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Cardiovascular Research 2004 62(1):122-134; doi:10.1016/j.cardiores.2004.01.005
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to β-adrenergic agonists

Uwe Kirchhefer*,a, Larry R Jonesb, Frank Begrowa, Peter Boknika, Lutz Heinc, Martin J Lohsec, Burkhard Riemannd, Wilhelm Schmitza, Jörg Stypmanne and Joachim Neumanna

aInstitut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Domagkstr. 12, 48149 Münster, Germany
bDepartment of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
cInstitut für Pharmakologie, Universität Würzburg, 97078 Würzburg, Germany
dKlinik und Poliklinik für Nuklearmedizin, Westfälische Wilhelms-Universität, 48149 Münster, Germany
eMedizinische Klinik und Poliklinik C, Westfälische Wilhelms-Universität, 48149 Münster, Germany

* Corresponding author. Tel.: +49-251-8355510; fax: +49-251-8355501. Email address: kirchhef{at}uni-muenster.de

Received 23 April 2003; revised 12 December 2003; accepted 2 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Ca2+ release from the cardiac junctional sarcoplasmic reticulum (SR) is regulated by a complex of proteins, including the ryanodine receptor (RyR), calsequestrin (CSQ), junctin (JCN), and triadin 1 (TRD). Moreover, triadin 1 appears to anchor calsequestrin to the ryanodine receptor. Methods: To determine whether triadin 1 overexpression alters excitation–contraction coupling, we examined the effects of cardiac-specific overexpression of triadin 1 on SR Ca2+ handling and contractility in transgenic (TG) compared to wild-type (WT) mice. Results: The overexpression of triadin 1 was associated with an enhanced SR Ca2+ load, reflected by a 22% higher amplitude of caffeine-induced Ca2+ transients. The decline of Ca2+ transients during caffeine exposure was prolonged by 57%. The detection of resting spontaneous SR Ca2+ release events (Ca2+ sparks) revealed an increased amplitude (by 16%), decline (by 47%), and width (by 47%) in TG. This was associated with a redistribution of Ca2+ spark amplitudes from one population to two populations. Measurement of cardiac function by echocardiography and left ventricular (LV) catheterization revealed a decreased cardiac contractility in vivo. The impaired response to β-adrenergic receptor (β-AR) stimulation in TG hearts was associated with an increased protein expression of β-AR kinase 1. In addition, the increase of the L-type Ca2+ peak current and the increase of phospholamban (PLB) phosphorylation at Thr17 were reduced under β-AR stimulation. Conclusion: Taken together, our data suggest that triadin 1 overexpression results in a complex modulation of SR Ca2+ handling, which may contribute, at least in part, to the depressed basal contractility and the blunted response to β-adrenergic agonists in TG mice.

KEYWORDS Calcium (cellular); SR (function); e–c Coupling; Contractile function; Protein phosphorylation

Abbreviations: EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid • BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid • HEPES, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Alterations in the expression level and/or functional state of proteins of the free sarcoplasmic reticulum (SR), namely SERCA2a and phospholamban (PLB) [1,2], cause profound changes in cardiac contractility and pathophysiology [3,4]. The regulation of the SR Ca2+ release both under normal conditions and in the diseased heart is not completely understood [5,6]. The Ca2+ release process is maintained by a quaternary protein complex at the junctional SR membrane consisting of the ryanodine receptor (RyR), calsequestrin (CSQ), junctin (JCN), and triadin 1 (TRD) [7–9].

The RyR or Ca2+ release channel is a tetramer which is associated with the FK-506 binding protein stabilizing the interaction between single ryanodine receptor subunits [10]. The activation of the RyR is initiated by Ca2+ which enters the cytosol via voltage-dependent L-type Ca2+ channels. This process is called the Ca2+-induced Ca2+ release [11]. Furthermore, the open probability of the RyR is tightly controlled by the phosphorylation of the cAMP-dependent protein kinase (PKA) and the Ca2+/calmodulin-dependent protein kinase (CaM kinase) during β-adrenergic receptor (β-AR) stimulation [12]. The Ca2+-dependent interaction between the RyR and CSQ at the lumenal face of the junctional SR membrane is mediated by two anchoring proteins, TRD and JCN. CSQ is a Ca2+-binding protein localized in the lumen of the junctional SR, which stores the Ca2+ required for Ca2+ release during the systole [9]. Both JCN and TRD are integral membrane proteins of the junctional SR, which have structural similarities.

