Sodium accumulation promotes diastolic dysfunction in end-stage heart failure following Serca2 knockout

Abstract

Alterations in trans-sarcolemmal and sarcoplasmic reticulum (SR) Ca²⁺ fluxes may contribute to impaired cardiomyocyte contraction and relaxation in heart failure. We investigated the mechanisms underlying heart failure progression in mice with conditional, cardiomyocyte-specific excision of the SR Ca²⁺-ATPase (SERCA) gene. At 4 weeks following gene deletion (4-week KO) cardiac function remained near normal values. However, end-stage heart failure developed by 7 weeks (7-week KO) as systolic and diastolic performance declined. Contractions in isolated myocytes were reduced between 4- and 7-week KO, and relaxation was slowed. Ca²⁺ transients were similarly altered. Reduction in Ca²⁺ transient magnitude resulted from complete loss of SR Ca²⁺ release between 4- and 7-week KO, due to loss of a small remaining pool of SERCA2. Declining SR Ca²⁺ release was partly offset by increased L-type Ca²⁺ current, which was facilitated by AP prolongation in 7-week KO. Ca²⁺ entry via reverse-mode Na⁺–Ca²⁺ exchange (NCX) was also enhanced. Up-regulation of NCX and plasma membrane Ca²⁺-ATPase increased Ca²⁺ extrusion rates in 4-week KO. Diastolic dysfunction in 7-week KO resulted from further SERCA2 loss, but also impaired NCX-mediated Ca²⁺ extrusion following Na⁺ accumulation. Reduced Na⁺-K⁺-ATPase activity contributed to the Na⁺ gain. Normalizing [Na⁺] by dialysis increased the Ca²⁺ decline rate in 7-week KO beyond 4-week values. Thus, while SERCA2 loss promotes both systolic and diastolic dysfunction, Na⁺ accumulation additionally impairs relaxation in this model. Our observations indicate that if cytosolic Na⁺ gain is prevented, up-regulated Ca²⁺ extrusion mechanisms can maintain near-normal diastolic function in the absence of SERCA2.

Introduction

Contraction in adult ventricular myocytes is triggered by a transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) during the action potential (AP). This rise in [Ca²⁺]_i results from Ca²⁺ influx through L-type Ca²⁺ channels, which in turn triggers release of a much larger amount of Ca²⁺ from the sarcoplasmic reticulum (SR) (Bers, 2001). Relaxation occurs as Ca²⁺ is recycled into the SR by the SR Ca²⁺-ATPase (SERCA) and as Ca²⁺ is extruded from the cell, primarily by the Na⁺–Ca²⁺ exchanger (NCX).

Altered cardiomyocyte Ca²⁺ homeostasis is believed to be an important mechanism underlying heart failure (HF), and may contribute to both systolic and diastolic dysfunction in this condition. Systolic dysfunction results, at least in part, from reduced magnitude of Ca²⁺ transients due to lower Ca²⁺ content of the SR (Bers, 2006). Reduced SR content is believed to be caused by depressed SERCA function and greater diastolic SR Ca²⁺ leak. Increased expression and function of the NCX may also impair SR Ca²⁺ reuptake since NCX competes with SERCA for extrusion of Ca²⁺ from the cytosol (Pogwizd et al. 2001). Less is known about the cellular mechanisms that underlie diastolic HF. However, Hasenfuss et al. (1999) observed that hearts from human HF patients with diastolic dysfunction had decreased SERCA expression but unchanged NCX. In contrast, patients with preserved diastolic function exhibited increased NCX levels. Thus, alterations in the relationship between trans-SR and trans-sarcolemmal Ca²⁺ fluxes may be critical in determining diastolic function in the failing heart, and progression to end-stage failure. The ability to increase transport of Ca²⁺ and Na⁺ across the sarcolemma may be an important compensatory mechanism for reduction in SR function, and a progressive decline in this ability may cause deterioration of diastolic function in HF patients.

We recently investigated the consequences of SERCA down-regulation in adult mice with cardiomyocyte-specific, conditional excision of the Serca2 gene (Andersson et al. 2009a). SERCA protein expression declined rapidly following gene deletion, and was below 5% of control values after 4 weeks. Surprisingly, we observed only moderately impaired cardiac performance with a relatively minor reduction in both systolic and diastolic function (Andersson et al. 2009a). Cardiomyocytes were observed to compensate for loss of SR function by increasing Ca²⁺ cycling across the sarcolemma. In the present study, we examine the cellular mechanisms underlying the eventual progression to end-stage HF in these mice. Our results indicate that while reduction of SR function following SERCA loss contributes to both systolic and diastolic dysfunction, impaired relaxation also results from slowing of NCX-mediated Ca²⁺ extrusion due to cytosolic Na⁺ accumulation. Importantly, we show that when such Na⁺ increases are prevented, up-regulated Ca²⁺ extrusion mechanisms can maintain near-normal diastolic function in the complete absence of a functional SR.

Methods

Ethical approval

The investigation was approved by the Norwegian National Committee for Animal Welfare under the Norwegian Animal Welfare Act, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996), and ethical guidelines from The Journal of Physiology (Drummond, 2009).

