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CARDIOVASCULAR
Dipartimento di Biotecnologie e Bioscienze, Università Milano-Bicocca, Milano, Italy (M.R., M.A., G.M., C.A., R.C., A.Z.); and Prassis Sigma-Tau Research Institute, Settimo Milanese, Italy (P.B., R.M., P.F.)
Received for publication
March 3, 2008
Accepted
June 4, 2008.
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Abstract |
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). The two actions are simultaneously exerted by the compound PST2744 (istaroxime), whose therapeutic index (inotropy/proarrhythmia) largely exceeds the one of digoxin in single-cell and whole-animal studies (Micheletti et al., 2002; Rocchetti et al., 2003). The favorable therapeutic profile of istaroxime has been confirmed in animal models of heart failure (Mattera et al., 2007; Sabbah et al., 2007) and in man (Ghali et al., 2007). However, whether this can still be attributed to SERCA2 stimulation is an open question.
Dysfunction of the sarcoplasmic reticulum (SR) is a key feature in myocardial remodeling and is considered as a central mechanism in a wide spectrum of hypertrophy/failure etiologies. Such functional impairment has been variably attributed to down-regulation of SERCA2 protein transcription and/or to an increase in the inhibitory (unphosphorylated) form of phospholamban (PLB) (Bers, 2006). Thus, the expression and conformation of the molecular target of istaroxime may be changed in the failing myocardium, with unknown consequences on its effect. At a more general level, the question is how molecular remodeling may affect the response of drugs acting through SERCA2 modulation.
The present study aims to test whether istaroxime is capable of stimulating SR Ca2+ uptake also in the presence of cardiac hypertrophy/failure. To this end, modulation of Ca2+ handling by istaroxime was tested in an experimental model of cardiac dysfunction in which chronic pressure overload was induced by aortic constriction in the guinea pig. The results obtained show that SR impairment can be largely reversed by pharmacological means in this model. This leads to a "functional" interpretation of SERCA2 abnormality, potentially relevant to the therapy of contractile dysfunction. Such an interpretation suggests that istaroxime may act by preventing the interaction between SERCA and PLB. To obtain a preliminary evaluation of this hypothesis, we also tested istaroxime effect on SERCA activity in skeletal muscle microsomes devoid of PLB. This collateral observation is reported in the supplemental data.
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Materials and Methods |
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) and to the guidelines for animal care endorsed by the hosting institution.
Aortic Banding Model
Chronic pressure overload was induced in guinea pigs after banding of the ascending aorta (AoB) under ketamine (100 mg/kg)-xylazine (5 mg/kg) i.p. Sham-operated littermates (sham) were used as controls.
Myocyte Preparation and Recording Solutions
Guinea pigs were killed by cervical dislocation under ketamine-xylazine anesthesia 12 weeks after AoB. Cardiac hypertrophy/heart failure was evaluated through the heart weight/body weight (HW/BW) and lung weight/body weight (LW/BW) ratios. Ventricular myocytes were isolated by using a retrograde coronary perfusion method previously published (Zaza et al., 1998), with minor modifications. Rod-shaped, Ca2+-tolerant myocytes were used within 12 h from dissociation.
During measurements, myocytes were superfused at 2 ml/min with Tyrode's solution containing 154 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES-NaOH, and 5.5 mM D-glucose, adjusted to pH 7.35. The pipette solution contained 110 mM K+-aspartate, 23 mM KCl, 0.2 mM CaCl2 (calculated free Ca2+ = 10-7 M), 3 mM MgCl2, 5 mM HEPES KOH, 0.5 mM EGTA KOH, 0.4 mM GTP-Na salt, 5 mM ATP-Na salt, and 5 mM creatine phosphate Na salt, pH 7.3. Tyrode and pipette solutions were modified for the SR reloading protocol, as detailed in Experimental Protocols (below). A thermostated manifold, allowing for a fast (electronically timed) solution switch, was used for cell superfusion. All measurements were performed at 35 ± 0.5°C. Throughout the present study, istaroxime was tested at the concentration of 4 µM, corresponding to the steep portion of the concentration-response curve for inotropy (Micheletti et al., 2002) and shown to be effective on SR function of normal myocytes of the same species (Rocchetti et al., 2005).