The highly charged C-terminal region of cardiac TRD, localized in the lumen of the junctional SR, contains a single "KEKE motif" and avidly binds to CSQ [13]. This anchoring function of TRD may facilitate the transfer of Ca2+ from the lumen of the cardiac junctional SR into the RyR and beyond [8,9,14]. Further support for an important role of TRD in the regulation of SR Ca2+ release is provided by observations from electron microscopy [15]: TRD is found near the inner surface of the RyR, where it appears to aid in condensation and concentration of CSQ. Moreover, the purified skeletal muscle isoform of triadin was shown to decrease the open state probability of the skeletal muscle RyR in planar lipid bilayers [16,17]. Recently, fivefold overexpression of TRD in mouse hearts resulted in cardiac hypertrophy as well as contractile abnormalities [18]. This phenotype was associated with down-regulation of RyRs and elevated diastolic Ca2+ levels, suggesting a physiological role for the protein in regulating the SR Ca2+ handling in heart. This was tested in more detail in the present study with the aid of TRD transgenic (TG) compared to wild-type (WT) mice. We show that TG mice have an increased peak amplitude of caffeine-induced Ca2+ transients, suggesting a higher SR Ca2+ content. The reduced rate of decay of Ca2+ transients under caffeine application may be caused, in part, by a down-regulation of the Na+–Ca2+ exchanger (NCX). The altered Ca2+ spark activity may contribute to an enhanced diastolic SR Ca2+ leak in TG. The blunted contractile response to β-AR stimulation can be explained by desensitization of β-ARs, a reduced L-type Ca2+ peak current, and a lower Thr17 phosphorylation of PLB.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Materials
[125I]ICYP was purchased from DuPont NEN (Boston, MA, USA). ICI 89,406, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), and Indo-1/AM were supplied by Sigma-Aldrich (Munich, Germany). Fluo-3/AM was obtained from Molecular Probes (Eugene, OR, USA).

2.2. Generation and identification of transgenic mice
TG mice were produced as described previously [19]. We obtained three TG mouse lineages expressing similar triadin 1 protein levels. Therefore, we saved only one TG mouse lineage on which we performed the following experiments. However, a caveat is in order: We cannot completely rule out the possibility that the transgene was inserted probably at different locations of the mouse genome. Age-matched TG and WT mice (16–20 weeks old) of either sex were used in the following experiments. Animals were handled according to approved protocols of the animal welfare committees of the University of Münster, Indiana University, and the University of Würzburg. This investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.3 Measurement of SR Ca2+ load
Single ventricular myocytes were enzymatically isolated from TG and WT mouse hearts and loaded with Indo-1/AM as described [18]. Indo-1 was excited at 365 nm, and the emitted fluorescence signal was measured at 405 and 495 nm. The cytosolic Ca2+ concentration was estimated by calculating the ratio of fluorescence signals emitted by excitation at 405 and 495 nm. The diastolic and systolic Ca2+ transients were defined as the minimal and maximal Indo-1 fluorescence ratios, respectively. We cannot completely rule out the possibility that Indo-1 concentrations maybe different in cardiomyocytes from WT and TG animals. However, this is unlikely because we used the same procedure for labeling WT and TG cardiomyocytes. Moreover, experiments were randomized between WT and TG animals. Moreover, the ratiometric procedure we used with Indo-1 has the advantage that changes in cell labeling should not easily affect the measurement [20]. The measurement of caffeine-induced Ca2+ transients [Ca2+]i was performed as described [21]. This protocol was varied by additional application of 5 mM Ni2+ to block the NCX current.

2.4 Detection of Ca2+ sparks by confocal microscopy
Cardiomyocytes were placed in a perfusion chamber and loaded for 5 min at room temperature with 5 µM Fluo-3/AM, followed by a 20-min wash in dye-free Tyrode solution. A confocal laser scanning head (MRC 2000, Biorad, Hemel Hempstead, United Kingdom) connected to an inverted microscope (Nikon, Düsseldorf, Germany) was used to image the cells with the help of Laserpix software (Bio Rad, Hercules, CA, USA). The excitation wavelength of 488 nm was provided by an argon ion laser. Spontaneous increases in Fluo-3 fluorescence (Ca2+ sparks) were recorded in the line-scan mode (x–y). This mode scanned a single line through the cardiomyocyte longitudinally every 1.3 ms. The steps in the analysis algorithm included (1) locating single increases in fluorescence that are two times the standard deviation above the mean background fluorescence intensity of the line-scan image; (2) drawing a box of 100 x 180 pixels around each background-substracted Ca2+ spark; and (3) extracting peak amplitude and kinetics of the Ca2+ spark. The peak amplitude of the Ca2+ spark was set as the ratio of the maximal increase of fluorescence (F) to the resting fluorescence (F0) before the elevation of the Ca2+ spark (F/F0). The resting fluorescence was determined as the average fluorescence between the first line scan and the threshold. The background was subtracted from F and F0. The time to peak was defined as the time from resting fluorescence to the peak level. The time to 50% decay of the Ca2+ spark was defined as the time from peak level to 50% of the peak amplitude.

