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Am J Physiol Heart Circ Physiol 293: H3165-H3174, 2007. First published September 21, 2007; doi:10.1152/ajpheart.00799.2007
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Triadin is a critical determinant of cellular Ca cycling and contractility in the heart

Uwe Kirchhefer,1 Jan Klimas,2 Hideo A. Baba,3 Igor B. Buchwalow,4 Larissa Fabritz,5 Marion Hüls,5 Marek Matus,1 Frank U. Müller,1 Wilhelm Schmitz,1 and Joachim Neumann6

1Institut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Münster, Germany; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic; 3Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen, Essen; 4Gerhard-Domagk-Institut für Pathologie and 5Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster, Münster; and 6Institut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany

Submitted 10 July 2007 ; accepted in final form 17 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of beta-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([Ca]i) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [Ca]i peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to beta-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function.

transgenic mice; sarcoplasmic reticulum; hypertrophy; Ca signaling; cardiac function; force-frequency relationship; beta-adrenergic receptor stimulation

HEART FAILURE REPRESENTS the most important cause of death in Western countries. Its high mortality originates in part from severe complications like premature death caused by ventricular arrhythmias (40). The induction of arrhythmias by abnormal automaticity is forced by a cardiac remodeling in heart failure. There is now increasing evidence that propagation of arrhythmias by delayed afterdepolarizations (DADs) and/or depression of contractility in heart failure is linked to a malfunction of sarcoplasmic reticulum (SR) Ca release.

SR Ca release is maintained by a macromolecular protein complex consisting of the ryanodine receptor (RyR), calsequestrin (CSQ), triadin, and junctin that is activated by L-type Ca current (ICa,L) (3, 47). Aside from cytosolic Ca, SR Ca release channel activity is also regulated by SR luminal Ca (13, 39). Its storage and release are under the control of CSQ (13), whereas triadin and junctin may serve as linker proteins between CSQ and the RyR (24, 47). The tethering of CSQ to the inner surface of the SR allows it to sequester Ca in the vicinity of the RyR during SR Ca cycling (16, 43). CSQ may act as a Ca sensor that inhibits the RyR at low SR luminal Ca via interaction with triadin/junctin (42). An increase of SR luminal Ca disrupts the inhibition of the RyR because the CSQ Ca binding sites become more occupied with Ca, resulting in a weakened interaction between CSQ and triadin/junctin and an increased open probability of the channel (12). Thus the interaction between these proteins appears to be critical for the regulation of SR Ca release.

Indeed, several disorders of the SR Ca release complex have been identified as causes of heart disease. Hyperphosphorylation of the RyR by PKA and Ca/calmodulin-dependent protein kinase II (CaMKII) induces a Ca leak during diastole, which can cause heart failure and lead to fatal arrhythmias (30–32, 44). Moreover, increases in the expression of triadin and junctin were associated with contractile failure and an increased propensity for ventricular automaticity. The forced expression of triadin or junctin in rat myocytes resulted in an increase of the RyR open probability or a depressed contractility, respectively (10, 41). Although more difficult to decipher because of various adaptive changes, overexpression of triadin or junctin in mice also caused an impaired cardiac performance, coincident with an enhanced diastolic SR Ca leak (18–20). Thus the reduced expression levels of triadin and junctin in failing human hearts may help to maintain the SR Ca release as close to normal as possible (10). Consistently, the ablation of junctin was associated with enhanced cardiac function and increased Ca cycling parameters in mice (45). Thus a better understanding of the cross talk between the proteins of the SR Ca release complex may lead to new therapeutics for the treatment of heart failure and ventricular arrhythmias.

Hence, here we used transgenic mice with co-overexpression of triadin and junctin resulting in a stable approximately threefold expression of triadin associated with an unchanged junctin expression. This strategy allows a more precise definition of the role of triadin in excitation-contraction coupling by the circumvention of expressional changes of junctin. Here we found that the forced expression of triadin leads to cardiac hypertrophy, prolonged relaxation, a blunted response to beta-adrenergic agonists, and impaired Ca cycling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Breeding of transgenic mice. Generation of transgenic mice with heart-directed overexpression of either dog triadin-1 (TRD) or junctin was described previously (25, 46). These transgenic mouse lines were crossbred (JxT). Age- and sex-matched double-transgenic JxT and wild-type (WT) mice were used in the following experiments. Here we studied adult mice at 14–18 wk of age. Animals were handled and maintained according to protocols approved by the animal welfare committees of the University of Münster, Indiana University, and the University of Halle-Wittenberg. This investigation conformed to the National Research Council's Guide for the Care and Use of Laboratory Animals.

SDS-PAGE and immunoblotting. Hearts were homogenized for 1 min in a buffer containing 50 mM MOPS (pH 7.0) and 0.25 M sucrose using a Polytron PT-10 homogenizer (Kinematica, Lucerne, Switzerland). Homogenates were solubilized in 5% SDS buffer containing 62.5 mM Tris·HCl (pH 6.8), 5% glycerol, and 40 mM dithiothreitol. For immunoblot analysis, 40 or 200 µg [detection of RyR, RyR phosphoserine-2809, and Na/Ca exchanger (NCX)] of homogenate protein were separated on SDS-PAGE (37). After transfer of proteins to nitrocellulose, the blots were incubated with different antibodies. The amount of bound protein was detected by 125I-labeled protein A and quantified using Storm 860 (Molecular Dynamics, Sunnyvale, CA). We used various antibodies raised against the following proteins: dog and mouse junctin (20), dog and mouse triadin (25), sarco(endo)plasmic reticulum Ca-ATPase-2a (SERCA2a) (17), CSQ (29), phospholamban (PLB) (15), PLB phosphoserine-16 (Badrilla, Leeds, UK), PLB phosphothreonine-17 (Badrilla), RyR (47), NCX (19), RyR phosphoserine-2809 (Badrilla), and sarcolemmal Ca pump (Ca pump of plasma membrane; PMCA ATPase) (ABR, Golden, CO).

