INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

AN IMPORTANT TRANSMEMBRANE TRANSPORT protein, the Na⁺/Ca²⁺ exchanger (NCX1) regulates cellular Ca²⁺ homeostasis in many tissues. In cardiac muscle, by exchanging 3 extracellular Na⁺ for 1 intracellular Ca²⁺ (forward mode), NCX1 is universally accepted as the dominant Ca²⁺ efflux mechanism during diastole. The reverse mode operation of NCX1 (3 Na⁺ out: 1 Ca²⁺ in) has been proposed to load the sarcoplasmic reticulum (SR) with Ca²⁺ (17), trigger SR Ca²⁺ release (15), and directly activate the contractile elements (17). We (32) previously demonstrated that NCX1 overexpression by adenovirus (Adv)-mediated gene transfer in adult rat myocytes resulted in altered patterns of contraction and intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients, changed SR Ca²⁺ contents, and lower resting and diastolic [Ca²⁺]_i. In agreement with studies of transgenic mice overexpressing NCX1 (23, 26), the most consistent explanation of our observations is that NCX1 is functional in both Ca²⁺ influx and efflux modes (32).

In myocytes isolated from rat hearts 3-8 wk after a moderate myocardial infarction (MI), in addition to abnormal contractile function (4, 29), both Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles (7) and NCX1 currents (33) were depressed. It is unclear whether the abnormal contractility could be caused by the depressed NCX1 activity in MI myocytes. The effects of decreased NCX1 expression/function on adult myoycte contractility have not been investigated. In the absence of a specific inhibitor of the NCX1, one approach to downregulate and eliminate NCX1 function is by knocking out the NCX1 gene. Unfortunately, despite the demonstrated absence of both forward and reverse NCX1 activity, homozygous NCX1 deficiency (knockout) resulted in nonbeating hearts during development and lethality between embryonic days 9 and 10 (24). Another approach is to use an antisense (AS) to downregulate the expression of the NCX1 in adult rat myocytes. Previous studies of NCX1 downregulation employed AS oligonucleotides (AS-oligos) in embryonic (22), neonatal (16, 19), and adult cardiac myocytes (8). In none of these studies were the effects of NCX1 downregulation on myocyte contractility examined. The present study was undertaken to examine the functional consequences of NCX1 downregulation and to test the hypothesis that in adult rat cardiac myocytes, NCX1 downregulation alters cardiac myocyte contraction and [Ca²⁺]_i transient dynamics in a manner similar to that observed in MI myocytes.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Myocyte isolation and culture. Cardiac myocytes were isolated from the septum and left ventricular free wall of male Sprague-Dawley rats (~280 g) by successive perfusion with collagenase and hyaluronidase (5). Portions of freshly isolated myocytes were seeded on laminin-coated coverslips and used within 2 h of isolation for contractility measurements (6). For myocyte culture, myocytes were either seeded on laminin-coated coverslips, which were subsequently placed in four-well trays (Nuclone), or placed directly on laminin-coated four-well trays. Culture medium consisted of serum-free media 199 (Earle's balanced salts without L-glutamine and NaHCO₃) supplemented with (in mM) 25 NaHCO₃, 5 creatine, 5 taurine, 2 carnitine, and 0.1 ascorbic acid. We also added 100 U/ml insulin, 31 µg/ml 5-bromo-2-deoxyuridine, 0.2% bovine serum albumin, 500 U/ml penicillin, and 4 µg/ml gentamicin to the culture medium. After 2 h, the media were changed to remove nonadherent myocytes. The myocytes were incubated for an additional 3-4 h before initiation of electrical stimulation. Myocytes were electrically paced (1 Hz) in culture medium containing 1.8 mM extracellular Ca²⁺ concentration ([Ca²⁺]_o), as previously described (32). Culture media were changed daily over the course of the experiments. Under continuous pacing culture conditions, we (20) have previously demonstrated that myocyte contractility did not decline for at least 72 h.

NCX1 AS-oligos. A pair of chimeric AS-oligos (5'-TGAGacttccaatTGTT-3' and 5'-AAGCatgttgtACAA-3') targeted to contiguous regions of rat NCX1 mRNA around the start codon (nucleotides 26 to 10 and 9 to +6) was purchased from Oligos (Wilsonville, OR). These chimeric AS-oligos have four phosphorothioate-modified nucleotides (capital letters) at both the 5'- and 3'-ends.