2.5. Electrophysiology
L-type Ca2+ currents (ICa) were recorded using the whole-cell patch-clamp technique [18]. Ca2+ currents were elicited by applying 200-ms depolarizing pulses every 10 s from a holding potential of –40 mV. Kinetics of ICa were measured at a test potential of +10 mV. The extracellular solution contained 130 mM TEA-Cl, 4 mM 4-aminopyridine, 1 mM MgCl2, 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 10 mM dextrose, 2 mM CaCl2 (pH 7.3). K+ currents were suppressed under these conditions. The intracellular solution was composed of (in mM): potassium aspartate 80, KCl 50, KH2PO4 10, MgCl2 0.5, MgATP 3, HEPES 10, ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 1 (pH 7.4). Where indicated, 2 mM BaCl2 instead of 2 mM CaCl2 was used in the extracellular solution. In addition, where indicated Ca2+ currents were monitored using 10 mM BAPTA in the internal solution instead of 1 mM EGTA. The effects of SR Ca2+ release on ICa were measured using rapid application of 10 mM caffeine. Caffeine-induced Ca2+ release was initiated by fast exchanging the extracellular solution for 2 s (Warner Instrument). Ca2+ currents were measured immediately (200 ms) after application of caffeine. The response to β-AR stimulation of ICa was tested by using 0.1 µM isoproterenol in the extracellular solution.

2.6. Surface electrocardiogram (ECG)
TG and WT mice were anesthetized with 15 ml/kg of 2.5% tribromoethanol via intraperitoneal injection. ECG signals were received by electrodes in all four extremities and amplified (MVU-0608, Föhr Medical Instruments, Seeheim, Germany) allowing the measurement of bipolar limb leads in standard mode. Signals were digitized with an PowerLab recording unit (AD Instruments, Castle Hill, Australia). RR, PQ, QRS, and QT intervals were measured from six consecutive beats of lead I and II, and averaged.

2.7. β-AR density
Membranes were prepared from individual ventricles of TG and WT hearts for the measurement of β-AR density as described previously [21]. Total β-AR density was determined as reported by us [21]. For competition binding studies, 15 µg of membranes was incubated with a saturating concentration of [125I]ICYP (80 pM) and with varying concentrations of ICI 89,406 (1 pM–100 µM). Reactions were conducted at 37 °C for 60 min. Samples were filtered onto Whatman GF/B filters, washed, and the membrane bound activity was determined in a {gamma}-scintillation counter. Competition binding curves were analyzed by nonlinear regression analysis as described [22].

2.8. Echocardiography and Doppler studies
Transthoracic echocardiographic measurements were performed on TG and WT mice as described [23]. The percentage shortening of the heart was calculated from the M-mode left ventricular (LV) dimensions as described previously [23]. In addition, Doppler flow measurements of aortic outflow and mitral inflow were performed.

2.9. Heart perfusion
Hearts from TG and WT mice were subjected to anterograde perfusion using the work-performing modus with an oxygenized Krebs–Henseleit buffer [18]. After a 30-min stabilization period, isoproterenol (0.01 µM) was administered into the perfusion system for 10 min. Preparations were freeze-clamped immediately either after the perfusion with buffer or after the stimulation period.

2.10. Immunoblotting
Perfused hearts from TG and WT mice were homogenized in 10 mM NaHCO3, 50 mM NaF, and 5 mM NaP2O7 (pH 7.4) at 4 °C for 90 s, using a Polytron PT-10 homogenizer (Kinematica, Lucerne, Switzerland). Crude homogenates were centrifuged at 10,000 x g for 20 min at 4 °C. Aliquots of supernatants (100 µg) were solubilized at 30 °C for 10 min in 5% SDS–stop solution [18]. Samples were subjected to 10% SDS–PAGE and blotted onto nitrocellulose membranes. The membranes were then reacted with polyclonal antibodies recognizing PLB phosphorylated both at Ser16 and at Thr17 (Upstate, Lake Placid, NY, USA) or a mouse monoclonal antibody recognizing βARK1 (Upstate). The immunoreactivity was visualized by 125I-labeled protein A (for Ser16-PLB and Thr17-PLB) or by an anti-mouse secondary antibody conjugated with alkaline phosphatase (for βARK1) and quantified using a PhosphorImager (Amersham). In addition, 50 µg of membranes (see Section 2.7) was electrophoresed in a 8% SDS–polyacrylamide gel and transferred to nitrocellulose [24]. The nitrocellulose was incubated with a mouse monoclonal antibody C2C12 raised to purified canine cardiac NCX (ABR, Golden, CO, USA). Antibody-reacting bands were detected by an anti-mouse secondary antibody conjugated with alkaline phosphatase, and then quantified using a PhosphorImager (Amersham). NCX was identified by co-migration of a molecular mass standard at 120 and 160 kDa.

2.11. Hemodynamic performance
Left ventricular catheterization was performed in closed-chest TG and WT mice as described previously [25]. Increasing doses of dobutamine were injected into the left jugular vein. Heart rate and the first derivative of left intraventricular pressure (+dP/dt and –dP/dt) were measured continuously.

