AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 292: H1607-H1618, 2007. First published November 22, 2006; doi:10.1152/ajpheart.00525.2006
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Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Antonis A. Armoundas,1,* Jochen Rose,2,* Rajesh Aggarwal,3 Bruno D. Stuyvers,4 Brian O'Rourke,2 David A. Kass,2 Eduardo Marbán,2 Stephen R. Shorofsky,3 Gordon F. Tomaselli,2 and C. William Balke5

1Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts; 2Department of Medicine, Division of Molecular Cardiobiology, Johns Hopkins University, and 3Department of Medicine, Division of Cardiology, University of Maryland, Baltimore, Maryland; 4Department of Medicine, Division of Cardiology, University of Calgary, Calgary, Alberta, Canada; and 5Departments of Medicine and Physiology and Institute of Molecular Medicine, University of Kentucky College of Medicine, Lexington, Kentucky

Submitted 22 May 2006 ; accepted in final form 8 November 2006


   ABSTRACT
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 ABSTRACT
METHODS
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Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca2+ concentration ([Ca2+]i) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca2+ handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (ICa,L) density (–0.964 ± 0.172 vs. –0.745 ± 0.128 pA/pF at +10 mV) and Na+/Ca2+ exchanger (NCX) currents (2.1 ± 0.8 vs. 2.3 ± 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca2+]i was depressed (at +10 mV, 0.72 ± 0.07 and 0.56 ± 0.04 µM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca2+ spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca2+ in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the {alpha}1C-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). {alpha}1C protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca2+ uptake and release synergistically contribute to the depressed [Ca2+]i and the altered AP profile phenotype.

calcium; ion channels; sarcoplasmic reticulum; pacing tachycardia

THE REDUCED CONTRACTILITY and altered repolarization of the failing human heart and many animal models of heart failure (HF) are, in part, the result of abnormal intracellular Ca2+ cycling (4, 34, 35, 57, 58, 66). However, despite extensive investigation, the precise molecular mechanisms responsible for altered Ca2+ handling in the failing heart remain controversial. Although there is a general consensus that sarcoplasmic reticulum (SR) Ca2+ handling is abnormal in HF, some studies suggest that these abnormalities are primarily the result of the abnormal density/function of Ca2+ uptake and extrusion mechanisms [i.e., sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a and Na+/Ca2+ exchanger (NCX)] (34, 66), while others attribute these abnormalities primarily to alterations in SR Ca2+ release [via the SR Ca2+ release channel, ryanodine receptor (RyR)] (57, 58, 75). Furthermore, while the different animal models of HF exhibit a similar phenotype reminiscent of human HF, emerging information supports the notion that the common phenotype of HF may represent a final common pathway for unique model (or injury)-specific molecular phenotypes.

The pacing-induced HF model is characterized by abnormal beta-adrenergic receptor signaling (46) and elevated renin-angiotensin-aldosterone stimulation (56) that result in chamber dilation with markedly elevated diastolic pressures, depressed systolic function (83), and increased apoptosis (48). Thus we hypothesized that in a pacing-induced HF model the altered autonomic tone results in abnormal SR Ca2+ release and related defects in SR Ca2+ uptake as the primary mechanism of abnormal Ca2+ homeostasis. To better understand the mechanisms responsible for abnormal Ca2+ homeostasis and cellular electrophysiology, we studied the L-type Ca2+ (ICa,L) and NCX currents, whole cell intracellular Ca2+ concentration ([Ca2+]i) transients, local Ca2+ transients (i.e., Ca2+ sparks), and mRNA and protein levels of the major Ca2+-handling molecules in the rabbit pacing-induced HF model.

To examine the hypothesis that the failing myocyte phenotype, manifested by depressed Ca2+ transients and potentially arrhythmogenic prolonged action potential (AP) duration (APD), is the result of a single mechanism versus a multiplicity of mechanisms, we used a comprehensive approach to isolate and study the molecular and functional phenotype of potentially abnormal Ca2+ homeostatic processes, involving the primary Ca2+ entry, release, uptake, and extrusion mechanisms in myocytes from failing hearts.


   METHODS
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Rabbit pacing-induced HF model. Nine New Zealand White rabbits of either sex underwent sterile implantation of a bipolar pacemaker. Rabbits were anesthetized with intravenous thiopental sodium, intubated, and volume ventilated. A laparotomy was performed, and the diaphragm and the pericardium were opened to expose the heart. Two custom-made pacing wires were placed in the apex of the heart. A VVI pacemaker (Minix 8340 or Thera SR 8962, Medtronic) was inserted into a pocket formed between the abdominal muscles. Rapid pacing was maintained by permanently attaching a magnet to the posterior surface of the pacemaker pulse generator. Animals were allowed to recover from surgery for 3–4 days, after which pacing was initiated at 400 beats/min for 2–4 wk. Heart failure was verified by transthoracic echocardiography. After 2.5 ± 0.7 wk of pacing, the left ventricular end-diastolic diameter increased (normal 13 ± 3 mm vs. failing 18 ± 3 mm; P < 0.05), left ventricular end-diastolic pressure increased (normal 2.0 ± 3 mmHg vs. failing 19.0 ± 7.1 mmHg; P < 0.05), systolic fractional shortening (in % of end-diastolic diameter) decreased (normal 46 ± 8% vs. failing 21 ± 7%; P < 0.05), and the rate of pressure rise decreased (normal 6,900 ± 1,620 mmHg/s vs. failing 3,600 ± 270 mmHg/s; P < 0.05), consistent with severe HF (35). All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.

