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Cardiovascular Research 2004 62(3):538-547; doi:10.1016/j.cardiores.2004.01.038
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Enhanced sarcolemmal Ca2+ efflux reduces sarcoplasmic reticulum Ca2+ content and systolic Ca2+ in cardiac hypertrophy

M.E Díaz, H.K Graham and A.W Trafford*

Unit of Cardiac Physiology, The University of Manchester, 1.523 Stopford Building, Manchester M13 9PT, UK

* Corresponding author. Tel.: +44-161-275-7969; fax: +44-161-275-2703. Email address: trafford{at}man.ac.uk

Received 4 November 2003; revised 14 January 2004; accepted 29 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Recent work has identified reductions in the systolic Ca2+ transient in cardiac disease states. The aim of the present study was to identify the mechanisms responsible for perturbations of intracellular calcium homeostasis in isolated cardiac myocytes and determine if such changes can quantitatively explain the reduced systolic Ca2+ transient. Methods: Left ventricular hypertrophy (LVH) was induced by aortic coarctation in adult ferrets. Changes in intracellular Ca2+ regulation, sarcolemmal Ca2+ fluxes and SR function were measured in single left ventricular cardiac myocytes. Results: Cardiac hypertrophy was associated with a 29% increase in action potential duration (APD90); a 48% reduction in the amplitude of and 19% slowing in the rate of decay of the systolic Ca2+ transient; a 20% decrease in SR Ca2+ content and a 36% increase in inward Na+–Ca2+ exchange current for a given change in [Ca2+]i (all P<0.05). Peak L-type Ca2+ current density, integrated Ca2+ influx and SERCA2a protein levels remained unchanged in hypertrophy. By determining the relationship between SR Ca2+ content and systolic Ca2+, the reduction in SR Ca2+ content quantitatively explained the smaller systolic Ca2+ transient. The reduced SR Ca2+ content also accounted for a smaller fractional release of Ca2+ from the SR and lower gain of excitation contraction coupling in cardiac hypertrophy. The increased sarcolemmal-mediated Ca2+ efflux was sufficient to explain the reduction in SR Ca2+ content. Conclusions: The findings indicate that the primary mechanism underlying the smaller systolic Ca2+ transient amplitude in cardiac hypertrophy is decreased SR Ca2+ content occurring as a consequence of reduced SR Ca2+-ATPase-mediated Ca2+ uptake and increased sarcolemmal-mediated Ca2+ efflux from the cell. The increased Na+–Ca2+ exchange-mediated current for a given change in intracellular Ca2+ concentration provides a mechanism for the development of arrhythmias in the face of a reduced SR Ca2+ load in cardiac hypertrophy.

KEYWORDS e-C coupling; Hypertrophy; SR (function); Na/Ca-exchanger; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Diseases of the cardiovascular system are a major health problem associated with significant morbidity and mortality [1]. Contributory factors include the genesis of arrhythmias and contractile dysfunction. It is likely that many of the observed alterations are due to changes in the processes leading to contraction and relaxation at the cellular level [2,3].

Previous heart failure (HF) studies have found a reduced amplitude of the systolic Ca2+ transient, contraction and a slowed rate of decay of [Ca2+]i [4–7]. The situation during the period of compensated cardiac hypertrophy is more controversial where systolic Ca2+ and contractility may be unaltered, decreased or increased [8] likely reflecting the stage and extent of the underlying pathology. However, where dysfunction is present potential mechanisms include: (i) impaired triggering of Ca2+ release from the SR as a consequence of a reduced SR Ca2+ loading [9,10] and (ii) altered myofilament Ca2+ sensitivity.

Given the steep dependence of systolic [Ca2+]i on SR loading [11,12], SR function is probably key to altered Ca2+ homeostasis in disease. While very few studies have quantitatively examined SR Ca2+ content in HF, many have found abnormalities in SR-related proteins, e.g., SERCA, RyR, phospholamban (PLB) and FKBP12.6. SR Ca2+ content is ultimately controlled by the fluxes of Ca2+ across the sarcolemma (SL) [13]. Whereas Ca2+ influx via ICa-L is often unaltered in HF, Na+–Ca2+ exchange (NCX) expression and function is typically enhanced [9,14,15]. This has at least three important consequences: (i) if Ca2+ entry is unaltered then Ca2+ flux balance can only be achieved by a smaller systolic rise of [Ca2+]i; (ii) it is potentially arrhythmogenic since a given change of [Ca2+]i should generate a greater depolarising current; and (iii) in reverse mode, it can trigger SR Ca2+ release especially if [Na+]i is increased [16].

