5'-Terminal deletions occur in coxsackievirus B3 during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negative-strand viral RNA.

Adult human enteroviral heart disease is often associated with the detection of enteroviral RNA in cardiac muscle tissue in the absence of infectious virus. Passage of coxsackievirus B3 (CVB3) in adult murine cardiomyocytes produced CVB3 that was noncytolytic in HeLa cells. Detectable but noncytopathic CVB3 was also isolated from hearts of mice inoculated with CVB3. Sequence analysis revealed five classes of CVB3 genomes with 5' termini containing 7, 12, 17, 30, and 49 nucleotide deletions. Structural changes (assayed by chemical modification) in cloned, terminally deleted 5'-nontranslated regions were confined to the cloverleaf domain and localized within the region of the deletion, leaving key functional elements of the RNA intact. Transfection of CVB3 cDNA clones with the 5'-terminal deletions into HeLa cells generated noncytolytic virus (CVB3/TD) which was neutralized by anti-CVB3 serum. Encapsidated negative-strand viral RNA was detected using CsCl-purified CVB3/TD virions, although no negative-strand virion RNA was detected in similarly treated parental CVB3 virions. The viral protein VPg was detected on CVB3/TD virion RNA molecules which terminate in 5' CG or 5' AG. Detection of viral RNA in mouse hearts from 1 week to over 5 months postinoculation with CVB3/TD demonstrated that CVB3/TD virus strains replicate and persist in vivo. These studies describe a naturally occurring genomic alteration to an enteroviral genome associated with long-term viral persistence.

Anti-oxidant effects of estrogen reduce [Ca2+]i during metabolic inhibition.

We previously reported that 17beta-estradiol (betaE2) inhibits the rise in [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in mouse cardiomyocytes, but the mechanism has not yet been clarified. Estrogen has been reported to have anti-oxidant properties. We, therefore, have investigated whether interaction with the estrogen receptor (ER) is involved, or whether estrogen reduces free-radical-induced impairment of Na(+)-K(+) ATPase in cardiac myocytes, and whether this effect reduces [Ca(2+)](i) rise. Male mouse ventricular myocytes were studied. Flow cytometry was used with fluo-3 for [Ca(2+)](i) measurement. Dead cells were excluded from analysis by propidium iodide fluorescence. betaE2 reduced the increase in [Ca(2+)](i) during MI even in the presence of the ER blocker tamoxifen. A similar effect on [Ca(2+)](i) was produced by its non-estrogenic isomer, betaE2-estradiol. Other hormones (estrone and estriol) with a phenolic structure also inhibited Ca(2+) overload during MI, but testosterone without the structure did not. The betaE2 effect was attenuated by inhibition of Na(+)-Ca(2+) exchanger (KB-R7943) or Na(+)-K(+) ATPase (low K(+) or ouabain), but not by block of L-type Ca(2+) channel (nifedipine). Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), a superoxide scavenger, decreased the rise in [Ca(2+)](i) and abolished the betaE2 effect during MI. We conclude that the acute cardioprotective effect of estrogen during MI may be mediated by an ER-independent anti-oxidant action, which results in improved function of Na(+)-K(+) ATPase.

Myocyte function and [Ca 2+ ]i homeostasis during early allogenic heart transplant rejection.

BACKGROUND: Recent studies from our laboratory have demonstrated that in vivo contractile function of rejecting mouse heterotopic abdominal heart allografts 5 days after transplantation is depressed to 40% of that of syngenic controls, and that this depression of function is prevented by the nitric oxide synthase (NOS) inhibitor NG-monomethyl-l-arginine. However, the mechanisms of altered myocyte function caused by nitric oxide production in this setting are not established. METHODS: We measured intracellular calcium concentration ([Ca2+]i) transients (fluo-3, confocal microscopy), fractional shortening (video motion), and L-type Ca2+ currents (whole-cell patch clamp) 5 days after transplantation in ventricular myocytes freshly isolated from syngenic (Balb/C into Balb/C) and allogenic (Balb/C into C3H) transplants. RESULTS: L-type Ca2+ currents, [Ca2+]i transient amplitudes, and fractional shortening did not differ between nonrejecting, syngenic and rejecting, allogenic transplants. Catecholamine responsiveness as analyzed by the change in the peak [Ca2+]i transient induced by 100 nM isoproterenol was also similar. Superfusion with l-arginine, an NOS substrate, caused decreased shortening with no change in [Ca2+]i transients in allogenic myocytes, but had no effect in syngenic myocytes. CONCLUSIONS: Depressed contractile function of rejecting allogenic heart transplants in vivo appears to be caused in part by an NOS-dependent decrease in myofilament Ca2+ sensitivity.

Activation of connexin-43 hemichannels can elevate [Ca(2+)]i and [Na(+)]i in rabbit ventricular myocytes during metabolic inhibition.