Morphology, immunohistochemistry, and electron microscopy. Heart tissue probes were fixed in buffered 4% formaldehyde and routinely embedded in paraffin. Deparaffinized sections were routinely stained with Mayer's hematoxylin-eosin and according to the trichrome method of Masson-Goldner. Paraffin-embedded sections were also dewaxed in xylene, rehydrated in graded alcohols, and transferred into PBS. After antigen retrieval (Reveal, Biocarta, Hamburg, Germany) and blocking of nonspecific binding sites with BSA basic blocking solution (Aurion, Wageningen, The Netherlands) as previously described (4), sections were incubated with primary antibodies o/N at 4°C. Rabbit polyclonal antibodies to triadin (25) and junctin (20) were applied. For fluorescence visualization of bound primary antibodies, sections were treated with a biotinylated goat anti-rabbit antibody (Dianova, Hamburg, Germany) and visualized with streptavidin-Alexa 488 (Molecular Probes, Leiden, The Netherlands). Immunostained sections were examined on a Zeiss Axiophot2 microscope. For electron microscopy, small tissue pieces of left ventricle were fixed by immersion with 2.5% glutaraldehyde in 0.1 M PBS. After fixation, the specimens were fixed in buffered 1% osmium tetraoxide for 2 h, dehydrated in graded ethanol series, and embedded in Epon. Ultrathin sections were cut and stained with uranyl acetate and lead citrate. The sections were studied under a Zeiss EM 902A transmission electron microscope.

Left ventricular catheterization. Left ventricular catheterization was performed in closed-chest mice by modification of a method described previously (19). Increasing doses of dobutamine were injected into the left jugular vein. Heart rate and the first derivatives of left intraventricular pressure (+dP/dt and –dP/dt) were monitored continuously.

Doppler echocardiography. Doppler echocardiography was performed using a 12-MHz Doppler (Philips Sonos 5500) as described (21). Diastolic function is even more frequency dependent than systolic function. If mouse lineages differ in heart rate, pacing, preferably of the right atrium, may be helpful for proper evaluation. Right atrial pacing was performed via an octapolar catheter in anesthetized closed-chest mice.

Cell shortening and Ca transients. Myocytes were enzymatically isolated from mouse ventricles and loaded with Indo-1/AM (Sigma-Aldrich, St. Louis, MO) as described previously (20). Myocytes were stimulated with 0.5 Hz at 23°C, and intracellular Ca transients were determined as follows. Indo-1 was excited at 365 nm. The emitted fluorescence was detected at 405 and 495 nm. The cytosolic Ca concentration ([Ca]i) was estimated by calculating the ratio of fluorescence signals. The maximum and minimum Indo-1 fluorescence ratios were detected, and their difference was defined as {Delta}[Ca]i. Moreover, the time to 50% decay of Ca transients was measured. The shortening of myocytes and the rate of 50% relaxation were recorded simultaneously using a video edge detection system (21). The response of myocytes to beta-adrenergic stimulation was tested by application of isoproterenol (Iso; 1 nM to 0.1 µM). Where indicated, stimulation frequencies were stepwise increased from 0.5 to 5 Hz. Parameters were recorded at steady-state conditions. These were achieved normally after 5 min. SR Ca load was estimated by measurement of {Delta}[Ca]i in the presence of 10 mM caffeine for 1 min in electrically unstimulated myocytes. The amplitude of the electrically evoked Ca transient, expressed as the percentage of the caffeine-induced Ca transient, has been referred to as fractional SR Ca release (2). The single application of caffeine may underestimate the peak Ca transient because of Ca extrusion by the NCX at the onset of the Ca transient rise (1). The additional application of 5 mM Ni, which blocks effectively the Na/Ca exchange current (14, 19), circumvents this problem. In addition, the time constant of decline of Ca transients ({tau}) was determined under these conditions.

Electrophysiology. ICa,L was recorded in isolated cardiomyocytes using the whole cell patch-clamp technique (20). Ca currents were elicited by applying 200-ms depolarizing pulses every 10 s from a holding potential of –40 mV. The extracellular solution contained 130 mM tetraethylammonium (TEA)-Cl, 4 mM 4-aminopyridine, 1 mM MgCl2, 10 mM HEPES, 10 mM dextrose, and 2 mM CaCl2 (pH 7.3). Potassium currents were suppressed under these conditions. The intracellular solution was composed of 80 mM potassium aspartate, 50 mM KCl, 10 mM KH2PO4, 0.5 mM MgCl2, 3 mM MgATP, 10 mM HEPES, and 1 mM EGTA (pH 7.4). The response to beta-adrenergic receptor stimulation of ICa,L was tested by using 0.1 µM Iso in the extracellular solution. Kinetics of ICa,L were measured at a test potential of +10 mV.