Construction of recombinant replication-deficient Adv. The basic protocol is described by He et al. (11). Briefly, the coding sequence of rat heart NCX1 was released from pcDNA3.1(+) by sequential digestion with HindIII and XhoI. The NCX1 sequence (3,067 bp) was inserted in the AS direction (ASNCX1) into the shuttle vector pAdTrack-cytomegalovirus (CMV), using the same HindIII and XhoI restriction sites on the shuttle vector. The resulting shuttle vector plasmid was linearized with PmeI, mixed with supercoiled pAdEasy-1, and used to electroporate BJ5183 cells. The recombinant plasmids usually generated smaller colonies that were selected and screened by restriction endonuclease mapping (PacI). Once recombination was confirmed, supercoiled plasmid DNA (isolated from minipreps) was transformed into DH10B cells for large-scale amplification. The recombinant construct was linearized with PacI and used to transfect HEK-293 cells. Recombinant Adv containing both green fluorescent protein (GFP) and ASNCX1 (each under a separate CMV promoter) (Adv-GFP-ASNCX1) was harvested at 7 days, purified with CsCl gradient, and stored at 20°C in 5 mM Tris (pH 8.0), 50 mM NaCl, 0.05% bovine serum albumin, and 25% glycerol. The presence of ASNCX1 in recombinant Adv was confirmed by polymerase chain reaction with the use of primers for the CMV promoter and the polyadenylation site of pAdTrack-CMV.

Adenoviral infection of cardiac myocytes. Two hours after isolation, myocytes seeded in four-well trays were infected with either Adv-GFP-ASNCX1 or Adv expressing GFP alone (Adv-GFP) at a multiplicity of infection of 1 for 3 h. Media were then changed and myocytes were studied after 6, 24, 48, and 72 h in continued pacing culture. Over 95% of myocytes fluoresced green (excitation 478 nm, emission 535 nm) within 6 h, indicating successful Adv infection and GFP expression. We (20) have previously demonstrated that Adv infection of myocytes had no effect on myocyte contractility when examined after 72 h of continuous pacing culture. For the sake of brevity, the myocytes infected with Adv-GFP and Adv-GFP-ASNCX1 are referred to as GFP and ASNCX1 myocytes, respectively.

Myocyte shortening measurements. Myocytes adherent to the coverslips were bathed in 0.6 ml of air- and temperature-equilibrated (37°C) HEPES-buffered (20 mM, pH 7.4) medium 199 containing either 0.6, 1.8, or 5.0 mM [Ca²⁺]_o and were placed on a temperature-controlled stage (37°C) of a Zeiss IM35 microscope. Measurements of myocyte contraction (1 Hz) were performed as previously described (27, 29, 32). In another series of experiments designed to simulate more physiological conditions, myocyte shortening was measured in 1.1 mM [Ca²⁺]_o and pacing frequency was varied between 1 and 3 Hz.

Caffeine-induced contractures. Myocytes bathed in 5.0 mM [Ca²⁺]_o were paced at 1 Hz. At 200 ms after the 21st beat, a trigger transistor-transistor logic pulse of 2.4-s duration was generated by a programmable multichannel stimulator (STIM-6, Ionoptix; Milton, MA) to initiate caffeine (5 mM) application by puffer (Ionoptix) superfusion, which allowed rapid solution changes around a single cell (29, 32, 33). The caffeine-induced contracture and subsequent relaxation were captured by charge-coupled device video camera (Ionoptix) and analyzed (29).

[Ca²+]_i transient measurements. Myocytes were exposed to 0.67 µM of fura 2-AM for 15 min at 37°C. Fura 2-loaded myocytes mounted in a Dvorak-Stotler chamber situated in a temperature-controlled stage (37°C) of a Zeiss IM35 inverted microscope were field stimulated to contract at 1 Hz between platinum wire electrodes (30, 32). [Ca²⁺]_o was varied between 0.6 and 5.0 mM. [Ca²⁺]_i transient measurements, calibration of fura 2 fluorescence signals, and [Ca²⁺]_i transient analyses were performed as previously described (30, 32).

NCX1, sarco(endo)plasmic reticulum Ca²+-ATPase, and calsequestrin immunoblotting. Cultured myocytes were harvested for immunoblotting on days 3 and 6. Cultured myocytes in a four-well tray were rinsed three times with ice-cold phosphate-buffered saline. They were then scraped into 1 ml of ice-cold lysis buffer containing (in mM) 50 Tris (pH 8.0), 150 NaCl, 1 Na⁺ orthovanadate, 1 phenylmethylsulfonyl fluoride, 100 NaF, 1 EDTA, and 1 EGTA, and 0.5% NP40, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cell lysate was snap-frozen with dry ice-ethanol and stored at 80°C.