The purpose of the present work was to develop a chronic model of left ventricular hypertrophy (LVH) and determine at the cellular level how changes in systolic Ca2+ arise. We have then extended our investigations to specifically determine the role of adaptations in SL Ca2+ fluxes to altering SR Ca2+ content and thus if the changes in Ca2+ handling offer a quantitative and mechanistic explanation for the reduced amplitude of the systolic Ca2+ transient. Such integrative analysis reveals that the reduced amplitude of the systolic Ca2+ transient in LVH is exactly accounted for by the lower SR Ca2+ content and mechanistically this is explained by a greater SL-mediated Ca2+ efflux.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation 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 The UK Animals (Scientific Procedures) Act 1986).

2.1. Induction of cardiac hypertrophy
Thirty-three adult male ferrets (912±34 g, no difference between sham or clipped animals) were anaesthetised with intramuscular medetomidine (80 µg kg–1) and ketamine (8 mg kg–1), intubated and ventilated with oxygen. Analgesia was provided using carprofen (4 mg kg–1) and antibiosis with enrofloxacin (5 mg kg–1). Pressure overload of the left ventricle was produced in 17 animals by placement of an ameroid occluder (Research Instruments, CA, USA) on the ascending aorta. Sixteen sham-operated animals served as controls undergoing the same procedure excepting occluder placement. Echocardiography was performed on isoflurane (1–2% in oxygen) anaesthetised animals to monitor the development of cardiac hypertrophy and contractile dysfunction. Left ventricular free wall thickness and ejection fraction were assessed just below the tips of the mitral valves using a 14-MHz linear array probe (Siemens, CA, USA).

2.2 Cell isolation, measurement of [Ca2+]i, contraction and voltage clamp
Single myocytes were isolated from the left ventricular free wall using an enzymatic digestion as previously described [13]. [Ca2+]i was measured using Fluo-3AM (Molecular Probes, OR, USA) (5 µmol l–1, 5 min loading and 30 min de-esterification) and calibrated by obtaining the Ca2+ saturated fluorescence at the end of the experiment by impalement of the cell with a micropipette [17].

Voltage clamp control was achieved using the perforated patch clamp technique with amphotericin-B (240 µg ml–1) [17]. The switch clamp facility (frequency 1–3 kHz and gain 0.7–2.3) of the Axoclamp 2B voltage clamp amplifier (Axon Instruments, CA, USA) was used to overcome the access resistance of the perforated patch. The experimental solution contained (in mmol l–1): NaCl, 140; HEPES, 10; glucose, 10; KCl, 4; CaCl2, 2; Probenecid, 2; MgCl2, 1. For voltage clamp experiments, K+ and Cl– currents were blocked by addition of (in mmol l–1): 4-aminopyridine, 5; BaCl2, 0.1; and DIDS, 0.1 titrated to pH 7.4. Micropipettes (<2 M{Omega}) were filled with (in mmol l–1): KCH3O3S, 125; KCl, 20; NaCl, 10; HEPES, 10; MgCl2, 5 titrated to pH 7.2 with KOH. The temperature of the superfusate was controlled at 23 or 37±0.1 °C (Cell Microcontrols, CA, USA). Action potentials were elicited at 0.5 Hz with a 2 nA depolarising pulse.