Electrophysiology Techniques
Ventricular myocytes were voltage-clamped in the whole-cell configuration (Axopatch 200-A; Molecular Devices, Sunnyvale, CA). Membrane capacitance (Cm) and series resistance were measured in every cell but left uncompensated; the average values of series resistance in sham and AoB experiments were 5.1 ± 0.2 (n = 48) and 5.0 ± 0.2 (n = 63) (N.S.) M
, respectively. Current signals were filtered at 2 kHz and digitized at 5 kHz (Axon Digidata 1200). Trace acquisition and analysis was controlled by dedicated software (Axon pClamp 8.0). Guinea pig ventricular myocytes do not express Ito (Zicha et al., 2003); thus, peak inward current measured upon depolarizations from a holding potential of -40 mV (INa fully inactivated) essentially reflects Ca2+ influx through ICaL and Na+/Ca2+ exchanger current (INCX); this was confirmed in preliminary experiments (see Supplemental Fig. 1). It is fair to stress that inward current, albeit adequate to calculate Ca2+-induced Ca2+ release (CICR) gain (see below), cannot be assumed to reflect ICaL. Accurate measurement of ICaL requires series resistance compensation, estimation of time-dependent run-down, and intracellular K+ substitution by Cs+, none of which was implemented in the present experiments. In particular, K+ substitution by Cs+ was avoided because it affects SR function (Kawai et al., 1998), the main object of this study.
Measurements of Intracellular Ca2+
Single-myocyte intracellular Ca2+ activity was measured fluorometrically using the membrane-permeable dye Indo1-AM (8 µM; Invitrogen, Carlsbad, CA) as described previously. Indo1-AM fluorescent emission was measured at two wavelengths (410 and 490 nm) (Grynkiewicz et al., 1985). The signals at the two wavelengths (F410 and F490) were separately low-pass filtered (200 Hz) and digitized at 2 kHz. Cytosolic Ca2+ activity was calculated from the F410/F490 ratio after low-pass digital filtering (FFT, 100 Hz) and subtraction of the background luminescence. Conversion of F410/F490 ratio to free cytosolic Ca2+ concentration (Caf) was performed as described by Sipido and Callewaert (1995) after dye calibration in ionomycin permeabilized myocytes (Rocchetti et al., 2005).
Measurement of SERCA Activity in SR Microsomes
The methods for this set of experiments are reported in the supplemental data, where the relevant results are also presented.
Experimental Protocols
Protocol 1 (Caffeine Pulse Protocol). Transmembrane current (holding potential -80 mV) and cytosolic Ca2+ were simultaneously recorded (in Tyrode's solution) during a 5-s caffeine pulse (10 mM) applied 10 s after a loading train of voltage steps (-40 to 0 mV, 200 ms, 0.37 Hz).
Protocol 2 (SR Reloading Protocol). The Na+/Ca2+ exchanger (NCX) was inhibited by 30-min cell incubation in an Na+- and Ca2+-free solution (replaced by equimolar Li+ and 1 mM EGTA). SR was initially depleted by a brief caffeine pulse (with 154 mM Na+ to allow Ca2+ extrusion through the NCX) and then progressively reloaded by a train of depolarizing pulses (-40 to 0 mV, 200 ms, 0.25 Hz) in the presence of 1 mM Ca2. The pipette solution was Na+-free (Na+ salts were replaced by K+ or Tris salts).
Estimation of Functional Parameters
Total SR Ca2+ content (CaSRT; in micromoles per liter of cytosolic volume) was estimated by integrating the INCX elicited by the caffeine pulse (protocol 1) and dividing the nanomoles of Ca2+ by the estimated cell volume (Cm/6.44) (Bers, 2002d) (Table 1). INCX was defined as the transient component of caffeine-induced membrane current; thus, the steady-state current present during caffeine superfusion (pedestal) was subtracted before integration.
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The NCX function was evaluated by plotting INCX as a function of Caf during caffeine pulses (protocol 1). The slope of this relation was obtained by linear interpolation of the points in the final third of Ca2+ transient relaxation, when bulk cytosolic Caf values more closely reflect subsarcolemmal ones (Bers, 2002c) (cells in which the INCX/Caf relation was entirely nonlinear were not used for this analysis). The steady-state value of Caf measured at holding potential just before caffeine application will be referred to as "resting" Ca2+ (Carest).
The Ca2+ uptake function of SR (SERCA2 uptake flux minus leak flux) was dynamically tested by the SR reloading protocol (protocol 2). The rate of SR reloading was determined from the increment of Ca2+ transient amplitude in subsequent voltage steps delivered after caffeine-induced depletion. The time constant of Ca2+ transient relaxation (
decay), reflecting the rate of net SR Ca2+ uptake, was measured during each step of the reloading process by monoexponential fit of the Ca2+ transient decay. In consideration of the dependence of SERCA2 activity from cytosolic Ca2+ (Bers and Berlin, 1995), decay was also plotted as a function of peak Caf achieved during each step (see Supplemental Fig. 2).
