Cardiovascular Research

Cardiovascular Research 2005 67(4):636-646; doi:10.1016/j.cardiores.2005.05.006

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Copyright © 2005, European Society of Cardiology

Increased SR Ca²⁺ cycling contributes to improved contractile performance in SERCA2a-overexpressing transgenic rats

Lars S. Maier^a, Christian Wahl-Schott^b,d, Wiebke Horn^a, Stefan Weichert^a, Christian Pagel^a, Stefan Wagner^a, Nataliya Dybkova^a, Oliver J. Müller^c, Michael Näbauer^d, Wolfgang-M. Franz^d,*,1 and Burkert Pieske^a,1

^aAbt. Kardiologie and Pneumologie/Herzzentrum, Georg-August-Universität Göttingen, Germany
^bPharmakologie für Naturwissenschaften, Dept. Pharmazie, LMU München, Germany
^cInnere Medizin III, Universitätsklinikum Heidelberg, Germany
^dMed. Klinik and Poliklinik I-Großhadern, Marchioninistr. 15, 81377 München, Germany

^* Corresponding author. Email address: wolfgang.franz{at}med.uni-muenchen.de

Received 7 January 2005; revised 18 April 2005; accepted 1 May 2005

	Abstract

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Objective: Heart failure is associated with reduced function of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA2a) but increased function of sarcolemmal Na⁺/Ca²⁺ exchanger (NCX), leading to decreased SR Ca²⁺ content and loss of frequency-potentiation of contractile force. We reported that SERCA2a-overexpression in transgenic rat hearts (TG) results in improved contractility. However, it was not clear whether TG have improved contractility due to frequency-dependent improved SR Ca²⁺ handling.

Methods: Therefore, we characterized TG (n = 35) vs. wild-type (WT) control rats (n = 39) under physiological conditions (37 °C, stimulation rate <8 Hz). Twitch force, intracellular Ca²⁺ transients ([Ca²⁺]_i), and SR Ca²⁺ content were measured in isolated muscles. The contribution of transsarcolemmal Ca²⁺ influx (I_Ca) through L-type Ca²⁺ channels (LTCC) and reverse mode NCX (I_Na/Ca) to Ca²⁺ cycling were studied in isolated myocytes.

Results: With increasing frequency, force increased in TG muscles by 168 ± 35% (8 Hz; P<0.05) and SR Ca²⁺ content increased by maximally 118 ± 31% (4 Hz; P<0.05). In WT, there was a flat force-frequency response without changes in SR Ca²⁺ content. Relaxation parameters of force and [Ca²⁺]_i decay were accelerated at each frequency in TG vs. WT by 10%. At prolonged rest intervals (<240 s), force and SR Ca²⁺ content increased significantly more in TG. Consequently, absolute SR Ca²⁺ content measured in myocytes was increased 2-fold in TG. Transsarcolemmal Ca²⁺ fluxes estimated by I_Ca (at 0 mV –10.2 ± 1.1 vs. –16.9 ± 1.3 pA/pF) and I_Na/Ca (0.17 ± 0.02 vs. 0.46 ± 0.05 pA/pF) were decreased in TG vs. WT (P<0.05), whereas NCX and LTCC protein expression was only slightly reduced (P = n.s.).

Conclusion: In summary, SERCA2a-overexpression improved contractility in a frequency-dependent way due to increased SR Ca²⁺ loading whereas transsarcolemmal Ca²⁺ fluxes were decreased.

KEYWORDS Calcium (cellular); Contractile function; E–C coupling; Transgenic animal models; SR (function)

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This article is referred to in the Editorial by J.W.M. Bassani and R.A. Bassani (pages 581–582) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Intracellular Ca²⁺ homeostasis and excitation–contraction (E–C) coupling are mainly regulated by the sarcoplasmic reticulum (SR): upon stimulation, Ca²⁺ enters the cells through L-type Ca²⁺ channels (LTCC), thereby inducing SR Ca²⁺ release and leading to contraction [1,2]. Ca²⁺ is eliminated from the cytosol by SR Ca²⁺-ATPase (SERCA2a) and to a smaller extent by sarcolemmal Na⁺/Ca exchanger (NCX). Under steady-state conditions, the same amount of Ca²⁺ as had been previously released from the SR is taken up by SERCA2a, while NCX extrudes that amount of Ca²⁺ that had previously entered the cell through LTCC [3]. Sarcolemmal Ca²⁺ pumps and mitochondrial Ca²⁺ uniporter eliminate only a small fraction of Ca²⁺ ions (1–2%) and may be more important in long-term Ca²⁺ regulation [2].