Data analysis. Data are reported as means ± SE. Comparisons between the means of two groups were performed by unpaired Student's t-test. For multiple comparisons, a two-way repeated-measure ANOVA was used followed by a Newman-Keuls test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Stable expression of triadin in JxT double-transgenic mice. Here we used the {alpha}-myosin heavy chain promoter to drive heart-directed expression of both dog triadin and junctin in mouse hearts (11). Samples of heart homogenates of JxT and WT mice were probed with antibodies recognizing both dog and mouse triadin or junctin. A substantial overexpression of triadin was evident in the double-transgenic JxT line (Fig. 1, Table 1). Two mobility forms of triadin were detected and correspond to the deglycosylated and glycosylated protein (Fig. 1). Interestingly, we found that the ratio of glycosylated to deglycosylated triadin was fourfold lower in JxT compared with WT hearts (Table 1). The protein level of junctin remained constant in JxT (Fig. 1, Table 1) but was reduced in single-transgenic TRD hearts (20). Dog junctin was recognized by immunoblotting as a 26-kDa molecular mass protein, whereas mouse junctin ran at 24 kDa in SDS-PAGE (Fig. 1). Thus we found a higher expression of triadin in JxT mice, whereas the expression of junctin remained unchanged. This allows the study of a model with overexpression of triadin without any adaptation processes by an altered expression of junctin, in contrast to TRD mice. The double-transgenic JxT mice were further investigated by use of biochemical, histological, and (electro)physiological analyses. To test whether overexpression of triadin in JxT hearts altered the expression of other regulatory SR and sarcolemmal proteins, we utilized immunoblotting analysis (Fig. 1, Table 1). Expression of SERCA2a, calsequestrin, PLB, and PMCA ATPase was not different in JxT homogenates. The phosphorylation state of PLB at both Ser16 and Thr17 was similar in JxT and WT hearts. In contrast, the expression of the RyR was decreased by 28% in JxT hearts, whereas the phosphorylation of the RyR at Ser2809 was increased by 57% in this group. The expression of the NCX was reduced by 25% in JxT mice. Immunohistochemical staining of ventricular sections confirmed data from immunoblotting analysis showing abundant expression of triadin in JxT myocytes (data not shown). In addition, the expression of junctin was unchanged in JxT myocytes (data not shown).


Figure 1
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  		Fig. 1. Identification of triadin, junctin, and regulatory cardiac proteins. Heart homogenates of mice with overexpression of triadin and junctin (JxT) and wild-type mice (WT) were separated on SDS-PAGE. After transfer of proteins to nitrocellulose, the blots were incubated with specific antibodies, and the amount of bound protein was detected. Blots were probed with an antibody recognizing canine and mouse triadin and an antibody recognizing dog (d.) and mouse (m.) junctin. The glycosylated form of triadin runs at 40 kDa ({psi}). Note the higher molecular mass of dog junctin compared with the endogenous mouse protein. Moreover, blots were incubated with antibodies specific for calsequestrin (CSQ), ryanodine receptor (RyR), phosphorylated ryanodine receptor (RyR-P), CaATPase [sarco(endo) plasmic reticulum Ca-ATPase-2a; SERCA2a], phospholamban (PLB), Na/Ca exchanger (NCX), and sarcolemmal Ca pump (Ca pump of plasma membrane; PMCA ATPase) as described in MATERIALS AND METHODS.

 

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  		Table 1. Comparison between WT and JxT

 
Cardiac hypertrophy. JxT mice exhibited a mild degree of hypertrophy as indicated by an increase in heart-to-body weight ratio relative to the ratio in WT mice (5.7 ± 0.1 vs. 5.0 ± 0.1 mg, respectively, n = 16, P < 0.05). In addition, the left atrial weight was increased in JxT (4.5 ± 0.2 mg) compared with WT (3.7 ± 0.2 mg, n = 16, P < 0.05) mice. The histological examination showed an increase of myocyte diameters in JxT (11.5 ± 0.2 µm) compared with WT (10.0 ± 0.3 µm, n = 3, P < 0.05) mice. Electron microscopy demonstrated that the structure of myofibrils was disrupted, with partial separations occurring in single fibers, and the myofibrils were frequently displaced by an electron-dense matrix in JxT myocytes (Fig. 2), indicating a diseased myocardium in an early stage of contractile failure. However, JxT mice had a normal mortality and did not show overt signs of heart failure.


Figure 2
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  		Fig. 2. Electron microscopy. Ultrastructural electron microscope analysis of myocytes in JxT and WT hearts. Note the structural disorganization and myofibrillar disruption in JxT myocytes. Magnification = x7,500.

 
Impaired cardiac performance of JxT hearts. To assess the effects of a stable triadin expression on cardiac function in JxT mice, we examined the left ventricular function in response to beta-adrenergic stimulation using cardiac catheterization. The maximal rates of pressure development (+dP/dt) were reduced under basal conditions in JxT (by 26%) hearts (Fig. 3A). JxT hearts showed a blunted contractility in response to maximum dobutamine administration compared with WT (Fig. 3A). The measurements revealed depressed rates of relaxation (–dP/dt) under basal conditions (by 26%) and under stimulation of beta-adrenergic receptors in JxT hearts (Fig. 3B). Interestingly, the heart rate was also reduced under both basal and Iso-stimulated conditions in JxT compared with WT hearts (Fig. 3C), suggesting frequency-dependent effects on the measurement of cardiac hemodynamics. However, an impaired relaxation was also measured with Doppler echocardiography under right atrial pacing in JxT mice. This procedure allows the assessment of cardiac function independent of differences in the heart rate occurring between the mouse lines. Here, early and atrial mitral waves conflated at accelerating pacing cycle lengths in JxT mice (Fig. 4). The left ventricular ejection time was prolonged by 70% at fixed frequencies during right atrial pacing in JxT compared with WT mice (Fig. 4). The fractional shortening was unchanged under fixed frequency between JxT and WT hearts, suggesting a comparable systolic function in JxT mice.