The amplification factor in CICR gain was calculated according to two methods. In the first one, peak amplitude of Ca2+ transient was divided by "peak inward current"; in the second one, the maximum velocity of Ca2+ rise (dCa/dtmax) was divided by peak inward current. Both methods use peak inward current in lieu of Ca2+ influx, thus yielding a value in arbitrary units (Bers, 2002b). This approach was preferred because an absolute estimate of CICR gain was beyond the aims of this study, and the peak value of inward current may more accurately reflect Ca2+ influx under the present experimental conditions. It should be stressed that, because Ca2+ release and Ca2+ influx are linearly related (Bers, 2002b), their ratio is independent of their absolute value.
Substances
Stock Indo1-AM solution (1 mM in dry dimethyl sulfoxide) was diluted in Tyrode's solution. Istaroxime was dissolved in water. Istaroxime (PST2744; chemical structure in Micheletti et al., 2002; Rocchetti et al., 2003) was synthesized at Prassis Sigma-Tau (Settimo Milanese, Italy), Indo-1AM was from Molecular Probes, and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Statistical Analysis
Sham and AoB conditions are represented by distinct experimental groups; to minimize the effect of intersubject variability, data were collected from more than five animals in each condition. Sham and AoB animals were studied in alternate sequence. Individual means were compared by paired or unpaired Student's t test as appropriate; in the SR loading protocol, differences were tested by two-way ANOVA, applied to either absolute values or istaroxime-induced changes. Statistical significance was defined as p < 0.05 (N.S., not significant). The least-square method was used for linear and nonlinear fitting and parameter estimation. Data are expressed as mean ± S.E. of independent determinations; the coefficient of variation (CV) was calculated as the ratio between S.D. and mean. Sample size (number of cells) is specified for each experimental condition in the tables and figure legends.
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Results |
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Functional Characterization of the AoB Model. This section of results compares myocardial function of animals 12 weeks after AoB with that of sham of the same gender. HW/BW, LW/BW, and Cm of shams and AoB animals are compared in Table 2. HW/BW was significantly increased by AoB. There was a tendency to increase the LW/BW ratio in AoB. Because of the large scatter, this change did not achieve statistical significance; however, in 25% of AoB animals, the LW/BW ratio was more than 2-fold the average of sham animals. Cm was significantly larger after AoB to indicate an increase in cell size. Within the study period, mortality was null in both sham and AoB groups. As shown in Fig. 1, CaSRT was decreased by approximately 32% after AoB (p < 0.05 versus sham) (Fig. 1B).
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During the SR reloading protocol, the parameters of Ca2+ transients (amplitude and decay) and the CICR gain changed over subsequent depolarizing pulses (Fig. 2), reflecting a progressive increase in the SR Ca2+ content. Thus, the analysis of the time course of these parameters provides information on the SR Ca2+ uptake function (Rocchetti et al., 2005). After AoB, the time courses of Ca2+ transient amplitude and CICR gain were markedly slowed compared with sham animals; decay was uniformly increased over the whole reloading train (Fig. 2B). Differences between sham and AoB myocytes in the time course of all variables were significant (p < 0.05), as tested by two-way ANOVA. The change in decay was evident also when compared at similar cytosolic Ca2+ concentrations (measured at the beginning of the decay of Ca2+ transient; see Supplemental Fig. 2). The changes in Ca2+ transient amplitude occurring during the loading protocol and between sham and AoB myocytes (Fig. 2A) are accompanied by changes in amplitude and inactivation rate of inward current, as expected from Ca2+-dependent inactivation of ICaL (Lee et al., 1985).
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In contrast to the marked depression of SR function detected by the reloading protocol (protocol 2), during steady-state stimulation in normal Tyrode (protocol 1) the amplitudes of V-induced Ca2+ transients (111.2 ± 11 versus 106.8 ± 9 nM, N.S.), their dCa/dtmax (12.7 ± 1.3 versus 13.0 ± 1.0 nM/ms, N.S.), and peak inward current (-4.44 ± 0.32 versus -3.99 ± 0.36 nA, N.S.) were similar between sham and AoB myocytes. Accordingly, CICR gain was unchanged between the two conditions, independently of the method used for its evaluation (Fig. 3). The slope of the INCX/Caf relation and Carest were unchanged by AoB (Fig. 4).