2.12. Statistical analysis
Data are reported as means±S.E.M. Statistical differences between the different types of mice were calculated by ANOVA followed by Bonferroni's t test. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characteristics of caffeine-induced Ca2+ transients
To test the effects of overexpression of TRD on the SR Ca2+ content, caffeine-induced Ca2+ transients were measured in isolated cardiomyocytes (Fig. 1A). The twitch Ca2+ transient peak amplitude was unchanged between TG and WT (Fig. 1B, left panel), consistent with previous results [18]. Interestingly, the caffeine-triggered peak amplitude of [Ca2+]i was increased by 22% in TG compared to WT, indicating a higher SR Ca2+ load in TG (Fig. 1B, left panel). The time integral of the caffeine-induced Ca2+ transient was enhanced by almost twofold in TG (Fig. 1A). Furthermore, the time constant of decline of caffeine-dependent [Ca2+]i was prolonged by 57% in TG (Fig. 1B, right panel), suggesting a depressed NCX function. Consistently, immunoblot analysis revealed that the expression level of total NCX was diminished by 23% in TG. Here, we quantified both the 120-kDa (21,355±1826 and 29,065±2284 phosphorimager units in TG and WT, respectively, n=6, p<0.05) and the 160-kDa (8446±1490 and 9504±1261 phosphorimager units in TG and WT, respectively, n=6, n.s.) molecular mass forms of NCX (Fig. 1C). Moreover, when 5 mM Ni2+ was added, which effectively inhibited INa/Ca but allowed the Ca2+ transient to develop [26], the peak amplitude of [Ca2+]i remained higher in TG (Fig. 1B, left panel). Furthermore, the rate of decline of [Ca2+]i during the Ca2+ transient was still prolonged by 69% in TG (Fig. 1B, right panel). The application of Ni2+ was accompanied by a 3.6-fold and a 3.3-fold reduction in the rate of decline of caffeine-induced Ca2+ transients in TG and WT, respectively (Fig. 1B, right panel), suggesting the involvement of additional factors in the slower Ca2+ extrusion process in TG.


Figure 1
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  		Fig. 1 Effects of caffeine (±Ni2+) on Ca2+ transients and expression of the NCX. Ca2+ transients [Ca2+]i were measured in response to either 10 mM caffeine (left panel) or 10 mM caffeine plus 5 mM Ni2+ (right panel) in WT and TG cardiomyocytes (A). The data were summarized for the peak amplitude of [Ca2+]i (B, left panel) and the time constant of decline (B, right panel). The basal twitch [Ca2+]i peak amplitude was measured in paced cardiomyocytes (0.5 Hz). The caffeine-induced Ca2+ transients in the presence or absence of Ni2+ were obtained in quiescent TG and WT cardiomyocytes. The [Ca]i peak amplitude indicates the difference between the maximal and minimal Indo-1 fluorescence ratio. The protein expression of the NCX was determined in TG and WT membranes (C). Immunoblotting was performed with the use of an antibody raised to canine cardiac NCX. Shown is a representative immunoblot. The 120- and 160-kDa molecular mass forms of NCX are indicated (arrowheads).

 
3.2 Ca2+ sparks in isolated cardiomyocytes
Because characteristics of local spontaneous increases of intracellular Ca2+ (Ca2+ sparks) might be influenced by the enhanced SR Ca2+ load, we measured the temporal and spatial properties of Ca2+ sparks by use of the confocal microscope in the longitudinal line-scan mode. Ca2+ spark amplitude was increased by 16% in TG (Table 1). Moreover, Ca2+ spark frequency was increased by 29% in TG, although it did not reach statistical significance (Table 1). More significantly, the time to maximum peak intensity and the time to 50% decay of Ca2+ sparks were prolonged by 51% and 47%, respectively, in TG (Table 1). The altered kinetics of decline are visualized in the two-dimensional image (Fig. 2A) and the 3-D waterfall plot (Fig. 2B). Additionally, the spread width of Ca2+ sparks was increased by 47% in TG (Table 1). The rate of diastolic SR Ca2+ leak was estimated by the product of amplitude x frequency x duration x spatial size of Ca2+ sparks [27]. This results in a ~3-fold higher resting SR Ca2+ leak in TG. The distribution of Ca2+ spark amplitudes in WT revealed only one population (Fig. 2C). The maximum was reached at a F/F0 ratio of 2.3. In contrast, the amplitudes of Ca2+ sparks in TG were best described by two distinct distributions (Fig. 2C). Whereas a larger number of Ca2+ sparks (69%) had a maximum amplitude at a F/F0 ratio of 2.8, a smaller number of Ca2+ sparks (31%) reached its maximum amplitude at a F/F0 ratio of 5.5. The altered distribution of Ca2+ spark amplitudes was not accompanied by similar characteristics of the time to 50% decay (Fig. 2D). Here, we observed only one population of Ca2+ sparks in both groups.


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  		Table 1 Ca2+ sparks in TG and WT cardiomyocytes

 

Figure 2
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  		Fig. 2 Ca2+ sparks in WT and TG cardiomyocytes. Representative line-scan images of Ca2+ sparks (A) with the corresponding waterfall plots (B) were obtained from myocytes of WT and TG hearts. Histograms of Ca2+ spark amplitudes (C) and time to 50% decay (D) from WT and TG.