Figure 3
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  		Fig. 3. Left ventricular in vivo hemodynamics. The maximum rate of left ventricular pressure development (+dP/dt; A), the maximum rate of left ventricular pressure decline (–dP/dt; B), and the heart rate (C) were measured at basal conditions and in response to increased doses of dobutamine in closed-chest anesthetized JxT and WT mice by cardiac catheterization. bpm, Beats/min.

 

Figure 4
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  		Fig. 4. Doppler echocardiography. Representative example of mitral valve flow at accelerating pacing from 100- to 90-ms cycle length in JxT mice. The conflation of early (E) and atrial (A) waves occurring at a pacing cycle length of 90 ms (arrow at far right). Note the prolonged left ventricular ejection time (LVET) in JxT compared with WT mice, as summarized in the appended table. FS, fractional shortening; Vcf, velocity of circumferential fiber shortening.

 
Blunted response to beta-adrenergic stimulation of JxT myocytes. To test whether the impaired in vivo cardiac function in JxT hearts was paralleled by changes at the cellular level, we determined the shortening and relengthening of isolated myocytes under basal conditions and in the presence of increasing doses of Iso (Fig. 5A). The maximum shortening (Fig. 5C) and the rate of 50% relaxation (Fig. 5D) were unchanged under basal conditions (0.5 Hz) between both groups. The administration of maximum doses of Iso (0.1 µM) resulted in a lower extent of shortening in JxT (by 32%) compared with WT myocytes (Fig. 5C). Moreover, myocytes of JxT mice relaxed more slowly (by 50%) in response to administration of maximum Iso than WT cells (Fig. 5D). To test whether the Iso-stimulated effects on myocyte shortening and relaxation were paralleled by alterations in Ca transients, {Delta}[Ca]i and the time to 50% decay of [Ca]i were measured simultaneously (Fig. 5B). We found that {Delta}[Ca]i was comparable under nonstimulated conditions between both groups (data not shown) but diminished in the presence of maximum doses of Iso in JxT mice (Fig. 5E). Although we observed a hastening of the Ca transient decay in both groups, this parameter was prolonged under basal (by 19%) and maximum Iso-stimulated (by 21%) conditions in JxT myocytes (Fig. 5F).


Figure 5
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  		Fig. 5. Myocyte shortening and Ca transients in response to beta-adrenergic receptor stimulation. Shown are representative traces of edge detection (A) and Ca transient (B) measurements recorded at different isoproterenol (Iso) concentrations in WT myocytes. The shortening (C), the rate of 50% relaxation (D), {Delta}[Ca]i (E), and the time to 50% decay of [Ca]i (F) were measured at 0.5-Hz stimulation frequency in response to increasing doses of Iso in myocytes of JxT and WT mice; n = no. of myocytes/hearts. Indo-1, indomethacin-1; [Ca]i, cytosolic Ca concentration; {Delta}[Ca]i, change in cytosolic Ca concentration.

 
Flat staircase and no frequency-dependent acceleration of relaxation in JxT mice. We analyzed the maximum shortening and the rate of 50% relaxation of myocytes in response to increasing stimulation frequencies ("Treppe" phenomenon, Fig. 6A). The shortening-frequency relationship was positive in WT compared with JxT myocytes, which exhibited no staircase phenomenon (Fig. 6C). Moreover, increasing stimulation frequencies accelerated the rate of 50% relaxation in WT but not in JxT myocytes (Fig. 6D). Here, {Delta}[Ca]i and the time to 50% decay of [Ca]i were measured in parallel in myocytes of all groups (Fig. 6B). Fig. 6E shows that the relationship between {Delta}[Ca]i and frequency was positive in WT, whereas this relationship was negative in JxT myocytes. A frequency-dependent acceleration of the 50% decay time of Ca transients was clearly apparent in both groups but less pronounced in JxT myocytes (Fig. 6F).


Figure 6
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  		Fig. 6. Frequency-dependent effects on myocyte shortening and Ca transients. Representative edge detection (A) and Ca transient (B) recordings in response to different stimulation frequencies are shown for WT myocytes. Frequency was stepwise increased every 5 min from 0.5 to 5 Hz, and effects on shortening (C), the rate of 50% relaxation (D), {Delta}[Ca]i (E), and the time to 50% decay of [Ca]i (F) were monitored in myocytes of JxT and WT mice; n = no. of myocytes/hearts.