Animal care

The Serca2^flox/flox Tg(αMHC-MerCreMer) mouse (KO) employed in our experiments allows for inducible, cardiomyocyte-specific disruption of the Serca2 gene in adult mice (Andersson et al. 2009a). Serca2 gene excision in 8- to 10-week-old mice was accomplished by inclusion of tamoxifen base powder (RM1 FG SQC, 811004, Scanbur BK) in the feed (100 mg (200 g)⁻¹) for 7 days (Andersson et al. 2009c). Serca2^flox/flox mice (FF) served as controls (Andersson et al. 2009a,b;) and were subjected to the same feeding regime. Tamoxifen administration results in Serca2 gene excision exclusively in the cardiomyocytes of KO animals (Andersson et al. 2009a). A total of 171 anaesthetized (∼2% isoflurane) mice were killed by cervical dislocation, and hearts were harvested at 4 and 7 weeks following tamoxifen administration. Echocardiographic characterization of heart function was performed on anaesthetised (∼2% isoflurane), self-breathing mice (Andersson et al. 2009a).

Histology

Following fixation in 4% formaldehyde, hearts were transected at the midventricular level, routinely processed and embedded in paraffin. Serial 3.5 μm sections were stained with haematoxylin and eosin, acid fuchsin orange G (AFOG) and van Gieson stains, and were assessed blindly by two investigators. The degree of fibrosis in each heart was given a score on a scale of 0–3.

Western blotting

Protein levels were determined by Western blot as described in Andersson et al. (2009a) but with images detected by a LAS-4000 Mini CCD detection system (Fujifilm, Tokyo, Japan). Antibodies used for protein detection were: SERCA2a (MA3-919), L-type Ca²⁺ channel α₂δ₁subunit (MA3-921), pan-plasmalemmal Ca²⁺-ATPase (PMCA, MA3-914) (all Affinity BioReagents, Golden, CO, USA), NCX1 (Thomas et al. 2003), Na⁺–K⁺-ATPase (NKA) α₂ isoforms (Millipore, Billerica, MA, USA), and L-type Ca²⁺ channel α_1C subunit (AAC-003, Alomone Laboratories, Jerusalem, Israel). SERCA2b antiserum (Campbell et al. 1993) was the kind gift of Frank Wuytack, Katholieke Universiteit Leuven, Leuven, Belgium. Calsequestrin was used as loading control.

Ca²⁺-ATPase content

Total left ventricular (LV) Ca²⁺-ATPase was quantified as thapsigargin-sensitive calcium-dependent incorporation of [³²P] using whole-tissue homogenates and [γ³²P]ATP (Everts et al. 1989; Andersson et al. 2009a).

Cardiomyocyte experiments

Cardiomyocytes were enzymatically isolated (Andersson et al. 2009a) and placed in a perfusion chamber on the stage of an inverted microscope. Experiments were performed at 37°C. Basic cell shortening and Ca²⁺ transient measurements were recorded during field stimulation (3 ms biphasic pulse, 25% above threshold) during superfusion with Hepes Tyrode solution containing (in mm): 140 NaCl, 1 CaCl₂, 0.5 MgCl₂, 5.0 Hepes, 5.5 glucose, 0.4 NaH₂PO₄ and 5.4 KCl (pH 7.4). Contractions were measured using a video edge detector (Crescent Electronics, Sandy, UT, USA) coupled to a television monitor. Ca²⁺ transients were recorded in fluo-4 AM (Invitrogen Molecular Probes, Eugene, OR, USA) loaded myocytes using an LSM 510 confocal microscope (Zeiss GmbH, Jena, Germany) in line-scan mode (Louch et al. 2006). Time constants (τ) and rate constants (defined as 1/τ) were calculated from single exponential fits of Ca²⁺ transient decays (Mork et al. 2009). Time to 50% decline was measured in experiments with very slow changes in [Ca²⁺]_i which were difficult to fit exponentially.

Diastolic [Ca²⁺]_i was determined by whole-cell photometry (Photon Technology International, Monmouth Junction, NJ, USA) in cells pipette-loaded with a solution containing (in mm): 0.1 fura-2 salt (Invitrogen Molecular Probes), 120 potassium aspartate, 0.5 MgCl₂, 10 NaCl, 0.06 EGTA, 10 Hepes, 10 glucose, 25 KCl and 4 K₂-ATP (pH 7.2). Fura-2 fluorescence ratios (F₃₄₀/F₃₈₀) were calibrated to [Ca²⁺]_i, and calculated diastolic values were then used to calibrate Ca²⁺ transients recorded in fluo-4-loaded cells (Cheng et al. 1993; Andersson et al. 2009a).

Patch-clamp experiments were conducted with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA), pCLAMP software (Axon Instruments) and low resistance pipettes (1–2 MΩ). AP recordings were made in bridge-mode, and representative tracings employed as voltage-clamp waveforms as described (Mork et al. 2009). L-type Ca²⁺ current was recorded during APs in Na⁺- and K⁺-free internal (in mm: 135 CsCl₂, 3 Mg-ATP, 5 EGTA and 10 Hepes) and external (in mm: 136 TEA-Cl, 1.1 MgCl₂, 10 glucose, 10 Hepes, 4 KCl and 1 CaCl₂) solutions, and calculated as the current sensitive to 20 μm nifedipine (Mork et al. 2009). L-type Ca²⁺ current was also recorded during depolarizing 200 ms voltage steps from −50 mV, with an internal solution containing (in mm) 130 CsCl, 0.33 MgCl₂, 4 Mg-ATP, 0.06 EGTA, 10 Hepes and 20 TEA, and an extracellular solution containing (in mm) 20 CsCl, 1 MgCl₂, 135 NaCl, 10 Hepes, 10 d-glucose, 4 4-aminopyridine and 1 CaCl₂ (Andersson et al. 2009a).