 
3.3 Modulation of L-type Ca2+ channel current
Here, we tested whether the altered cellular Ca2+ handling in TG influenced the properties of inactivation of ICa. We used Ba2+ as the charge carrier instead of Ca2+ (Fig. 3A). Ba2+ ions permeating through the L-type Ca2+ channel substitute poorly for Ca2+ in the Ca2+-induced inactivation of the L-type channel, and also are ineffective in inducing SR Ca2+ release. The normalized IBa current density was comparable between TG and WT (5.7±0.6 and 8.6±0.9 pA/pF, respectively, n=10–14, n.s.). Consistent with previous results [28], with Ba2+ as the charge carrier, the inactivation of the L-type Ca2+ channel was prolonged and was best fit by a monoexponential function in WT and TG (Fig. 3A). Nonetheless, {tau}Ba was increased in TG (Fig. 3B). With Ca2+ as the charge carrier when cells were loaded with BAPTA instead of EGTA (Fig. 3C), giving a more effective buffering of intracellular Ca2+ [29,30], the normalized ICa current density was unchanged between both groups (Fig. 3D, left panel). The fast and slow time constants of inactivation, {tau}1 and {tau}2, respectively, were prolonged in myocytes of TG hearts (Fig. 3D, middle and right panel). When the SR was depleted of Ca2+ by rapid application of caffeine (Fig. 3E), the inactivation of ICa exhibited two components [31]. The fast time constant of inactivation of ICa, {tau}1, was slower in TG (Fig. 3F, left panel), whereas the slow component of inactivation of ICa, {tau}2, remained unchanged between both groups (Fig. 3F, right panel). Moreover, we tested the effects of β-AR stimulation on L-type Ca2+ current. At depolarizing pulses between +10 to +60 mV, the normalized ICa current densities were decreased in TG (Fig. 3G, left panel). However, the fast and slow components of ICa inactivation remained unchanged between TG and WT (Fig. 3G, middle and right panel). The cell capacitance was increased by 12% in TG compared to WT (241±8 and 216±7 pF, respectively, n=10–14, p<0.05).


Figure 3
Figure 3
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  		Fig. 3 Electrophysiology in isolated cardiomyocytes. Electrophysiological parameters were determined in WT and TG cardiomyocytes using the whole-cell patch clamp method. Shown are typical recordings of whole-cell L-type Ca2+ currents in WT and TG and the quantified data for different conditions. ICa was recorded and measured at a test potential of +10 mV. The L-type Ca2+ current was registered when 2 mM Ca2+ in the external solution was changed to 2 mM Ba2+ (A). The time constant of inactivation was increased in TG (B). Traces show recordings using BAPTA and Ca2+ in the internal and external solution, respectively (C). The current density remained unchanged in the presence of BAPTA between TG and WT (D, left panel). The fast (D, middle panel) and slow (D, right panel) time constants of inactivation, {tau}1 and {tau}2, respectively, were higher in TG under BAPTA. Note the prolonged inactivation kinetics of ICa after rapid application of caffeine with Ca2+ in the external solution (E). This is reflected by an enhanced {tau}1 (F, left panel) in TG. However, {tau}2 was not different between TG and WT (F, right panel). ICa was also measured in the presence of 0.1 µM isoproterenol (Iso). The current density was decreased in TG under Iso (G, left panel). The time constants of inactivation, {tau}1 and {tau}2, were unchanged between both groups (G, middle and right panel, respectively).

 
3.4. ECG measurement
Here, we investigated whether alterations in the SR Ca2+ handling in TG mice were associated with abnormalities in the depolarization and the repolarization of these hearts. To this end, surface ECGs were measured by recording limb-leads. The heart rate was not different between TG and WT (366±29 and 397±20 bpm, respectively, n=7, each, n.s.). Consistently, an unchanged heart rate was observed in echocardiographic measurements (Table 2). Furthermore, the PQ interval (25±4 and 23±3 ms, respectively, n=7, each, n.s.) and the duration of the QRS interval (33±4 and 35±3 ms, respectively, n=7, each, n.s.) remained unchanged between TG and WT.


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  		Table 2 Echocardiography and Doppler measurements in TG and WT mice

 
3.5. In vivo assessment of cardiac performance by echocardiography and cardiac catheterization
To assess the left ventricular function in vivo, we determined cardiac dimensions, indices of systolic cardiac function, and diastolic filling parameters by M-mode (Fig. 4) and Doppler echocardiography. Data are summarized in Table 2. The intraventricular septum and LV posterior wall thicknesses were increased in TG. LV end-systolic dimension was reduced, whereas fractional shortening was decreased by 35% in TG (Fig. 4). The end-diastolic dimension (Fig. 4) and diastolic filling parameters (e.g., E-wave, A-wave) were not different between TG and WT. To further investigate cardiac function in vivo, we examined the β-AR response using cardiac catheterization (Fig. 5A). The maximal rate of pressure development (+dP/dt) was reduced under basal conditions in TG. TG showed a blunted contractility in response to dobutamine administration compared to WT. The rate of pressure development was decreased by 48% under maximal β-AR stimulation (Fig. 5B). Cardiac catheterization also revealed depressed rates of relaxation (–dP/dt) under basal conditions, and under dobutamine stimulation in TG. At the maximal dose of dobutamine, there was a 49% decrease in the rate of relaxation (Fig. 5C).