 
Increased SR Ca load in JxT mice. To determine the effects of stable expression of triadin in JxT mice on the SR Ca content, caffeine-induced Ca transients were measured in the absence (Fig. 7A) and presence of Ni (Fig. 7B) in isolated myocytes. The twitch {Delta}[Ca]i was not different between both groups (Fig. 7C). We found an increased caffeine-induced {Delta}[Ca]i in JxT (by 23%) compared with WT myocytes (Fig. 7C), indicating a higher SR Ca load in JxT mice. The rapid application of caffeine plus Ni had no additional effect compared with the single administration of caffeine, revealed by a 27% higher {Delta}[Ca]i in JxT myocytes (Fig. 7C). The fractional SR Ca release, reflecting the percentage of Ca stored within the SR that is released on electrical stimulation, was not different between both groups (70 ± 4% in JxT vs. 69 ± 3% in WT). The time constant of decline of caffeine-triggered Ca transients was prolonged by 65% in JxT myocytes (Fig. 7C), suggesting a depressed NCX function. The additional administration of Ni (Fig. 7C) was associated with a comparable percentage reduction in the decay kinetics of caffeine-induced Ca transients in JxT (by 237 ± 27%) and WT myocytes (by 226 ± 23%). This suggests that the altered cytosolic Ca extrusion in JxT is not influenced by Ca transport/buffering mechanisms other than an impaired NCX function. Indeed, the protein expression of the PMCA ATPase was not different between JxT and WT hearts (Table 1).


Figure 7
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  		Fig. 7. Caffeine-induced Ca transients. Representative tracings of Ca transients in JxT and WT myocytes under basal electrical stimulation (0.5 Hz) and under stimulation with rapid application of 10 mM caffeine in the absence (A) and presence of 5 mM Ni (B). The amplitude of Ca transients, {Delta}[Ca]i, was measured at basal conditions and in response to caffeine (±Ni) in myocytes of JxT and WT mice (C, left). The time constant of decay ({tau}) of caffeine-triggered [Ca]i (±Ni) was determined simultaneously (C, right); n = no. of myocytes/hearts.

 
Unchanged ICa,L in JxT mice. To test whether the altered Ca signaling in JxT was accompanied by changes in the trigger for SR Ca release, we measured the ICa,L under basal and Iso-stimulated conditions in isolated myocytes (Fig. 8A). At depolarizing potentials between –30 and +60 mV, the normalized ICa,L densities were unchanged between JxT and WT myocytes (Fig. 8B). At all depolarizing pulses tested, the normalized ICa,L densities were not different after beta-adrenergic stimulation between JxT and WT myocytes (Fig. 8B). The fast and slow components of ICa,L inactivation remained unchanged between JxT and WT myocytes (data not shown).


Figure 8
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  		Fig. 8. L-type Ca currents (ICa). Shown are typical recordings of whole cell L-type Ca currents at a test potential of +10 mV under basal and Iso-stimulated conditions in JxT and WT myocytes (A). The peak current-voltage relationships (±Iso) were obtained from JxT and WT myocytes (B). Ca channels were activated by 200-ms depolarizing pulses from a holding potential of –40 mV to the indicated test potentials. Ordinate is given in current divided by the cell capacitance (pA/pF); n = no. of myocytes/hearts.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown for the first time that the stable expression of triadin in the myocardium of double-transgenic JxT mice was associated with hypertrophy, impaired basal relaxation, depressed cardiac function after stimulation of beta-adrenergic receptors or high-stimulation frequencies, and impaired SR Ca cycling. Importantly, these effects were observed on the background of an unchanged expression of junctin, which shares structural and functional similarities with triadin and appears to anchor CSQ to the RyR (17, 47). In contrast, the overexpression of triadin in single-transgenic mice was associated with a 73% downregulation of junctin, limiting the interpretation of physiological alterations, which were thus ascribed directly to triadin. Moreover, the separate investigation of both models is stringently necessary because recent studies have revealed new insight into the important role of junctin in regulating SR Ca homeostasis and contractility (9, 10, 45). Thus characterization of the present co-overexpression model allowed a more precise definition of triadin function in SR Ca signaling.

Here we observed a mild cardiac hypertrophy at both the whole organ and myocyte level in JxT mice. Interestingly, heart hypertrophy occurred in transgenic mice with overexpression of triadin, junctin, or CSQ (20, 38, 46) and in knockout mice with ablation of CSQ (23). Thus overexpression or deletion of one of the partners within the macromolecular SR Ca release complex leads to an altered stoichiometry and, therefore, a modulated interaction of the proteins. The result is an impaired cellular Ca cycling, manifested in a prolonged decay of [Ca]i in JxT myocytes. This may activate either calcineurin or CaMKII, leading to the transcription of several genes (e.g., GATA-4, MEF2) that are involved in the induction of cardiac hypertrophy (6, 33, 35). Indeed, transgenic overexpression of calcineurin or the CaMKII was associated with severe cardiac hypertrophy (5, 31). Alternatively, Ca may be transported from the SR to the nucleus (36), thereby directly inducing the transcription of hypertrophy-associated genes.

Consistent with the prolonged decay of [Ca]i in JxT mice, we measured an impaired relaxation under basal and stress-provoked conditions. The single overexpression of triadin resulted in a similar contractile phenotype in TRD mice (19, 20). This suggests that the impaired relaxation in both transgenic models was not influenced by an altered expression level of junctin under these conditions, although it is very critical in regulating the SR Ca release. Application of junctin to the luminal side of the purified RyR enhanced the open probability of the channel (12). This effect may explain the reduced SR Ca load and the lower Ca spark frequency in junctin-overexpressing mice (18). In line with these findings, overexpression of junctin in isolated myocytes was associated with a prolonged relaxation (9, 10). In contrast, the deletion of junctin was accompanied by a higher SR Ca content and a hastened relaxation in knockout mice (45). Because of an unchanged expression of the SR Ca uptake proteins SERCA2a and PLB and a comparable phosphorylation of PLB at Ser16 and Thr17 in JxT hearts, we suggest that the lower expression and/or activity of the NCX results in a prolonged [Ca]i decay and an impaired relaxation in JxT mice, although the extent of cytosolic Ca removal during diastole by the NCX is limited in mouse heart (28). Here mechanisms of Ca extrusion/buffering other than the NCX are not involved in the impaired decay kinetics of [Ca]i in JxT mice, supported by the fact of an unchanged expression of the sarcolemmal Ca pump.