Intracellular Na⁺ concentration ([Na⁺]_i) was assessed in myocytes loaded with SBFI AM as described previously (Swift et al. 2007 & Baartscheer et al. 1997). Total NKA current was obtained by rapidly elevating extracellular [K⁺] from 0 mm to 5.4 mm (Baartscheer et al. 1997; Swift et al. 2007). The α₂ NKA isoform component of the total current was calculated as the difference current following treatment with 5 μm ouabain, which selectively blocks the α₂ isoform (Berry et al. 2007). Remaining current was attributed to the α₁ isoform.

Statistics

Data are expressed as mean values ±s.e.m. Statistical significance was calculated using paired or unpaired t tests. P values < 0.05 were considered significant.

Results

Development of end-stage HF

Kaplan Meyer survival curves (Fig. 1A) indicated that KO mice died over a period of 3.4 weeks, starting at 6.9 weeks following induction of Serca2 gene deletion by tamoxifen. KO mice were killed at 7 weeks (7-week KO) in further experiments due to ethical considerations. To examine the phenotype and mechanisms of HF development, data from 7-week KO were compared with the 4 week time point (4-week KO). Cardiac output was reduced in 7-week, but not 4-week KO (Fig. 1B), as we have observed previously (Andersson et al. 2009a). Similarly, left atrial diameter (LAD), a sensitive parameter for congestive HF in mice (Finsen et al. 2005), was increased between 4-week and 7-week KO (Fig. 1C, P < 0.05 within KO). Lung weight/body weight ratios also indicated greater pulmonary congestion in 7-week KO than 4-week KO (167%vs. 130% of FF values, P < 0.05, Table 1). Tissue Doppler measurements showed that HF development involved impairment of both systolic and diastolic function (Fig. 1D and E, respectively). We also observed depressed heart rate in KO mice (Table 1), which we believe resulted from Serca2 ablation in the sinoatrial node (Andersson et al. 2009a).

Figure 1. Progression to end-stage HF in KO mice involved impairment of systolic and diastolic function.

A, survival curves (FF, n= 26; KO, n= 27). Echocardiographic measurements of cardiac output (B), left atrial diameter (C), systolic tissue velocity (D), and diastolic tissue velocity (E) (FF, n= 6; KO, n= 5). F, whole heart histological sections at 20× (top panels, hematoxylin/eosin) and 100× magnification (bottom panels, AFOG, with blue staining indicating fibrosis). *P < 0.05 vs. equivalent FF.

Table 1.

Animal and cell characteristics

	4-week FF	4-week KO	7-week FF	7-week KO
Age (weeks)	14.0 ± 0.1	13.9 ± 0.2	17.1 ± 0.1	16.8 ± 0.2
Heart rate (min−1)	487 ± 18	433 ± 24*	498 ± 19	395 ± 11*
BW (g)	23.7 ± 0.6	26.4 ± 0.7*	27.0 ± 0.7	24.6 ± 0.7*
LVW (mg)	75.7 ± 2.4	87.5 ± 2.7*	89.0 ± 3.0	83.5 ± 3.0
LW (mg)	134 ± 2	182 ± 6*	153 ± 5	232 ± 9*
LVW/BW	3.2 ± 0.1	3.3 ± 0.0	3.3 ± 0.0	3.4 ± 0.1
LW/BW	5.4 ± 0.3	7.0 ± 0.2*	5.7 ± 0.2	9.5 ± 0.4*
Cell length (μm)	129.6 ± 1.8	127.6 ± 1.8	127.6 ± 1.7	131.9 ± 1.9
Cell width (μm)	22.0 ± 0.4	21.8 ± 0.4	22.2 ± 0.4	21.5 ± 0.3
Fract. short. at 6 Hz (%)	3.76 ± 0.54	2.92 ± 0.43	2.98 ± 0.32	1.43 ± 0.11*
dl/dtmin at 6 Hz (μm ms−1)	0.101 ± 0.020	0.048 ± 0.007*	0.075 ± 0.010	0.019 ± 0.004*

BW, body weight; LVW, left ventricular weight; LW, lung weight; Fract. short., fractional shortening, *P < 0.05 vs. FF; n_animals: 4-week FF = 29, 4-week KO = 32; 7-week FF = 35; 7-week KO = 25; n_cells (length/width): 4-week FF = 115, 4-week KO = 121; 7-week FF = 139; 7-week KO = 143; n_cells (6 Hz function): 4-week FF = 13, 4-week KO = 13, 7-week FF = 15, 7-week KO = 11.

LV weight/body weight ratios and LV myocyte dimensions were similar in FF and KO at both time points (Table 1), indicating that LV hypertrophy was not present in KO. Histological examination of heart sections showed no evidence of inflammatory cell infiltration, generalized myocyte degeneration or disarray in KO hearts (Fig. 1F). However, a modest degree of fibrosis was observed with no apparent progression between the two time points (mean score = 1.3 ± 0.5 in 4-week KO vs. 1.3 ± 0.8 in 7-week KO, P= NS). Since we could not detect time-dependent alterations in LV geometry or histology, we hypothesized that HF progression in KO resulted from declining systolic and diastolic cardiomyocyte function.

Isolated myocyte contractions and Ca²⁺ transients

Representative myocyte contraction recordings during 1 Hz stimulation are shown in Fig. 2A. Contraction magnitude was reduced in 4-week KO from 4-week FF values, and further decreased in 7-week KO (Fig. 2B). Cardiomyocyte relaxation was also progressively slowed between 4-week and 7-week KO cells (Fig. 2C). Similar changes were observed in Ca²⁺ transients (Fig. 2D). Mean transient magnitude was markedly decreased in 4-week KO myocytes, and further decreased in 7-week KO (Fig. 2E). The declining phase of the Ca²⁺ transient was progressively slowed in KO cells (Fig. 2F).