Figure 4
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  		Fig. 4 M-mode echocardiography. Representative examples of two-dimensional guided M-mode echocardiography in anaesthetized WT and TG mice. Arrows indicate LV end-diastolic and end-systolic dimensions.

 

Figure 5
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  		Fig. 5 In vivo measurement of LV hemodynamic parameters in response to β-AR stimulation. The contractile parameters of WT and TG closed chest-anaesthetized mice were studied by cardiac catheterization. Parameters were measured under basal conditions (Ctr) and in response to increased dobutamine administration. HR, the heart rate (A); +dP/dt, maximal rate of LV pressure development (B); –dP/dt, maximal rate of LV pressure decline (C).

 
3.6. β-AR signaling and PLB phosphorylation
To evaluate whether the decreased response to β-AR stimulation reflected an impaired adrenergic signaling, we measured the β-AR density using 125ICYP binding in membrane preparations. The total density of β-ARs was similar in preparations from TG and WT hearts (28.1±1.1 and 27.9±1.8 fmol/mg, respectively, n=5–6, n.s.). Membranes from TG and WT exhibited similar numbers of high affinity receptors. We measured 65.3% and 72.4% of high affinity receptors in TG and WT, respectively. The dissociation constant KD was somewhat increased in TG compared to WT (26.0±1.0 and 20.2±0.3 pM, respectively, n=5–6, p<0.05). Immunoblotting was utilized for detection of the βARK1 protein expression in homogenates of TG and WT hearts. The expression level of βARK1 was increased by 23% in TG compared to WT (3408±167 and 2763±115 phosphorimager units, respectively, n=5–8, p<0.05), which may desensitize β-ARs resulting in the functional uncoupling of β-ARs (Fig. 6A). Furthermore, the phosphorylation of PLB at Ser16 was unchanged between TG and WT. The effect of isoproterenol on the phosphorylation status of PLB on Ser16 was similar between TG and WT (by 138% and by 153%, respectively) hearts (Fig. 6B,C, upper panel). In addition, the phosphorylation of PLB at Thr17 was comparable between both groups. Interestingly, the effect of isoproterenol on the phosphorylation of PLB on the Thr17 site was lower in TG compared to WT (by 7% and 85%, respectively) hearts (Fig. 6B,C, lower panel).


Figure 6
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  		Fig. 6 Expression level of βARK1 and phosphorylation state of PLB. Shown are representative immunoblots. Proteins in ventricular homogenates from WT and TG mice were separated by SDS–PAGE and transferred to nitrocellulose membranes. βARK1 (A), PLB phosphorylated at Ser16 (B, upper panel), or at Thr17 (B, lower panel) were identified with specific antibodies as described in the Methods. Hearts were perfused in the absence or presence of 0.01 µM isoproterenol (+Iso). Antibody-reacting bands for phosphorylated PLB at Ser16 (C, upper panel) or at Thr17 (C, lower panel) were quantified under basal and stimulated (+Iso) conditions using a Phosphorimager.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. Background
Several in vitro studies indicated that TRD is involved in the interaction with the RyR, JCN, and CSQ, forming a quaternary complex in the cardiac junctional SR [9,13]. Furthermore, TRD anchors CSQ to the RyR in a Ca2+-dependent manner giving a first indication of a putative role of TRD in regulating SR Ca2+ handling [8,9,13,14]. We initiated a strategy in which this protein was overexpressed in transgenic mouse hearts [18]. TG mice exhibited a reduced protein expression of the RyR and JCN, whereas CSQ, SERCA2a, and PLB remained unchanged. We found an increased heart weight with induction of the hypertrophic signaling program in TG mice. This was attributed in part to the increased diastolic Ca2+ transients and the prolonged decay of [Ca2+]i in TG. We observed an impaired relaxation and a blunted contractility with increased pressure loading in work-performing TG hearts. To gain further insight into the underlying mechanisms of an altered cellular Ca2+ handling and impaired contractility, we extended our study of this transgenic model.