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.

Gender influences [Ca(2+)](i) during metabolic inhibition in myocytes overexpressing the Na(+)-Ca(2+) exchanger.

BACKGROUND: The Na(+)-Ca(2+) exchanger (NCX) may contribute to Ca(2+) overload and injury in ischemic cardiomyocytes. Recently, NCX overexpression was reported to increase ischemia/reperfusion injury in male and oophorectomized female but not in female mice. We therefore measured the effects of gender and estrogen on [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in myocytes from wild-type (WT) and transgenic (TG) mice overexpressing NCX. METHODS AND RESULTS: Flow cytometry was used with fluo 3 for [Ca(2+)](i) and sodium green for [Na(+)](i) measurements. Male TG mouse myocytes had higher [Ca(2+)](i) after 30 minutes of MI (1086+/-160 nmol/L, n=8) than male WT (688+/-104 nmol/L, n=9, P=0.01). The increase in [Ca(2+)](i) during MI induced by NCX overexpression in female myocytes was not significant, however (TG 552+/-62 nmol/L, n=9; WT 426+/-44 nmol/L, n=7). The magnitude of rise in [Ca(2+)](i) during MI was greater in male than female myocytes. KB-R7943, an NCX inhibitor, abolished the effect of NCX overexpression but did not totally eliminate the effect of gender on [Ca(2+)](i) during MI. NCX current density and basal Na(+) pump function were not influenced by gender. The rise in [Na(+)](i) during MI was greater in male than in female myocytes. Estrogen attenuated the increase in [Ca(2+)](i) and [Na(+)](i) in male myocytes during MI and abolished the gender difference in [Na(+)](i) during MI. CONCLUSIONS: Increased expression of NCX results in a more marked rise in [Ca(2+)](i) during MI in male than in female mouse myocytes. This gender difference appears to be mediated in part by an inhibitory effect of estrogen on the rise in [Na(+)](i), an NCX modifier, during MI.

A novel contractile phenotype with cardiac transgenic expression of the human P2X4 receptor.

The P2X4 receptor is a newly identified receptor expressed in the heart cell. Its function was elucidated with cardiac transgenic (TG) expression of the receptor by using the myocardium-specific a-myosin heavy chain promoter. The presence of the transgene was determined by polymerase chain reaction by using primers specific to the receptor and the vector linker region, by Southern blotting of the genomic DNA, and by immunoblotting and immunohistochemistry of both isolated cardiac myocytes and intact hearts. In intact heart study, the P2X4 receptor TG mouse exhibited significantly elevated basal cardiac contractility with greater rates of contraction and relaxation, left ventricular developed pressure, and cardiac output compared with nontransgenic (NTG) animals but showed no evidence of hypertrophy or heart failure. The TG heart also showed a greater increase of cardiac contractility in response to the P2X receptor agonist 2-methylthioATP, consistent with overexpression of a functional P2X4 receptor with consequent increase in the receptor-mediated response. In isolated cardiac cell study, the TG heart cell showed a similar level of basal contraction amplitude as the NTG heart cell while exhibiting a threefold greater increase in contractility during stimulation by 2-methylthioATP. Thus, an increased responsiveness of the overexpressed P2X4 receptor to endogenous ATP is responsible for the enhanced basal cardiac performance in the intact TG heart. The sustained enhanced contractile function with no associated heart pathology in the P2X4 receptor TG mouse suggests a novel physiologic role of the P2X4 receptor, that of stimulating the cardiac contractility.

Functional importance and caffeine sensitivity of ryanodine receptors in primary lymphocytes.

Calcium signaling patterns are important for the specific regulation of activation and effector function in lymphocytes. Studies of [Ca2+]i regulation in lymphocytes, including the involvement of ryanodine receptors (RyR) and the importance of caffeine-sensitive pools, have been carried out mainly in lymphocyte cell lines and the presence and functional importance of these pools in primary lymphocytes has not been addressed. Here we show by confocal microscopy that caffeine caused a prompt but transitory increase of [Ca2+]i in primary lymphocytes, an effect that was inhibited by pre-treatment with ryanodine. Furthermore, the increase of [Ca2+]i in CD4+ and CD8+ MLR T lymphocytes stimulated by 5 microg/ml concanavalin A was significantly inhibited by pretreatment with caffeine. In functional studies, caffeine decreased cytotoxicity against myocyte target cells which is probably related to an altered calcium signaling in CD8+ MLR lymphocytes. Caffeine also terminated spontaneous Ca2+ oscillations and induced a rise in [Ca2+]i in CD4- and CD8- MLR lymphocytes probably of B cell origin. These results demonstrate that caffeine alters Ca2+ signaling in primary lymphocytes, and suggest that RyR, probably the skeletal muscle receptor (RyR-1) and brain receptor (RyR-3), are involved in mediating this effect. It is also possible that blocking of inositol-1,4,5-triphosphate (IP3) receptors is involved in the effects of caffeine on lymphocyte activation.

