Association of annexin A5 with Na+/Ca2+ exchanger and caveolin-3 in non-failing and failing human heart

Annexin A5 is a Ca2+ dependent phosphatidylserine binding protein mainly located in the T-tubules and sarcolemma of cardiomyocytes. Our objectives were to determine whether annexin A5 was associated with various protein(s) and whether such an association was modified in failing (F) hearts. The association between annexin A5 and the cardiac Na+/Ca2+ exchanger (NCX) was demonstrated by immunohistofluorescence, annexin A5-biotin overlay and co-immunoprecipitations (IPs) performed with microsomal preparations (MPs) from non-failing (NF) (n = 8) and F (dilated cardiomyopathy, n = 7) human hearts. We moreover found caveolin-3 in the immunoprecipitates, indicating the presence of multimolecular subsarcolemmal complexes. Surface plasmon resonance assays in NF MPs allowed us to demonstrate direct interaction between the NCX and caveolin-3 and immobilized annexin A5. Interaction was Ca2+-dependent and inhibited by the specific antibody. In addition, dissociation by zwittergent 3-14 (ZW 3-14) of the complexes from MPs increased specific interactions. In F hearts, specific interactions were blunted in native MPs but were fully recovered after treatment with ZW 3-14. In conclusion, we demonstrated that a direct interaction between annexin A5 and the cardiac NCX occurs in complexes including caveolin-3. In F hearts, despite the increase in the exchanger level, almost all of the NCX was involved in complexes. These interactions probably occurred in the intracytoplasmic regulatory loop of the exchanger, suggesting a different regulation of the exchanger in heart failure, consistent with a role in altered Ca2+ handling.     

Role of cyclic ADP-ribose in Ca2+-induced Ca2+ release and vasoconstriction in small renal arteries

Cyclic-ADP-ribose (cADPR) has been reported to serve as a second messenger to mobilize intracellular Ca2+ independent of IP3 in a variety of mammalian cells. This cADPR-mediated Ca2+ signaling pathway importantly participates in the regulation of various cell functions. The present study determined the role of endogenous cADPR in mediating ryanodine-sensitive Ca2+-induced Ca2+ release (CICR) in vascular myocytes from small renal arteries and vasomotor response of these arteries. In freshly-isolated renal arterial myocytes, addition of CaCl2 (0.01, 0.1, and 1 mM) into the Ca2+-free bath solution produced a rapid Ca2+ release response from the sarcoplasmic reticulum (SR), with a maximal increase of 237+/-25 nM at 1 mM CaCl2. This CaCl2 response was significantly blocked by a cell-membrane permeant cADPR antagonist, 8-bromo-cADP-ribose (8-br-cADPR) (30 microM) or ryanodine (50 microM). Caffeine, a classical CICR or ryanodine receptor activator was found to stimulate the SR Ca2+ release (Delta[Ca2+]i: 253+/-35 nM), which was also attenuated by 8-br-cADPR or ryanodine. Using isolated and pressurized small renal arteries bathed with Ca2+-free solution, both CaCl2 and caffeine-induced vasoconstrictions were significantly attenuated by either 8-br-cADPR or ryanodine. Biochemical analyses demonstrated that CaCl2 and caffeine did not increase cADPR production in these renal arterial myocytes, but confocal microscopy showed that a dissociation of the accessory protein, FK506 binding protein 12.6 (FKBP12.6) from ryanodine receptors was induced by CaCl2. We conclude that cADPR importantly contributes to CICR and vasomotor responses of small renal arteries through enhanced dissociation of ryanodine receptors from their accessory protein.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

Relative abundance of alpha2 Na(+) pump isoform influences Na(+)-Ca(2+) exchanger currents and Ca(2+) transients in mouse ventricular myocytes