4.2 Increased SR Ca2+ load in TG
The SR Ca2+ content was increased by 22% in TG as judged by a higher caffeine-triggered Ca2+ transient amplitude. Although the NCX plays only a minor role in the competition of diastolic Ca2+ transport in mouse ventricle [32], an altered NCX function may influence the rise respectively the amplitude of the caffeine-induced Ca2+ transient as shown for other species [33]. Again, when Ni2+ was applied to inhibit INa/Ca, the amplitude of the caffeine-induced Ca2+ transient was still increased by 28% in TG. Considering the unchanged protein expression and activity of SERCA2a in TG [18], we suggest that the enhanced SR Ca2+ load is caused by an altered functional interaction of the junctional SR proteins. However, there is no evidence from our experiments for a direct inhibitory role of TRD on the RyR activity [16,17] because other proteins were changed in their expression level (e.g., down-regulation of the RyR and JCN). The cardiac-specific overexpression of CSQ was accompanied by an increased SR Ca2+ load [34]. These data indicate that an increase in CSQ protein expression can lead to an increased storage of Ca2+ in the SR. However, this mechanism cannot be operational in the present model: The CSQ protein expression was identical in WT and TG cardiomyocytes. We speculate here that there may be a higher SR Ca2+ content because increased TRD may increase Ca2+ binding to CSQ. We observed a prolonged decay of the caffeine-induced Ca2+ transients in TG, suggesting an impaired NCX function. Consistently, the protein expression of the NCX was diminished by 23% in TG. However, the slowing of the decline by the additional application of Ni2+, which inhibits effectively the NCX, was rather similar in TG (3.6-fold) and WT (3.3-fold). Therefore, we suggest that the diminished expression of the NCX provides only a minor contribution to the prolonged decay of [Ca2+]i in TG. The hypertrophy may lead to a diminished surface–volume relationship of the TG cardiomyocytes as observed in a model of myocardial infarction [35]. These authors explained the discrepancy between a higher NCX expression and an unchanged Ca2+ efflux via the NCX by a reduced surface–volume ratio in hypertrophied cells. While INa/Ca depends on the surface, the amount of Ca2+ transport correlates with the volume of the cardiomyocyte. Thus, we suggest that the apparent Ca2+ efflux via the NCX may be slowed in cardiomyocytes of hypertrophied TG caused by a reduced surface–volume relationship.

4.3 Altered Ca2+ spark characteristics in TG
The SR Ca2+ content is a critical factor in regulating the gating of RyRs which can be tested by measurement of Ca2+ spark parameters. Several studies reported an enhanced Ca2+ spark frequency with higher SR Ca2+ loading [36,37]. However, the Ca2+ spark rate was unchanged between both groups. Thus, we suspect that the increased amplitude of spontaneous Ca2+ sparks in TG may result from a higher SR Ca2+ load. The higher Ca2+ spark amplitude could be a compensation for the reduced expression of the RyR in TG [18]. This compensation might explain why we measured an unchanged global [Ca]i peak amplitude in electrically driven TG cardiomyocytes [18]. However, this explanation is not necessarily complete, because twitch Ca2+ transients are not caused by spontaneous SR Ca2+ release events but instead due to depolarization-induced Ca2+ sparks. In future studies, it will be informative to measure depolarization-driven Ca2+ sparks. We speculate here that in TG, there may be an increased sensitivity of RyRs to the higher lumenal Ca2+. This could explain why we determined only a 30% decrease of open RyRs in TG although the protein expression of RyRs was down-regulated by 55% [18]. The increased spread width of Ca2+ sparks in TG suggests an enhanced number of recruited RyRs. Furthermore, an increased sensitivity of RyRs may lead to a longer opening of the channel [26]. This could explain why time to peak of Ca2+ sparks was prolonged in TG. The prolonged decay of Ca2+ sparks in TG, exhibiting a normal distribution, may result from a combination of several factors [38,39]. For example, an increased sensitivity of RyRs may decelerate the termination of the SR Ca2+ release. Interestingly, we observed a redistribution of Ca2+ sparks to a population with smaller (69%) and a population with larger amplitudes (31%) in TG. This variation of Ca2+ spark amplitudes is unlikely to be due to detection of out-of-focus events because there was no correlation between the amplitude and spread width, time to peak, or time to 50% decay (data not shown). Spontaneous SR Ca2+ release events depend on the SR Ca2+ load. Therefore, we suggest that the population of Ca2+ sparks with the higher maximum F/F0 amplitude (31%) in TG could be attributable to alterations of the RyR characteristics. The higher SR Ca2+ load in TG may contribute to an increased sensitivity of a fraction of RyRs. This ends up in the recruitment of a larger number of RyRs within a defined cluster. Furthermore, triadin 1 is known to anchor calsequestrin to the RyR in a Ca2+-dependent manner. Thus, triadin 1 overexpression may alter per se the single-channel properties of RyRs.