2.3 Sarcoplasmic reticulum and sarcolemmal Ca2+ flux measurements
SR Ca2+ content and sarcolemmal Ca2+ fluxes were quantified as described [11,18]. Briefly, following steady-state stimulation, membrane potential was held at –40 mV and SR Ca2+ discharged by application of 10 mM caffeine. The resulting Na+–Ca2+ exchange current was integrated and corrected for non-electrogenic Ca2+ efflux. Under voltage clamp, sarcolemmal Ca2+ influx was determined by integrating the L-type Ca2+ current (100 ms step from –40 to +10 mV) and Ca2+ efflux by integrating the Na+–Ca2+ exchange tail current upon repolarisation [11]. All sarcolemmal fluxes and SR Ca2+ measurements are expressed relative to total cell volume obtained from capacitance measurements in each cell [13].

2.4. Immunoblotting
Full thickness wedges of the left ventricular free wall were snap frozen and stored in liquid nitrogen until use. Samples were homogenised on ice in RIPA buffer with protease inhibitors (1 g ml–1 leupeptin, 10 µg ml–1 PMSF and 2 µg ml–1 aprotonin). A total of 5 µg of total protein was separated through an 8% denaturing polyacrylamide gel and transferred to PVDF membrane. SERCA2a was then blotted using a mouse anti-SERCA2a (Affinity BioReagents, USA) primary antibody and HRP conjugated secondary antibody. Luminescence (Supersignal. Pierce, UK) was captured and quantified using a chemiluminescent camera (Syngene, UK). Primary and secondary antibodies were then stripped (Restore Stripping Buffer, Pierce, UK) and the membrane reprobed for GAPDH. SERCA2a intensity was normalised to GAPDH signal intensity to control for loading/transfer differences.

2.5. Statistics
All data are expressed as mean±standard error for n experiments. Comparisons between groups of data are made with Student's t-test or analysis of variance and considered significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Model characteristics
At the time of sacrifice (LVH, 112±17; sham, 139±21 days following surgery; P>0.25) none of the LVH animals exhibited clinical symptoms of heart failure, i.e., lethargy or dyspnoea. Three of the clipped animals died suddenly from aortic aneurysms 51±8 days following surgery. The end diastolic left ventricular wall thickness increased from 3.7±0.1 mm in shams to 5.4±0.1 mm in LVH (n=7 each, P<0.001) and ejection fraction decreased from 0.68±0.02 to 0.53±0.02 (P<0.001).

Table 1 summarises the gross organ and cellular parameters. The heart/body weight ratio increased by 67% in LVH. Despite the absence of clinical symptoms of heart failure, there was a small (27%) but significant increase in the lung/body weight ratio suggesting that these animals were at a very early stage of cardiac decompensation. However, in a separate series of experiments (not shown), animals with overt heart failure showed a 386% increase in the lung/body weight ratio and 210% increase in heart/body weight ratio indicating considerable scope for additional cardiac hypertrophy before clinical symptoms are evident.


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  		Table 1 Organ and cellular parameters

 
3.2 [Ca2+]i and contraction
Echocardiography indicates decreased contractility in vivo; the next series of experiments determined if this was maintained in vitro. Experiments were performed under voltage clamp conditions thus controlling for any effect changes in action potential duration have on [Ca2+]i [5,19,20]. Fig. 1A shows typical measurements. The data of Fig. 1A and normalised data of Fig. 1B suggest that both the amplitude of systolic [Ca2+]i and contractility are reduced and that the rate of decay of [Ca2+]i and relaxation are slowed in LVH, the extent of the alterations in [Ca2+]i and contraction are summarised in the mean data of Table 2. In LVH, the amplitude of the systolic Ca2+ transient is reduced to 52.1% of sham levels. In addition, the changes in Ca2+ transient amplitude and relaxation are maintained under more physiological conditions (37 °C).


Figure 1
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  		Fig. 1 Systolic [Ca2+]i, contractile and electrophysiological alterations in LVH. (A) Original records of [Ca2+]i (top) and contraction (bottom) from sham (left panels) and LVH (right panels) myocytes. (B) Normalised Ca2+ transients (upper) and contraction records (lower). (C) Typical ICa-L for sham (bullet) and LVH ({triangleup}) myocytes. (D) Relationship between peak ICa-L density and cell capacitance for sham (bullet) and LVH ({triangleup}) myocytes. Lines through the data are best-fit linear regressions to the sham data (solid), LVH data (dashed) and combined data (dotted). (E) Representative action potentials recorded from a sham (bullet) and LVH ({triangleup}) myocyte. (F) Mean repolarisation times for action potentials in sham (bullet) and LVH ({triangleup}) myocytes. *P<0.05.

