Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms.

The cardiac Na(+)/Ca(2+) exchanger (NCX), an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7 degrees C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents (I(NCX)) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I(NCX) of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 degrees C using the giant excised patch technique. The I(NCX) for both isoforms exhibited Na(+)-dependent inactivation and Ca(2+)-dependent positive regulation. The I(NCX) of NCX1.1 exhibited typical mammalian temperature sensitivities with Q(10) values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I(NCX) of NCX-TR1.0 was relatively temperature insensitive with Q(10) values of 1.2 and 1.1 for peak and steady-state currents, respectively. I(NCX) current decay was fit with a single exponential, and the resultant rate constant of inactivation (lambda) was determined as a function of temperature. As expected, lambda decreased monotonically with temperature for both isoforms. Although lambda was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on lambda was not different between the two isoforms. These data suggest that the disparities in I(NCX) temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after alpha-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I(NCX) temperature dependence between the two isoforms are not due to potential disparities in either the I(NCX) regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.

Evolutionary and physiological variation in cardiac troponin C in relation to thermal strategies of fish.

Striated muscle contraction is initiated when troponin C (TnC) binds Ca(2+), which activates actinomyosin ATPase. We investigated (i) the variation between cardiac TnC (cTnC) primary structure within teleost fish and (ii) the pattern of TnC expression in response to temperature acclimation. There were few differences between rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares), yellow perch (Perca flavescens), goldfish (Carassius auratus), white sucker (Catostomus commersoni), and icefish (Chaenocephalus aceratus) in cTnC amino acid sequence. No variation existed in the regulatory Ca(2+)-binding site (site 2). The site 3 and 4 substitutions were limited to residues not directly involved in Ca(2+) coordination. Fish cTnC primary structure was highly conserved between species (93%-98%) and collectively divergent from the highly conserved sequence seen in birds and mammals. Northern blots and polymerase chain reaction showed that thermal acclimation of trout (3 degrees, 18 degrees C) did not alter the TnC isoform pattern. While cardiac and white muscle had the expected isoforms-cTnC and fast troponin C (fTnC), respectively-red muscle unexpectedly expressed primarily ftnC. Cold acclimation did not alter myofibrillar ATPase Ca(2+) sensitivity, but maximal velocity increased by 60%. We found no evidence that TnC variants, arising between species or in response to thermal acclimation, play a major role in mitigating the effects of temperature on contractility of the adult fish heart.

Ca(2+) binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms.

A reduction in temperature lowers the Ca(2+) sensitivity of skinned cardiac myofilaments but this effect is attenuated when native cardiac troponin C (cTnC) is replaced with skeletal TnC. This suggests that conformational differences between the two isoforms mediate the influence of temperature on contractility. To investigate this phenomenon, the functional characteristics of bovine cTnC (BcTnC) and that from rainbow trout, Oncorhynchus mykiss, a cold water salmonid (ScTnC), have been compared. Rainbow trout maintain cardiac function at temperatures cardioplegic to mammals. To determine whether ScTnC is more sensitive to Ca(2+) than BcTnC, F27W mutants were used to measure changes in fluorescence with in vitro Ca(2+) titrations of site II, the activation site. When measured under identical conditions, ScTnC was more sensitive to Ca(2+) than BcTnC. At 21 degrees C, pH 7.0, as indicated by K(1/2) (-log[Ca] at half-maximal fluorescence, where [Ca] is calcium concentration), ScTnC was 2.29-fold more sensitive to Ca(2+) than BcTnC. When pH was kept constant (7.0) and temperature was lowered from 37.0 to 21.0 degrees C and then to 7.0 degrees C, the K(1/2) of BcTnC decreased by 0.13 and 0.32, respectively, whereas the K(1/2) of ScTnC decreased by 0.76 and 0.42, respectively. Increasing pH from 7.0 to 7.3 at 21.0 degrees C increased the K(1/2) of both BcTnC and ScTnC by 0.14, whereas the K(1/2) of both isoforms was increased by 1.35 when pH was raised from 7.0 to 7.6 at 7.0 degrees C.

Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy.

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.

Effects of diabetes and hypertension on myocardial Na+-Ca2+ exchange.

