steady state rapid cooling contracture remained unchanged when both agents were present simultaneously, while the post-rest rapid cooling contracture was significantly depressed compared to that observed with Bay K 8644 alone. 7. Bay K 8644 and ryanodine appear to have similar actions with respect to promoting diastolic loss of calcium from the sarcoplasmic reticulum. Although caffeine also decreases post-rest potentiation, it antagonizes rest-depression caused by Bay K 8644. The data from these experiments suggest that this reversal is a result of depressed intracellular calcium buffering and enhanced myofilament sensitivity produced by caffeine in the presence of increased transmembrane calcium influx promoted by Bay K 8644.

Control of interval-force relation in canine ventricular myocardium studied with ryanodine.

1. The mechanism of post-extrasystolic, rest and frequency potentiation was studied in canine isolated ventricular muscle. 2. Ryanodine, which impairs Ca availability from the sarcoplasmic reticulum (SR), reduced the amplitude of the extrasystole less than that of the steady state contraction. Ryanodine also inhibited post-extrasystolic potentiation and converted rest-potentiation into rest depression. Rest-potentiation was blocked preferentially by ryanodine compared to post-extrasystolic potentiation. An increase in the contribution of extracellular Ca to the extrasystolic contraction could not entirely account for the post-extrasystolic potentiation. 3. Prolonged rest, by itself, also caused depression of the first post-rest contraction. During rest-potentiation, SR Ca seemed to play a greater role in contraction than transmembrane Ca influx. However, the ability of the 'release pool' of Ca in the SR to be reprimed after a contraction was reduced. This was seen as a decrease in post-extrasystolic potentiation elicited immediately after rest. 4. A decrease in stimulus interval was associated with a transient decrease in contraction amplitude followed by an increase. An abrupt increase in stimulus interval had the opposite effect. Ryanodine blocked the initial transient changes and accelerated the delayed changes. These results suggest that the transient changes in contraction after sudden changes in drive interval are dependent on the SR. 5. Transmembrane Ca entry and the rate of recovery of the Ca release process (repriming) in the SR after a contraction seem to be interval-dependent. The data also indicate that different mechanisms are involved in post-extrasystolic and rest-potentiation. 6. The results are consistent with a model which proposes 'recirculation' of activator Ca within the SR after a contraction or of the presence of an appreciable amount of inactivation of the SR Ca release process during normal stimulation. An increased pool of releasable Ca due to longer recirculation time or a time-dependent decay in the level of inactivation of Ca release from the SR may give rise to rest-potentiation.

Citrate decreases Ca current and changes selectivity of cardiac Ca channels.

Previous studies using citrate as a low affinity Ca buffer have shown that this agent inhibits myocardial contractions and neurotransmitter release. Using several different low affinity Ca buffers we have demonstrated that these effects are unlikely to be attributable to Ca buffering per se. Here we demonstrate that citrate can directly inhibit Ca current (ICa). The decrease in ICa appears to be due to shifts in channel gating (due to surface charge effects) and also to a decrease in channel selectivity for Ca over monovalent ions.

Citrate alters Ca channel gating and selectivity in rabbit ventricular myocytes.

Addition of 10 mM citrate at constant free extracellular Ca concentration [( Ca]o; 2 mM) reduced contraction in rabbit ventricular muscle and isolated myocytes. We have recently shown that extracellular citrate decreases contraction and Ca current (ICa) in cardiac muscle by a direct effect on Ca channels rather than by Ca buffering per se [D. M. Bers, L. V. Hryshko, S. M. Harrison, and D. Dawson. Am. J. Physiol. 260 (Cell Physiol. 29): C900-C909, 1991]. Citrate rapidly depressed peak ICa and shifted both the peak ICa and the apparent reversal potential (Erev) to more negative potentials. When the impermeant cations, tetraethylammonium or N-methylglucamine were used instead of intracellular Cs, the citrate-induced shift in Erev was reduced or eliminated but depression of ICa was still observed. Thus citrate appears to alter the selectivity (PCa/PCs) of the Ca channel and reduce ICa. We also studied the effects of citrate on Na current through the Ca channel, observed when the divalent cation concentration is submicromolar. This current, termed INS for nonspecific, also exhibited leftward shifts in peak INS and smaller changes in Erev in the presence of citrate. However, neither peak INS nor single-channel conductance were affected by citrate. Thus the reduced PCa/PCs is due primarily to alteration of Ca permeation rather than monovalent cation permeation. Activation and inactivation curves for both ICa and INS were shifted toward more negative potentials by citrate. The shifts in gating and peak current to more negative membrane potentials would be consistent with a surface charge effect. The much larger shift in Erev for ICa (than for INS) is consistent with a reduction in Ca selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-calcium exchange: recent advances.

Na-Ca exchange proteins are involved in Ca homeostasis in a wide variety of tissues. Unique Na-Ca exchangers have been identified by molecular biological approaches and it appears that these may represent a superfamily of ion transporters, similar to that identified for ion channels. Major advances in our understanding of these transporters have occurred in the past decade by combining molecular approaches with electrophysiological analyses. The regulatory and transport properties of Na-Ca exchangers are beginning to become understood in molecular detail. It also appears that the physiological roles of Na-Ca exchange may be quite complex. This brief review highlights some recent advances in Na-Ca exchange research obtained through the combination of molecular biological and electrophysiological approaches.

Current-voltage relations and steady-state characteristics of Na+-Ca2+ exchange: characterization of the eight-state consecutive transport model.