 

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  		Table 2 [Ca2+]i and contraction measurements

 
3.3 Sarcolemmal Ca2+ entry and action potentials
The reduced systolic Ca2+ transient in LVH could arise through altered properties of trigger Ca2+ entry (ICa-L). This was examined under voltage clamp conditions (0.5 Hz, 23 °C, –40 to +10 mV, 100 ms). Fig. 1C shows representative L-type Ca2+ currents and Table 3 summarises the properties of the L-type Ca2+ current and shows that in LVH there was a tendency for the peak current density at +10 mV to be reduced (not significant) and there was no difference in the total amount of Ca2+ entering the cell on ICa-L. The slight decrease in ICa-L density may reflect a change in the relationship between cell capacitance and ICa-L density. This is examined in greater detail in Fig. 1D where the data have been fitted with linear regressions. The fits to the data have slopes significantly different from zero (P<0.02). However, neither the slopes of the data sets (sham: –0.019±0.008 and LVH: –0.013±0.004 pA pF–2) or their intercepts (sham: 8.70±1.8 and LVH: 7.84±1.4 pA pF–1) are different from each other (P>0.1) and the pooled data can be equally well fit with a single regression line having slope –0.014 pA pF–2 and intercept 7.96 pA pF–1. Thus, while there is a relationship between ICa-L density measured at +10 mV and cell capacitance this does not differ between control and LVH; however, this can account for the slightly reduced ICa-L density observed in hypertrophied cells.


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  		Table 3 Calcium current and action potential properties

 
Fig. 1E shows typical action potentials and Fig. 1F illustrates the properties of repolarisation. Times to 50%, 75%, 90% and 95% repolarisation are increased in LVH (P<0.05, Table 3). There is a small, yet significant change in the resting membrane potential in LVH; however, the peak amplitude of the action potential is unaltered (Table 3). Despite the increased action potential duration, in LVH the Ca2+ transient is reduced to a similar extent (57%) as under voltage clamp conditions (461±85 to 261±36 nmol l–1, P<0.05, n=6–9 cells, 3–4 hearts).

3.4 Sarcoplasmic reticulum Ca2+ content
In the next series of experiments, we have determined if the reduction in Ca2+ transient amplitude is due to a change in SR content. In LVH at 23oC, SR Ca2+ content is reduced to 80.3% of sham values (38.5±1.7–30.9±1.6 µmol l–1, P<0.005, n=29–39 cells, 5–6 hearts). The mean data of Fig. 2B show that this reduction in SR Ca2+ content is maintained at physiological temperatures and rates of stimulation (P<0.05, n=15–39 cells, 5–6 hearts). The above method assumes that the relationship between cell volume and surface area does not change in LVH. In both human and rat, this was reported to be unaltered [21,22]. We have also quantified SR Ca2+ loading indirectly by measuring the amplitude of the caffeine-evoked Ca2+ transient (Fig. 2C). Using this volume/surface area independent approach, SR loading is reduced to 57% of sham levels (394±35 vs. 686±138 nmol l–1, P<0.05).


Figure 2
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  		Fig. 2 Sarcoplasmic reticulum Ca2+ content in LVH. (A) Calculation of SR Ca2+ content. Following steady-state stimulation, membrane potential is held at –40 mV and 10 mM caffeine applied (solid bar). The resulting inward Na+–Ca2+ exchange current (middle panel) is then integrated (lower panel) to yield SR Ca2+ content. (B) Mean data for SR Ca2+ contents measured in sham (bullet) and LVH ({triangleup}) cells following steady-state stimulation with 100 ms pulses from –10 to +10 mV at the frequencies and temperatures given on the abscissa. (C) Mean data showing the amplitude of the caffeine-evoked Ca2+ transient in sham (solid) and LVH (open) cells at 23 °C. *P<0.05.