In the mouse, genetic reduction in the Na(+), K(+)-ATPase alpha1 or alpha2 isoforms results in different functional phenotypes: heterozygous alpha2 isolated hearts are hypercontractile, whereas heterozygous alpha1 hearts are hypocontractile. We examined Na(+)/Ca(2+) exchange (NCX) currents in voltage clamped myocytes (pipette [Na(+)]=15 mM) induced by abrupt removal of extracellular Na(+). In wild-type (WT) myocytes, peak exchanger currents were 0.59+/-0.04 pA/pF (mean+/-S.E.M., n=10). In alpha1(+/-) myocytes (alpha2 isoform increased by 54%), NCX current was reduced to 0.33+/-0.05 (n=9, P<0.001) indicating a lower subsarcolemmal [Na(+)]. In alpha2(+/-) myocytes (alpha2 isoform reduced by 54%), the NCX current was increased to 0.89+/-0.11 (n=8, P=0.03). The peak sarcolemmal Na(+) pump currents activated by abrupt increase in [K(+)](o) to 4 mM in voltage clamped myocytes in which the Na(+) pump had been completely inhibited for 5 min by exposure to 0 [K(+)](o) were similar in alpha1(+/-) (0.86+/-0.12, n=10) and alpha2(+/-) myocytes (0.94+/-0.08 pA/pF, n=16), and were slightly but insignificantly reduced relative to WT (1.03+/-0.05, n=24). The fluo-3 [Ca(2+)](i) transient (F/F(o)) in WT myocytes paced at 0.5 Hz was 2.18+/-0.09, n=34, was increased in alpha2(+/-) myocytes (F/F(o)=2.56+/-0.14, n=24, P=0.02), and was decreased in alpha1(+/-) myocytes (F/F(o)=1.93+/-0.08, n=28, P<0.05). Thus the alpha2 isoform rather than the alpha1 appears to influence Na(+)/Ca(2+) exchanger currents [Ca(2+)](i) transients, and contractility. This finding is consistent with the proposal that alpha2 isoform of the Na pump preferentially alters [Na(+)] in a subsarcolemmal micro-domain adjacent to Na(+)/Ca(2+) exchanger molecules and SR Ca(2+) release sites.     

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).     

Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.

In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Extreme sarcoplasmic reticulum volume loss and compensatory T-tubule remodeling after Serca2 knockout

Cardiomyocyte contraction and relaxation are controlled by Ca(2+) handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca(2+) fluxes. Here we investigate whether altered Ca(2+) fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na(+)/Ca(2+)-exchanger proximal to ryanodine receptors in the SR but did not contain Ca(2+)-channels. Ca(2+) measurements demonstrated a switch from SR-driven to Ca(2+) influx-driven Ca(2+) transients in KO. Still, SR Ca(2+) release constituted 20% of the Ca(2+) transient in KO. Mathematical modeling suggested that Ca(2+) influx via Na(+)/Ca(2+)-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca(2+) release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.     

Calcium regulation of a slow post-spike hyperpolarization in vagal afferent neurons.

Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow) that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPslow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from <0.1 to >10 Hz. After a single action potential, the AHPslow in nodose neurons displays a slow rise time to peak (0.3-0.5 s) and a long duration (3-15 s). The slow kinetics of the AHPslow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ influx via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPslow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPslow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency adaptation in vagal afferent neurons.     

Functional relevance of the stretch-dependent slow force response in failing human myocardium

Stretch induces immediate and delayed inotropic effects in mammalian myocardium via distinct mechanosensitive pathways, but these effects are poorly characterized in human cardiac muscle. We tested the effects of stretch on immediate and delayed force response in failing human myocardium. Experiments were performed in muscle strips from 52 failing human hearts (37 degrees C, 1 Hz, bicarbonate buffer). Muscles were stretched from 88% of optimal length to 98% of optimal length. The resulting immediate and delayed (ie, slow force response [SFR]) increases in twitch force were assessed without and after blockade of the sarcoplasmic reticulum (SR; cyclopiazonic acid and ryanodine), stretch-activated ion channels (SACs; gadolinium, streptomycin), L-type Ca2+-channels (diltiazem), angiotensin II type-1 (AT1) receptors (candesartan), endothelin (ET) receptors (PD145065 or BQ123), Na+/H+ exchange (NHE1; HOE642), or reverse-mode Na+/Ca+ exchange (NCX; KB-R7493). We also tested the effects of stretch on SR Ca2+ load (rapid cooling contractures [RCCs]) and intracellular pH (in BCECF-loaded trabeculae). Stretch induced an immediate (<10 beats), followed by a slow (5 to 10 minutes), force response. Twitch force increased to 232+/-6% of prestretch value during the immediate phase, followed by a further increase to 279+/-8% during the SFR. RCC amplitude significantly increased, but pHi did not change during SFR. Inhibition of SACs, L-type Ca2+ channels, AT1 receptors, or ET receptors did not affect the stretch-dependent immediate or SFR. In contrast, the SFR was reduced by NHE1 inhibition and almost completely abolished by reverse-mode NCX inhibition or blockade of sarcoplasmic reticulum function. The data demonstrate the existence of a functionally relevant, SR-Ca2+-dependent SFR in failing human myocardium, which partly depends on NHE1 and reverse-mode NCX activation.     

Effects of Na(+)/Ca(2+)-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.     

Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex

OBJECTIVE: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. METHODS: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. RESULTS: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. CONCLUSION: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.     