An analytical expression for Na+-Ca2+ exchange currents in cardiac cells has been obtained for an eight-state model. The equation obtained has been used to derive theoretical expressions for current-voltage relationships, maximum Na+-Ca2+ exchange currents, and half-saturating concentrations for Na+ and Ca2+. These equations were analyzed over a wide range of cytoplasmic and extracellular Na+ and Ca2+ concentrations, under forward and reverse "zero-trans" conditions. Correspondence of theoretical results with those obtained from giant excised patch experiments are presented. Rate constants from published reports were used to evaluate turnover rates for Na+-Ca2+ exchange in the forward and reverse directions. A factor, epsilon, is introduced that permits prediction of the extent to which the Na+-Ca2+ exchange cycle is under voltage or diffusion control. This factor can be conveniently used for data interpretation and comparison. The derived equations also provide a foundation for continuing experimental evaluation of the fidelity of this model.

Ca current facilitation during postrest recovery depends on Ca entry.

Whole cell Ca current (ICa) recovery after periods of rest was examined in voltage-clamped rabbit ventricular myocytes with Na and K currents suppressed. To evaluate rest-dependent changes in ICa independent of the effects of sarcoplasmic reticular (SR) Ca release, the intracellular Ca ([Ca]i) transients were usually buffered by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (10 mM) in the patch pipette. When voltage-clamp pulses were resumed (at 0.5 Hz) after a period of rest, several pulses were required to reattain steady-state peak ICa levels. From depolarized holding potentials between -40 and -50 mV, peak ICa of the first pulse was large and gradually decayed to steady-state levels (negative ICa staircase). This potentiation of postrest ICa was mediated by increased recovery from inactivation of Ca channels during the rest period. In contrast, with more negative holding potentials (-70 to -90 mV), the initial postrest ICa was relatively small (rest depression) and facilitation of ICa was then observed for subsequent pulses (positive ICa staircase). This ICa facilitation was mediated by a progressive decrease in the ICa inactivation rate. Depression of the initial postrest ICa required 10-15 s of rest to fully develop and became relatively constant for longer rest intervals (30-300 s). Postrest ICa depression (i.e., subsequent ICa facilitation) was abolished by replacement of extracellular Ca ([Ca]o) with either Ba or Sr. Thus ICa facilitation depends on Ca entry. Increasing [Ca]o increased postrest ICa facilitation and reducing [Ca]o had an opposite effect. When ICa was altered by changing step potential, maximal ICa facilitation occurred when ICa was maximal. Thus ICa facilitation can be graded by the amount of Ca entry. As ICa facilitation was not altered by ryanodine, this response is not likely to be due to SR Ca release. However, increasing [Ca]i buffering by using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette abolished the ICa staircase. Our results indicate that Ca entry can facilitate subsequent ICa, presumably through actions occurring near the sarcolemma. These local changes in [Ca]i lead to a progressive slowing in the rate of ICa inactivation.

Rapid cooling contractures as an index of sarcoplasmic reticulum calcium content in rabbit ventricular myocytes.

Rapid cooling contractures (RCCs) were used to assess changes in sarcoplasmic reticulum (SR) Ca content in both isolated rabbit ventricular myocytes and multicellular preparations. The main difference observed between these preparations was the magnitude of RCCs relative to twitches, apparently due to differences in measured parameters, i.e., unloaded shortening vs. isometric tension. When multicellular preparations were unloaded, RCC shortening was similar to that observed in myocytes. RCC magnitude decreased as the time between the last electrical stimulation and the RCC was increased (rest decay). RCC rest decay closely paralleled that of postrest twitches, suggesting that SR Ca loss is responsible for this process. Paired RCC experiments were used to investigate RCC relaxation and rest decay. When a second RCC (RCC2) was induced immediately after the first (RCC1), a large contracture was still observed (RCC2/RCC1 x 100 = 77.8 +/- 7.3%, mean +/- SD), indicating that the SR resequestered the majority of Ca on rewarming. This fraction was increased (to 92.9 +/- 5.5%) if Na and Ca-free solution was used during RCCs and rewarming, indicating that Na-Ca exchange also contributes to RCC relaxation. Increasing the interval between paired RCCs led to a decrease in RCC2, analogous to rest decay. This rest decay was abolished by inhibiting Na-Ca exchange, indicating that SR Ca loss during rest is mediated primarily by this process. RCCs were abolished by 10 mM caffeine. Ryanodine (1 microM) greatly accelerated RCC rest decay but had less effect on RCCs generated immediately after a train of stimulation. This accelerated rest decay was also dependent on Na-Ca exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of rest potentiation in canine ventricular muscle by BAY K 8644: comparison with caffeine.

Rest potentiation, believed to be due to increased utilization of sarcoplasmic reticular calcium, was converted to rest depression by BAY K 8644 (1 microM). Plateau height and duration of the postrest beat were enhanced by BAY K 8644, suggesting an enhancement of extracellular calcium entry. Caffeine (3 mM) also produced depression at all rest intervals, although to a lesser extent than BAY K 8644. Compared with BAY K 8644, treatment with caffeine resulted in an elevation of plateau amplitude and a shortening of action potential duration. Action potential configuration changes induced by rest were unaltered by caffeine despite reduction in rest potentiation. Caffeine-induced rest depression was associated with an increase in the time to peak tension. This was not observed with BAY K 8644. Treatment with both caffeine (3 mM) and BAY K 8644 (1 microM) greatly prolonged time to peak tension. Action potential duration and plateau height were either maintained or increased. Less rest depression was observed with the combination than with either agent alone. These results suggest that 1) BAY K 8644 and caffeine inhibit rest potentiation by different mechanisms, and 2) caffeine-induced inhibition of calcium uptake by the sarcoplasmic reticulum may enhance the effect of BAY K 8644-induced increase in calcium influx on the contractile apparatus.