Contractile dysfunction in heart failure (HF) [4,5] was related to defective SR Ca²⁺ uptake in animal models [6] and humans [1]. Direct relationships between impaired force-frequency relation (FFR), intracellular Ca²⁺ transients ([Ca²⁺]_i) [7,8], SR Ca²⁺ content [9,10], and SERCA2a expression [11] were reported in human HF. SERCA2a appears to play a key role for Ca²⁺ homeostasis, but little information regarding subcellular consequences of SERCA2a-overexpression is available.

Previous studies with SERCA2a-overexpressing transgenic mice [12,13], adenovirus-mediated gene transfer in neonatal rat myocytes [14,15], and animal models of HF reported enhanced contractility [16–18]. However, these studies investigated contractility under baseline conditions (force, shortening, ventricular pressure at low stimulation rates) but neither isometric twitch force nor SR Ca²⁺ content under the most physiological experimental conditions possible (37 °C) at increasing stimulation frequencies (up to 8 Hz). No detailed quantitative electrophysiological analyses of SR Ca²⁺ content, L-type Ca²⁺ currents (I_Ca), and NCX function (I_Na/Ca) were performed, and the effects of transgenic upregulation of SERCA2a on the expression and function of other Ca²⁺ regulatory proteins is completely unknown. Excessive SERCA2a-overexpression may even decrease contractility due to immediate Ca²⁺ reuptake before troponin C binding can occur ("futile" Ca²⁺ cycling) [19].

We recently reported that transgenic SERCA2a-overexpression in rat hearts (TG) leads to improved relaxation and contractility under normal and pressure overload conditions [20]. The aim of the present study was a detailed analysis of the effects of TG on E–C coupling processes under physiological conditions. Here, we demonstrate improved frequency-potentiation of force related to increased SR Ca²⁺ loading whereas transsarcolemmal Ca²⁺ cycling is reduced.

	2. Methods

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Ca²⁺ handling was investigated using TG (n = 35) and age-matched wild-type (WT) rats (n = 39) from one strain generated as reported recently [20]. SERCA2a protein level was overexpressed by 45.8 ± 6.1% in TG vs. WT. Data on heart and body weights did not differ between groups (Table 1a). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US NIH (Publication No. 85-23, revised 1996). Experiments were performed in isolated left (n = 39) and right (n = 6) ventricular trabeculae or papillary muscles obtained from 17 WT and 28 TG rat hearts.

View this table: [in this window] [in a new window]   Table 1a Body weight, heart weight, muscle, and myocyte parameters

 

	2.1. RCC experiments

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Muscles were mounted into a chamber, superfused with Krebs-Henseleit-buffer (KHB) containing (in mmol/L): NaCl 127, KCl 2.3, NaHCO₃ 25, KH₂PO₄ 1.3, MgSO₄ 0.6, CaCl₂ 1.5, glucose 11, insulin 10 IU/L (37 °C), and electrically stimulated. In order to measure SR Ca²⁺ loading, rapid cooling contractures (RCCs) were elicited by a decrease in chamber temperature to 1 °C [10,21].

	2.2. Intracellular Ca2+ transients

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Ca²⁺ transients were assessed using aequorin which was injected into the muscles [8]. Aequorin light emission was analyzed using the amplitude (mV amplifier output) and time course of signals. Since aequorin is consumed when Ca²⁺ is bound we do not calibrate our signals.

	2.3. Experimental protocol

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
FFR were tested by increasing frequency from 0.5–8 Hz. At each frequency recordings of isometric force were obtained followed by RCCs. In order to investigate the extent of which the SR gains Ca²⁺ during rest, rest intervals between 1–240 s were instituted from a basal frequency of 1 Hz and post-rest force or RCCs were measured. RCCs were normalized to basal values (0.5 Hz and 1 s rest, respectively) and only compared within each group. In a subset of experiments, isoproterenol (10^–9–10^–5 mol/L) was added to the solutions.