 
The relationship between systolic [Ca2+]i and SR Ca2+ content is given by [11]:



Formula 1

(1)

Thus, from the reduction in SR content to 80.3% of control, Eq. (1) predicts that the Ca2+ transient amplitude should be reduced to 48.5% of control levels. This is in very close agreement with the 52% measured experimentally and is strongly supportive of a causal link between the changes in Ca2+ transient amplitude and the SR Ca2+ content.

3.5 Fractional SR Ca2+ release and EC coupling gain
The fractional release of Ca2+ from the SR can be estimated from the ratio of the amplitude of the systolic Ca2+ transient to that of the caffeine-evoked Ca2+ transient [12]. Using this method, SR fractional release is reduced in LVH from 0.65±0.03 to 0.54±0.04 (P<0.05, n=16–23 cells). An alternative method to determine fractional release is to derive the buffering properties of each cell and then convert changes in free cytosolic Ca2+ to total Ca2+ fluxes ({Delta}CaT) and thence calculate the amount of Ca2+ released from the SR [11,17]:



Formula 2

(2)

Using this method, the difference in SR fractional release still exists although is less than that obtained from the ratio of the amplitudes of the evoked Ca2+ transients (0.54±0.06 in sham vs. 0.37±0.04 in LVH, P<0.05). This is explained by the latter method accounting for sarcolemmal Ca2+ entry, which equates to ~11% of the SR content.



Formula 3

(3)

An alternative to fractional release when considering the efficiency of EC coupling is to examine the gain of EC coupling [12,23]. When gain is calculated using the method given in Eq. (3), it is found to be less in LVH (5.6±0.6 vs. 3.7±0.5, P<0.02). However, SR Ca2+ content markedly affects both gain [12,24] and fractional release and when both parameters are normalised to SR Ca2+ content the difference between sham and LVH is lost (P=0.3). Alternative approaches using {delta}[Ca2+]i/{delta}t normalised to ICa-L entry also failed to detect differences in gain between sham and LVH.

3.6 Ca2+ removal
In the next experiments, the mechanisms responsible for the reduced SR Ca2+ content were investigated. The relative contributions made to Ca2+ removal from the cytoplasm by SERCA, NCX and SL Ca2+-ATPase (PMCA) were determined using the protocol of Fig. 3A. When all Ca2+ removal processes are active the rate constant of decay of [Ca2+]i was 4.59 s–1 (ksystolic). In caffeine, where SERCA activity is effectively negated, the decay rate (kcaffeine) slowed to 1.26 s–1. In the presence of Ni2+ and caffeine, where both SERCA and NCX are ineffective, the rate of decay of [Ca2+]i (kNi) due to PMCA was 0.29 s–1.


Figure 3
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  		Fig. 3 Relative contributions to Ca2+ removal. (A) Experimental protocol. Time-course showing [Ca2+]i. Following steady-state stimulation (sys.), membrane potential is held at –40 mV and 10 mM caffeine applied as indicated. Following caffeine removal stimulation is resumed (–40 to +10 mV, 100 ms, 0.5 Hz, 23 °C) to steady state before 10 mM Ni2+ is applied. Stimulation is then stopped and caffeine applied in the maintained presence of Ni2+. (B) Expanded records of systolic, caffeine-evoked and Ni2++caffeine-evoked Ca2+ transients fitted with single exponentials (y=a+e–kt). Values of the rate constant (1/k) are provided for the individual fits. (C) Summary of relative contributions to Ca2+ removal made by the SR Ca2+-ATPase, Na+–Ca2+ exchanger and slow mechanisms (PMCA and mitochondria) in sham (solid) and LVH (open) myocytes. *P<0.05.

 
From these rate constants, the relative contribution to Ca2+ removal by SERCA, NCX and PMCA can be calculated. Fig. 3C summarises this data. Consistent with a number of biochemical studies [9,15,25], in LVH the contribution made by SERCA decreased (82.5±1.7–76.1±1.1%), NCX increased (14.3±1–18.5±0.8 %) and PMCA increased (3.2±0.3–5.5±0.4 %). All values P<0.01, n=19–30 cells, 5–6 hearts. The rate constant data also yields the value of the factor required to correct the NCX measurements for non-electrogenic, PMCA, mediated Ca2+ efflux when quantifying SR Ca2+ content and SL Ca2+ efflux [11,13,18]. In the current study, the correction factor for sham conditions is 1.21 and for LVH is 1.29.