We observed a blunted response of the myocardium to beta-adrenergic stimulation in JxT mice, as shown by a depressed contractility in catheterized mice and a decreased shortening of isolated myocytes. An increase in stimulation frequencies resulted in a similar depression of myocyte shortening. These effects were accompanied by parallel changes of {Delta}[Ca]i. This was not due to changes in the ICa,L. The normalized ICa,L densities were unchanged in JxT myocytes. In contrast, the single overexpression of triadin was associated with a lower peak amplitude of ICa,L after administration of Iso (19). Thus it is more likely that the disarrangement and disruption of muscle fibers in JxT hearts, possibly reflecting the onset of a cardiac remodeling, contribute to the diminished contractile response to beta-adrenergic stimulation. The importance of an intact myofilament structure for maintenance of contractile function was widely demonstrated in genetically induced cardiomyopathies, ischemia/reperfusion-injured hearts, and heart failure (7).

Here we found an increased SR Ca load in myocytes of JxT mice. This effect also occurred in single-transgenic triadin-overexpressing mice (19), suggesting that an altered expression level of junctin does not contribute to the enhanced SR Ca storage capacity under these conditions. In contrast, adenovirus-mediated expression of triadin in rat myocytes was associated with a lower SR Ca load, consistent with an increased open probability of RyRs in lipid bilayers and a diminished amplitude of spontaneous Ca sparks (41). The expression level of the RyR was unchanged, which might explain the observed differences in the cellular Ca cycling between the models. In the present study, the reduced expression of the RyR may represent a compensatory mechanism to limit the Ca release from the SR, which is overloaded with Ca. However, the fractional SR Ca release was not different between both groups. What are the mechanisms underlying the increased SR Ca load in the present double-transgenic model with stable expression of triadin? It has been shown by in vitro studies that skeletal muscle triadin can reduce the activity of the RyR (27, 34). Furthermore, application of triadin to the luminal side of the RyR, reassociated with CSQ, produced a decrease in the channel activity (12). These authors proposed a model in which CSQ stabilizes SR Ca release by inhibiting the RyR through interaction with triadin (42). Thus, at present, controversy exists regarding the question of whether triadin functions as a repressor or rather a stimulator of SR Ca release. It remains to be elucidated whether the degree of glycosylation of triadin can influence its functional state. In this study, we measured a reduced glycosylation of triadin, suggesting partial glycosylation of a heterologously expressed dog protein in mouse heart due to a limited capacity of oligosaccharyltransferase or an inaccessibility of the glycosylation sequon to this enzyme (25). However, triadin overexpression obviously favors the occurrence of leaky Ca release channels by a hyperphosphorylation of RyRs, cytosolic Ca waves, DADs, and stress-provoked arrhythmias in both adenovirus-infected myocytes and transgenic mice (22, 41), whereas a reduced expression of triadin, as shown in end-stage human heart failure, may increase the threshold for malignant ventricular arrhythmias (10). Similar to changes in triadin overexpression models, RyR and CSQ mutations, altering the properties of the channel gating and reducing luminal SR Ca binding, respectively, can promote spontaneous SR Ca release and the generation of cytosolic Ca waves. This results in the development of DAD-triggered ventricular arrhythmias, impressing clinically as catecholamine-induced polymorphic ventricular tachycardia (8, 26).

In summary, the stable expression of triadin was associated with cardiac hypertrophy, a depressed contractile function under basal and stress-provoked conditions, and a higher SR Ca content in double-transgenic JxT mice. These effects were observed under an unchanged expression level of junctin, the homologous partner protein of triadin in the SR Ca release complex. Thus we suggest that triadin is a critical determinant in regulating cellular Ca cycling and contractility. However, the characterization of a triadin-deficient model may further elucidate the role of triadin in normal and diseased hearts.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Deutsche Forschungsgemeinschaft (U. Kirchhefer, J. Neumann; Ki 653/13-1/2) and by the IZKF Münster ZPG4 (to L. Fabritz).


   ACKNOWLEDGMENTS
 
We thank Nicole Hinsenhofen for technical assistance. We are grateful to Dr. L. R. Jones for providing several antibodies (anti-junctin, anti-triadin, anti-SERCA2a, anti-RyR).