	2.4. Myocyte isolation

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Hearts were removed and perfused with a Tyrode's solution containing (in mmol/L): NaCl 138, KCl 4, MgCl₂ 1, glucose 10, NaH₂PO₄ 0.33, HEPES 10 (37 °C), to which collagenase (70 mg/50 mL) and protease (6 mg/50 mL) were added for myocyte isolation [22].

	2.5. Recording techniques for patch-clamp experiments

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Experiments were carried out with microelectrodes having resistances of 2–3 M (37 °C). Cell capacitance was calculated by applying 5 mV steps from –80 mV in the hyperpolarizing direction and integrating the current required to charge the membrane when stepping back to –80 mV.

	2.6. SR Ca2+ content

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Absolute SR Ca²⁺ content was determined by measuring the integral of caffeine-induced inward I_Na/Ca. Myocytes were held at –80 mV with an electrode containing (mmol/L): NaCl 5, CsCl 110, tetraethylammonium chloride (TEA) 20, MgCl₂ 1, MgATP 2, HEPES 10, EGTA 0.05. The cell was superfused in a microstream containing (mmol/L): NaCl 135, MgCl₂ 1, CaCl₂ 2, CsCl 10, glucose 10, HEPES 10. After a sequence of conditioning pulses (eight 200 ms pulses to 0 mV), followed by a 60 s rest to load the SR maximally with Ca²⁺, the cell was abruptly superfused for 6 s with caffeine (10 mmol/L) to release SR Ca. The resulting inward I_Na/Ca was integrated (nA*ms=pC) and normalized to cell volume (pC/pL) and capacitance (pC/pF).

	2.7. ICa

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Myocytes were superfused with solution containing (mmol/L): 135 NaCl, 10 CsCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and voltage-clamped [23] with an electrode filled with (mmol/L) 120 CsCl, 20 TEA, NaCl 1, MgCl₂ 1, EGTA 10, MgATP 2, HEPES 10. Myocytes were held at –80 mV and a prepulse to –45 mV inactivated Na⁺ channels. I_Ca was then activated by stepwise depolarizing from –30 to +80 mV (200 ms). Steady-state inactivation curves were determined by depolarizing the membrane (–60 to +60 mV) with conditioning pulses of 2 s, followed by a pulse to +10 mV. The current amplitudes during the second pulse were normalized to the maximal current amplitude, plotted as a function of the preceding membrane potential (Boltzmann function). Time constants of channel inactivation (₁, ₂) were determined by biexponential function fitting the current evoked during a pulse to 0 mV.

	2.8. INa/Ca

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
For recordings of NCX activity, reverse-exchange current was measured. Myocytes were held at –40 mV. The pipette contained (mmol/L): CsCl 130, TEA 20, NaCl 20, MgCl₂ 1, EGTA 14, MgATP 2, HEPES 10, CaCl₂ 0.1. To activate outward currents, cells were superfused in a microstream containing (mmol/L): 145 NaCl, CsCl 10, 1 MgCl₂, 2 CaCl₂, 10 glucose, 10 HEPES. Outward exchange current was activated when the cell was immersed for 5 s in an adjacent microstream of solution containing Li⁺ instead of Na⁺.

	2.9. Western blots

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
For measuring protein expression of NCX and LTCC we performed standard Western blot techniques [11,19]. Briefly, equal amounts of protein were subjected to SDS-PAGE and blotted to nitrocellulose. The blots were blocked in 5% nonfat milk dissolved in TBS (20 mmol/L Tris–Cl, pH 7.5, 50 mmol/L NaCl), then probed with NCX antibodies (ABR), antibodies against the _1C subunit of LTCC (Alamone), or GAPDH (Bio Trend) in TBS containing 0.5% nonfat milk and 0.1% Tween 20. The membranes were incubated for 1 h with horse radish peroxidase-labeled antibody (Amersham). Immunoreactive bands were visualized and exposed to x-ray film.

	2.10. Statistics

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Data are expressed as mean ± S.E.M. Statistical analysis was performed with Student's unpaired t-test and one or two-way ANOVA followed by Student–Newman–Keuls test where appropriate. Statistical significance was taken as P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
3.1. Frequency-dependent effects on force and SR Ca²⁺ content
Fig. 1A shows representative isometric twitches and RCCs in muscles from WT and TG. When the muscles were rapidly cooled from 37 to 1 °C, Ca²⁺ was released from the SR and RCCs developed rapidly [2,24]. Twitch force marginally increased upon raising frequency in WT without changes in RCCs. In contrast, force and RCCs greatly increased in TG showing a frequency-dependent increase in SR Ca²⁺ load in TG but not WT.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Frequency-dependent changes in twitch force and SR Ca2+ content. A. Representative isometric steady-state twitches and RCCs in muscles from a WT and a TG heart. B. Average values for twitch and RCC amplitudes in WT (n = 10) and TG (n = 20) muscles (%basal values at 0.5 Hz). *P<0.05 vs. 0.5 Hz, #P<0.05 WT vs. TG.