Isolation of ventricular myocytes. Isolated myocytes were obtained from failing and control left ventricles by enzymatic dissociation as previously described (34, 35). The apex (1–3 g) was excised and immediately frozen in liquid nitrogen for later protein and mRNA analysis. Only Ca2+-tolerant rod-shaped myocytes with clear cross striations and without spontaneous contractions or signification granulation were selected for experiments.

Solutions. The external solution for AP measurements contained (in mmol/l) 138 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 0.33 NaH2PO4, and 10 HEPES (pH 7.4 with NaOH). The pipette solution for recording APs contained (in mmol/l) 140 KCl, 5 NaCl, 1 MgCl2, 10 HEPES, 2 EGTA, and 4 MgATP (pH 7.4 with KOH).

For the measurement of ICa,L and [Ca2+]i,the extracellular solution contained (in mmol/l) 140 NaCl, 10 dextrose, 10 HEPES, 10 CsCl, 1 MgCl2, and 1 CaCl2 (pH adjusted to 7.4 with NaOH). The intracellular solution contained (in mmol/l) 130 Cs-glutamate, 10 HEPES, 0.33 MgCl2, 4 Mg2ATP, and 0.05 indo-1 (Molecular Probes, Eugene, OR), and the pH was adjusted to 7.2 with CsOH.

For the measurement of NCX current, the extracellular solution contained (in mmol/l) 140 NaCl, 2 CaCl2, 2 MgCl2, 5 HEPES, 10 glucose (pH 7.4, adjusted by NaOH). In addition, Na+-K+-ATPase, K+ channels, and Ca2+ channels were blocked with 20 µmol/l ouabain, 1 mmol/l BaCl2, and 1 µmol/l nifedipine, respectively. The electrogenic NCX current was inhibited by addition of 5 mmol/l Ni2+. The pipette solution contained (in mmol/l) 146 CsOH, 20 NaCl, 42 L-aspartic acid, 3 MgCl2, 10 HEPES, 20 TEA, 21 CaCl2, 42 EGTA, and 10 MgATP (pH 7.4, adjusted by CsOH). The free Ca2+ concentration was 67 nmol/l (18).

[Ca2+]i in the presence and absence of thapsigargin were measured in an extracellular solution containing (in mmol/l) 138 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 0.33 NaH2PO4, and 10 glucose (pH 7.4, adjusted by NaOH). The pipette solution contained (in mmol/l) 130 K glutamate, 9 KCl, 10 NaCl, 0.5 MgCl2, and 5 MgATP (pH 7.2, adjusted by KOH) with 80 µmol/l indo-1.

Electrophysiological recording techniques. The whole cell configuration of the patch-clamp technique was used. Myocytes were transferred to the stage of an inverted microscope and superfused with external solution at a rate of 1–2 ml/min. All recordings were performed at 37°C. Whole cell currents were recorded with an Axopatch 200A amplifier (Axon Instruments), low-pass filtered at 5 kHz, and digitized at 10 kHz via a Digidata 1200 A/D (Axon Instruments) interface for off-line analysis. Myocyte capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 10-mV depolarizing pulse from a holding potential of –80 mV.

A xenon arc lamp was used to excite indo-1 fluorescence at 365 nm, and the ratio (R = F405nm/F495nm) of the emitted fluorescence after subtraction of cellular autofluorescence was used to calculate free [Ca2+]i according to the equation [Ca2+]i = Kd x beta[(R – Rmin)/(Rmax – R)], where beta is ratio of free to bound indo-1 fluorescence at 495 nm, using a Kd of 844 nmol/l, as reported for rabbit cardiomyocytes (7). The average Rmin, Rmax, and beta for the fluorescence system were determined to be 0.4 ± 0.4, 9.8 ± 2.9, and 7.8 ± 3.2 (n = 6), respectively, as previously described (66). The rate of Ca2+ removal ({tau}Ca) was determined by fitting a single exponential to the [Ca2+]i time course 20 ms after return to the holding potential.

Local SR Ca2+ release events, Ca2+ sparks. Spontaneous Ca2+ sparks were recorded with the fluorescence of the Ca2+ indicator fluo-3 and a custom-built laser scanning confocal microscope (82). The bath solution was a modified Tyrode solution containing (in mmol/l) 135 NaCl, 4 CsCl, 0.33 NaHPO4, 1 MgSO4, 10 HEPES, 10 glucose, and 1 CaCl2, with pH 7.3. Myocytes were loaded with the membrane-permeant form of the Ca2+ indicator fluo-3 AM (Molecular Probes), by incubating the myocytes for 30 min at 21–23°C in the bath solution containing fluo-3 AM (10 µmol/l) and pluronic acid (0.05% wt/vol). Fluo-3 was excited with an argon laser at a wavelength of 488 nm. The fluorescence emission was measured at wavelengths >515 nm.