Figure 2. Contractions and Ca²⁺ transients became progressively smaller and slower to decline in KO cardiomyocytes.

A, representative contraction traces, from isolated myocytes during 1 Hz field stimulation. Mean fractional shortening (B) and maximum relaxation velocity measurements (C). Representative Ca²⁺ transients recorded by fluo-4 fluorescence (D). Mean Ca²⁺ transient magnitudes (E) and decay kinetics (F). (n for panels B and C, E and F: 4-week FF = 13, 16; 4-week KO = 13, 20; 7-week FF = 15, 19; 7-week KO = 11, 19; *P < 0.05 vs. equivalent FF, ‡P < 0.05 vs. equivalent 4-week).

Increasing stimulation frequency to 6 Hz increased contraction magnitude to near-normal values in 4-week KO (Table 1). In 7-week KO, cell shortening was also improved by increasing the stimulation rate (1 Hz, 14% FF; 6 Hz, 48% FF, P < 0.05), but mean measurements remained significantly below control values. Similarly, relaxation kinetics were improved in both KO groups at higher stimulation frequency, but remained more markedly below FF values in 7-week KO (25% FF vs. 47% FF in 4-week KO, P < 0.05, Table 1). Thus, during stimulation in the physiological frequency range, myocyte contraction magnitude and relaxation kinetics roughly paralleled in vivo measurements of systolic and diastolic function. While we observed important differences in contraction amplitudes with frequency, Ca²⁺ transient amplitudes were similarly depressed in KO myocytes at 1 and 6 Hz (4-week KO: 25 ± 4%, 27 ± 4% FF, respectively; 7-week KO: 15 ± 2%, 16 ± 3% FF, P= NS for 1 Hz vs. 6 Hz, P < 0.05 for 4-week vs. 7-week KO). Diastolic [Ca²⁺]_i in KO was not altered from FF values at either time point or frequency (data not shown).

Declining SR function

We examined changes in cellular Ca²⁺ fluxes underlying alterations in Ca²⁺ transients and contraction/relaxation in KO myocytes. Ca²⁺ release elicited by rapid application of 10 mm caffeine was reduced to 27% of FF values in 4-week KO and 4% in 7-week KO, indicating declining SR Ca²⁺ content (Fig. 3A and B). To estimate SR Ca²⁺ release during each beat, we compared steady-state Ca²⁺ transients in the presence and absence of caffeine (Andersson et al. 2009a) (Fig. 3D). In FF cells, Ca²⁺ transient amplitudes were markedly reduced during caffeine, confirming that the Ca²⁺ transient was predominantly derived from SR (Fig. 3E). In contrast, SR Ca²⁺ release was minute in 4-week KO (11% FF values), and was not detectable in 7-week KO. Similarly, calculated rate constants for SR Ca²⁺ re-uptake were markedly reduced in 4-week KO, and were not significantly different from zero in 7-week KO (Fig. 3F). Using the measurements of SR-dependent and caffeine-elicited Ca²⁺ transients, we estimated that fractional release was reduced to 21 ± 3% in 4-week KO compared with 51 ± 10% in 4-week FF controls (P < 0.05). Very low SERCA2a expression levels were observed in 4-week KO; however, a small but significant further down-regulation was observed in 7-week KO (Fig. 3C). SERCA2b expression was also markedly reduced in KO, but similar in 4-week and 7-week cells (10 ± 1% and 10 ± 1% FF values, respectively, P= NS). Although Western blot sensitivity is below optimal at low protein expression levels, a reduction in SERCA2a levels between 4- and 7-week KO could account for declining SR function. Indeed, we observed that thapsigargin-sensitive Ca²⁺-ATPase content in whole-heart homogenates was reduced from 15.8 ± 3.0% to 7.9 ± 1.6% of FF values between 4-week and 7-week KO (P < 0.05). Also, the small degree of SR function present in 4-week KO cells was blocked by 1 μm thapsigargin, as caffeine-elicited transients were reduced to 4 ± 4 nm (vs. 167 ± 42 nm in untreated, P < 0.05) and SR Ca²⁺ release did not occur (data not shown). Seven-week KO transient magnitudes were unaffected by thapsigargin application (23 ± 4 nmvs. 26 ± 4 nm in untreated, P= NS). Thus, very low SERCA levels appear capable of partially refilling the SR in 4-week KO myocytes, and this residual SERCA activity is lost by the 7-week time point.

Figure 3. Minute SR function was present in 4-week, but not 7-week KO.

Representative caffeine-elicited Ca²⁺ transients (A) and magnitude measurements (B). Expression levels of SERCA2a (arrow) vs. calsequestrin (lower bands) (C). SR Ca²⁺ release during each beat was assessed by comparing Ca²⁺ transients in the presence and absence of caffeine (D). SR Ca²⁺ release (E) and reuptake (F). (n for panels B and C, E and F: 4-week FF = 13, 5, 9; 4-week KO = 20, 6, 19; 7-week FF = 20, 6, 18; 7-week KO = 19, 7, 19; *P < 0.05 vs. equivalent FF, ‡P < 0.05 vs. equivalent 4-week).