 
Average data for twitch force and RCCs in WT (Fig. 1B) did not change significantly when stimulation frequency was increased. In contrast, in TG force frequency-dependently increased by 168 ± 35% at 8 Hz (vs. 0.5 Hz; P<0.05). This positive FFR was accompanied by an increase in RCC amplitude by 88 ± 31% (P<0.05). Interestingly, absolute force values for twitches at 0.5 Hz did not differ significantly between WT and TG. However, increasing frequency to 8 Hz leads to higher force in TG compared to WT (Table 1b). It should be mentioned that WT rat myocardium usually shows a flat or only slightly positive FFR, and even negative FFR were reported [2,21,24].

View this table: [in this window] [in a new window]   Table 1b Force-frequency data

 
3.2. Increasing stimulation rate accelerates twitch contractions and Ca²⁺ transients
The time course of twitches and Ca²⁺ transients critically depends on SR Ca²⁺ uptake and is physiologically accelerated at high stimulation rates [2,25]. From typical twitch contractions and Ca²⁺ transients at 1 Hz (Fig. 2A) it can be seen that time to peak amplitudes is decreased, and relaxation and Ca²⁺ transient decline are shortened in TG. Fig. 2B shows average results for 50% and 90% relaxation of twitch force (RT_50%, RT_90%). At each frequency relaxation was faster by 10% in TG compared to WT. RT_50% decreased in WT from 35.1 ± 0.9 ms at 0.5 Hz to 26.4 ± 1.6 ms at 8 Hz (P<0.05), and in TG from 32.2 ± 0.6 to 25.1 ± 1.5 ms (P<0.05), and similarly in RT_90%. In addition, values for 90% decay of Ca²⁺ transients are presented for WT and TG showing muscles with significant faster Ca²⁺ transient decay in TG at 0.5 Hz (44.0 ± 1.3 vs. 35.0 ± 1.1 ms, P<0.05) and 8 Hz (42.4 ± 1.1 vs. 30.8 ± 0.6 ms, P<0.05). Similarly, the time to peak tension and total twitch time decreased with increasing stimulation rates (Table 1b). Again, at all frequencies parameters were smaller in TG vs. WT.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Influence of frequency on kinetics of twitches and Ca2+ transients. A. Normalized twitches and Ca2+ transients in muscles from a WT and TG heart (1 Hz). B. Average values for 50% and 90% relaxation (RT50%, RT90%) of force (WT n = 15, TG n = 19), and Ca2+ transients (inset: Aequorin RL90%; WT n = 5, TG n = 8) at increasing frequencies. *P<0.05 vs. 0.5 Hz, #P<0.05 WT vs. TG.

 
3.3. β-adrenergic stimulation increases force and accelerates Ca²⁺ transients
Fig. 3 summarizes the effects of β-adrenergic stimulation. TG and WT showed a concentration-dependent increase in force with isoproterenol (10^–5 mol/L vs. control) with a maximum of 199 ± 50% for WT and 279 ± 75% for TG (P<0.05). This inotropic response is associated with typical acceleration of relaxation parameters of force as well as Ca²⁺ transient decay. RT_90% in WT decreased from 70.3 ± 2.9 ms at control to 50.9 ± 2.5 ms with 10^–5 mol/L isoproterenol (P<0.05), and in TG from 65.9 ± 2.6 to 43.4 ± 1.9 ms (P<0.05) with similar changes in RL_90%. At each concentration of isoproterenol time parameters were shorter in TG vs. WT rats (by 10%). These differences were even more pronounced at higher isoproterenol concentrations (15%).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Influence of isoproterenol on amplitude and kinetics of twitches and Ca2+ transients. Average values for twitch amplitude, RT90% of force (WT n = 15, TG n = 22), and Ca2+ transients (inset: Aequorin RL90%; WT n = 6, TG n = 6) when increasing isoproterenol concentration. *P<0.05 vs. 0.5 Hz. #P<0.05 WT vs. TG.