The lateral and axial spatial resolutions of the confocal system equipped with a x63 1.4-numerical aperture oil immersion objective lens were 0.25 and 0.52 µm. Line scan images were created from a series of 512 lines with 256 pixels per line, 0.1 µm per pixel, and 3.0 ms per line (including 0.5-ms reset time per scan); the pixel dwell times were 10.0 µs. The scan line positions were selected to be in the middle of the myocyte, away from the edges and avoiding the nucleus. Sparks were sampled in rabbit ventricular myocytes according to the protocol used by Satoh et al. (71). Briefly, myocytes were field stimulated every 6 s. Ca2+ sparks were determined from the line scan images and sampled during the resting period following the overall Ca2+ transient (see GoGoFig. 3A). Amplitude, duration, and width (spread in the axial direction) were determined for each Ca2+ spark. Data from control animals were compared with those from failing animals, using the Mann-Whitney test for comparison of nonnormal distributions. The line scan images were analyzed with custom-made IDL routines.


Figure 1
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  		Fig. 1. Stimulation frequency-dependent differences in the action potential (AP) duration (APD) and membrane resting potential of ventricular myocytes isolated from control and failing ventricles [control: no. of animals (Nc) = 12, no. of myocytes (nc) = 18; failing: no. of animals (Nf) = 5, no. of myocytes (nf) = 11]. A: APD at 50% repolarization (APD50). B: APD at 90% repolarization (APD90). There is an overall prolongation of APD of myocytes isolated from failing compared with control hearts. In Figs. 1, 2, and 4–7 circles represent data from control and squares from failing hearts. Values are means ± SD. *P < 0.05 vs. control.

 

Figure 2
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  		Fig. 2. Records of representative intracellular Ca2+ concentration ([Ca2+]i) transients (indo-1 fluorescence ratios of 405 to 485 nm) from control and failing hearts (A), elicited by voltage steps to –40, –20, 0, 20, and 40 mV from a holding voltage of –80 mV. B: summary Ca2+ transient data (*P < 0.05 failing vs. control). C: peak current-voltage (I-V) relationships for L-type Ca2+ currents. Values are means ± SD.

 

Figure 3
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  		Fig. 3. Local sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) in rabbit ventricular myocytes from control and failing animals. A: 3-dimensional representation of spatiotemporal variations of [Ca2+]i along a 25-µm line scan during ~9 s. Each frame represents a series of 500 successive scans (3 ms/scan; 1.5 s/frame); the number of scans was translated into a horizontal timescale (horizontal arrow). Ca2+ sparks were sampled during the resting period (frames 1, 2, and 3) following the stimulated twitch (frame 0). Variations of the ratio of peak to background fluorescence intensities (F/Fo) reflected the local variations of [Ca2+]i as indicated by the pseudocolor bar (see text). B: representative sparks in cells isolated from control (left) and failing (right) hearts. C: distribution of the amplitudes of the Ca2+ sparks as reflected in the ratio between peak and background. Ca2+ sparks from failing animals are smaller than Ca2+ sparks from control animals (P < 0.0001, Mann-Whitney test of medians). D: comparison of the frequency distribution of spark amplitudes in myocytes from control and failing hearts showed that the 2 distributions were not statistically different (total no. of observations was 474 and 289 in myocytes from control and failing hearts, respectively).

 
mRNA measurements. The DNA fragments used to generate rabbit-specific riboprobes were amplified from reverse-transcribed total rabbit ventricular RNA with the polymerase chain reaction (PCR). The PCR products were cloned into pCR2.1 (Invitrogen) and, if necessary, subcloned into the EcoRI site of pSP70 (Promega). All constructs were confirmed by DNA sequencing. The Na+ channel template was designed to protect a fragment in the I–II linker of the Na+ channel that is unique to the cardiac isoform.

The rabbit cardiac Na+ channel (rabNav1.5) probe spans nucleotides 1655 to 1801 (146 bp), and NCX (rabNCX) spans nucleotides 443 to 585 (142 bp) (Table 1). For better discrimination of the fragment sizes, the probes were cut at the unique restriction sides (NgomI for rabNav1.5, BsaI for rabNCX), resulting in protected sequences of 78 and 116 bp for rabNav1.5 and rabNCX, respectively.


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  		Table 1. Riboprobe amplification and RT-PCR primers and probes

 
Ribonuclease protection assays (RPAs) were performed as previously described (39). Total RNA was prepared with TRIzol reagent (GIBCO) according to the manufacturer's instructions. The integrity of all RNA samples was confirmed by analysis on a denaturing agarose gel and quantified by optical density measurements at 260 nm. Determinations were performed in duplicate on 10 µg of RNA from each ventricular sample.

Steady-state mRNA levels were quantified by exposing the gels on a storage phosphor screen and then scanning on a phosphoimager (Molecular Dynamics); quantification of the transcript levels was performed with ImageQuant software (Molecular Dynamics). The level of the rabNCX gene expression is given as the relative density of the protected fragment normalized to the density of the control protected fragment (rabNav1.5), the cardiac isoform of the Na+ channel, thereby normalizing for both RNA loading and the fraction of the sample that is cardiac myocytes.

Fluorescence-based kinetic real-time PCR was performed, using a Perkin-Elmer Applied Biosystems model 7900 sequence-detection system to quantify mRNA levels as previously described (14). Total RNA was isolated from the rabbit ventricle with Qiagen RNeasy with on-column DNase digestion. Each reporter signal was then divided by the fluorescence of an internal reference dye (ROX) to normalize for non-PCR-related fluorescence and to 18S rRNA to normalize for loading. All primers and probes are shown in Table 1. The level of gene expression was normalized to a reference sample to permit comparison among samples.