Enhanced Ca²⁺ influx

Representative recordings of L-type Ca²⁺ currents during voltage-clamp steps are shown in Fig. 4A. Mean measurements of peak current in 4-week and 7-week KO were 140% and 149% of FF values, respectively (P < 0.05 vs. FF, P= NS within KO). No marked shift in the current–voltage relationship was observed. Slower Ca²⁺ current decay kinetics in KO (half-decay time: 4-week FF = 4.7 ± 0.2 ms, 4-week KO = 8.5 ± 0.8 ms P < 0.05, 7-week FF = 5.6 ± 0.5 ms, 7-week KO = 8.6 ± 0.9 ms P < 0.05) resulted in very large integrated Ca²⁺ currents, especially in 7-week KO (4-week KO = 188% FF, 7-week KO = 220% FF, P= NS within KO, Fig. 4B). Parallel alterations were observed in L-type Ca²⁺ channel expression. Levels of the pore-forming subunit, α_1C, were 146% of FF values in 4-week KO (Fig. 4C), and there was a strong tendency for further up-regulation in 7-week KO (178% FF). Levels of the α₂/δ₁ regulatory subunit were also increased to 133 ± 10% and 147 ± 10% of FF levels in 4-week and 7-week KO, respectively (data not shown; P= NS within KO).

Figure 4. Increased L-type Ca²⁺ current in KO myocytes.

A, representative L-type Ca²⁺ currents recorded during a voltage step from −50 mV to −10 mV (left panel). L-type current–voltage relationship (right panel). B, integrated current measurements during the step to –10 mV. C, protein levels of the Ca²⁺ channel α_1C subunit (arrow) vs. calsequestrin (lower bands). (n for panels A and B, and C: 4-week FF = 14, 5; 4-week KO = 12, 6; 7-week FF = 8, 6; 7-week KO = 13, 7; *P < 0.05 vs. equivalent FF).

We examined whether alterations in AP configuration might additionally modify L-type Ca²⁺ influx and the Ca²⁺ transient. Representative recordings (Fig. 5A) and mean data (Fig. 5B) show that APs were prolonged at 50% and 70% repolarization in 7-week KO. Resting membrane potential and transient outward K⁺ current measurements were similar in all groups (data not shown), suggesting that AP prolongation in KO resulted primarily from augmented Ca²⁺ current. Also, AP prolongation was not observed in 7-week KO during treatment with 20 μm nifedipine (data not shown). The representative APs shown in Fig. 5A were employed as voltage-clamp waveforms in 7-week FF and 7-week KO cells. Figure 5C and D show that steady-state stimulation with the KO AP resulted in a larger Ca²⁺ transient than stimulation with the FF AP. This difference in amplitude was small in absolute terms (27 nm in 7-week FF cells, 14 nm in 7-week KO cells, P= NS), but proportionally very large in KO (% increase, 67%vs. 19% in FF, P < 0.05). In 7-week KO myocytes, where the entire transient results from Ca²⁺ influx, augmentation of transients during the KO AP predominantly resulted from a 43% increase in integrated L-type Ca²⁺ current (Fig. 5E). Thus, overall L-type Ca²⁺ entry in 7-week KO was dramatically increased from 7-week FF values by approximately 3- to 3.5-fold, when AP alterations were considered (Fig. 5E). This indicates that AP alterations can be an important compensatory mechanism for augmenting Ca²⁺ influx when SR function is inhibited.

Figure 5. AP prolongation facilitated Ca²⁺ entry in 7-week KO.

A, representative AP recordings at 1 Hz. B, times to 20%, 50% and 70% repolarization (APD₂₀, APD₅₀ and APD₇₀) (n= 16, 17, 11, 16; *P < 0.05 vs. corresponding FF). C, representative Ca²⁺ transients recorded during voltage-clamp stimulation with AP waveforms from A. Insets illustrate an expanded time scale with APs superimposed. Transient magnitude (D), integrated L-type Ca²⁺ current (E), and decay kinetics of the Ca²⁺ transient (F). (n for panels D, E and F: 7-week FF = 9, 8, 9; 7-week KO = 12, 4, 12; *P < 0.05 vs. equivalent FF AP, ‡P < 0.05 vs. equivalent 7-week FF).

NCX-mediated Ca²⁺ entry was also examined in 7-week FF and KO, by treating myocytes with 5 μm KB-R7943 (Tocris/Bio Nuclear AB, Bromma, Sweden) to preferentially block reverse-mode function (Iwamoto et al. 1996; Satoh et al. 2000). KB-R7943 treatment did not alter Ca²⁺ transient magnitude in 7-week FF cells (134 ± 23 nmvs. 140 ± 23 nm in untreated, P= NS), but markedly reduced transient size in 7-week KO (14 ± 2 nmvs. 23 ± 4 nm in untreated, P < 0.05). This suggests that the Ca²⁺ transient in 7-week KO is supported by enhanced NCX-mediated Ca²⁺ entry, in addition to increased L-type Ca²⁺ current (described above). Reverse-mode NCX function might be expected to be further facilitated by AP prolongation. However, switching between the FF and KO AP stimulus during KB-R7943 treatment augmented Ca²⁺ transients (increase of 17 ± 8% in 7-week FF, 67 ± 10% in 7-week KO, P < 0.05) to a similar extent as was observed in untreated cells (Fig. 5D). Thus, the prolonged AP in 7-week KO predominantly amplifies Ca²⁺ transients by increasing L-type current.