 
3.4. Increasing rest intervals raises SR Ca²⁺ content
Since RCCs are a semiquantitative measure of SR Ca²⁺ content, quantitative SR Ca²⁺ load was assessed using caffeine-induced inward I_Na/Ca [2]. Fig. 4A shows representative traces for I_Na/Ca with increased current in TG vs. WT. On average, I_Na/Ca was 4.98 ± 0.43 pA/pL in WT and increased in TG to 8.80 ± 2.07 pA/pL (P<0.05). The integrated charge transferred was also significantly higher in TG (8.13 ± 1.38 pC/pL or 1.28 ± 0.22 pC/pF) compared to WT (3.99 ± 0.28 pC/pL or 0.73 ± 0.05 pC/pF; P<0.05). When normalizing to cell volume, absolute SR Ca²⁺ load was 84.3 ± 14.3 in TG vs. 41.4 ± 2.9 µmol/L cytosol in WT (P<0.05). These results suggest that there is not only a frequency-dependent increase in SR Ca²⁺ load (as measured by RCCs), but also the absolute [Ca²⁺] in the SR is higher in TG.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effects on SR Ca2+content. A. Quantitative assessment of SR Ca2+ load measured as caffeine-induced inward INa/Ca in myocytes from WT (n = 10) and TG (n = 6) hearts. The inset shows the average SR Ca2+ load. *P<0.05 WT vs. TG. B. Average values for rest-dependent changes in twitch and RCC amplitudes in WT (n = 10) and TG (n = 10) muscles (%basal value 1 s rest). *P<0.05 vs. 1 s. #P<0.05 WT vs. TG.

 
We also analyzed the potential of the SR to accumulate Ca²⁺ during rest periods (Fig. 4B). Rats typically show potentiation of force after increasing rest intervals [2,21]. In WT, post-rest force increased by 126 ± 44% (P<0.05) after 240 s, but RCCs did not change. In contrast, TG showed much larger post-rest twitches at any rest interval (after 240 s by 198 ± 52%; P<0.05) associated with clear increases in RCCs (by 153 ± 55%; P<0.05). This suggests large rest-dependent SR Ca²⁺ accumulation in TG.

3.5. Decreased I_Ca and NCX function
Representative current–voltage relations show reduced current density at most voltages applied in TG vs. WT (Fig. 5A). Average data (Fig. 5B) depicts decreased peak I_Ca density at 0 mV of –10.2 ± 1.1 for TG vs. –16.9 ± 1.3 pA/pF for WT myocytes (P<0.05). However, Western blot analysis revealed no change in LTCC protein expression in TG (n = 10) vs. WT (n = 9) hearts (–3.8 ± 6.7%). Inactivation parameters did not differ between WT and TG (Table 2). However, there is a clear trend of faster I_Ca decline in TG which might be attributable to feedback through the increased SR Ca²⁺content in TG.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effects on L-type Ca2+ currents (ICa). A. Representative ICa in WT and TG myocytes when depolarizing stepwise from –30 to +80 mV. B. IV relation with average values for WT (n = 24) and TG (n = 6) myocytes. #P<0.05 WT vs. TG.

 

View this table: [in this window] [in a new window]   Table 2 ICa parameters

 
Representative traces for I_Na/Ca density measured by I_Na/Ca reverse mode were clearly reduced in TG compared to WT (Fig. 6A). Average data (Fig. 6B) show significantly lower I_Na/Ca outward current density in TG (0.17 ± 0.02 pA/pF) vs. WT (0.46 ± 0.05 pA/pF). This underlines that NCX function is reduced in TG vs. WT. Interestingly, we found only a slight reduction in NCX protein expression in TG (n = 10) vs. WT (n = 9) hearts of –12.0 ± 7.4%.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 A. Representative caffeine-induced outward INa/Ca. B. Average values for WT (n = 24) and TG (n = 6) myocytes. *P<0.05 WT vs. TG.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
This is the first detailed report on E–C coupling processes after transgenic SERCA2a-overexpression in rat myocardium under physiological conditions. Our main findings are that 1) a 45% increase in SERCA2a protein levels is associated with improved contraction and relaxation kinetics and pronounced frequency or rest potentiation of force; 2) the functional effects were related to enhanced SR Ca²⁺ content and increased [Ca²⁺]_i; and 3) I_Ca and NCX activity are reduced indicating a shift from transsarcolemmal to intracellular Ca²⁺ cycling.