Protein measurements. Quantification of SERCA2a, phospholamban (PLB), and NCX proteins was performed as previously described (14). Frozen tissue samples were homogenized on ice in 10 ml/g of tissue (wt/wt) in lysis buffer containing (in mmol/l) 145 NaCl, 0.1 MgCl2, 15 HEPES, and 10 EGTA (pH 7.0) and protease inhibitors (Complete, Boehringer Mannheim). The protein concentration was assayed with Protein-Assay (Bio-Rad). Quantification of RyR was performed on membrane preparations as previously described (58). For separation of SERCA2a, PLB, and NCX lysates were run in triplicate on a 4–15% polyacrylamide gradient gel (Bio-Rad), and for RyR membrane preparations were run on 3–8% Tris-acetate gel (Invitrogen). The same control sample was run on every gel to be used as a reference for normalization across blots. Nonspecific antibody binding was blocked for 1 h in phosphate-buffered saline (PBS) with Tween 20 and nonfat milk. The membranes were then incubated in Tween-PBS with the primary antibodies (polyclonal anti-Ca2+ channel {alpha}1c antibody ACC-003, Alomone Labs; monoclonal anti-SERCA2a antibody MA3–910, monoclonal anti-PLB antibody MA3–922, monoclonal anti-NCX antibody MA3–926, and monoclonal anti-RyR antibody MA3–916, Affinity BioReagents) and the appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected with chemiluminescence on Hyperfilm-ECL (Amersham Life Science) or Lumi-Light Western Blotting Substrate (Roche). Films were digitally scanned; band densities were normalized to the average density of the reference lanes.

Statistical analysis. Pooled electrophysiological data are presented as means ± SD, while protein and mRNA data are presented as means ± SE. Statistical comparisons were made with unpaired t-tests (unless otherwise specified), with P < 0.05 considered to be statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
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APD in control and failing myocardium. We measured APD at 50% and 90% repolarization (APD50 and APD90) from 18 myocytes isolated from 12 control ventricles and 11 myocytes from 5 failing ventricles over a range of stimulation frequencies from 0.1 to 1.0 Hz. APD50 and APD90 of myocytes isolated from the midmyocardial layer of failing hearts were significantly longer than those of myocytes isolated from normal hearts at stimulation frequencies of 0.1 and 0.2 Hz (Fig. 1). The resting membrane potential was not significantly different in myocytes isolated from failing compared with control hearts (–86.8 ± 6.7 mV in control vs. –87.7 ± 2.9 mV in failing hearts at 1 Hz).

Measurement of ICa,L, [Ca2+]i transients, and sparks. Contraction in the ventricular myocardium is initiated by Ca2+ entry through the ICa,L. Representative [Ca2+]i transients from control and failing hearts, elicited by voltage steps to –40, –20, 0, 20, and 40 mV from a holding potential of –100 mV with a prepulse to –50 mV to inactivate the Na+ current, are shown in Fig. 2A. The voltage dependence and kinetics of ICa,L do not differ between myocytes isolated from control and failing hearts. The current-voltage relationships for ICa,L measured in ventricular myocytes isolated from control and failing hearts reveal no significant difference in ICa,L density (Fig. 2C).

In contrast, the peak [Ca2+]i in myocytes isolated from failing hearts were significantly depressed at test voltages ≥0 mV (Fig. 2B). To investigate whether changes in [Ca2+]i transient amplitude are related to changes in local Ca2+ release events we measured spontaneous Ca2+ sparks. Representative Ca2+ sparks measured in myocytes isolated from control and failing hearts are shown in Fig. 3B. The amplitudes of the Ca2+ sparks are reflected in the ratio of the peak to background fluorescences and were analyzed with the distribution histograms shown in Fig. 3C. Ca2+ sparks in myocytes from the failing hearts were significantly smaller than those from control hearts (P < 0.0001, Mann-Whitney test of medians). There were no differences in Ca2+ spark kinetics (i.e., width or duration) in cells isolated from control and failing animals (1.1 ± 0.7 vs. 1.0 ± 0.8 µm and 22.6 ± 11.8 vs. 22.4 ± 13.8 ms, respectively). Finally, there was no difference in Ca2+ spark frequency (obtained by dividing each of the histograms of Fig. 3B by its corresponding number of frames) between the two myocyte groups (Fig. 3D), albeit the distribution corresponding to the failing myocytes appeared to be smaller.

Measurement of NCX current. NCX significantly contributes to the [Ca2+]i and AP profile. We measured the NCX current with the voltage protocol shown in Fig. 4A. Rampwise changes in voltage (+60 to –120 mV, 90 mV/s) were applied at a rate of 0.1 Hz from a holding potential of –30 mV. Under the experimental conditions used, the Ni2+-sensitive current is primarily through the exchanger (44). Figure 4, B and C, show representative current recordings in myocytes isolated from a control and a failing heart, respectively. After application of 5 mmol/l Ni2+, the current decreased (tracing b) compared with that in the absence of Ni2+ (tracing a) (Fig. 4, B and C, top). The difference currents (a – b) are shown in Fig. 4, B and C, bottom, respectively. As demonstrated in the representative recordings and as summarized in Fig. 4D and Table 2, there was no significant difference in the NCX current density in myocytes isolated from control and failing hearts. Reversal potentials did not differ between myocytes isolated from control and failing hearts (–28.7 ± 5.2 mV vs. –30.1 ± 5.0 mV); both values were close to the calculated value of –27.2 mV, consistent with the Ni2+-sensitive current representing NCX current and indicating effective buffering of intracellular Ca2+ under our experimental conditions (Table 2).