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; Micheletti et al., 2007). Istaroxime increased SR Ca2+ content by 79.2 ± 21.1% (p < 0.05 versus control; Fig. 5). Stimulation of SR Ca2+ uptake function by istaroxime was also evident during the SR reloading protocol, in which NCX contribution was absent (see Materials and Methods, protocol 2). The rate of change of Ca2+ transient amplitude and CICR gain during the reloading process was increased, and decay was shortened by the drug (Fig. 6). Differences between baseline and istaroxime superfusion in the time course of all variables were significant (p < 0.05), as tested by two-way ANOVA. Consistently with stimulation of SR uptake function, istaroxime increased CICR gain measured under normal Tyrode superfusion (functioning NCX; Fig. 7); istaroxime also increased the slope of the INCX/Caf relationship by 93.3 ± 35% (p < 0.05 versus control; Fig. 8) and Carest by 41.3 ± 9.9% (p < 0.05 versus control; Fig. 8).
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decay and CICR gain), istaroxime effect, although highly significant in both groups, was not significantly different between sham and AoB myocytes, probably because of the larger scatter of values. The absolute SR performance achieved under istaroxime in AoB myocytes approached that observed in sham myocytes (Fig. 6B) to indicate substantial recovery of AoB-induced dysfunction.
CICR gain (measured by protocol 1) was similarly increased by istaroxime in AoB and sham myocytes (Fig. 7). The slope of INCX/Caf relationship (+173 ± 89%) and Carest (+29 ± 5%) were also increased by istaroxime; the effect was again similar between sham and AoB myocytes (Fig. 8).
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Discussion |
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); the present ex vivo observations (Table 2) are substantially in agreement with in vivo ones. HW/BW and Cm were consistently increased after AoB, thus reflecting clear-cut myocardial hypertrophy in all cases. Lung congestion was present in a minority of cases. The absence of mortality in the AoB groups rules out the possibility that the hearts used for cell studies may come from a surviving subpopulation, i.e., one with particularly mild abnormalities. Cardiac decompensation observed in the present work was mild compared with that observed in guinea pigs studied up to 8 weeks after banding of descending aorta (Kiss et al., 1995; Ahmmed et al., 2000).
The main functional derangement observed in myocytes from AoB animals concerned SR Ca2+ uptake function. This was manifested by a marked depression of SR reloading (after caffeine-induced depletion) (Fig. 2). In contrast, reduction in SR Ca2+ content was relatively milder (-32%; Fig. 1), and CICR gain during steady-state stimulation under normal Tyrode superfusion was unchanged (Fig. 3). Considering that SR reloading was measured starting from very low cell Ca2+ content and during NCX blockade, the discrepancy might suggest that the derangement in SR uptake function may be unveiled by low Ca2+ content and partially compensated by NCX function. The observation that the reduction of SR Ca2+ content was not associated with a decrease in CICR gain might suggest that the ryanodine receptor (RyR) sensitivity to cytosolic Ca2+ was increased in AoB myocytes, as commonly observed in the failing heart (Yano et al., 2005).
Abnormalities of the SR uptake function, as those detected in this study, can be due to reduced SERCA2 activity or to increased Ca2+ leak through RyR channels. Previous biochemical evaluations showed that maximal ATPase activity of SERCA2 is decreased in this model, despite normal expression of SERCA2 protein (Micheletti et al., 2007). Reduced SERCA2 activity might result from an increase in the unphosphorylated (monomeric) form of PLB, previously described in this model (Micheletti et al., 2007). In this case, SERCA2 abnormality would be functional, rather than structural, a view also supported by the effect of istaroxime discussed below. This pattern differs from that more often described in hypertrophy, in which a decrease in SERCA2 expression contributes to down-regulation of SR Ca2+ transport (Bers, 2006).