	4.1. FFR and relaxation

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Depending on experimental conditions or strain, rat FFR can be negative, positive, or flat due to differences in SR Ca²⁺ handling [26,27]. In the present study, force and SR Ca²⁺ content in trabeculae of WT rats did not increase with increasing frequencies. This indicates that despite frequency-induced increases in I_Ca (due to increased frequency of action potentials) [2], the SR of WT rats is unable to respond to this extra amount of Ca²⁺ with enhanced SR Ca²⁺ load. This phenomenon was previously explained with the inability of WT rat heart to overcome the negative effects of refractoriness of SR Ca²⁺ release at high stimulation rates [24]. In contrast, in TG myocardium refractoriness can be easily overcome due to increased SR Ca²⁺ load resulting in a clear positive FFR. Most likely the increase in SR Ca²⁺ load is attributable to a frequency-dependent activation of SERCA2a (as previously shown by paired RCCs [10]), e.g. by CaMKII in combination with a decrease of Ca²⁺ extrusion via NCX (with less activity in TG) as a consequence of increased [Na⁺]_i [28]. Another aspect is that phospholamban (PLB) expression is not altered in TG vs. WT [20]. Therefore, SERCA2a/PLB ratio is greatly increased with less PLB inhibiting SERCA2a leading to increased SR Ca²⁺ uptake in TG. Consequently, SERCA2a-overexpression leads to a greatly enhanced SR Ca²⁺ uptake and subsequent positive FFR.

Similarly, Hajjar et al. [29] reported for isolated rat myocytes that SERCA2a-overexpression can restore impaired FFR initially induced by PLB overexpression. The present data also agree with our observations in failing human myocardium that the FFR may be improved by low concentrations of forskolin [30] or isoproterenol [31]. However, stimulation of the cAMP–PKA axis not only stimulates SERCA2a activity by PLB phosphorylation, but has multiple further effects such as phosphorylation of LTCC, ryanodine receptors (RyR), or troponin C. Therefore, in our previous studies stimulation of the cAMP–PKA axis was far from normalizing contractility, and higher concentrations of forskolin or isoproterenol even had detrimental effects related to Ca²⁺ overload. In contrast, specific overexpression of SERCA2a protein selectively improves SR Ca²⁺ uptake [29].

It is important to note that the beneficial effect of SERCA2a-overexpression on contractility is most prominent at higher stimulation rates. This was previously only incompletely appreciated in muscles from transgenic mice at 22 °C which were stimulated up to 2 Hz [32]. Since our experiments in TG were performed at physiological temperature and frequencies (37 °C, up to 8 Hz), we believe that our results of a frequency-dependent increase in twitch force (and SR Ca²⁺ content) are of major importance for the understanding of the physiological consequences of SERCA2a-overexpression.

Twitch parameters at all frequencies were accelerated in TG rats. This observation can be explained by increased amounts of SERCA2a proteins in TG and CaMKII-dependent activation of SR Ca²⁺ uptake [33]. This effect was also observed in studies using PLB knockout mice [34] or SERCA2a transfected rabbit myocytes [35]. Intrinsic myofilament properties might also change relaxation kinetics [36].

β-adrenergic stimulation increases force and shortens activation and relaxation parameters of force and Ca²⁺ transients [37]. Similarly, in the present study force increased while relaxation parameters decreased. In addition, relaxation at each isoproterenol concentration was accelerated in TG vs. WT and increased concentration-dependently (from 10% to 15%). Interestingly, RT_90% at the highest isoproterenol concentration was faster as compared to the relaxation at the highest stimulation frequency. Similar results were recently shown in SERCA2a transfected rabbit myocytes [38].

	4.2. SR Ca2+content

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
During rest Ca²⁺ is removed from the cytosol into the SR by SERCA2a and across the sarcolemma mainly by NCX [2]. There is a finite rate of SR Ca²⁺ leak which can either be taken up by the SR (resulting in constant SR Ca²⁺ load) or it can be partly removed from the cytosol by NCX (decreasing SR Ca²⁺ content). In the absence of SR Ca²⁺depletion rest potentiation of twitches appears to be normal in rat myocardium. This has been interpreted as a recovery of E–C coupling mechanism from a refractory state with increased fractional SR Ca²⁺ release [39,40].