Figure 4
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  		Fig. 4. Measurement of Ni2+-sensitive Na+/Ca2+ exchanger (NCX) current density in rabbit ventricular myocytes. A: representative current records shown on a slow timescale elicited by the ramp protocol on left (+60 to –120 mV, 90 mV/s, 0.1 Hz, holding at –30 mV). Horizontal bars depict application of 5 mmol/l Ni2+. Note the rapid time course of the Ni2+-induced block, the reproducibility of the Ni2+-insensitive component, and the relative stability of recording without significant rundown. B and C, top: measurement of Ni2+-sensitive NCX current density in rabbit ventricular myocytes isolated from control (B) and failing (C) hearts. Representative current traces were elicited by the same ramp protocol used in A. I-V relationships before (tracing a) and after (tracing b) application of 5 mmol/l Ni2+ are shown. Bottom: the difference is the NCX current. D: there were no differences in the Ni+-sensitive current in myocytes isolated from control and failing hearts. Values are means ± SD.

 

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  		Table 2. Current densities of Na+/Ca2+ exchanger at selected voltages and reversal potentials in myocytes isolated from control and failing ventricular myocytes

 
Measurement of decay of [Ca2+]i transient. The contribution of NCX to Ca2+ removal was assessed by measurement of [Ca2+]i in the presence and absence of the SERCA2a inhibitor thapsigargin. [Ca2+]i transients were elicited by 200-ms voltage-clamp steps to +20 mV from a holding potential of –80 mV at a rate of 0.5 Hz before and 3 min after exposure to 10 µmol/l thapsigargin.

As illustrated in Fig. 5A, thapsigargin reduced the amplitude and prolonged the decay of the [Ca2+]i transient. [Ca2+]i transients in the myocytes isolated from the failing ventricles (Fig. 5B) were characterized by smaller peak amplitudes and slower decays compared with the myocytes isolated from control hearts. Thapsigargin further reduced the peak and prolonged the decay of [Ca2+]i transients of the myocytes from failing hearts. In the baseline state, the time constant of decay of [Ca2+]i ({tau}Ca) was significantly larger in myocytes isolated from failing myocardium. After inhibition of SERCA2a, {tau}Ca was comparable in myocytes from both groups (Fig. 5C). The absolute increase in {tau}Ca was greater in myocytes isolated from the control hearts, but this difference did not reach statistical significance (Fig. 5D). The relative increase in {tau}Ca with thapsigargin, however, was significantly larger in myocytes isolated from control compared with failing hearts (P < 0.05, Fig. 5D). This result suggests that myocytes from failing hearts rely more on NCX for cytosolic Ca2+ removal than myocytes isolated from control hearts, consistent with a defect in SR Ca2+ uptake.


Figure 5
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  		Fig. 5. Effect of SR Ca2+-ATPase inhibition on [Ca2+]i transients. A and B: [Ca2+]i transients were evoked by 200-ms voltage-clamp steps to +20 mV from a holding potential of –80 mV at a rate of 0.5 Hz in rabbit ventricular myocytes isolated from control (A) and failing (B) ventricular myocardium in the absence and presence of 10 µmol/l thapsigargin. C: summarized data (Nc = 3, nc = 13; Nf = 3, nf = 16) demonstrate a substantial increase in the time constant of Ca2+ removal from the myocyte ({tau}Ca) in myocytes isolated from failing compared with control ventricles under baseline conditions. Blocking Ca2+-ATPase with thapsigargin, however, increased the absolute {tau}Ca to a comparable value in both groups. D: there was a slightly but not statistically significantly larger increase in {tau}Ca in the control group. The relative increase in {tau}Ca with thapsigargin was significantly larger in control compared with failing myocardium Values are means ± SD (*P < 0.05).

 
Measurement of Ca2+-handling mRNA and protein. We measured mRNA and immunoreactive protein levels of the major Ca2+-handling proteins to better understand the molecular mechanisms of altered excitation-contraction coupling (ECC) in this model of HF.

ICa,L {alpha}1c-subunit, RyR, SERCA2a, and PLB mRNA levels were measured with kinetic RT-PCR (Fig. 6B). There was no significant difference in the level of {alpha}1c (Fig. 6A) or RyR (Fig. 6B) mRNA in ventricular myocardium from control and failing hearts. However, the mRNA levels of SERCA2a (Fig. 6C) and PLB (Fig. 6D) were significantly reduced in failing compared with control hearts (P < 0.05).