Abnormalities in cardiac function have been extensively documented in experimental and clinical diabetes. These aberrations are well known to be exaggerated when hypertension and diabetes co-exist. The objective of the present study was to examine whether alterations in the activity of the myocardial Na+-Ca2+ exchanger (NCX) can account for the deleterious effects of diabetes and (or) hypertension on the heart. To this aim, the following experimental groups were studied: (i) control; (ii) diabetic; (iii) hypertensive; and (iv) hypertensive-diabetic. Wistar rats served as the control group (C) while Wistar rats injected with streptozotocin (STZ, 55 mg/kg) served as the diabetic (D) group. Spontaneously hypertensive (SH) rats were used as the hypertensive group (H) while SH rats injected with STZ served as the hypertensive-diabetic (HD) group. Sarcolemma was isolated from the ventricles of the C, D, H, and HD groups and NCX activity was examined using rapid quenching techniques to study initial rates over a [Ca2+]o range of 10-160 microM. The Vmax of NCX was lower in the D group when compared with the C group (D, 2.96 +/- 0.26 vs. C, 4.0 +/- 0.46 nmol x mgprot(-1) x s(-1), P < 0.05), however combined diabetes and hypertension (HD) did not affect the Vmax of NCX activity (HD, 3.84 +/- 0.88 vs. H, 3.59 +/- 0.24 nmol x mgprot(-1) x s(-1), P > 0.05). However, analysis of the Km values for Ca2+ indicated that both the D and HD groups exhibited a significantly lower Km when compared with their respective control groups (D, 42 +/- 4 vs. C, 56 +/- 4 microM, P < 0.05; HD, 33 +/- 7 vs. H, 51 +/- 8 microM, P < 0.05). Immunoblotting using polyclonal antibodies (against canine cardiac NCX) exhibited the typical banding of 160, 120, and 70 kDa. The 120 kDa band is believed to represent the native exchanger with its post-translational modifications. Examination of the blots revealed a lower intensity of the 120 kDa band in the D group when compared with the C group, however, no significant difference in the HD group was observed. We speculate that the lower Vmax in the D group may be due to a reduced concentration of exchanger protein in the membrane. The absence of this defect in the HD group may be a result of compensatory mechanisms to the overall hemodynamic overload, however, this remains to be determined. The increased affinity for Ca2+ in both the D and HD groups (determined by the lower Km values) is an interesting finding and may be due to changes in sarcolemmal lipid bilayer composition secondary to diabetes-induced hyperlipidemia.

Cloning, expression, and characterization of the trout cardiac Na(+)/Ca(2+) exchanger.

Isoform 1 of the cardiac Na(+)/Ca(2+) exchanger (NCX1) is an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation. Studies with trout heart sarcolemmal vesicles have shown NCX to have a high level of activity at 7 degrees C, and this unique property is likely due to differences in protein structure. In this study, we describe the cloning of an NCX (NCX-TR1) from a Lambda ZAP II cDNA library constructed from rainbow trout (Oncorhynchus mykiss) heart RNA. The NCX-TR1 cDNA has an open reading frame that codes for a protein of 968 amino acids with a deduced molecular mass of 108 kDa. A hydropathy plot indicates the protein contains 12 hydrophobic segments (of which the first is predicted to be a cleaved leader peptide) and a large cytoplasmic loop. By analogy to NCX1, NCX-TR1 is predicted to have nine transmembrane segments. The sequences demonstrated to be the exchanger inhibitory peptide site and the regulatory Ca(2+) binding site in the cytoplasmic loop of mammalian NCX1 are almost completely conserved in NCX-TR1. NCX-TR1 cRNA was injected into Xenopus oocytes, and after 3-4 days currents were measured by the giant excised patch technique. NCX-TR1 currents measured at approximately 23 degrees C demonstrated Na(+)-dependent inactivation and Ca(2+)-dependent activation in a manner qualitatively similar to that for NCX1 currents.

Cloning and sequencing of complementary DNA for fatty acid binding protein from rainbow trout heart.

A cDNA encoding a rainbow trout homologue of mammalian heart fatty acid binding protein (H-FABP) was isolated. The deduced protein sequence is 75% identical to that of rat H-FABP. The structural conservation of H-FABPs and their evolutionary relationship are discussed.

Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish.