The current results indicate that WT rat myocardium shows some increase in post-rest force associated with a marginal increased SR Ca²⁺ content. It appears that this rest potentiation of force is due to a very slow phase of recovery of SR Ca²⁺ release processes from a previous activation. This may be related to a slow phase of RyR recovery of normal Ca²⁺ sensitivity from an adapted or inactivated state and has been observed at the level of Ca²⁺ spark availability in rat and rabbit myocytes [2]. In contrast, TG is characterized by pronounced increases in post-rest force associated with marked increases in SR Ca²⁺ content and release. The reason for increased SR Ca²⁺ content during rest remains controversial. One reason could be that the SR content increases Ca²⁺ by means of decreased Ca²⁺ efflux via NCX [2]. While a slightly higher intracellular [Na⁺] in the cells from TG rats (even by 1 mM) could also explain this in principle, there is no evidence indicating that this is the case. Nevertheless, increased SERCA2a protein expression results in more effective SR Ca²⁺ reloading during rest. Similarly we could recently show that positive inotropic compounds which stimulate SERCA2a can improve the impaired post-rest behavior in failing human myocardium [31].

To supplement the semiquantitative RCC results with more quantitative analysis we quantified SR Ca²⁺ load by patch-clamp experiments. We confirmed that SR Ca²⁺ load in TG vs. WT is 2-fold increased. This is in agreement with previous findings in SERCA2a transgenic mice [41]. However, the difference in SR Ca²⁺ load reported by these authors between TG vs. WT (25%) was much smaller to our study. The most important differences between the two studies may be the different levels of SERCA2a-overexpression (20% vs. 46% in our study), the experimental conditions, as well as species. Yao et al. [41] used SERCA2a transgenic mice and measured at low temperature (25 °C) and low [Ca²⁺]_o (1 mmol/L). In the present experiments we used myocytes from SERCA2a transgenic rats at 37 °C and [Ca²⁺]_o of 2 mmol/L.

As a limitation of the study, it should be pointed out that the total amount of Ca²⁺ extruded by the NCX even might underestimate SR Ca²⁺ content since Ca²⁺ removal by sarcolemmal Ca²⁺ pumps and mitochondrial Ca²⁺ uptake were not taken into account [2]. Unfortunately, we do not have any information about these pathways in TG. Of note, decreased NCX activity measured in TG (although integral I_Na/Ca to assess SR Ca²⁺ load was used) might contribute to a possible underestimated SR Ca²⁺ content. Thus, NCX current measurements are qualitative in nature and may not always exactly reflect quantitative changes. Finally, a contribution of Ca²⁺ activated Cl currents and non-selective cation currents cannot be ruled out. However, when applying nickel to block NCX the current was completely abolished. Moreover, since no differences in these currents are expected to occur in TG vs. WT we do not believe that this has an impact on our main conclusions.

	4.3. ICa and NCX

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
It was previously reported that I_Ca was unchanged in TG vs. WT mice, whereas I_Na/Ca was decreased by 10% [41]. We suggest in the present report that I_Na/Ca is reduced even 2.5-fold and I_Ca by 40% in TG vs. WT rats. As discussed in the previous section, these discrepancies may be due to differences in animal models and experimental conditions. However, these data also demonstrate that even in rat myocardium where Ca²⁺ cycling already in WT predominantly depends on SR Ca²⁺ transport [2], SERCA2a-overexpression further enhances intracellular vs. transsarcolemmal Ca²⁺ cycling.

When measuring Ca²⁺ influx and efflux and comparing these data we found very similar values. Not surprisingly to us, when calculating absolute values we found that I_Ca (Ca²⁺ influx) was 17.4 µmol/L for WT and 7.3 µmol/L for TG, i.e. the amount of Ca²⁺ entering the cell in TG via LTCC was only 42% compared to WT. Given the balance of fluxes, Ca²⁺ efflux via NCX should meet Ca²⁺ entry via LTCC. Indeed, when NCX outward current density (I_Na/Ca) was measured, there was a reduction of the transport capacity to 37% in TG vs. WT. Although not measured during regular E–C coupling, the latter result suggests the NCX activity seems to be reduced to a very similar amount as compared to the reduced I_Ca. In consequence, in the light of unchanged protein expression for NCX and LTCC, functional data shows reduced transsarcolemmal Ca²⁺ cycling vs. SR function. Nevertheless, further investigations in the presence of a functional SR are needed to fully understand the consequences of SERCA2a-overexpression.