Ca²⁺ extrusion: compensation and de-compensation

We investigated whether alterations in Ca²⁺ extrusion contributed to slowing of Ca²⁺ transient decline during development of diastolic dysfunction between 4- and 7-week KO. Decay kinetics were examined for transients recorded in the presence of caffeine (as in Fig. 3D), when Ca²⁺ removal is dependent solely on trans-sarcolemmal flux. Representative Ca²⁺ transients (Fig. 6A) and mean data (Fig. 6B) show that while Ca²⁺ extrusion was more rapid than FF in 4-week KO, extrusion was slowed to control values in 7-week KO. By comparing rate constants of Ca²⁺ reuptake and extrusion, we can calculate that 60% of the overall slowing of Ca²⁺ transient decline between 4-week and 7-week KO resulted from loss of SR re-uptake, while the remaining 40% resulted from slowing of Ca²⁺ extrusion. Importantly, slowing of extrusion in KO occurred despite a progressive up-regulation in NCX and PMCA protein levels between 4 and 7 weeks (Fig. 6C).

Figure 6. Ca²⁺ extrusion was enhanced in 4-week, but not 7-week KO.

A, steady-state Ca²⁺ transients recorded in the presence of 10 mm caffeine. B, decay rate measurements. C, protein levels of NCX and PMCA (arrows) vs. calsequestrin (lower bands). (n for panels B and C: 4-week FF = 16, 5; 4-week KO = 18, 6; 7-week FF = 20, 6; 7-week KO = 18, 7; *P < 0.05 vs. equivalent FF, ‡P < 0.05 vs. equivalent 4-week).

In AP clamp experiments, we observed that the 7-week KO AP did not alter Ca²⁺ transient decline (Fig. 5F), since even the prolonged AP was repolarized shortly after the transient peak (insets in Fig. 5C). We hypothesized that slowing of Ca²⁺ extrusion instead resulted from inhibition of forward-mode NCX function by elevation of [Na⁺]_i. Indeed, SBFI ratios were increased in 7-week myocytes, indicating cytosolic Na⁺ accumulation (Fig. 7A). Interestingly, this was associated with reduced expression levels of NKA α₁ and α₂ isoforms (Fig. 7B). Total NKA current was also reduced in 7-week KO, which included a 40% reduction in α₂ current and a tendency for decreased α₁ current (Fig. 7C). We have previously shown that reduced NKA activity increases [Na⁺]_i (Swift et al. 2007). As proof of principle that such changes can also impair Ca²⁺ extrusion, we examined Ca²⁺ removal during NKA blockade by ouabain. In 7-week FF cells, ouabain significantly slowed the half-decay time of Ca²⁺ transients recorded during caffeine (Fig. 7D). Ouabain treatment did not, however, slow Ca²⁺ extrusion in 7-week KO, suggesting that baseline elevations in [Na⁺]_i may have maximally inhibited NCX function. Interestingly, Ca²⁺ extrusion during ouabain was more rapid in 7-week KO than FF, which probably resulted from the marked up-regulation of PMCA protein expression in KO.

Figure 7. Elevated [Na⁺]_i impaired Ca²⁺ extrusion in 7-week KO.

A, SBFI F₄₁₀/F₅₉₀ ratios were increased in 7-week KO indicating cytosolic Na⁺ accumulation (n= 9, 9, 13, 10 (order as in key here and in E)). B, expression of NKA α₁ and α₂ isoforms (n= 6 all groups). C, NKA currents obtained by rapid elevation of extracellular [K⁺]_i were subdivided into α₁ and α₂ components based on sensitivity to 5 μm ouabain (n for 7-week FF = 10, 7-week KO = 6). D, Ca²⁺ extrusion during NKA blockade by 1 mm ouabain, as estimated by half-decay time of Ca²⁺ transients during caffeine (n for 7-week FF = 10, 7-week KO = 10). E, Ca²⁺ transients following dialysis with 6 mm[Na⁺]_i (n= 19, 11, 20, 19). (For panels A–C, *P < 0.05 vs. equivalent FF; panel D, *P < 0.05 vs. untreated; ‡P < 0.05 vs. equivalent 7-week FF; panel E, *P < 0.05 vs. equivalent FF, ‡P < 0.05 vs. equivalent 4-week).

To further investigate the importance of elevated [Na⁺]_i for slowing of Ca²⁺ extrusion, we examined Ca²⁺ transients in cells dialysed with patch pipettes containing a low [Na⁺] (6 mm). Figure 7E shows that under such conditions the decay of Ca²⁺ transients was significantly enhanced in 7-week KO (compare Fig. 2F), and became even more rapid than in 4-week KO. These data support a detrimental role of Na⁺ accumulation in promoting diastolic dysfunction in 7-week KO.

Discussion

In this study, we observed that mice with cardiomyocyte-specific ablation of SERCA2 developed end-stage HF by 7 weeks following knockout. Declining systolic and diastolic function between 4-week and 7-week KO was associated with reduction in cardiomyocyte contraction magnitude and slowing of relaxation, caused by parallel changes in Ca²⁺ transients. Progressive reduction in SR Ca²⁺ release was partially offset by AP prolongation, which increased L-type Ca²⁺ current, and by greater Ca²⁺ entry via reverse-mode NCX function. Diastolic dysfunction in end-stage HF resulted from a combination of declining SERCA activity and accumulation of intracellular Na⁺ which impaired NCX-mediated Ca²⁺ extrusion. Increased [Na⁺]_i in 7-week KO was caused, at least in part, by down-regulation of NKA.