3.7. SERCA2a protein levels
The previous data indicates that SERCA-mediated Ca2+ uptake is reduced in LVH. This could arise through at least three routes: (i) reduced SERCA2a protein expression, (ii) altered regulation by PLB and (iii) dysfunctional trafficking of SERCA2a to the SR. Fig. 4A shows a representative immunoblot of SERCA2a protein levels. Normalised to GAPDH, the SERCA2a protein levels are unchanged in LVH (Fig. 4B, SERCA2a/GAPDH ratio=0.98±0.2 in 7 sham hearts and 1.29±0.2 in 9 LVH hearts, P>0.3). The SR dependent rate constant, ksystolic–kcaffeine, decreases in LVH from 4.17±0.3 to 2.94±0.2 s–1 (P<0.005) showing abnormal SR function even in the presence of unaltered SERCA2a protein levels.


Figure 4
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  		Fig. 4 SERCA2a protein expression is unaltered in LVH. (A) Representative immunoblots for SERCA2a (top) and GAPDH (lower) in sham (lanes 1–3) and LVH (lanes 4–8). Following SERCA2a detection, the blot was stripped and probed for GAPDH. (B) Mean data showing SERCA2a expression normalised to GAPDH expression in sham (solid bar) and LVH (open bar) hearts.

 
3.8 Na+–Ca2+ exchange function in LVH
The increase in NCX-mediated Ca2+ removal in LVH could occur directly through an increase in NCX protein/function or indirectly as a consequence of reduced SERCA2a expression/activity. In the absence of direct quantitative measurements of NCX protein, this can be addressed in two ways: firstly, from the Ni2+ sensitive rate constant (kcaffeine–kNi) which is greater in LVH (Fig. 5D, 0.78±0.04 vs. 0.66±0.04 s–1, P<0.05) suggesting increased NCX function.


Figure 5
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  		Fig. 5 Na+–Ca2+ exchanger activity is increased in LVH. (A) Caffeine-evoked Ca2+ transients (top) and inward Na+–Ca2+ exchange currents (bottom) for sham (left) and LVH (right) myocytes. Caffeine was applied following steady-state stimulation (23 °C, 0.5 Hz, –40 to +10 mV, 100 ms step). The relationship between [Ca2+]i and Na+–Ca2+ exchange current (at –40 mV) was obtained during the decay phase (80–10% of peak [Ca2+]i) of the caffeine-evoked transient (highlighted sections). (B) Na+–Ca2+ exchange current vs. [Ca2+]i for the records shown in A for sham (bullet) and LVH ({triangleup}) cells. The data are fit with linear regressions. (C–D) Mean data for sham (solid) and LVH (open) myocytes showing the relationship between Na+–Ca2+ exchange current and [Ca2+]i (C) and the Ni2+ sensitive (Na+–Ca2+ exchange dependent) rate constant for Ca2+ removal from the cytosol (D). *P<0.05.

 
The second method of identifying increased NCX function in LVH is to investigate the relationship between [Ca2+]i and Na+–Ca2+ exchange current. This is summarised in Fig. 5A–C. During the decay phase (from 80% to 10% maximum [Ca2+]i, highlighted regions of Fig. 5A) of the caffeine-evoked Ca2+ transient when [Ca2+]i and INCX are expected to be in equilibrium [26,27] the slope is 0.18 pA (nmol l–1)–1 in shams and 0.35 pA (nmol l–1)–1 in LVH. On average, in LVH, there is 36% more Na+–Ca2+ exchange-mediated inward current for a given change in [Ca2+]i (P=0.01, n=16–23 cells, 3–5 hearts, Fig. 5C).