Interestingly, there are striking similarities between our study (decreased I_Ca and increased SR Ca²⁺ content) and a study by Henderson et al. [42] who showed a 50% reduction in I_Ca in NCX-knockout mice. It may be that the same general phenomenology is occurring in our TG rats. Specifically, increased SR Ca²⁺ loads will lead to reduced I_Ca without changes in LTCC expression (as also shown by Henderson et al.). One potential mechanism for these observations might be via Ca²⁺ mediated inactivation of LTCC (i.e. faster Ca²⁺ efflux from the SR might promote rapid I_Ca inactivation). However, neither in our study nor in their study differences in inactivation kinetics were found (although we saw an almost significant trend with faster kinetics in TG vs. WT). Most likely, Ca²⁺ induced inactivation from increased SR Ca²⁺ release in TG does play a role. However, Henderson et al. speculated that the cells rather limit Ca²⁺ influx than upregulate Ca²⁺ efflux. Increased cytosolic/SR Ca²⁺ cycling might activate Ca²⁺ dependent transcription pathways leading to synthesis of non-functional channel protein as a means of protection from Ca²⁺ overload. However since this is completely speculative the unknown mechanisms of I_Ca and I_Na/Ca reduction are a limitation of the present study.

	4.4. Clinical relevance for HF

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
SERCA2a-overexpression might be useful in human HF [43] especially in patients with a negative FFR or diastolic dysfunction due to cytosolic Ca²⁺ overload. Because disturbed Ca²⁺ handling seems to play a role in the pathophysiology of HF, the relevance of SERCA2a-overexpression to rescue disturbed Ca²⁺ cycling and myocardial function is of particular importance. As shown by del Monte et al. [44] gene transfer of SERCA2a can improve contractility and can restore the impaired FFR in myocytes from failing human hearts. Nevertheless, further experimental work will be necessary to investigate the effects of SERCA2a-overexpression in transgenic animal models and SERCA2a gene transfer in human cardiac tissue on Ca²⁺ handling and myocardial performance.

However, an increased amount of SR Ca²⁺ may also be detrimental due to an increased potential for Ca²⁺ leak through RyR, potentially resulting in delayed afterdepolarizations and arrhythmias. This is even more important in HF where RyR may be hyperphosphorylated [33,45]. Indeed, we found increased mortality of SERCA2a TG rats after myocardial infarction, as reported recently by our group [46]. Therefore, SERCA2a-overexpression may improve Ca²⁺ handling but also induce arrhythmias under certain conditions. Future therapeutic approaches in HF may therefore be targeted to both improved SR Ca²⁺ uptake and stabilization of RyR.

In summary, whether and to what extent overexpressing SERCA2a might be beneficial in the treatment of HF has to be further investigated, since recent reports by Teucher et al. [19] and Chen et al. [46] in contrast to earlier work [12–18] showed negative effects on E–C coupling and increased arrhythmias under certain conditions.

	Acknowledgments

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 
Dr. Franz (SFB320/B-6) and Dr. Pieske are supported by DFG grants (PI414/2). Dr. Pieske is also supported by BMBFT (KHN-TP8). Dr. Wahl-Schott was supported by FoFöLe grant from LMU. Dr. Maier is supported by DFG Emmy-Noether-Programm (MA1982/1–2 and 1–4), by GlaxoSmithKline Research Foundation and by a grant from the Medizinische Fakultät (Forschungsförderung).

	Notes

 
¹ Equal contribution.

Time for primary review 24 days

	References

Top Abstract 1. Introduction 2. Methods 2.1. RCC experiments 2.2. Intracellular Ca2+... 2.3. Experimental protocol 2.4. Myocyte isolation 2.5. Recording techniques for... 2.6. SR Ca2+ content 2.7. ICa 2.8. INa/Ca 2.9. Western blots 2.10. Statistics 3. Results 4. Discussion 4.1. FFR and relaxation 4.2. SR Ca2+content 4.3. ICa and NCX 4.4. Clinical relevance for... Acknowledgments References

 

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