Elevation of [Na⁺]_i has been previously reported in both human HF (Pieske et al. 2002) and animal models (Despa et al. 2002; Baartscheer et al. 2003). The mechanisms proposed to underlie this Na⁺ accumulation include increased late Na⁺ current (Sossalla et al. 2008) and increased Na⁺–H⁺ exchange (Baartscheer et al. 2003). As well, in agreement with our observations, several previous studies have reported reduced NKA expression in failing myocytes (Schwinger et al. 1999; Muller-Ehmsen et al. 2001; Despa et al. 2002; Swift et al. 2008). Two of these studies have additionally reported decreased NKA activity (Schwinger et al. 1999; Swift et al. 2008). We observed reduced expression of both the α₁ and α₂ NKA isoforms. Based on our previous work (Swift et al. 2007), we believe that the α₁ isoform plays a more important role in controlling global [Na⁺] than the α₂ isoform. With a reduction in α₁ expression, it might be hypothesized that remaining pumps could simply increase their activity to maintain normal [Na⁺]_i. However, our preliminary data indicate that this is not the case, as we have observed that even partial blockade of the α₁ isoform results in increased [Na⁺]_i. With reduced NKA pumping capacity, we postulate that [Na⁺]_i rises to a new steady-state level in 7-week KO, which stimulates remaining pumps, resulting in a normal rate of Na⁺ extrusion to balance Na⁺ influx. However, we actually measured reduced NKA current. This discrepancy is probably due to the fact that NKA current was measured in patch-clamped, dialysed cells which allowed comparison of pump current in KO and FF with [Na⁺]_i normalized. Interestingly, we observed that the α₂ component of the total NKA current was significantly decreased in 7-week KO. In our recent work, we have shown that the α₂ isoform is preferentially localized to the T-tubules, where it regulates local [Na⁺] near NCX (Swift et al. 2007). Therefore, it follows that down-regulation of α₂ in 7-week KO could impair NCX-mediated Ca²⁺ extrusion. Indeed, we observed that NKA blockade by ouabain treatment slowed Ca²⁺ extrusion in FF. Our finding that ouabain did not alter Ca²⁺ extrusion in KO suggests that Na⁺ accumulation in the dyad may have maximally impaired forward-mode NCX in these cells.

PMCA function is believed to normally play a minor role in Ca²⁺ removal. However, data from the NCX knockout mouse indicate that the PMCA can have a surprisingly large capacity to extrude Ca²⁺ when challenged (Henderson et al. 2004). We presently observed a striking up-regulation of PMCA expression by more than 200% in 7-week KO, which would be expected to increase the rate of Ca²⁺ extrusion. As well, the Ca²⁺ affinity of PMCA might theoretically also be increased in KO myocytes, due to the action of regulatory molecules such as calmodulin (Brini, 2009). Thus, with NCX-mediated Ca²⁺ extrusion inhibited in these cells, PMCA may become an important mechanism for Ca²⁺ removal. In support of this view, we observed that in the presence of ouabain and high [Na⁺]_i (Swift et al. 2008), Ca²⁺ extrusion was significantly faster in 7-week KO than FF. Also, normalization of [Na⁺]_i by dialysis increased the rate of decay of 7-week KO Ca²⁺ transients beyond 4-week KO values. Therefore, with NCX function uninhibited, Ca²⁺ extrusion via NCX and PMCA may be able to maintain diastolic function at near-normal levels in the complete absence of SERCA function.

The mechanisms that compensate for declining SR Ca²⁺ content and release are intriguing. We previously reported up-regulation of the L-type Ca²⁺ channel and increased Ca²⁺ current magnitude in 4-week KO myocytes (Andersson et al. 2009a). We presently confirmed these findings, and observed that increases in Ca²⁺ channel expression and current magnitude persist in 7-week KO. In fact, integrated currents tended to be even larger in 7-week than 4-week KO. Our AP clamp experiments showed that the longer AP in 7-week KO additionally facilitated L-type Ca²⁺ current, resulting in Ca²⁺ currents that were 3- to 3.5-fold larger than those recorded in FF cells during FF APs. Additional Ca²⁺ current during the prolonged AP substantially increased Ca²⁺ transient magnitude in 7-week KO. However, these effects were more modest in FF cells, which have functional SR, since AP prolongation is known to reduce the efficiency of Ca²⁺-induced Ca²⁺ release (Sah et al. 2003). Ca²⁺ currents may have been additionally enhanced in 7-week KO by Ca²⁺–calmodulin-dependent kinase II phosphorylation, as has been reported in other heart failure models (Wang et al. 2008).

AP prolongation might also be expected to enhance Ca²⁺ entry via reverse-mode NCX. However, we observed that the 7-week KO AP induced a similar increase in Ca²⁺ transient magnitude in the presence and absence of KB-R7943. It appears then that the benefits of AP prolongation in 7-week KO mice stem primarily from greater L-type Ca²⁺ current. Compared to the untreated condition, however, Ca²⁺ transient magnitude was decreased by KB-R7943 in 7-week KO, but unaltered in 7-week FF. This suggests that NCX-mediated Ca²⁺ entry is enhanced in KO on a beat-to-beat basis, as predicted when Na⁺ levels are increased and Ca²⁺ transients are reduced. At the tested dose of 5 μm, KB-R7943 is reported to block 90% of reverse-mode NCX function without reducing forward-mode activity (Satoh et al. 2000). Although some have suggested that KBR is not mode-selective, recent evidence indicates that bi-modal NCX blockade increases Ca²⁺ transient magnitude (Ozdemir et al. 2008). We observed that KBR reduced transient magnitude and did not alter decay kinetics (data not shown), indicating preferential reverse-mode blockade.