L-type Ca²+ current measurements. Whole cell patch-clamp recordings were performed at 30°C, as described in detail previously (28, 31-34). Briefly, fire-polished pipettes (tip diameter 4-6 µm) with resistances of 0.8-1.4 M when filled with standard internal solution were used. Pipette solution consisted of (in mM) 120 Cs-aspartate, 10 EGTA, 10 HEPES, 4 MgATP, and 4 MgCl₂ (pH 7.2 with CsOH). External solution contained (in mM) 135 choline Cl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). Before myocyte stimulation was started, holding potential was changed from 70 to 40 mV to inactivate fast inward Na⁺ current. To ensure steady-state Ca²⁺ loading in the SR, six conditioning pulses (from 40 to 0 mV, 300 ms, 1 Hz) were delivered before arrival of each test pulse (from 30 to +70 mV, 10-mV increments, 400 ms). After the last test pulse at +70 mV, the myocyte was held at 40 mV for 1 s before being returned to holding potential of 70 mV. Currents were filtered at 2 kHz, and data were acquired at 2 kHz. Leak-subtracted inward currents were used in analysis for L-type Ca²⁺ current (I_Ca,L) amplitudes and inactivation kinetics (28). Whole cell capacitance (C_m) for each myocyte was measured by application of a small hyperpolarizing pulse (10 mV, 16 ms) and integration of the resulting current change (digitized at 50 kHz, 0.5-kHz filter) over time (28). To facilitate comparison of I_Ca,L among cells, maximal I_Ca,L amplitude of each myocyte was divided by its C_m to account for variations in cell sizes.

Action potential measurements. Action potentials from GFP and ASNCX1 myocytes were recorded after 72 h of continual pacing culture with the use of current-clamp configuration at 1.5× threshold stimulus and 4-ms duration (34). Pipette solution consisted of (in mM) 125 KCl, 4 MgCl₂, 0.06 CaCl₂, 10 HEPES, 5 potassium EGTA, 3.1 Na₂ATP, and 5 Na₂-creatine phosphate (pH 7.2). External solution consisted of (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 0.6 NaH₂PO₄, 15 HEPES, and 5 glucose, pH 7.4.

Measurement of SR Ca²+ content. SR-releasable Ca²⁺ content was estimated by integrating forward NCX1 current (I_Na,Ca) induced by caffeine exposure, as described by us previously (31-33). The pipette solution consisted of (in mM) 100 Cs⁺ glutamate, 1 MgCl₂, 30 HEPES, and 2.5 MgATP; pH 7.2. The external solution was composed of (in mM) 130 NaCl, 5 CsCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20 HEPES, and 10 glucose; pH 7.4, 30°C. [Ca²⁺]_o was either 0.6 or 5 mM. Holding potential was 80 mV. At 200 ms after the 11th conditioning pulse (from 80 to 0 mV, 300 ms, 1 Hz), with membrane potential (E_m) held at 80 mV, caffeine (5 mM; 2.4 s) was applied by puffer superfusion. Currents were digitized at 0.5 kHz and collected for ~5 s.

Statistics. All results are expressed means ± SE. For analysis of a parameter (e.g., systolic [Ca²⁺]_i, maximal contraction amplitude, SR Ca²⁺ content) as functions of group (GFP vs. ASNCX1) and [Ca²⁺]_o, two-way ANOVA was used to determine statistical significance. A linear model-fitted standard least squares (JMP version 4, SAS Institutes; Cary, NC) was used. Student's t-test was used to compare protein abundance, I_Ca,L, action potential parameters, and half-times of relaxation from caffeine-induced contracture between GFP and ASNCX1 myocytes. In all analyses, P < 0.05 was taken to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of NCX1 AS-oligos on myocyte contractility, caffeine contracture, and NCX1 protein abundance. Because the half-life of NCX1 protein was estimated to be ~33 h in neonatal cardiomyocytes (19), we reasoned that at least 72 h (2 half-lives) of exposure to ASNCX1 would be required to observe an effect.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Antisense (AS) oligonucleotides (AS-oligos) to Na+/Ca2+ exchanger (NCX1) depresses adult rat cardiac myocyte contractility. Isolated myocytes were exposed to a pair of chimeric AS-oligo (0.5 µM each; see METHODS) to NCX1 for 72 h in continuous pacing culture (). Myocytes cultured without AS-oligos served as controls (). Myocyte contraction was measured at extracellular Ca2+ concentrations ([Ca2+]o) of 0.6, 1.8, and 5.0 mM. Each point represents the mean ± SE of 18-58 observations. Error bars are not shown if they fall within the boundaries of the symbol. Statistical analyses are given in RESULTS.