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Kirchhefer, Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Domagkstr. 12, 48149 Münster, Germany (e-mail: kirchhef{at}uni-muenster.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bassani JWM, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells–species-dependent differences in cellular mechanisms. J Physiol 476: 279–293, 1994.[Abstract/Free Full Text]
  2. Bassani JWM, Yuan WL, Bers DM. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol Cell Physiol 268: C1313–C1319, 1995.[Abstract/Free Full Text]
  3. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  4. Buchwalow IB, Podzuweit T, Samoilova VW, Wellner M, Haller H, Grote S, Aleth S, Boecker W, Schmitz W, Neumann J. An in situ evidence for autocrine function of NO in the vasculature. Nitric Oxide 10: 203–212, 2004.[CrossRef][Web of Science][Medline]
  5. Chu G, Carr AN, Young KB, Lester JW, Yatani A, Sanbe A, Colbert MC, Schwartz SM, Frank KF, Lampe PD, Robbins J, Molkentin JD, Kranias EG. Enhanced myocyte contractility and Ca2+ handling in a calcineurin transgenic model of heart failure. Cardiovasc Res 54: 105–116, 2002.[Abstract/Free Full Text]
  6. Colomer JM, Means AR. Chronic elevation of calmodulin in the ventricles of transgenic mice increases the autonomous activity of calmodulin-dependent protein kinase II, which regulates atrial natriuretic factor gene expression. Mol Endocrinol 14: 1125–1136, 2000.[Abstract/Free Full Text]
  7. Day S, Westfall M, Metzger J. Tuning cardiac performance in ischemic heart disease and failure by modulating myofilament function. J Mol Med 85: 911–921, 2007.[CrossRef][Web of Science][Medline]
  8. Dirksen WP, Lacombe VA, Chi M, Kalyanasundaram A, Viatchenko-Karpinski S, Terentyev D, Zhou Z, Vedamoorthyrao S, Li N, Chiamvimonvat N, Carnes CA, Franzini-Armstrong C, Györke S, Periasamy M. A mutation in calsequestrin, CASQ2 D307H, impairs sarcoplasmic reticulum Ca2+ handling and causes complex ventricular arrhythmias in mice. Cardiovasc Res 75: 69–78, 2007.[Abstract/Free Full Text]
  9. Fan GC, Yuan QY, Zhao W, Chu GX, Kranias EG. Junctin is a prominent regulator of contractility in cardiomyocytes. Biochem Biophys Res Commun 352: 617–622, 2007.[CrossRef][Web of Science][Medline]
  10. Gergs U, Berndt T, Buskase J, Jones LR, Kirchhefer U, Müller FU, Schlüter KD, Schmitz W, Neumann J. On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure. Am J Physiol Heart Circ Physiol 293: H728–H734, 2007.[Abstract/Free Full Text]
  11. Gulick J, Subramaniam A, Neumann J, Robbins J. Isolation and characterization of the mouse cardiac myosin heavy-chain genes. J Biol Chem 266: 9180–9185, 1991.[Abstract/Free Full Text]
  12. Gyorke I, Hester N, Jones LR, Gyorke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J 86: 2121–2128, 2004.[Web of Science][Medline]
  13. Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S, Wiesner TF. Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7: D1454–D1463, 2002.[CrossRef][Web of Science][Medline]
  14. Hobai IA, Bates JA, Howarth FC, Levi AJ. Inhibition by external Cd2+ of Na/Ca exchange and L-type Ca channel in rabbit ventricular myocytes. Am J Physiol Heart Circ Physiol 272: H2164–H2172, 1997.[Abstract/Free Full Text]
  15. Jones LR, Field LJ. Residues 2–25 of phospholamban are insufficient to inhibit Ca2+ transport ATPase of cardiac sarcoplasmic reticulum. J Biol Chem 268: 11486–11488, 1993.[Abstract/Free Full Text]
  16. Jones LR, Suzuki YJ, Wang W, Kobayashi YM, Ramesh V, Franzini-Armstrong C, Cleemann L, Morad M. Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin. J Clin Invest 101: 1385–1393, 1998.[Web of Science][Medline]
  17. Jones LR, Zhang L, Sanborn K, Jorgensen AO, Kelley J. Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum. J Biol Chem 270: 30787–30796, 1995.[Abstract/Free Full Text]
  18. Kirchhefer U, Hanske G, Jones LR, Justus I, Kaestner L, Lipp P, Schmitz W, Neumann J. Overexpression of junctin causes adaptive changes in cardiac myocyte Ca2+ signaling. Cell Calcium 39: 131–142, 2006.[CrossRef][Web of Science][Medline]
  19. Kirchhefer U, Jones LR, Begrow F, Boknik P, Hein L, Lohse MJ, Riemann B, Schmitz W, Stypmann J, Neumann J. Transgenic triadin 1 overexpression alters SR Ca2+ handling and leads to a blunted contractile response to beta-adrenergic agonists. Cardiovasc Res 62: 122–134, 2004.[Abstract/Free Full Text]
  20. Kirchhefer U, Neumann J, Baba HA, Begrow F, Kobayashi YM, Reinke U, Schmitz W, Jones LR. Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1. J Biol Chem 276: 4142–4149, 2001.[Abstract/Free Full Text]
  21. Kirchhefer U, Neumann J, Bers DM, Buchwalow IB, Fabritz L, Hanske G, Justus I, Riemann B, Schmitz W, Jones LR. Impaired relaxation in transgenic mice overexpressing junctin. Cardiovasc Res 59: 369–379, 2003.[Abstract/Free Full Text]
  22. Kirchhof P, Klimas J, Fabritz L, Zwiener M, Jones LR, Schäfers M, Hermann S, Boknik P, Schmitz W, Breithardt G, Kirchhefer U, Neumann J. Stress and high heart rate provoke ventricular tachycardia in mice expressing triadin. J Mol Cell Cardiol 42: 962–971, 2007.[CrossRef][Web of Science][Medline]