Effects of adenovirus-mediated sorcin overexpression on excitation-contraction coupling in isolated rabbit cardiomyocytes

To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10+/-0.05% [n=311] versus 2.42+/-0.06% [n=312], respectively; P<0.001). Action potential duration (at 20 degrees C) was significantly less in the Ad-sorcin group (458+/-22 ms, n=11) compared with the control group (520+/-19 ms, n=10; P<0.05). In voltage-clamped, fura 2-loaded myocytes (20 degrees C), a reduced peak-systolic and end-diastolic [Ca2+]i was observed after Ad-sorcin transfection. L-type Ca2+ current amplitude and time course were unaffected. Caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+-Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+-extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. beta-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+-transient amplitude predominantly by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.     

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.     

Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes

Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivation of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by I(Ca) and deranged the smoothly graded I(Ca)-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.     

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.     

Stretch-dependent slow force response in isolated rabbit myocardium is Na+ dependent

OBJECTIVE: Stretch induces functional and trophic effects in mammalian myocardium via various signal transduction pathways. We tested stretch signal transduction on immediate and slow force response (SFR) in rabbit myocardium. METHODS: Experiments were performed in isolated right ventricular muscles from adult rabbit hearts (37 degrees C, 1 Hz stimulation rate, bicarbonate-buffer). Muscles were rapidly stretched from 88% of optimal length (L88) to near optimal length (L98) for functional analysis. The resulting immediate and slow increases in twitch force (first phase and SFR, respectively) were assessed at reduced [Na+]o or without and with blockade of stretch activated ion channels (SACs), angiotensin-II (AT1) receptors, endothelin-A (ET(A)) receptors, Na+/H+-exchange (NHE1), reverse mode Na+/Ca2+-exchange (NCX), or Na+/K+-ATPase. The effects of stretch on sarcoplasmic reticulum Ca2+-load were characterized using rapid cooling contractures (RCCs). Intracellular pH was measured in BCECF-AM loaded muscles, and action potential duration (APD) was assessed using floating electrodes. RESULTS: On average, force increased to 216+/-8% of the pre-stretch value during the immediate phase, followed by a further increase to 273+/-10% during the SFR (n=81). RCCs significantly increased during SFR, whereas pH and APD did not change. Neither inhibition of SACs, AT1, or ET(A) receptors affected the stretch-dependent immediate phase nor SFR. In contrast, SFR was reduced by NHE inhibition and almost completely abolished by reduced [Na+]o or inhibition of reverse-mode NCX, whereas increased SFR was seen after raising [Na+]i by Na+/K+-ATPase inhibition. CONCLUSIONS: The data demonstrate the existence of a delayed, Na+- and Ca2+-dependent but pH and APD independent SFR to stretch in rabbit myocardium. This inotropic response appears to be independent of autocrine/paracrine AT1 or ET(A) receptor activation, but mediated through stretch-induced activation of NHE and reverse mode NCX.     

Excitation-dependent intracellular Ca2+ waves at the border zone of the cryo-injured rat heart revealed by real-time confocal microscopy

Intracellular Ca2+ waves, which develop under Ca2+-overloaded conditions of the injured myocardium, are regarded as an important substrate for triggered arrhythmias. However, little is known about whether Ca2+ waves arise or become proarrhythmic in the injured heart in situ. On the hypothesis that injured myocardium manifests frequent Ca2+ waves and produce an oscillatory [Ca2+]i rise leading to triggered activity, we applied cryo-injury to the epicardial surface of fluo 3-AM-loaded perfused rat hearts and analyzed spatiotemporal [Ca2+]i changes at border zones of the injured myocardium using real-time confocal microscopy. In intact regions Ca2+ waves barely emerged, whereas the border zone myocardium exhibited frequent Ca2+ waves, propagating randomly within the individual cells. Two different types of Ca2+ waves were identified: highly frequent waves (159.6+/-86.5 waves/min/cell, n=266) adjacent to the cryo-ablated regions, and less frequent waves (79.0+/-50.1 waves/min/cell, n=160) slightly farther (>2 cells) away from the ablated regions (vicinities). The former Ca2+ waves emerged asynchronously to Ca2+ transients. Contrariwise, the latter depended on ventricular excitation: they vanished instantaneously on Ca2+ transients, but emerged more frequently and propagated more swiftly after cessation of higher-frequency pacing. Immediately after 3-Hz pacing, some cryo-injured hearts exhibited oscillatory [Ca2+]i rises; an instantaneous and synchronous elevation of [Ca2+]i followed by burst occurrence of Ca2+ waves with a gradual decrease in incidence and propagation velocity in a considerable number of cells. These observations indicate that myocardial injury induces Ca2+ waves in the heart, and that their synchronous occurrence could become a substrate for triggered arrhythmias.     

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.