Effects of Adv-GFP-ASNCX1 infection on NCX1 abundance. Another method to introduce ASNCX1 into adult cardiac myocytes was infection with recombinant, replication-defective Adv (12, 13, 32). Seventy-two hours after infection with either Adv-GFP or Adv-GFP-ASNCX1, cardiac myocyte lysates were collected to analyze for NCX1 abundance by immunoblotting. Under nonreducing gel conditions, NCX1 was detected as a band of apparent molecular mass of 160 kDa (18, 31, 32). Compared with GFP myocytes, ASNCX1 myocytes had significantly (P < 0.01) less NCX1 protein (45.8 ± 4.0 vs. 31.9 ± 0.8 arbitrary units, n = 5) after 72 h of infection (Fig. 2). When examined after 6 days of infection, ASNCX1 myocytes exhibited further reduction in NCX1 protein compared with GFP myocytes (42.5 ± 4.0 vs. 14.6 ± 2.0 arbitrary units, n = 5; P = 0.0003) (Fig. 2). As an internal control for protein loading, we measured calsequestrin in each of the myocyte lysates (Fig. 2). Calsequestrin expression has been shown to be unchanged during ontogenic development, aging, cardiac hypertrophy, and failing human myocardium (10). There were no significant (P = 0.80) differences in calsequestrin protein amounts between GFP and ASNCX1 myocytes (946 ± 14 vs. 951 ± 13 arbitrary units) (Fig. 3). In addition, there were no (P = 0.17) differences in SERCA2 amounts between ASNCX1 and GFP myocytes (45.4 ± 1.1 vs. 48.5 ± 1.8 arbitrary units) (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Immunoblots of NCX1, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2), and calsequestrin. Proteins in myocyte homogenates (50 µg/lane) were separated by gel electrophoresis and transferred to Immun-Blot polyvinylidene difluoride membranes, and NCX1, SERCA2, and calsequestrin were identified by immunoblotting, as described in METHODS. Composite results are presented in RESULTS. Numbers at left refer to apparent molecular mass. GFP, green fluorescent protein.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Downregulation of NCX1 alters contractile function in adult rat myocytes. Isolated myocytes were infected with recombinant adenovirus (Adv) expressing either GFP alone or GFP and AS to NCX1 (ASNCX1) and were then cultured for 72 h under continuous electrical stimulation (1 Hz) conditions. For contraction studies, cultured myocytes were paced (1 Hz) to contract at 37°C and [Ca2+]o of 0.6 (A), 1.8 (B), or 5.0 mM (C). Shown are steady-state paced twitches from myocytes expressing either GFP (left) or ASNCX1 (right). Results are summarized in Table 1.

Effects of NCX1 downregulation on myocyte contractile function. At 0.6 mM [Ca²⁺]_o, ASNCX1 myocytes shortened more than GFP myocytes (Fig. 3 and Table 1). In contrast, at 5.0 mM [Ca²⁺]_o, ASNCX1 myocytes shortened less than GFP myocytes (Fig. 3 and Table 1). The differences in twitch amplitudes were no longer apparent at intermediate [Ca²⁺]_o levels (Fig. 3 and Table 1). These conclusions are supported by highly significant (P < 0.0001) [Ca²⁺]_o and group × [Ca²⁺]_o interaction effects, indicating that the magnitude and/or direction of the effects of [Ca²⁺]_o on cell shortening was different across experimental groups (GFP vs. ASNCX1).

Effects of NCX1 downregulation on relaxation from caffeine-induced contractures. After steady-state twitch amplitude was achieved, application of 5 mM caffeine to a myocyte at end diastole caused a large contracture due to SR Ca²⁺ release and then relaxation to a shorter resting cell length in the continued presence of caffeine (Fig. 4). The incomplete relaxation is thought to be due to increased myofilament sensitivity to Ca²⁺ by caffeine. Relaxation in the continued presence of caffeine was mediated primarily by forward NCX1 because SR Ca²⁺ accumulation was inhibited (1, 2, 30, 33). The half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was significantly (P < 0.0002) slower in ASNCX1 (3.09 ± 0.26 s, n = 15; Fig. 4B) than GFP (1.55 ± 0.24 s, n = 13; Fig. 4A) myocytes.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Downregulation of NCX1 slows relaxation from caffeine-induced contractures in adult rat myocytes. After 21 twitches (1 Hz, 37°C, 5.0 mM [Ca2+]o) to ensure steady-state sarcoplasmic reticulum (SR) Ca2+ load, caffeine (5 mM) was "puffed" onto a myocyte for 2.4 s (index bar) to induce contracture and myocyte relaxation was followed until complete. We (33) have previously shown that 2.4 s of caffeine exposure was sufficient to release all SR Ca2+ content in rat myocytes. At low (5 mM) caffeine concentrations, rapid transient phase of contraction largely reflects changes in intracellular [Ca2+] ([Ca2+]i). Relaxation in continued presence of caffeine was primarily due to forward Na+/Ca2+ exchange because SR Ca2+ accumulation was inhibited. Note that 72 h after Adv exposure, myocytes infected with recombinant Adv expressing both GFP and ASNCX1 (B) had significantly prolonged relaxation compared with myocytes infected with Adv expressing GFP only (A).