Figure 6
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  		Fig. 6. mRNA levels of Ca2+-handling proteins in control and failing hearts. A–D: scatter plots of normalized {alpha}1c (A), ryanodine receptor (RyR)2 (B), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a (C), and phospholamban (PLB) (D) mRNA in control and failing hearts measured by kinetic RT-PCR. Steady-state levels of {alpha}1c and RyR2 mRNAs are not altered in failing compared with control hearts, while SERCA2a and PLB are significantly decreased in failing hearts. E: representative NCX ribonuclease protection assay showing the expected bands for the NCX (probe 180 bp, protected fragment 118 bp) and the cardiac Na+ channel (Nav1.5; probe 170 bp, protected fragment 78 bp) in control and failing hearts. Probes lane, probes alone; yeast tRNA lane and remaining lanes, 10 µg of total RNA from different control and failing ventricles. F: scatter plot of normalized NCX mRNA in 10 control and 9 failing hearts; values plotted are the average of duplicate determinations. Steady-state level of mRNA encoding NCX is not altered in failing compared with control hearts (*P < 0.05). AU, arbitrary units.

 
NCX mRNA was quantified by RPA; Fig. 6E shows a representative RPA comparing left ventricular apical samples from normal and failing hearts. There is sample-to-sample variation in both normal and failing hearts in rabNCX- as well as rabNav1.5-protected fragments; however, the summarized data (Fig. 6F) reveal no significant difference in the steady-state level of NCX mRNA in failing compared with control myocardium.

Western blotting was used to measure the levels of immunoreactive Ca2+-handling proteins. We observed no difference in the level of {alpha}1c protein expression (Fig. 7A) in tissues from control and failing hearts. There was a modest but statistically significant downregulation of the level of RyR (Fig. 7B), SERCA2a (Fig. 7C), and PLB monomer (Fig. 7D) proteins in failing compared with control ventricles. The antibody used in the NCX Western blots recognized three specific bands of ~160, ~120, and ~70 kDa (23); there is a 48% overall increase in immunoreactive protein in failing myocardium (Fig. 7E; P < 0.05).


Figure 7
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  		Fig. 7. Protein levels of the major Ca2+-handling proteins in control and failing hearts. Representative Western blots and scatter plots of the {alpha}1c-subunit of the Ca2+ channel (A), RyR (B), SERCA2a (C), PLB (D), and NCX (E) in control and failing hearts are shown. Scatter plots for each of the Ca2+-handling proteins demonstrate a significant downregulation of immunoreactive RyR, SERCA2a, and PLB monomer, and an upregulation of NCX. There is no change in immunoreactive {alpha}1c (A). *P < 0.05.

 
DISCUSSION

The pacing tachycardia HF model typically yields dilated hearts, with depressed basal contractility and elevated diastolic pressures. Other dominant features of this model include a marked decline in systolic function, chamber dilation, a marked increase in end-diastolic pressure, and chamber remodeling. This model is also characterized by abnormal beta-adrenergic receptor signaling (46), elevated activity of the renin-angiotensin-aldosterone pathway (56), apoptosis (48), and atrial and ventricular arrhythmias (67). Therefore, this model recapitulates the phenotype of certain types of human HF manifested by depressed [Ca2+]i transients and prolonged APs.

The overall goal of this study was to probe the mechanism(s) responsible for the altered Ca2+ homeostasis and cellular electrophysiology in a clinically relevant rabbit model of HF. We found that in myocytes from failing hearts the APD was longer and the ICa,L density and NCX currents were unaltered, yet the Ca2+ spark amplitude and [Ca2+]i transients were smaller. Decay of [Ca2+]i in myocytes isolated from control hearts exhibited a greater reliance on SR Ca2+ uptake, consistent with aberrant uptake in cells from failing hearts (Fig. 5). At the molecular level, we found that in the failing heart SERCA2a and PLB mRNA levels were downregulated as well as RyR, SERCA2a, and PLB protein levels, while NCX protein was upregulated. Overall, our data suggest that the development of HF in this model is associated with interrelated defects in both SR Ca2+ uptake and release mechanisms that synergistically contribute to the phenotype observed in myocytes from failing hearts.

Trigger of SR Ca2+ release. Compared with controls, we did not find any significant changes in the Ca2+ trigger for SR Ca2+ release in this model; namely, there were no significant reductions in ICa,L density (Fig. 2C), mRNA (Fig. 6A), and the immunoreactive {alpha}1C-subunit (Fig. 7A).

Our data are entirely consistent with those in the canine pacing-induced HF model (40, 65), human dilated cardiomyopathy (DCM), and ischemic cardiomyopathy (ICM) (8, 9, 60, 69), all of which exhibit no change in ICa,L density. However, some studies in the rabbit pacing-induced (80) and rat post-myocardial infarction (MI) (54) HF models have shown a reduction in ICa,L density, and studies in human DCM and ICM have shown a frequency-dependent decrease in ICa,L density (77). Also, studies in human DCM and ICM (27, 79, 80), the rabbit pacing-induced HF model (12), and the rat (17) and canine (24) post-MI HF models have shown a decrease of the dihydropyridine (DHP) binding sites; however, they were found unaltered in the canine pacing-induced HF model (81). The steady-state mRNA {alpha}1c level in the canine pacing-induced HF model (40) and the failing human heart has been reported to be unaltered (72), while it was found to be decreased in another study in the failing human heart (79).

Overall, it appears that differences among studies in the density of the ICa,L or number of DHP binding sites can be attributed to differences in the severity of hypertrophy or failure.

SR Ca2+ release. In this study, we have observed that the decrease in [Ca2+]i (Figs. 2B and 5B) occurs despite the lack of a change in the trigger for Ca2+ release, the L-type Ca2+ current. There are several possible scenarios that could explain the depressed [Ca2+]i at the level of SR Ca2+ release.