In the adult mammalian heart, the majority of Ca2+ required for contraction is released from the sarcoplasmic reticulum (SR) via the Ca2+-release channel or ryanodine receptor (RyR). Such release is dependent upon a relatively small influx of Ca2+ entering the cell across the sarcolemma (SL) by means of the L-type Ca2+ channel or the dihydropyridine receptor (DHPR). In lower vertebrates, there is indirect evidence suggesting that Ca2+ influx across the SL may be sufficient to support contraction in the absence of Ca2+ release from the SR. This apparent difference in myocardial excitation-contraction (E-C) coupling was investigated further by determining DHPR and RyR densities in ventricular homogenate preparations from rat, trout, dogfish and hagfish. DHPR Bmax values (means +/- S.E.M.) were highest in rat (0.30 +/- 0.01 pmol mg-1), lower in trout (0.16 +/- 0.01 pmol mg-1) and dogfish (0.27 +/- 0.03 pmol mg-1), and slightly above the level of detection in hagfish (0.03 +/- 0.01 pmol mg-1). The DHPR dissociation constants (Kd) of 40-70 pmoll-1 in these three species were of similar magnitude. RyR binding revealed both high- and low-affinity sites in all species. RyR Bmax for the high-affinity site was greatest in the rat (0.68 pmol mg-1), lower in trout (0.19 pmol mg-1) and dogfish (0.07 pmol mg-1) and lowest in hagfish (0.01 pmol mg-1). The RyR Kd1 values for the high-affinity sites were comparable in all preparations (range 12-87 nmoll-1). The quantitative expression of RyRs in these species is consistent with the relative amount of SR present as indicated in physiological experiments and electron micrographs. Taking into consideration myocyte morphology of teleost and elasmobranch species, the data are consistent with a greater reliance on Ca2+ influx across the SL during E-C coupling in lower vertebrates, although a functional role for Ca2+ release from the SR in the more active species await further investigation.

Cloning and expression of salmon cardiac troponin C: titration of the low-affinity Ca(2+)-binding site using a tryptophan mutant.

Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

Effects of doxorubicin, 4'-epirubicin, and antioxidant enzymes on the contractility of isolated cardiomyocytes.

The purpose of this study was to determine the acute effects of doxorubicin and its less cardiotoxic epimer, 4'-epirubicin, on the contractile response of isolated myocytes, and to assess similarities or differences with respect to active oxygen-derived mechanisms. Calcium-tolerant myocytes from rat ventricle were field stimulated at 1.0 Hz, and the maximum extent of cell shortening, peak shortening velocity, and peak relaxation velocity of single twitches were measured by video edge detection. The contractile responses of the myocytes to the two anthracyclines were approximately equal. Exposure of the cells to 10 microM of either anthracycline for 20 min decreased all indices of contractility by 28% (p < 0.05). The active oxygen scavengers, superoxide dismutase and catalase, distinguished the extent to which active oxygen was involved in modifying cellular contractility. Paradoxically, superoxide dismutase alone (10 U/mL) decreased contractility by 21%. Nevertheless, superoxide dismutase (10 U/mL) prevented the decreases in contractility produced by doxorubicin. In contrast, superoxide dismutase only mildly (32%) protected against 4'-epirubicin. Catalase (10 U/mL), however, provided substantial (82-93%) protection against both anthracyclines. Hydrogen peroxide therefore, and presumably hydroxyl radicals, were involved in mediating the decreases in contractility from both doxorubicin and 4'-epirubicin. These results show that an acute exposure to clinically relevant concentrations of these anthracyclines significantly depresses myocyte contractility and that, in this respect, 4'-epirubicin is as potentially cardiotoxic as doxorubicin. The results with antioxidant enzymes also strongly support a free radical mechanism for the toxicity of doxorubicin and 4'-epirubicin to cardiomyocytes.

cGMP elevation does not mediate muscarinic agonist-induced negative inotropy in rat ventricular cardiomyocytes.