Effects of deletion of muscle LIM protein on myocyte function.

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes.

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.

Contractile reserve and intracellular calcium regulation in mouse myocytes from normal and hypertrophied failing hearts.

Mouse myocyte contractility and the changes induced by pressure overload are not fully understood. We studied contractile reserve in isolated left ventricular myocytes from mice with ascending aortic stenosis (AS) during compensatory hypertrophy (4-week AS) and the later stage of early failure (7-week AS) and from control mice. Myocyte contraction and [Ca(2+)](i) transients with fluo-3 were measured simultaneously. At baseline (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C), the amplitude of myocyte shortening and peak-systolic [Ca(2+)](i) in 7-week AS were not different from those of controls, whereas contraction, relaxation, and the decline of [Ca(2+)](i) transients were slower. In response to the challenge of high [Ca(2+)](o), fractional cell shortening was severely depressed with reduced peak-systolic [Ca(2+)](i) in 7-week AS compared with controls. In response to rapid pacing stimulation, cell shortening and peak-systolic [Ca(2+)](i) increased in controls, but this response was depressed in 7-week AS. In contrast, the responses to both challenge with high [Ca(2+)](o) and rapid pacing in 4-week AS were similar to those of controls. Although protein levels of Na(+)-Ca(2+) exchanger were increased in both 4-week and 7-week AS, the ratio of SR Ca(2+)-ATPase to phospholamban protein levels was depressed in 7-week AS compared with controls but not in 4-week AS. This was associated with an impaired capacity to increase sarcoplasmic reticulum Ca(2+) load during high work states in 7-week AS myocytes. In hypertrophied failing mouse myocytes, depressed contractile reserve is related to an impaired augmentation of systolic [Ca(2+)](i) and SR Ca(2+) load and simulates findings in human failing myocytes.

Cardiac unloading alters contractility and calcium homeostasis in ventricular myocytes.

Altered cardiac workload has an important effect on myocyte structure and function. Cardiac hypertrophy resulting from an increase in load has been studied extensively in the past. However, the effects of unloading and atrophy have recently become of more interest since devices for mechanical left ventricular unloading have been introduced into clinical practice for the treatment of patients with terminal heart failure, and a resulting improved cardiac and myocyte contractility have been reported. We used the heterotopic abdominal mouse heart transplant model in order to study the effects of 5 days of unloading on cell size (confocal microscopy), contractility (fractional shortening: video motion), calcium homeostasis ([Ca(2+)](i)transients, SR Ca(2+)content); and L-type Ca(2+)and sodium/calcium exchanger currents (whole cell patch clamp technique). We found unloading caused decreased cell volume consistent with atrophy. An increased fractional shortening and [Ca(2+)](i)transient were observed in myocytes from unloaded hearts as compared with controls. Transsarcolemmal I(Ca,L)and I(Na/Ca)densities, and SR Ca(2+)content were unaltered, as was membrane capacitance. A reduction in cell volume with mainteinance of internal and surface membrane areas, and/or a decrease in concentration of cellular protein Ca(2+)buffers, may contribute to the increase in the [Ca(2+)](i)transient in this model.

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes.

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.

Formation of planar and spiral Ca2+ waves in isolated cardiac myocytes.

A novel Nipkow-type confocal microscope was applied to image spontaneously propagating Ca2+ waves in isolated rat ventricular myocytes by means of fluo-3. The sarcolemma was imaged with di-8-ANEPPS and the nucleus with SYTO 11. Full frame images in different vertical sections were obtained at video frame rate by means of an intensified CCD camera. Three types of Ca2+ waves were identified: spherical waves, planar waves, and spiral waves. Both spherical waves and spiral waves could initiate a planar wave, and planar waves were not influenced by the presence of a nucleus. Spiral waves, however, were consistently found adjacent to a nucleus and displayed a slower propagation rate and slower rate of increase in Ca2+ concentration in the wave front than did spherical and planar waves. The planar waves were apparent throughout the vertical axis of the cell, whereas spiral waves appeared to have a vertical height of approximately 3 microm, less than the maximum thickness of the nucleus (5.0 +/- 0.3 microm). These results provide experimental confirmation of previous modeling studies which predicted an influence of the nucleus on spiral-type Ca2+ waves. When a spontaneous Ca2+ wave is small relative to the size of the nucleus, it appears that the Ca2+ buffering by the nucleus is sufficient to slow the rate of spontaneous propagation of the Ca2+ wave in close proximity to the nucleus. These findings thus support the idea that the nucleus can influence complex behavior of Ca2+ waves in isolated cardiac myocytes.