4.4 Prolonged inactivation of ICa in TG
SR Ca2+ release through RyRs contributes to the inactivation of the Ca2+ channel [40]. The involvement of intracellular Ca2+ in the inactivation kinetics of ICa has been demonstrated under basal conditions and in cardiomyocytes with altered Ca2+ levels [28,31,41]. We observed two components of inactivation of the L-type Ca2+ current in WT and TG under basal conditions [18], probably depending on Ca2+-dependent and voltage-dependent inactivation [42,43]. The fast constant of inactivation, which was prolonged by 20% in TG [18], was eliminated when Ca2+ was substituted with Ba2+ in the external solution both in WT and TG. Ba2+ enters the cardiomyocyte through the L-type Ca2+ channel. However, Ba2+ is not able to release Ca2+ from the SR. Hence, we used Ba2+ to uncouple the L-type Ca2+ channel from the SR. However, the time constant under Ba2+ was still prolonged in TG. This suggests to us that the longer inactivation of the L-type Ca2+ channel in TG cardiomyocytes was not due to a slower or smaller Ca2+ release from the SR. In contrast, a primary alteration in L-type Ca2+ current kinetics in TG appeared more likely. Therefore, we used BAPTA, which acts much more rapid than EGTA [18], to buffer Ca2+ near the L-type Ca2+ channel. When cellular Ca2+ was buffered with BAPTA, both inactivation constants remained slower in TG versus WT. This lends further support that uncoupling the SR from the L-type Ca2+ channel, by keeping Ca2+ constant after electrical depolarization of the cardiomyocyte, did not normalize Ca2+ channel inactivation in TG. Furthermore, when we used Ca2+ as charge carrier and released Ca2+ from the SR by rapidly applying caffeine the fast time constant of inactivation was prolonged. Based on these data, we suggest a possible link between the altered SR Ca2+ handling and the gating of the L-type Ca2+ channel. It is conceivable that the application of Ba2+ (i.e., Ca2+ is not releasable from the SR) or the total buffering of cytosolic Ca2+ by BAPTA "demasks" a situation in which the (voltage-dependent) inactivation of ICa has changed in TG resulting in a normal slow constant of inactivation at basal conditions. Consistently, when caffeine was applied the increase in cytosolic Ca2+ resulted in a comparable slow constant of inactivation in both groups. Sun et al. suggested that the slow component is also Ca2+-dependent [44]. Thus, the higher diastolic Ca2+ transient in TG might activate the CaM kinase locally thereby changing the gating of the L-type Ca2+ channel. Furthermore, we cannot exclude changes in the expression of subunits of the L-type Ca2+ channel in TG which could also cause altered Ca2+ channel kinetics [26].

4.5. Depressed contractility and blunted response to β-AR stimulation in TG
We observed a depressed cardiac function in vivo, reflected by a diminished fractional shortening in the echocardiographic measurements and decreased left ventricular pressures in catheterized, anaesthetized TG mice. Furthermore, we measured a blunted response of the heart to β-AR stimulation in TG mice. What are the mechanisms for these alterations observed? We detected an increased protein expression of βARK1. The βARK1 is a G-protein-coupled receptor kinase that specifically phosphorylates activated β-ARs, leading to their desensitization [45]. In human heart failure, the βARK1 protein levels and activity were increased by 2-fold [46]. Moreover, the cardiac-specific overexpression of βARK1 in transgenic mice resulted in an impaired cardiac function with a reduction of the maximum inotropic effect to β-AR agonists [47,48]. Thus, the increased expression levels of βARK1 may contribute in part to the abnormal response to β-AR stimulation in TG hearts. Furthermore, it is conceivable that the activity of βARK1 is directly modulated by the increased diastolic Ca2+ transient. It remains to be elucidated whether the altered intracellular Ca2+ level may activate the promoter for βARK1. In addition, the decreased peak ICa in TG under β-AR stimulation can be explained by a diminished Ca2+-dependent facilitation of ICa due to a reduced activation of CaM kinase and phosphorylation of the channel [49]. This may contribute to an impaired excitation–contraction coupling under β-AR stimulation. Moreover, while the phosphorylation status of PLB at Ser16, which is mediated by PKA, was unchanged under basal and stimulated conditions between TG and WT hearts, Thr17 phosphorylation of PLB hardly increased during isoproterenol stimulation in TG. As Thr17 is phosphorylated by CaM kinase, we suggest that the increased diastolic SR Ca2+ leak may lead to less Ca2+-dependent activation of CaM kinase under β-AR stimulation. This assumption would easily explain the reduced increase in force after β-AR stimulation, because unphosphorylated Thr17-PLB is known to inhibit cardiac contractility, in part, by inhibition of SERCA2a [2,50]. Finally, we cannot rule out the possibility of an attenuated Ca2+ sensitivity of the myofilaments contributing in part to the reduced contractility in TG.

In summary, overexpression of TRD is associated with profound changes in the interaction of the junctional SR Ca2+ release protein complex causing a higher SR Ca2+ load. This results in alterations of single Ca2+ release events (Ca2+ sparks) which may explain the increased diastolic SR Ca2+ leak. As a consequence, the altered cellular Ca2+ handling may promote the development of an impaired contractility and blunted response to β-AR stimulation in TG.


   Acknowledgements
 
This work was supported by NIH Grant HL-28556 (to L.R.J.), the Deutsche Forschungsgemeinschaft, and the SFB 556 (to J.N. and W.S.), the SFB 355 (to L.H. and M.J.L.), the Leibniz award (to M.J.L.), and the "Jung-Stiftung für Wissenschaft und Forschung", Hamburg, Germany (to B.R. and M.J.L.).


   Notes
 
Time for primary review 33 days


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

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