Guanosine 3',5'-cyclic monophosphate (cGMP) has been suggested to be involved in the negative inotropic effects of muscarinic receptor agonists in beta-adrenergic receptor agonist-stimulated ventricular preparations. To test this hypothesis, changes in contractility induced by acetylcholine or carbachol, or the nitrovasodilator sodium nitroprusside (SNP), in the presence of 1 nM isoproterenol were measured in electrically stimulated rat ventricular cardiomyocytes. In parallel experiments, cardiomyocytes were treated with the same agonists, and cGMP and adenosine 3',5'-cyclic monophosphate (cAMP) levels were estimated. After 2 min, isoproterenol increased the magnitude of cell shortening by 60% and accelerated contraction and relaxation rates. Carbachol (1 and 10 microM) and acetylcholine (1 and 10 microM) inhibited the positive inotropic effects of isoproterenol, whereas SNP (10 and 100 microM) had no inotropic effect. All three agents increased cGMP levels but had no effect on isoproterenol-stimulated cAMP levels. SNP caused the largest elevations in cGMP. These results suggest that the negative inotropic effects of muscarinic agonists observed in isoproterenol-stimulated rat ventricular cardiomyocytes are not mediated by alterations in cGMP and/or cAMP levels.

Temperature and pH effects on Ca2+ sensitivity of cardiac myofibrils: a comparison of trout with mammals.

Active salmonids maintain myocardial contractility at temperatures that are cardioplegic for mammals. We postulated that myofibrillar Ca2+ sensitivity in the trout heart might 1) exhibit lower temperature dependence and/or 2) be greater over the range of physiological temperatures. Temperature-induced changes in intracellular pH may also play a role as alkalosis typically increases calcium affinity of myofibrillar adenosinetriphosphatase (ATPase). Ca2+ sensitivities of ventricular myofibrillar ATPase were determined in rats and in rainbow trout (Oncorhynchus mykiss) over a physiological range of pH and temperatures. Maximal myofibrillar ATPase activities of each species were similar and equally affected by temperature. Trout myofibrillar ATPase lost Ca2+ dependence at 37 degrees C. At constant pH, reduced temperature decreased calcium affinity more in trout (0.35 pCa/10 degrees C) than in rat (0.08-0.16 pCa/10 degrees C). Under alpha-stat conditions, the effects of temperature were reduced in both trout (0.2 pCa/10 degrees C) and rat (no significant effect). Over trout physiological temperatures, Ca2+ sensitivity was greater than rat at 37 degrees C. Qualitatively similar results were observed in studies measuring tension in skinned trout ventricular fibers. One mechanism by which the trout heart is able to maintain contractility at low temperatures is through the inherent higher Ca2+ sensitivity of the contractile element compared with mammalian species.

Cellular functions of diabetic cardiomyocytes: contractility, rapid-cooling contracture, and ryanodine binding.

To study the mechanisms of cardiac dysfunction in experimental diabetes, adult rat cardiomyocyte shortening (measured with a video edge-detector system), the sarcoplasmic reticulum (SR) Ca2+ content [assessed by rapid-cooling contracture (RCC) and caffeine contracture (CC)] was examined. Ryanodine binding to the SR Ca(2+)-release channel of myocardium homogenate was also studied. Myocytes from diabetic rats showed depressed shortening (44% decrease compared with controls), reduced maximum rates of shortening and relengthening (58 and 56% decrease, respectively), and prolonged time to peak shortening (47% increase). RCCs and CCs from diabetic cells were 68 and 75% of the control values, respectively. Most of these cardiomyocyte abnormalities were corrected by daily insulin treatment in the diabetic rats. Ryanodine binding parameters indicated that the number of high-affinity binding sites was decreased in diabetic hearts. These data suggest that changes in contractile parameters as measured in diabetic myocytes are in good agreement with data obtained from intact heart or cardiac tissue preparations. Decreased SR Ca2+ content and reduced ryanodine binding sites indicate that the SR functions of storage and release of Ca2+ were depressed. This consequently may cause depressed contraction in diabetic hearts.

Temperature dependence of unitary properties of an ATP-dependent potassium channel in cardiac myocytes.

The temperature dependence of the properties of unitary currents in cultured rat ventricular myocytes has been studied. Currents flowing through an ATP-dependent K+ channel were recorded from inside-out patches with the bath temperature varied from 10 degrees to 30 degrees C. The channel conductance was 56 pS at room temperature (22 degrees C), and the amplitudes of unitary currents and the channel conductance exhibited a relatively weak (Q10 from 1.4 to 1.6) dependence on temperature. The temperature dependence of channel mean open times was biphasic with the low temperature (10-20 degrees C) range showing a relatively stronger temperature dependence (Q10 of 2.3) than the high temperature (20-30 degrees C) range (Q10 of 1.6). The activation energies for the two regions were determined from an Arrhenius plot with the activation energy, corresponding to the lower temperature range, near 16 kcal/mol. Thermodynamic analysis, using transition rate theory, indicated that the formation of a transition state prior to channel closure to be associated with a positive entropy component for the high Q10 region.