Experiments in ventricular myocytes (10, 50), membrane vesicles reconstituted in planar lipid bilayers (11), and isolated cardiac membrane vesicles (36) have demonstrated that SR lumen Ca2+ influences RyR gating such that RyRs are more likely to be triggered by cytosolic Ca2+ when SR luminal Ca2+ is elevated; thus the decrease in SR Ca2+ content (or the elevation of the activation threshold of RyR2 for luminal and/or cytosolic Ca2+) in the failing heart (34) could explain the reduction in Ca2+ release (Fig. 5). Alternatively, abnormal SR Ca2+ release could be attributed to RyR downregulation (Fig. 7B), or the ECC gain could be decreased in the failing myocytes. The role of the RyR in defective Ca2+ handling in the failing heart has been controversial at the levels of both expression (25, 38, 62) and function (38, 57). Although we found no significant difference in the steady-state levels of RyR mRNA (Fig. 6B) between control and failing hearts, the protein level of RyR was significantly downregulated in the failing hearts (Fig. 7B), suggesting that the downregulation of immunoreactive protein is not transcriptionally mediated in this model. On the other hand, while decreases in the ECC gain have been reported in failing rodent hearts (26), this observation has not been confirmed in large-animal models (34) or in human (51) HF studies.

Overall, the net result of the above scenarios would be smaller Ca2+ spark amplitude and depressed [Ca2+]i in myocytes from failing hearts. Ca2+ sparks in myocytes from failing human hearts are characterized by a slower time to peak and half-time of decay and a larger full width at half-maximum, compared with control hearts (53). In contrast to our previous findings in the hypertrophic heart (76), Ca2+ sparks in myocytes isolated from the failing rabbit ventricle are smaller compared with the control heart (P < 0.0001), although the kinetics of the individual sparks were unchanged. Furthermore, we have shown that while the decreased [Ca2+]i can be predominantly attributed to smaller Ca2+ spark amplitude, a nonsignificant decrease in the Ca2+ spark frequency (Fig. 3D) could synergistically contribute to smaller [Ca2+]i. The reduced frequency of sparks in myocytes from failing rabbit hearts is concordant with recently published data in human myocytes (53). It is also important to note that the hypertrophy-related (76) increases in Ca2+ spark amplitudes occurred without changes in SR Ca2+ content.

We did not directly measure the Ca2+ content of the SR in this study, and this is a potential limitation when comparing [Ca2+]i and Ca2+ sparks between the two groups. However, we do not believe that changes in SR Ca2+ content between control and failing myocytes can entirely explain our results for the following reasons. First, as noted above, hypertrophy-related increases in Ca2+ spark amplitudes occurred without significant changes in SR calcium content. Second, our experimental conditions, namely the low stimulation frequency and prepulses, were chosen specifically to minimize any potential changes in SR Ca2+ content and to insure a uniform, although not equal, SR Ca2+ load. Finally, differences in [Ca2+]i were observed at low stimulation frequencies when there is no difference in the SR Ca2+ load between myocytes from normal and failing hearts (75). Therefore, while we cannot entirely exclude the possibility that differences in SR Ca2+ load between myocytes from normal and failing hearts contribute to our observed changes in [Ca2+]i and Ca2+ spark amplitude, we believe that defects in SR Ca2+ release independently contribute to the altered Ca2+ handling in this model.

Cytosolic Ca2+ removal. Our data are consistent with HF-associated changes in Ca2+ handling (5, 8, 66) manifested by a reduction in the amplitude of [Ca2+]i (Figs. 2C and 5B). We have observed a significant reduction in SERCA2a (Fig. 6C) and PLB (Fig. 6D) mRNA levels, with unaltered NCX mRNA level. We also observed a reduction in SERCA2a protein (Fig. 7C; Refs. 31, 66) and PLB (Fig. 7C) protein levels and a significant upregulation of the NCX protein level.

Despite agreement that Ca2+ sequestration by the SR is defective in the failing myocardium, there is significant controversy about the molecular mechanisms. SERCA2a mRNA is decreased in a rat hypertrophy model (20) and human DCM and ICM (2, 20, 22, 28, 47, 52, 59, 73, 78, 79, 85), but some studies of human DCM have shown no change of the SERCA2a mRNA (63, 73, 74). Fewer studies have shown a reduction in the immunoreactive protein in the rat, rabbit, and canine post-MI HF models (13, 28, 31, 45, 66, 85), while some studies of human DCM have shown no change in SERCA2a protein (63, 73, 74).

Indeed, a decrease in the SERCA2a-to-PLB ratio has been associated with defective Ca2+ handling and contraction in several models of cardiac hypertrophy and failure (29, 30, 37, 61). While we found that the SERCA-to-PLB mRNA ratio was significantly decreased compared with controls (1.71 ± 0.46 vs. 2.37 ± 0.65, P < 0.05) the ratio of SERCA2a to PLB protein did not change between the two groups (1.08 ± 0.29 and 0.93 ± 0.16, respectively). Therefore, we cannot attribute the changes in Ca2+ handling we observed in our model to changes in the SERCA2a-to-PLB protein ratio.