3.9 Sarcolemmal Ca2+ efflux and SR Ca2+
To determine if the apparently modest changes in the balance between SR- and SL-mediated Ca2+ removal provide a mechanism for the reduced SR Ca2+ content we have re-expressed the data of Fig. 3C in terms of the ratio of SL- to SR-mediated Ca2+ removal. Sarcolemmal-mediated Ca2+ efflux increases by 46.2% in LVH from 22.1±2% to 32.3±2% of the amount removed by the SR (P<0.005). Using the approach illustrated in Fig. 6A, the relationship between SR content and SL Ca2+ efflux has been determined [11]. During the inotropic manoeuvre of refilling of the Ca2+ depleted SR, SL Ca2+ entry and efflux on each pulse have been measured [11,13]. From the SL Ca2+ flux measurements (Fig. 6A(ii)) net SL Ca2+ flux (Fig. 6A(iii)) and thus the change of SR Ca2+ content on each pulse (Fig. 6A(iv)) can be determined. Fig. 6B summarises the relationship between SL-mediated Ca2+ efflux and SR Ca2+ content; this is not a simple relationship and shows fractionally greater Ca2+ efflux as the SR Ca2+ loading increases. We have fit the data with a power function of the form:


Figure 6
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  		Fig. 6 Sarcolemmal Ca2+ efflux and SR Ca2+ content. (A) Experimental protocol. (i) Following depletion of the SR Ca2+ store with caffeine (not shown) cells were stimulated (–40 to +10 mV, 100 ms, 0.5 Hz, 23 °C) to steady state. (ii) Integrated Ca2+ fluxes via ICa-L (bullet) and the Na+–Ca2+ exchanger ({square}) are shown for each pulse. (iii) Net sarcolemmal Ca2+ movement ({blacktriangleup}) calculated from the integrated fluxes ({square} value subtracted from bullet value). (iv) Calculated change in SR Ca2+ content on each pulse ({diamond}) obtained by summing the net sarcolemmal fluxes (from iii). (B) Relationship between sarcolemmal Ca2+ efflux ({square} in panel A(ii)) and SR Ca2+ content ({diamond}, in panel A(iv)) fitted with a function (solid line) of the form y=a+b (SR Ca content)c.

 


Formula 4

(4)

The power function c is 2.8 and thus the decrease in SR Ca2+ content expected from the 46% increase in SL-mediated Ca2+ efflux is to 85.5% of control values. This is in very close agreement with that measured experimentally (80.3%) and implies that the increased SL-mediated efflux is responsible for decreasing SR Ca2+ load.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
To our knowledge, this is the first study that has quantitatively accounted for the reduction in systolic [Ca2+]i in LVH. We show that the reduced Ca2+ transient is entirely explained by a lower SR Ca2+ content. Furthermore, the change in SR content is mechanistically and quantitatively accounted for by a greater dependence on SL Ca2+ extrusion in LVH.

4.1 Systolic Ca2+, SR Ca2+ content and gain of EC coupling
The reduction of the systolic Ca2+ transient was maintained under current clamp conditions and thus cannot be explained simply by the short duration pulses used in the majority of experiments. The reduction in SR Ca2+ content to 80% of control values exactly accounted for the smaller Ca2+ transient. In addition to the Ca2+ transient changes, the decreased SR Ca2+ content also accounted for the apparent reduction in the fractional release of Ca2+ [12,24] and gain of EC coupling [10]. It is worth considering if a simple normalisation for SR Ca2+ content is suitable as gain and fractional release increase disproportionately with content. However, this only occurs as the SR becomes full and at lower contents appears linear [12,24]. Under these conditions, the SR is not showing evidence of being overloaded, e.g., spontaneous release and the contents measured here are less than those from some of our previous work with different conditions [13]. Thus, consistent with previous studies [9,22], we attribute the defective excitation contraction coupling (systolic Ca2+ transient amplitude) to reduced SR Ca2+ content and demonstrate that apparent changes in gain and fractional SR Ca2+ release are due to changes SR Ca2+ content rather than alterations in the coupling of SL Ca2+ entry to the RyR [10].