Characterization of myocardial Na(+)-Ca2+ exchange in rainbow trout.

This study compared Na(+)-Ca2+ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na(+)-Ca2+ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na(+)-Ca2+ exchange is stimulated approximately 200% after 30-min incubation with 10 micrograms/ml chymotrypsin at 21 degrees C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na(+)-Ca2+ exchange. While canine heart Na(+)-Ca2+ exchange exhibits a Q10 of greater than 2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of approximately 1.2. The absolute rates of Na(+)-Ca2+ exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7 degrees C. Furthermore, the temperature insensitivity of trout myocardial Na(+)-Ca2+ exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na(+)-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na(+)-Ca2+ exchanger with a similar pattern of bands (70, 120, and 160 kDa). Furthermore, a cDNA probe from canine Na(+)-Ca2+ exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na(+)-Ca2+ exchange exist between trout and mammalian hearts, the molecular basis is not yet known.

Regulation of myocardial contractility.

This symposium focuses on recent developments in the cellular and molecular basis of myocardial contractility. This topic is an essential element in modern exercise biology as both the acute and chronic responses to exercise are dependent on these mechanisms. The papers in this symposium reflect knowledge garnered from advances over the past decade in a variety of fields including: molecular biology, tissue culture (isolation of functional myocytes), organic chemistry (fluorescent Ca2+ indicators), and electrophysiology (patch clamp technology). In cardiac muscle, contractility is regulated essentially in two ways: the intracellular Ca2+ transient and the response of the myofilaments to that transient. This symposium addresses both of these mechanisms in some detail. The response of the myofilaments can be altered through both covalent modification (e.g., phosphorylation) of various contractile proteins and noncovalent (e.g., change in pH) mechanisms. The [Ca2+]i can increase from about 100 nM in diastole to the low microM range in systole. The peak [Ca2+]i observed in systole can vary several-fold owing to an orchestrated modulation of several different Ca2+ transport proteins on both the sarcolemma and sarcoplasmic reticulum. This modulation is quite distinct from that observed in skeletal muscles and underscores the physiological differences in these two types of striated muscle in varying contractile force. These proteins include: the L-type voltage-dependent Ca2+ channel and the Na+/Ca2+ exchanger in the sarcolemma, and the Ca2+ release channel and Ca2+ pump in the sarcoplasmic reticulum. Each of these proteins is subject to complex regulation by a variety of modulators, and aspects of this regulation are discussed in detail.

Ca2+ transport in myocardial sarcolemma from rainbow trout.

Sacrolemmal vesicles were isolated from trout ventricles with a yield of 0.51 mg protein/g wet wt of a fraction enriched approximately 15-fold over the crude homogenate as estimated by K(+)-stimulated p-nitrophenylphosphatase (K(+)-pNPPase) activity. Although the K(+)-pNPPase specific activity compared favorably with that of the rat heart, there were some striking differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and specific phospholipid content (mumol/mg protein) of the sacrolemmal fractions between the two species. Two major sarcolemmal Ca2(+)-transport proteins were investigated, the Na(+)-Ca2+ exchanger and the dihydropyridine (DHP) receptor, a component of the voltage-dependent L-type Ca2+ channel. From the initial rates of Na(+)-dependent Ca2+ uptake, it was determined that the exchanger has an apparent Km for Ca2+ of 14 +/- 1 microM and a maximum velocity of 7.7 +/- 1.1 nmol.mg protein-1.s-1 at 21 degrees C. Experiments using the DHP ligand [3H] (+) PN 200-110 to characterize the equilibrium binding to the DHP receptor in the sarcolemmal fraction yielded a Kd of 0.08 nM and maximum binding sites of 3.06 +/- 0.49 pmol/mg protein. Given the smaller dimensions of the trout myocyte and the resultant higher sarcolemmal surface to cytosolic volume compared with the mammalian myocyte, these in vitro findings are consistent with the notion that Ca2+ transport across sarcolemma is a quantitatively important contributor of Ca2+ delivery to and removal from the contractile element.