Ca2+ dependence of INCX was unchanged after AoB (Fig. 4). This finding is apparently at variance with up-regulation of NCX protein expression and enhancement of INCX reported in human heart failure (Studer et al., 1994; Pieske et al., 1999) and in hypertrophy models in various species, including guinea pig (Ahmmed et al., 2000). Although NCX protein expression was not evaluated in the present work, the functional observations are not necessarily in contrast with previous ones in the same species. In the work on guinea pig by Ahmmed et al. (2000), INCX was measured upon repolarization after long depolarizing steps (tail current). An increase in INCX tail current can be because of either genuine up-regulation of NCX function or simply because of an increase in cytosolic Ca2+ levels achieved during the depolarizing step (Barcenas-Ruiz et al., 1987). Under the conditions of the study by Ahmmed et al. (CICR suppression), the latter may be justified in hypertrophied myocytes by depressed SR Ca2+ uptake. In the present experiments, NCX function was defined through the relationship between INCX and cytosolic Ca2+, thus correcting for differences in cytosolic Ca2+ level. Nevertheless, in species other than guinea pigs, the same analysis detected an increase of NCX function in hypertrophy (Díaz et al., 2004); thus, it is difficult to rule out that differences in NCX function between this and previous studies may be real and possibly related to the severity of hemodynamic overload.
Istaroxime Effects in Sham and AoB Myocytes. In myocytes from sham-operated animals istaroxime improved Ca2+ handling, as previously reported in normal hearts of the same species (Rocchetti et al., 2005). Under ionic conditions in which all Ca2+ handling mechanisms were operative (normal Tyrode), istaroxime increased total SR Ca2+ content (Fig. 5), which implies a shift of the balance between Ca2+ uptake by SR and Ca2+ extrusion from the cell. In turn, SR Ca2+ uptake rate reflects the balance between active Ca2+ transport (by SERCA2) and passive Ca2+ leak through RyR channels, both fluxes being enhanced by high cytosolic Ca2+ (Meissner and Henderson, 1987; Shannon et al., 2000). As shown by previous studies in intact myocytes and isolated SR vesicles, istaroxime actions include inhibition of the Na+/K+ pump and stimulation of SERCA2 ATPase activity (Rocchetti et al., 2003, 2005) (also see the supplemental data). Although both these actions may concur to increase SR Ca2+ content, the former depends on the change in NCX electrochemical equilibrium, secondary to elevation of cytosolic Na+. When tested under conditions of complete inhibition of NCX function (Na+-free conditions), istaroxime was still able to stimulate SR Ca2+ uptake, as reflected by an increase in the rate at which SR reloads after depletion and by an acceleration of Ca2+ decay after voltage-induced transients (Fig. 6). These observations suggest that istaroxime-induced increase in SR Ca2+ content may also occur through SERCA2 stimulation, i.e., independently of Na+/K+ pump inhibition. Istaroxime also increased the efficacy by which Ca2+ influx triggers SR Ca2+ release (CICR gain) (Fig. 7), probably an effect secondary to the increase in luminal SR Ca2+ (Xu and Meissner, 1998; Rocchetti et al., 2005). Istaroxime increased both the x-axis intercept (Caf at INCX = 0) and the slope of the INCX/Caf relationship (Fig. 8). The intercept change may result from an increase in cytosolic Na+ and was expected from istaroxime effect on the Na+/K+ pump (Rocchetti et al., 2003). According to previous evidence, istaroxime does not directly stimulate NCX (Rocchetti et al., 2003); thus, the increase in the slope of the INCX/Caf relationship is more likely because of allosteric modulation of the exchanger by elevated Caf (Weber et al., 2001). The net effect of these two changes is a reduction in the rate of Ca2+ extrusion through NCX at resting membrane potential (-80 mV).
The rather severe SR dysfunction observed after AoB was almost completely reversed by istaroxime (Figs. 5 and 6). This implies that the dysfunction was exclusively functional and is consistent with the lack of SERCA2 protein down-regulation in this model (Micheletti et al., 2007).
Istaroxime Effect in Skeletal versus Cardiac SR Microsomes. The ability of istaroxime to recover the SR abnormality in AoB myocytes suggests that this agent may interfere with SERCA modulation by PLB. This is also consistent with the finding that istaroxime stimulates SERCA2 in cardiac microsomes from healthy guinea pig by increasing its affinity for cytosolic Ca2+ (Rocchetti et al., 2005; Micheletti et al., 2007) (see also supplemental data and Supplemental Fig. 4), which is limited by the interaction with PLB (Waggoner et al., 2007).
The observation reported in the supplemental data that istaroxime was unable to increase SERCA activity in skeletal muscle microsomes, which are naturally devoid of PLB, provides a preliminary support to this view (Supplemental Figs. 3 and 4). Albeit suggestive, this observation may not be conclusive and further experiments with a different strategy may be required to confirm the hypothesis.