Effects of NCX1 downregulation on [Ca²+]_i transients. [Ca²⁺]_i occupies a central role in cardiac myocyte excitation-contraction coupling. Thus the differences in contractile behavior between GFP and ASNCX1 myocytes may be related to differences in [Ca²⁺]_i homeostasis brought about by NCX1 downregulation in cardiac myocytes. Indeed, resting [Ca²⁺]_i measured in quiescent myocytes was significantly higher in ASNCX1 myocytes (Table 3; group effect, P < 0.0006). Changing [Ca²⁺]_o apparently had no significant effect on resting [Ca²⁺]_i (see Table 3) ([Ca²⁺]_o effect, P = 0.2286). Diastolic [Ca²⁺]_i comparisons between GFP and ASNCX1 myocytes essentially mirrored the results of resting [Ca²⁺]_i comparisons (see Table 3); significant group (P < 0.05) but insignificant [Ca²⁺]_o (P = 0.3523) effects.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Downregulation of NCX1 alters [Ca2+]i transients in adult rat myocytes. Seventy-two hours after adenovirus infection, myocytes were loaded with fura 2 and paced (1 Hz) to contract at 37°C and [Ca2+]o of 0.6 (A), 1.8 (B), and 5.0 mM (C). Shown are steady-state [Ca2+]i transients from GFP (left) and ASNCX1 myocytes (right). In another two groups of myocytes expressing either GFP or GFP and ASNCX1 but not loaded with fura 2, it was determined that autofluoresence from GFP accounted for <10% of the fura 2 signal obtained either at 360 or 380 nm excitation. Results are summarized in Table 3.

Effects of NCX1 downregulation on SR-releasable Ca²+ contents. Changes in [Ca²⁺]_i transient and contraction amplitudes with NCX1 downregulation may be due to alterations in SR Ca²⁺ content. The time integral of forward NCX1 current induced by caffeine (inward current in Fig. 6) was an estimate of SR Ca²⁺ content (31-33). At 0.6 mM [Ca²⁺]_o, both I_Na,Ca time integral (pC/cell) and SR-releasable Ca²⁺ (normalized to cell size; fmol/fF) were larger in ASNCX1 (Fig. 6B) than GFP myocytes (Fig. 6A and Table 4). In contrast, at 5 mM [Ca²⁺]_o, SR-releasable Ca²⁺ was smaller in ASNCX1 (Fig. 6D) than GFP myocytes (Fig. 6C and Table 4). For both I_Na,Ca time integral and SR-releasable Ca²⁺, two-way ANOVA indicated insignificant group (P > 0.36) but highly significant [Ca²⁺]_o (P < 0.0001) and group × [Ca²⁺]_o interaction (P < 0.0003) effects, indicating that changing [Ca²⁺]_o affected the inherent differences in SR Ca²⁺ contents between GFP and ASNCX1 myocytes.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Downregulation of NCX1 alters SR-releasable Ca2+ in adult rat myocytes. After 72 h of Adv infection, GFP (A and C) and ASNCX1 (B and D) myocytes were incubated with either 0.6 (A and B) or 5 mM (C and D) [Ca2+]o at 30°C and were voltage-clamped at 80 mV. At 200 ms after the 11th conditioning pulse (from 80 to 0 mV, 300 ms, 1 Hz), caffeine (5 mM) was puffed onto the myocyte for 2.4 s. Large transient inward current caused by caffeine-induced SR Ca2+ release was observed. This current represents Na+ entry accompanying Ca2+ extrusion by NCX1, and the time integral of this current provides an estimate of SR-releasable Ca2+ (31-33). Composite results are summarized in Table 4.