Effects of active oxygen generated by DTT/Fe2+ on cardiac Na+/Ca2+ exchange and membrane permeability to Ca2+.

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37 degrees C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 microM FeSO4. Exposure of the vesicles for 1 to 40 mins stimulated Na+/Ca2+ exchange about 2.5-fold. The DTT/Fe2+ treatment decreased the apparent Km for Ca2+ of Nai+-dependent Ca2+ uptake by 80% (from 63 to 13 microM). The effect on Vmax was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT/Fe2+ on the exchanger. Neither Zn2+ nor Ga3+ could replace Fe2+ in the stimulation of Na+/Ca2+ exchange. Passive Ca2+ efflux was determined by first allowing Na+/Ca2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca2+ leakage from the vesicles was slightly but significantly (P less than 0.05) increased by the action of DTT/Fe2+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na+/Ca2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the Km for Ca2+ prior to treatment.

Ca2+ transport across the plasma membrane of striated muscle.

In both types of striated muscle (skeletal and cardiac), calcium flux across the plasma membrane (sarcolemma) is regulated by at least three distinct membrane proteins; a voltage-dependent Ca2+ channel, Ca2+ pump (Ca2+ ATPase), and the Na+/Ca2+ antiporter. Each of these proteins is subject to regulation by intracellular second messengers. The magnitude and the role of this transsarcolemmal calcium flux are quite different between cardiac and skeletal muscle. In cardiac muscle, the influx is large, precedes, and is obligatory for contraction. There is general agreement that this influx is the trigger for Ca2+ release from the sarcoplasmic reticulum (SR) in the heart according to the Ca2+-induced Ca2+ release hypothesis. Variations in the transsarcolemmal Ca2+ influx have a profound effect on the strength of cardiac contraction, and it appears that this is the primary physiological strategy for regulation of contractility. In skeletal muscle, on the other hand, the T-tubules represent the richest source of dihydropyridine (DHP)-sensitive calcium channels known to exist, yet the influx of Ca2+ is proportionally much smaller and a significant portion enters the fiber following the twitch. While the majority of the Ca2+ influx is twitch dependent, it is quite clear that contraction in skeletal muscle is not predicated on this influx. It has been proposed that these DHP channels act as voltage sensors in order to initiate release of SR Ca2+; however, the link between the sensors and the opening of the SR Ca2+ (ryanodine-sensitive) channel is unknown. Transsarcolemmal Ca2+ transport appears to be subject to intense regulation to modify the acute response and demonstrates some plasticity in the adaptation to chronic perturbations.

Na+-Ca2+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise.

The high activity of the cardiac Na+-Ca2+ exchanger has led to the suggestion that it plays an important role in the regulation of myocardial contractility. We have proposed that exercise training increases stroke volume as a consequence of an enhanced contractility caused by an adaptation in Ca2+ transport across the cardiac plasma membrane (sarcolemma). The present study examined the possibility that the Na+-Ca2+ exchanger in heart muscle is modified in response to training. Sprague-Dawley rats (female, n = 72) were randomly divided into exercise-trained (T) and sedentary control (C) groups. As a result of the 11-wk treadmill-training paradigm, group T had a 7.6% higher (P less than 0.005) heart-to-body weight ratio and a 36% increase (P less than 0.01) in gastrocnemius mitochondrial enzyme activity. Na+-Ca2+ exchange was studied in highly purified sarcolemmal vesicles using rapid-quenching techniques. The absolute initial rate of uptake was significantly higher in T vs. C at calcium concentrations [( Ca2+]) ranging from 10 to 80 microM. This increased uptake appears to be due solely to the fact that the apparent Km of the myocardial Na+-Ca2+ exchanger for Ca2+ was significantly lower in T vs. C (15.7 +/- 1.1 vs. 36.1 +/- 2.6 microM), since the maximum velocity was unchanged. The observed increase in the affinity of the exchanger for Ca2+ is not attributable to group differences in vesicular purity, cross-contamination, or passive Ca2+ efflux. This observation is consistent with observed alterations in sarcolemmal composition in response to exercise training. We propose that the modification of the Na+-Ca2+ exchanger may play an important role in the adaptation of the heart to exercise.