  23. Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, Knollmann BEC, Horton KD, Weissman NJ, Holinstat I, Zhang W, Roden DM, Jones LR, Franzini-Armstrong C, Pfeifer K. Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia. J Clin Invest 116: 2510–2520, 2006.[CrossRef][Web of Science][Medline]
  24. Kobayashi YM, Alseikhan BA, Jones LR. Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction. J Biol Chem 275: 17639–17646, 2000.[Abstract/Free Full Text]
  25. Kobayashi YM, Jones LR. Identification of triadin 1 as the predominant triadin isoform expressed in mammalian myocardium. J Biol Chem 274: 28660–28668, 1999.[Abstract/Free Full Text]
  26. Kontula K, Laitinen PJ, Lehtonen A, Toivonen L, Viitasalo M, Swan H. Catecholaminergic polymorphic ventricular tachycardia: recent mechanistic insights. Cardiovasc Res 67: 379–387, 2005.[Abstract/Free Full Text]
  27. Kuniyasu A, Kawano S, Hirayama Y, Ji YH, Xu K, Ohkura M, Furukawa KI, Ohizumi Y, Hiraoka M, Nakayama H. A new scorpion toxin (BmK-PL) stimulates Ca2+-release channel activity of the skeletal-muscle ryanodine receptor by an indirect mechanism. Biochem J 339: 343–350, 1999.[CrossRef][Web of Science][Medline]
  28. Li L, Chu GX, Kranias EG, Bers DM. Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J Physiol Heart Circ Physiol 274: H1335–H1347, 1998.[Abstract/Free Full Text]
  29. Mahony L, Jones LR. Developmental changes in cardiac sarcoplasmic reticulum in sheep. J Biol Chem 261: 15257–15265, 1986.[Abstract/Free Full Text]
  30. Maier LS, Bers DM. Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart. Cardiovasc Res 73: 631–640, 2007.[Abstract/Free Full Text]
  31. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM. Transgenic CaMKII delta(C) overexpression uniquely alters cardiac myocyte Ca2+ handling. Reduced SR Ca2+ load and activated SR Ca2+ release. Circ Res 92: 904–911, 2003.[Abstract/Free Full Text]
  32. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101: 365–376, 2000.[CrossRef][Web of Science][Medline]
  33. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215–228, 1998.[CrossRef][Web of Science][Medline]
  34. Ohkura M, Furukawa KI, Fujimori H, Kuruma A, Kawano S, Hiraoka M, Kuniyasu A, Nakayama H, Ohizumi Y. Dual regulation of the skeletal muscle ryanodine receptor by triadin and calsequestrin. Biochemistry 37: 12987–12993, 1998.[CrossRef][Medline]
  35. Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, Overbeek P, Richardson JA, Grant SR, Olson EN. CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J Clin Invest 105: 1395–1406, 2000.[Web of Science][Medline]
  36. Poindexter BJ, Smith JR, Buja LM, Bick RJ. Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal and adult cardiomyocytes. Cell Calcium 30: 373–382, 2001.[CrossRef][Web of Science][Medline]
  37. Porzio MA, Pearson AM. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta 490: 27–34, 1977.[Medline]
  38. Sato Y, Ferguson DG, Sako H, Dorn GW, Kadambi VJ, Yatani A, Hoit BD, Walsh RA, Kranias EG. Cardiac-specific overexpression of mouse cardiac calsequestrin is associated with depressed cardiovascular function and hypertrophy in transgenic mice. J Biol Chem 273: 28470–28477, 1998.[Abstract/Free Full Text]
  39. Shannon TR, Wang F, Bers DM. Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model. Biophys J 89: 4096–4110, 2005.[CrossRef][Web of Science][Medline]
  40. Shin DD, Brandimarte F, De Luca L, Sabbah HN, Fonarow GC, Filippatos G, Komajda M, Gheorghiade M. Review of current and investigational pharmacologic agents for acute heart failure syndromes. Am J Cardiol 99: 4A–23A, 2007.[Web of Science][Medline]
  41. Terentyev D, Cala SE, Houle TD, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Williams SC, Gyorke S. Triadin overexpression stimulates excitation-contraction coupling and increases predisposition to cellular arrhythmia in cardiac myocytes. Circ Res 96: 651–658, 2005.[Abstract/Free Full Text]
  42. Terentyev D, Viatchenko-Karpinski S, Vedamoorthyrao S, Oduru S, Gyorke I, Williams SC, Gyorke S. Protein-protein interactions between triadin and calsequestrin are involved in modulation of sarcoplasmic reticulum calcium release in cardiac myocytes. J Physiol 583: 71–80, 2007.[Abstract/Free Full Text]
  43. Tijskens P, Franzini-Armstrong C, Jones LR. Overlapping roles of junctin and triadin in junctional SR. Biophys J 82: 177A–178A, 2002.
  44. Wehrens XHT, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: A critical mediator of heart failure progression. Proc Natl Acad Sci USA 103: 511–518, 2006.[Abstract/Free Full Text]
  45. Yuan QQ, Fan GC, Dong M, Altschafl B, Diwan A, Ren XP, Hahn HH, Zhao W, Waggoner JR, Jones LR, Jones WK, Bers DM, Dorn GW, Wang HS, Valdivia HH, Chu GX, Kranias EG. Sarcoplasmic reticulum calcium overloading in junctin deficiency enhances cardiac contractility but increases ventricular automaticity. Circulation 115: 300–309, 2007.[Abstract/Free Full Text]
  46. Zhang L, Franzini-Armstrong C, Ramesh V, Jones LR. Structural alterations in cardiac calcium release units resulting from overexpression of junctin. J Mol Cell Cardiol 33: 233–247, 2001.[CrossRef][Web of Science][Medline]
  47. Zhang L, Kelley J, Schmeisser G, Kobayashi YM, Jones LR. Complex formation between junction, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane. J Biol Chem 272: 23389–23397, 1997.[Abstract/Free Full Text]



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