Effects of NCX1 downregulation on I_Ca,L and C_m. Figure 7A shows a typical voltage-clamp experiment on an ASNCX1 myocyte, in which both the Na⁺ and K⁺ currents are either inactivated or blocked. With depolarization, inward currents that rapidly inactivate can clearly be observed. Under our experimental conditions, the inward current was carried by Ca²⁺ ions (3, 28). There were no differences in maximal I_Ca,L density (pA/pF), test potential at which maximal I_Ca,L occurred, and kinetic time constants of I_Ca,L inactivation between GFP and ASNCX1 myocytes (Table 5). The mean current-voltage relationships for GFP and ASNCX1 myocytes were virtually superimposable (Fig. 7B). In addition, C_m, a measure of cell surface area, was not different between GFP and ASNCX1 myocytes, indicating that NCX1 knockdown had no effect on myocyte size.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Downregulation of NCX1 does not affect L-type Ca2+ current in adult rat ventricular myocytes. A: family of leak-subtracted Ca2+ currents (ICa,L) from a myocyte measured 72 h after infection with Adv expressing both GFP and ASNCX1. [Ca2+]o was 1.8 mM and temperature was 30°C. Axopatch 1C amplifier was set at holding potential of 70 mV. Before stimulation was started, pCLAMP 6 was programmed to deliver +30 mV such that net holding potential was 40 mV to inactivate fast Na+ channels. Each test pulse (from 30 to +70 mV, 10-mV increments, 400 ms) was preceded by six conditioning pulses (from 40 to 0 mV, 300 ms, 1 Hz) to ensure steady-state SR Ca2+ loading. Composite data are shown in Table 5. B: current-voltage relationships of ICa,L in GFP and ASNCX1 myocytes. Peak amplitudes of ICa,L at each test potential was measured from current tracings depicted in A. Means ± SE for GFP (, n = 10) and ASNCX1 (, n = 10) myocytes incubated at 1.8 mM [Ca2+]o are shown. Error bars are not shown if they fall within boundaries of a symbol.

Effects of NCX1 downregulation on action potential. Because I_NaCa may contribute to action potential morphology, we measured action potential in GFP and ASNCX1 myocytes (Fig. 8). Resting E_m, action potential amplitude, and action potential duration at 50% repolarization measured in GFP myocytes (Table 6) were similar to those reported for adult rat myocytes cultured in serum-free medium for 72 h (9). There were no significant differences in resting E_m (P > 0.57), action potential amplitude (P > 0.56), and action potential duration at 50% (P > 0.13) and 90% repolarization (P > 0.21) between GFP and ASNCX1 myocytes (Fig. 8 and Table 6), indicating that NCX1 knockdown had no effect on myocyte resting E_m and action potential morphology.

View larger version (6K): [in this window] [in a new window]   Fig. 8.   Downregulation of NCX1 does not affect action potential amplitude and duration in adult rat myocytes. Seventy-two hours after Adv exposure, action potentials in GFP (A) and ASNCX1 (B) myocytes were measured at 30°C, as described in METHODS. Composite data are summarized in Table 6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our initial approach to knockdown NCX1 function in adult rat cardiac myocytes was to use a pair of chimeric NCX1 AS-oligos that have been shown to be effective in downregulating NCX1 in neonatal cardiomyocytes (19). After 72 h of NCX1 AS-oligos exposure, myocyte contractility was similar at 0.6 mM [Ca²⁺]_o and significantly decreased at 1.8 and 5.0 mM [Ca²⁺]_o (Fig. 1). However, neither NCX1 function (relaxation from caffeine-induced contractures) nor its abundance was different between control and AS-oligos-treated myocytes, suggesting that the decrease in myocyte contractility was due to nonsequence specific and/or toxic effects of the chimeric AS-oligos (21). Thus the first major finding of our current study is that AS-oligos were ineffective in knocking down NCX1 in adult cardiac myocytes, at least under the conditions used in our experiments. The discrepancy between our results and those of Slodzinski and Blaustein (19) may be because, unlike neonatal cardiomyocytes, uptake of naked DNA by adult rat cardiac myocytes is a very inefficient process (14).

Because gene transfer by Adv infection (12, 13, 32) was much more efficient than uptake of naked DNA by adult cardiac mycoytes (14), we then sought to downregulate NCX1 by Adv-mediated AS delivery. Infection of myocytes with Adv-GFP-ASNCX1 resulted in knockdown of NCX1 protein by 30% after 3 days and 66% after 6 days (Fig. 2). The time course of NCX1 protein disappearance with AS treatment in adult myocytes was similar to that observed in neonatal cardiomyocytes (57% knockdown after 7 days; Ref. 19).

Knockdown of NCX1 protein by AS was accompanied by decreased forward NCX1 activity, as indicated by prolonged relaxation from caffeine-induced contractures (Fig. 4). At physiological [Ca²⁺]_o of 1.1 mM and contraction frequencies of 1 and 3 Hz, contractile activity in ASNCX1 myocytes was not different compared with GFP myocytes (Table 2), suggesting that reducing NCX1 protein content had little apparent effect on contraction amplitudes measured at physiological [Ca²⁺]_o. In this regard, it is interesting to note that increasing NCX1 protein (~3-fold) via Adv-mediated gene transfer (32) or by the transgenic approach (23) also did not affect twitch sizes measured at [Ca²⁺]_o of 1 to 1.8 mM.