Despite markedly enhanced Ca²⁺ influx, declining SR function resulted in a progressive reduction in Ca²⁺ transient amplitude in KO cells, which was observed at both low and high stimulation frequency (1 Hz and 6 Hz). Four-week KO myocytes also exhibited reduced contraction magnitudes at 1 Hz, but contractile function was enhanced at high pacing rates as 6 Hz contractions were similar in magnitude to controls. This is in agreement with our previous observation that myofilament responsiveness to Ca²⁺ is increased in 4-week KO (Andersson et al. 2009a), which may become particularly pronounced at high stimulation rates when diastolic [Ca²⁺]_i is elevated. In addition, the slowed Ca²⁺ transient kinetics in KO may also improve contractile function by prolonging myofilament activation. Such alterations may continue to aid contractile function in 7-week KO, as contraction magnitude was increased between 1 Hz and 6 Hz despite the presence of very small Ca²⁺ transients at both frequencies. Nevertheless, it appears that such adaptations are insufficient to maintain systolic function at normal levels at this late time point when SR function fails completely.

Although there are clearly important differences between end-stage heart failure in humans and SERCA2 KO mice, some cellular mechanisms may be conceptually similar. Data from human HF appear to support our observation that slowing of Ca²⁺ extrusion is an important mechanism underlying diastolic dysfunction. Hasenfuss et al. (1999) observed that HF patients with impaired diastolic performance exhibited decreased SERCA expression but unchanged NCX. In contrast, NCX levels were increased in patients with preserved diastolic function. The authors suggested that greater NCX activity may compensate for reduced SERCA function to maintain near-normal Ca²⁺ decay and relaxation rates. Our results also support the notion that reduction in SERCA levels can promote systolic dysfunction. In human HF, SERCA expression is often reported to be reduced by 30–40% (for review, see Hasenfuss & Pieske, 2002), and this reduction is generally believed sufficient to impair systolic performance. Mice are clearly very adept at compensating for loss of SERCA, since we observed near-normal systolic function in 4-week KO despite a more than 95% reduction in SERCA2a protein. We have previously shown that activation of the sympathetic nervous system contributes to this compensation (Andersson et al. 2009a). In the present study, we observed that loss of a small remaining pool of SERCA2a by 7-week KO was associated with development of systolic HF. Since the half-life for SERCA protein is approximately 2–3 days (Andersson et al. 2009a), it is surprising that a functional amount of SERCA would be present beyond the first few weeks following knockout. However, it may be that there is normally an excessive amount of SERCA protein, much of which is either not required or not accessible for normal function. Alternatively, there may be two different pools of SERCA protein: one pool which has a short half-life, and a second perhaps smaller pool which has a longer half-life. Theoretically, declining SERCA activity between 4- and 7-week KO could result from gradual loss of this second pool of SERCA protein.

Compensatory mechanisms present in KO myocytes may not be present in human HF. For example, Ca²⁺ current is generally reported to be unaltered in failing human myocytes (Bers, 2006). As well, more profound AP prolongation in human HF is believed to reduce, not augment, Ca²⁺ transient magnitude (Sah et al. 2003). Therefore, relatively moderate reductions in SERCA expression such as those reported in HF patients may be sufficient to impair systolic function if Ca²⁺ influx is not increased. Our findings suggest then that strategies aimed at enhancing Ca²⁺ influx may be beneficial in treating systolic dysfunction. Diastolic function, on the other hand, would probably benefit from augmentation of NKA activity and Na⁺ unloading. Such therapies may, however, have the opposite effect on systolic function, since elevated [Na⁺]_i is known to increase Ca²⁺ transients by elevating SR Ca²⁺ load. Indeed, clinical trials have indicated that digoxin, a NKA inhibitor, is appropriate for treatment of systolic but not diastolic dysfunction in HF (Shammas et al. 2007). A therapeutic compromise may be attainable through agents such as istaroxime, a newly developed compound which simultaneously inhibits NKA and stimulates SERCA. Early clinical data suggest that istaroxime improves both contractility and diastolic relaxation in HF patients (Khan et al. 2009).

In conclusion, our data from the SERCA KO mouse indicate that declining SERCA function in end-stage HF contributes to the simultaneous development of both systolic and diastolic dysfunction. However, impaired relaxation also results from accumulation of intracellular Na⁺ following down-regulation of NKA. Our data indicate that Ca²⁺ extrusion mechanisms can dramatically compensate for SERCA loss to maintain diastolic function when Na⁺ gain is prevented.

Glossary

Abbreviations

AP

action potential

[Ca²⁺]_i

intracellular [Ca²⁺]

FF

Serca2^flox/flox mice

HF

heart failure

KO

Serca2^flox/flox Tg(αMHC-MerCreMer) mouse

LAD

left atrial diameter

LV

left ventricular

[Na⁺]_i

intracellular [Na⁺]

NCX

Na⁺–Ca²⁺ exchanger

NKA

Na⁺-K⁺-ATPase

PMCA

plasmalemmal Ca²⁺-ATPase

SERCA

SR Ca²⁺-ATPase

SR

sarcoplasmic reticulum

Author contributions

Conception and design of this study was conducted by W.E.L, I.S., K.B.A., G.C. and O.M.S. Experiments examining cardiomyocyte size, contractile function, Ca²⁺ and Na⁺ homeostasis, and action potential configuration, were performed by W.E.L., H.K.M., F.S. and J.M.A. Cardiomyocyte isolation was conducted by K.H. Echocardiographic analysis was performed by K.H. and I.S. Morphological and histological examinations were conducted by K.B.A., H.M.R. and B.R. Kaplan Meyer curves and LV Ca²⁺-ATPase activity were determined by K.B.A. All authors contributed to writing of the manuscript.