4.2 Roles of the SR and Na+–Ca2+ exchanger
Due to the steep dependence of systolic [Ca2+]i on SR loading, it is to be expected that the SR plays a major role in determining dysfunction in disease. While we do not find any evidence for reduced SERCA2a protein levels at this stage of LVH, it is not possible to determine if the protein that is present has been targeted correctly to the SR and is fully functional. We do however find that the SR dependent component of Ca2+ removal is decreased implying down-regulated function. This could also be due to altered interactions with the regulatory protein PLB, a situation likely if, as reported, PLB protein and Ser16 phosphorylation are reduced and phosphatase activity increased [28].

In the present study, we observe an increase in NCX-mediated function from three aspects: (i) the fractional contribution to Ca2+ removal, (ii) accelerated Ni2+ sensitive decay rate constant and (iii) greater NCX membrane current per change in [Ca2+]i. The latter two findings are supportive of an increased expression of functional NCX protein in this model consistent with previous studies [15,29]. The consequences of the increased NCX function are not easy to predict as operating in forward mode the exchanger would remove Ca2+ from the cytosol and thus help preserve diastolic function and hence the lower diastolic [Ca2+]i observed in LVH (Table 2); however, this would arise at the cost of an increased propensity for the development of arrhythmias due to the greater inward current for a given change of [Ca2+]i.

Despite the increase in non-SR-mediated Ca2+ efflux, the rate of decay of the systolic Ca2+ transient and relaxation of contraction are prolonged in LVH. Thus, it seems that even apparently modest decreases in SR Ca2+ uptake (from 82.5% to 76.1% of total Ca2+ removal) cannot be compensated for fully by increased SL-mediated efflux suggesting that perhaps therapeutic strategies that enhance SR function will be of greater benefit than those that accelerate SL Ca2+ removal.

The relative contributions to Ca2+ removal made by SERCA and SL mechanisms under control conditions in the ferret (81% and 19%, respectively) are similar to those reported for human [22] (77% and 23%) and rabbit myocardium [15] (71% and 29%). However, in heart disease, while human showed a similar increase in the contribution made by the SL and a fall in SR loading [22], the rabbit model showed a much larger increase in SL Ca2+ extrusion (250%) but had no change in SR Ca2+ content [15]. This discrepancy might be due to greater dependence on reverse mode NCX contributing to Ca2+ entry during the action potential and also reflect changes in [Na+]i [16]. Such differences highlight the importance of focussing on all the facets of Ca2+ handling when considering the underlying mechanisms of contractile dysfunction in disparate models of HF.

4.3. Study limitations
Much of the work was performed at 23 °C due to constraints in the experimental system. However, measurements performed at 37 °C also showed a similar effect of hypertrophy on Ca2+ transient amplitude and rate of decay of [Ca2+]i. The mechanisms responsible for removing Ca2+ from the cytosol show similar temperature dependencies [30]; therefore, it seems likely that the role of increased SL-mediated Ca2+ extrusion at 23 °C will be equally as important at 37 °C and thus explain the reduced SR Ca2+ contents and Ca2+ transient properties measured under these more physiological conditions.

It is also worth relating changes in intracellular Ca2+ homeostasis in unloaded cardiac myocytes to the contractile responses observed in vivo. Alterations in myofilament properties could influence the contractility responses to changes in [Ca2+]i in LVH; however, the ferret expresses the V3 or slow myosin isoform and therefore will not undergo the fast to slow switch observed in other species [31]. While preload and after-load alter the force–[Ca2+]i relationship in intact muscle and thus the contractile state of the heart [32], these effects are absent in the unloaded isolated myocyte. However, similar changes to those observed in this study (reduced contractility paralleled with a reduced Ca2+ transient amplitude) have been reported from experiments performed in intact cardiac preparations [4] thus indicating a connection between changes in Ca2+ homeostasis in isolated cells and contractile changes in intact muscle.

In summary, we offer the following explanation for smaller Ca2+ transients and slowed relaxation in LVH: (i) reduced SR Ca2+ content, (ii) decreased SERCA-mediated Ca2+ uptake and (iii) increased dependence on sarcolemmal-mediated Ca2+ efflux. We suggest that, consistent with recent gene transfer studies [33], the SR Ca2+ pump is useful therapeutic target for correcting the contractile dysfunction observed in heart failure.


   Acknowledgements
 
This work was supported by The British Heart Foundation.


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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