Altered NCX1 content or function, however, may have dramatic consequences on cardiac contractility and Ca²⁺ homeostasis when the myocyte is subjected to more stressful situations in vivo or in vitro. Indeed, a more complex pattern of contractile and [Ca²⁺]_i transient abnormalities in ASNCX1 myocytes is revealed when [Ca²⁺]_o was varied. Changing [Ca²⁺]_o may alter the direction of Ca²⁺ flux mediated by the NCX1, as suggested by the following energetics considerations. Assuming a 3 Na⁺: 1 Ca²⁺ stoichiometry for the NCX1, and with the use of resting cytosolic [Ca²⁺]_i (~100 nM; Table 3) and [Na⁺]_i (13 mM; Ref. 1) values, the equilibrium potential for the NCX1 (E_Na,Ca) can be calculated to be 41.6, 70, and 97.9 mV at 0.6, 1.8, and 5 mM [Ca²⁺]_o, respectively. With a measured E_m of around 75 mV (Table 6), it can be appreciated that at 0.6 mM [Ca²⁺]_o, E_Na,Ca exceeded E_m and Ca²⁺ efflux was thermodynamically favored. Conversely at 5 mM [Ca²⁺]_o, Ca²⁺ influx was favored because E_m was greater than E_Na,Ca. At 1.8 mM [Ca²⁺]_o, E_Na,Ca was very close to E_m and neither Ca²⁺ efflux nor influx was favored. At peak systole, E_m (~+55 mV; Table 6) exceeded E_Na,Ca (43.2, 67.6, and 90.6 mV at 0.6, 1.8, and 5 mM [Ca²⁺]_o, respectively). Therefore during systole Ca²⁺ influx was favored, although the duration of Ca²⁺ influx mediated by reverse NCX1 was uncertain due to ambiguities in changes in intracellular Na⁺ concentration ([Na⁺]_i) and [Ca²⁺]_i during the action potential. Experimentally, we found that at 0.6 mM [Ca²⁺]_o, maximal contraction and [Ca²⁺]_i transient amplitudes were significantly higher in ASNCX1 than GFP myocytes (Figs. 3 and 5 and Tables 1 and 3). Assuming no changes in myofilament Ca²⁺ sensitivity with NCX1 knockdown, a simple explanation for our experimental observations based on energetics considerations is that at 0.6 mM [Ca²⁺]_o, reduction in NCX1 resulted in less Ca²⁺ efflux at rest or diastole, leading to higher SR Ca²⁺ content (Fig. 6B), and higher peak [Ca²⁺]_i transient (Fig. 5A) and contraction (Fig. 3A) amplitudes. On the other hand, under conditions favoring Ca²⁺ influx ([Ca²⁺]_o = 5 mM), NCX1 reduction would result in less Ca²⁺ influx, lower SR Ca²⁺ content (Fig. 6D), decreased [Ca²⁺]_i transient (Fig. 5C) and contraction (Fig. 3C) amplitudes. At intermediate [Ca²⁺]_o of 1.8 mM, neither Ca²⁺ influx nor efflux was favored, and thus altering NCX1 amounts would not be expected to affect contraction and [Ca²⁺]_i transient amplitudes. Indeed, this was observed in both ASNCX1 myocytes (Figs. 3B and 5B and Tables 1 and 3) and myocytes overexpressing NCX1 (32). We emphasize that our simple interpretation did not take into account the controversies regarding NCX1 stoichiometry, other membrane transporters that likely affect [Na⁺]_i (Na⁺-K⁺-ATPase) and [Ca²⁺]_i (sarcolemmal Ca²⁺-ATPase) during an action potential, and that the bulk cytosolic [Na⁺]_i and [Ca²⁺]_i determined with fluorescent indicators may very well be different from those sensed by the NCX1.