Practical Implications. The majority of evidence available to date, mostly from studies in transgenic animals, identifies recovery of SR Ca2+ uptake function as a promising therapeutic strategy in heart failure (Schmidt et al., 2001; Haghighi et al., 2004); however, adverse effects have also been reported (Chen et al., 2004; Vangheluwe et al., 2006). The net outcome of this approach probably depends on the extent of SR uptake enhancement, which may be difficult to adjust if gene therapy is used. Under this aspect, availability of pharmacological tools for modulation of SR function would be highly desirable; however, it is unclear whether deranged SR function can be recovered by pharmacological means. Previous studies in animal models (Mattera et al., 2007; Micheletti et al., 2007; Sabbah et al., 2007) and man (Ghali et al., 2007) showed that the positive inotropic effect of istaroxime is retained in the failing myocardium, but it was unknown whether stimulation of SR Ca2+ uptake could still contribute to it. The present results not only prove that this is the case but show that almost complete recovery of failing SR uptake function might be achieved by pharmacological means. In addition to contractility, SR function may also affect myocardial electrical stability. Istaroxime is also a Na+/K+ pump inhibitor, but it is definitely less proarrhythmic than digoxin in animal studies (Micheletti et al., 2002; Rocchetti et al., 2003), and preliminary clinical evidence corroborates this finding (Ferrari et al., 2007; Ghali et al., 2007). The electrophysiological actions of the two substances have been thoroughly compared (Rocchetti et al., 2003), and the only mechanism found to account for the different arrhythmogenicity is stimulation of SR Ca2+ uptake (Rocchetti et al., 2005). This suggests that contractile recovery is not the only goal that may be achieved by modulation of SR function. The mechanism by which SR stimulation by istaroxime improves electrical stability is currently under evaluation.
Study Limitations. In the specific hypertrophy model used by this study, decreased SERCA2 activity occurs in the presence of normal SERCA2 protein expression (Micheletti et al., 2007), and this may represent a prerequisite for functional recovery by pharmacological means. This might prevent full extrapolation of the present findings to conditions in which SERCA2 expression is reduced. Nevertheless, a decrease in the ratio between SERCA2 and (unphosphorylated) PLB is a general feature of the failing myocardium (Haghighi et al., 2004), thus suggesting that functional down-regulation may have a general role in SR dysfunction. Thus, significant, even if incomplete, recovery might theoretically be achieved by pharmacological means in most cases.
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Acknowledgements |
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Footnotes |
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Parts of this work were presented in abstract form as follows: Rocchetti M, Alemanni M, Micheletti R, Ferrari P, and Zaza A (2007) Istaroxime modulation of sarcoplasmic reticulum function in cardiac hypertrophy/failure. In Winter Meeting on Translational Basic Science of the Heart Failure Association; 2007 Jan 24–27; Garmisch-Parten Kirchen, Germany.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: SERCA, sarco(endo)plasmic reticulum Ca2+ ATPase; PST2744, (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride); SR, sarcoplasmic reticulum; PLB, phospholamban; AoB, aortic banding; HW/BW, heart weight/body weight ratio; LW/BW, lung weight/body weight ratio; Cm, membrane electrical capacity; ICaL, L-type Ca2+ current; INCX, Na+/Ca2+ exchanger current; CICR, Ca2+-induced Ca2+ release; Caf, free cytosolic Ca2+ concentration; NCX, Na+/Ca2+ exchanger; CaSRT, total sarcoplasmic reticulum Ca2+ content; Carest, resting Ca2+ at -80 mV; decay, time constant of Ca2+ transient relaxation; dCa/dtmax, maximum velocity of Ca2+ rise; ANOVA, analysis of variance; CV, coefficient(s) of variation; RyR, ryanodine receptor; Im, membrane current; a.u., arbitrary unit(s); Lcyt, liters of cytosol.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Antonio Zaza, Dipartimento di Biotecnologie e Bioscienze, Università degli Studi Milano-Bicocca, P.zza della Scienza 2, 20126 Milano, Italy. E-mail: antonio.zaza{at}unimib.it
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) and AoB (
) myocytes; recordings were performed in the absence of NCX function (Na+-free Tyrode and pipette solutions). B, average values of Ca2+ transient parameters measured during each pulse (1–6) of the stimulation train in sham (
) effect on the caffeine-induced INCX (holding potential, -80 mV) and the corresponding cumulative INCX integrals in a sham and AoB myocyte. C, IST effect (
% increase of CaSRT) as a function of CaSRT level measured in control (cont, 
, n = 18); groups were pooled together]; continuous line represents the best exponential fit of the experimental data.