Quantitation of Na/Ca exchanger function in single ventricular myocytes.

A Na/Ca exchange current can be elicited in voltage clamped single ventricular myocytes by the abrupt removal of extracellular Na+ by means of a rapid switcher device. We measured this reverse Na/Ca exchange current in isolated mouse ventricular myocytes from wild-type mice, and from transgenic mice with hearts overexpressing the Na/Ca exchanger. In mouse ventricular myocytes, the current was sensitive to nickel, and was eliminated by removal of intracellular Na+. It was not influenced by 3 m m ouabain, and thus not contaminated by Na pump currents. The magnitude of the current reached a plateau within 10-15 min after obtaining a whole cell patch with the pipettes containing EGTA, to buffer [Ca2+]i and in zero extracellular K+ concentration to completely inhibit the Na pump, and allow equilibration of pipette Na+ with subsarcolemmal [Na+]. The magnitude of the current increased with increases in pipette [Na+]. Comparison of the current magnitudes in wild-type and transgenic myocytes showed a 2.5 and 2.7 fold increase in the current in transgenic myocytes at pipette [Na+] of 10 and 20 m m. The magnitude of this increase in Na/Ca exchanger currents in single transgenic myocytes compares well with the reported 2.5 fold increase in Na+-dependent 45Ca2+ uptake measured in ventricular sarcolemmal vesicles obtained from transgenic animals. With this approach, we found variation in exchanger current densities in different species, with values for mouse>rat>rabbit>dog>human. This technique should also be useful in quantifying changes in Na/Ca exchanger current density as a consequence of pathologic processes, and exposure to drugs.

Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts.

In this study we examined Na+/H+ exchange activity, Ca2+ transients, and contractility in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Myocytes from infarcted hearts (post-MI myocytes) were isolated from the peri-infarcted region of the left ventricle. Intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured with the fluorescent pH indicators seminaphthorhodafluor 1 and fluo 3, respectively, and contractility was assessed from changes in cell shortening during field stimulation. Experiments were performed at extracellular pH 7. 4 in the presence and absence (HEPES buffer) of CO2 and HCO-3. Our findings demonstrate that 1) myocytes after myocardial infarction (post-MI) were significantly larger than normal, 2) post-MI hypertrophy was not accompanied by changes in non-CO2 intracellular buffering power, 3) post-MI hypertrophy did not significantly affect the ability of Na+/H+ exchange to mediate pHi recovery from intracellular acidosis, 4) the stimulatory effect of ANG II (100 nM) on Na+/H+ exchange was significantly reduced in post-MI myocytes, 5) in HCO-3-buffered solutions, ANG II did not significantly stimulate pHi recovery from acidosis in post-MI myocytes, 6) the angiotensin AT1 receptor mediates the stimulatory action of ANG II on Na+/H+ exchange in normal and post-MI myocytes, and 7) the stimulatory effect of ANG II on the Ca2+ transient and contraction was blunted in post-MI myocytes bathed in HEPES-buffered solution. A suppressed ventricular responsiveness to ANG II may be beneficial in the intact myocardium by attenuating ATP consumption and by reducing intracellular Na+ accumulation during ischemia-reperfusion.

Influence of prior Na+ pump activity on pump and Na+/Ca2+ exchange currents in mouse ventricular myocytes.

We examined the dependence of peak Na+ pump and Na+/Ca2+ exchanger currents on prior Na+ pump inhibition induced by exposure to zero extracellular K+ in voltage-clamped adult murine ventricular myocytes. Abrupt activation of the Na+ pump by reexposure of myocytes to extracellular K+ with a rapid solution switcher resulted in the development of a transient peak current at approximately 500 ms, followed by a decline over 1-2 min to a steady-state level. The magnitudes of both the peak Na+ pump current (Ip) and the peak outward Na+/Ca2+ exchange current, activated by rapidly reducing extracellular Na+ to zero with the solution switcher, were dependent on previous Na+ pump activity. [Na+] gradients (Na+-binding benzofuran isophthalate fluorescence) between the patch pipette and the bulk cytosol were relatively small and could not account for the large differences between peak and steady-state Ip and reverse Na+/Ca2+ exchanger currents. Our results are consistent with the presence of a subsarcolemmal Na+ concentration gradient, which is similar for the Na+ pump and the Na+/Ca2+ exchanger. These findings also support the hypothesis that the Na+ pump and the Na+/Ca2+ exchanger are colocalized in the sarcolemma.

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure.

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+ concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current (ICa,L) and Na+/Ca2+ exchanger current (INa/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]i with the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]i transients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]i transients. ICa,L density was markedly decreased in PF myocytes. INa/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+ exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]i transient when the Na+/Ca2+ exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+ exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]i transients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.