The effects of ASNCX1 on myocyte contraction and [Ca²⁺]_i transients are directly related to the knockdown of NCX1 (Fig. 2) for the following reasons. First, Adv-mediated ASNCX1 delivery was not associated with changes in SERCA2 and calsequestrin protein levels (Fig. 2). Second, SR Ca²⁺ uptake, as estimated by the half-time of [Ca²⁺]_i decline (30), was not affected by downregulation of NCX1 (Table 3). Third, knockdown of NCX1 did not affect I_Ca,L (Fig. 7 and Table 5). Thus ASNCX1 exposure had no measurable effects on major Ca²⁺ regulatory pathways (other than the NCX1) involved in excitation-contraction coupling in cardiac myocytes. Fourth, knockdown of NCX1 did not affect resting E_m (Table 6) and action potential morphology (Fig. 8 and Table 6) in adult rat myocytes. This is an important observation because I_Na,Ca may contribute to action potential morphology and the abnormal pattern of contraction in ASNCX1 myocytes could be due to action potential changes rather than directly resulting from reduced NCX1 function in ASNCX1 myocytes. Finally, another argument in favor of a direct effect of ASNCX1 on myocyte contractility is our previous observation (32) that NCX1 overexpression produced contractility and [Ca²⁺]_i transient changes in exactly the opposite pattern observed in NCX1 downregulated myocytes.

Previous studies manipulating the expression of NCX1 in embryonic (22) and neonatal rat cardiomyocytes (16, 19) and adult guinea pig ventricular myocytes (8) were performed by exposure to AS-oligos. With the use of relatively high concentrations (3 µM) of AS-oligos directed at the 3' untranslated region of NCX1 mRNA, Lipp et al. (16) reported a 78% decrease of NCX1 function in neonatal rat myocytes after 24 h and 96% decrease after 48 h. Another study employing still higher concentrations (10 µM) of AS-oligos directed at the 3'-end of the canine NCX1 mRNA (nucleotides 2638-2657) demonstrated a 20-30% decrease in Na⁺-dependent Ca²⁺ uptake in embryonic rat myocytes after 24 h (22). A third study that used AS-oligos (2 µM) targeted to the start codon (nucleotides 11 to +9) of guinea pig cardiac NCX1 mRNA reported a significant 40% decrease in NCX1 expression after 2 days of exposure (8). Both NCX1 activity and protein expression were further reduced to ~10% of normal after 6 days of AS-oligos exposure (8). A fourth study used a pair of chimeric AS-oligos (0.5 µM of each oligo) targeted to contiguous regions of NCX1 mRNA around the start codon demonstrated that in neonatal rat cardiomyocytes, NCX1 function was decreased ~60% after 4 days and NCX1 protein was decreased ~60% after 7 days (19). In contrast to the present study, none of the previous studies (8, 16, 19, 22) examined the effects of NCX1 downregulation on contraction amplitudes. In addition, unlike Adv-mediated gene transfer, which has >95% efficiency in adult rat myocytes (32), it is not clear what percentage of cultured cardiac cells had taken up AS-oligos in the previous studies (8, 16, 19, 22).

In myocytes isolated from rat hearts 3-8 wk after MI, both I_NaCa (33) and Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles (7) were depressed. In addition, compared with myocytes isolated from sham-operated rats, contraction amplitudes were higher at 0.6 mM, not different at 1.8 and 3.0 mM, and lower at 5.0 mM [Ca²⁺]_o in post-MI myocytes (29). [Ca²⁺]_i transient amplitudes in post-MI myocytes stimulated at 1.1 mM [Ca²⁺]_o were not different from sham myocytes but were significantly lower at 4.9 mM [Ca²⁺]_o (4). SR Ca²⁺ content measured at 5 mM [Ca²⁺]_o was significantly reduced in post-MI myocytes (33). This pattern of contractile and [Ca²⁺]_i transient abnormalities, as well as lower SR Ca²⁺ content (measured at high [Ca²⁺]_o), in MI myocytes strongly resembles that observed in the present study, in which NCX1 was downregulated in normal adult rat myocytes, and suggests that depressed NCX1 function may partly account for the abnormalities in contraction and [Ca²⁺]_i homeostasis in post-MI myocytes.

In summary, we have demonstrated that downregulation of NCX1 by Adv-mediated AS delivery resulted in altered patterns of contraction and [Ca²⁺]_i transients, changed SR Ca²⁺ contents, higher resting and diastolic [Ca²⁺]_i levels, and slower relaxation from caffeine-induced contractures. Myocyte size, calsequestrin, SERCA2 expression, SR Ca²⁺ uptake, resting E_m, action potential amplitude and morphology, and I_Ca,L were unaffected by knockdown of NCX1. We conclude that the most consistent explanation of our observations is that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca²⁺ influx and efflux during a twitch. In addition, compared with their respective controls (GFP and sham myocytes, respectively), the patterns of contractile dysfunction and [Ca²⁺]_i transient abnormalities and reduced SR Ca²⁺ contents observed in NCX1-downregulated and post-MI myocytes were very similar. We therefore speculate that decreased NCX1 activity may partly account for the contractile dysfunction in postinfarction myocytes.