NCX constitutes the primary mode of extrusion of Ca2+ from the myocyte and is critically important to Ca2+ handling in the normal and the failing heart. In the rabbit pacing-induced HF model, NCX protein is significantly increased (Fig. 7E). This is qualitatively and quantitatively consistent with previous reports in failing human hearts from patients with ICM (21, 78) or DCM (70) and the canine pacing-induced (3, 35, 66) and rat post-MI (54) HF models. However, the increase in NCX protein was not associated with a change in the level of mRNA, in contrast to other studies (68, 84), which may be explained by altered turnover resulting in an increase in NCX protein.

Overall, our data support the conclusion that the abnormalities of Ca2+ handling that we observed in the rabbit rapid pacing model of HF can be attributed mainly to proximate abnormalities of the SR Ca2+ release mechanism that are further exacerbated by reduced SR Ca2+ uptake, for the following reasons. First, we observed changes in the peak amplitudes of [Ca2+]i transients (Fig. 2B) rather than changes in decay rates (Fig. 5D). Second, we observed decreases in the amplitudes of Ca2+ sparks under conditions where differences in the Ca2+ content of the SR are predicted to be small (75). Third, we found a downregulation of RyR, SERCA2a, and PLB, but an unaltered SERCA2a-to-PLB protein ratio and unchanged NCX current density.

Summary and significance. The heart failure literature is replete with multiple conflicting reports regarding the mechanisms that underlie the observed abnormalities in Ca2+ homeostasis, AP prolongation, and predisposition to life-threatening arrhythmias both in animal models of HF and in the failing human heart. The differences between these conflicting reports reflect, in part, the fact that distinct molecular abnormalities converge to a common HF phenotype. Such molecular abnormalities are often the result of distinct and divergent "insults" and "injuries" to the myocardium, which may produce HF phenocopies. The differential "trigger" events seen in animal HF models parallel that seen in human HF of different etiologies. Thus understanding the molecular mechanisms of these trigger events has important implications in the treatment of HF patients resulting from different etiologies. These differences are further compounded by species differences in the expression and function of major Ca2+ cycling molecules and by differences in the spatiotemporal progression and severity of the disease.

The findings of the present study support the notion that pacing-induced HF in the rabbit results from a distinct mixed mechanism (namely, prominent abnormalities in SR Ca2+ release and uptake) compared with HF that is a consequence of prolonged pressure and/or volume overload (where abnormalities in SR Ca2+ uptake predominate). These observations, then, challenge the conventional wisdom that cardiac hypertrophy and systolic dysfunction are two different set points on the continuous slippery slope of cellular failure that leads inexorably to symptomatic HF.

Therefore, this requires not only identification of the disease gene(s), but eventually connection of the gene(s) to molecular pathways that initiate, promote, suppress, and potentially reverse surrogate end points of the disease phenotype. Such a process is likely to clarify the distinction between true mechanisms and mere markers of HF, a distinction that has far-reaching preventive and therapeutic implications.

Study limitations. The single most important limitation of this study is the lack of direct information regarding the SR Ca2+ content. However, the fact that we observed only changes in the peak amplitude of [Ca2+]i, rather than in its decay rate, at low stimulation frequencies suggests no or minimal differences in the SR Ca2+ load among myocytes from normal and failing hearts. Furthermore, given that in slowly stimulated myocytes from normal and failing hearts the difference in SR Ca2+ content is minimized (75), we are inclined to believe that the SR Ca2+ content in the Ca2+ spark experiments is very similar. Thus it would not alter the interpretation of our results pertaining to the abnormal SR Ca2+ release, even if the SR Ca2+ content is profoundly reduced in these myocytes.

In the present study the whole cell patch-clamp technique was used to measure L-type Ca2+ and NCX current densities. The whole cell patch-clamp technique permits direct measurement of the currents in an intact cardiac myocyte; however, this method will alter the cytosolic composition. Although this artifact is equally applied to both control and failing myocytes, we cannot exclude the possibility that the Ca2+ and NCX currents are differentially modulated by cytoplasmic mediators in control and failing myocardium (15, 16).

Furthermore, in the experiments to measure NCX current density [Ca2+]i was clamped by a high concentration of EGTA in the pipette solution. Ca2+ is an important regulator of the activity of the NCX (33, 35) mediated by a high-affinity Ca2+ binding site composed of acidic residues in the large intracellular loop of the NCX (49). Since this Ca2+ affinity site is intrinsic to the NCX, it is unlikely that a difference in current density of the NCX between myocytes isolated from control and failing hearts would become apparent at higher [Ca2+]i. However, our data do not rigorously exclude this possibility.


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This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant P50-HL-52307 (G. F. Tomaselli, D. A. Kass, B. O'Rourke, E. Marbán). Support was also provided by NHLBI Grants 2RO1-HL-50435 and 1RO1-HL-071865 (C. W. Balke), Established Investigator Award C9740089N (C. W. Balke), American Heart Association Beginning Grant-in-Aid 0365304U (A. A. Armoundas), and Scientist Development Grant 0635127N (A. A. Armoundas).


   ACKNOWLEDGMENTS
 
We thank Dr. Yanli Tian, Debbie DiSilvestre, and Richard Tunin for technical support and Dr. Steven Reiken and Dr. Andrew R. Marks for advice in performance of RyR protein quantification.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. F. Tomaselli, Johns Hopkins Univ., Div. of Cardiology, 844 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205 (e-mail: gtomasel{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. A. Armoundas and J. Rose contributed equally to this work. Back


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