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ABSTRACT: Calcium is an important mediator in cardiac excitation and disorders in cardiac Ca(2+) homeostasis have great influence on the cardiac action potential. Therefore dysfunction in regulatory proteins that are involved in Ca(2+) handling can lead to the occurrence of severe arrhythmia. Although mutations in Ca(2+) regulating proteins are quite rare, they can offer general insights into arrhythmogenesis. Here, we briefly review some important aspects of arrhythmia-associated mutations in Ca(2+) regulating proteins with special emphasis to its associated pathophysiology.
Wiener Medizinische Wochenschrift 06/2012; 162(13-14):292-6.
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ABSTRACT: Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.
AJP Heart and Circulatory Physiology 03/2012; 302(10):H2008-17. · 3.71 Impact Factor
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Paul J M Wijnker,
Peter Boknik,
Ulrich Gergs,
Frank U Müller,
Joachim Neumann,
Cris dos Remedios,
Wilhelm Schmitz,
Jürgen R Sindermann,
Ger J M Stienen,
Jolanda van der Velden, Uwe Kirchhefer
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ABSTRACT: Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50=0.05±0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50=0.10±0.03) and dilated (DCM, ΔpCa50=0.06±0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.
Journal of Muscle Research and Cell Motility 09/2011; 32(3):221-33. · 1.98 Impact Factor
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Birgit Stallmeyer,
Sven Zumhagen,
Isabelle Denjoy,
Guillaume Duthoit,
Jean-Louis Hébert,
Xavier Ferrer,
Svetlana Maugenre,
Wilhelm Schmitz, Uwe Kirchhefer,
Ellen Schulze-Bahr,
Pascale Guicheney,
Eric Schulze-Bahr
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ABSTRACT: Very recently, mutations in the TRPM4 gene have been identified in four pedigrees as the cause of an autosomal dominant form of cardiac conduction disease. To determine the role of TRPM4 gene variations, the relative frequency of TRPM4 mutations and associated phenotypes was assessed in a cohort of 160 unrelated patients with various types of inherited cardiac arrhythmic syndromes. In eight probands with atrioventricular block or right bundle branch block--five familial cases and three sporadic cases--a total of six novel and two published TRPM4 mutations were identified. In patients with sinus node dysfunction, Brugada syndrome, or long-QT syndrome, no mutations were found. The novel mutations include six amino acid substitutions and appeared randomly distributed through predicted TRPM4 protein. In addition, eight polymorphic sites including two in-frame deletions were found. Mutations separated from polymorphisms by absence in control individuals and familial cosegregation in some families. In summary, TRPM4 gene mutations appear to play a major role in cardiac conduction disease but not for other related syndromes so far. The phenotypes are variable and clearly suggestive of additional factors modulating the disease phenotype in some patients.
Human Mutation 09/2011; 33(1):109-17. · 5.69 Impact Factor
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ABSTRACT: In the neonatal mammalian heart, the role of ryanodine receptor (=Ca(2+) release channel)-mediated sarcoplasmic reticulum (SR) Ca(2+) release for excitation-contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca(2+) transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca(2+) content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca(2+) storage but unchanged Ca(2+) signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation-contraction coupling in the developing mammalian heart.
Journal of Molecular and Cellular Cardiology 08/2011; 51(5):682-8. · 5.17 Impact Factor
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Andreas Blana,
Sven Kaese,
Lisa Fortmüller,
Sandra Laakmann,
Dierk Damke,
Kelly van Bragt,
Jens Eckstein,
Ilaria Piccini, Uwe Kirchhefer,
Stanley Nattel,
Günter Breithardt,
Peter Carmeliet,
Edward Carmeliet,
Ulrich Schotten,
Sander Verheule,
Paulus Kirchhof,
Larissa Fabritz
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ABSTRACT: Patients with long QT syndrome (LQTS) are at increased risk not only for ventricular arrhythmias but also for atrial pathology including atrial fibrillation (AF). Some patients with "lone" AF carry Na(+)-channel mutations.
The purpose of this study was to determine the mechanisms underlying atrial pathology in LQTS.
In mice with a heterozygous knock-in long QT syndrome type 3 (LQT3) mutant of the cardiac Na(+) channel (ΔKPQ-SCN5A) and wild-type (WT) littermates, atrial size, function, and electrophysiologic parameters were measured in intact Langendorff-perfused hearts, and histologic analysis was performed.
Atrial action potential duration, effective refractory period, cycle length, and PQ interval were prolonged in ΔKPQ-SCN5A hearts (all P < .05). Flecainide (1 μM) reversed atrial action potential duration prolongation and induced postrepolarization refractoriness (P < .05). Arrhythmias were infrequent during regular rapid atrial rate in both WT and ΔKPQ-SCN5A but were inducible in 15 (38%) of 40 ΔKPQ-SCN5A and 8 (29%) of 28 WT mice upon extrastimulation. Pacing protocols generating rapid alterations in rate provoked atrial extrasystoles and arrhythmias in 6 (66%) of 9 ΔKPQ-SCN5A but in 0 (0%) of 6 WT mice (P < .05). Atrial diameter was increased by nearly 10% in ΔKPQ-SCN5A mice > 5 months old without increase in fibrotic tissue.
Murine hearts bearing an LQT3 mutation show abnormalities in atrial electrophysiology and subtle changes in atrial dimension, including an atrial arrhythmogenic phenotype on provocation. These results support clinical data suggesting that LQTS mutations can cause atrial pathology and arrhythmogenesis and indicate that murine sodium channel LQTS models may be useful for exploring underlying mechanisms.
Heart rhythm: the official journal of the Heart Rhythm Society 12/2010; 7(12):1862-9. · 4.56 Impact Factor
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Larissa Fabritz,
Dierk Damke,
Markus Emmerich,
Susann G Kaufmann,
Kathrin Theis,
Andreas Blana,
Lisa Fortmüller,
Sandra Laakmann,
Sven Hermann,
Elena Aleynichenko, [......],
Daniela Volkery,
Burkhard Riemann, Uwe Kirchhefer,
Michael R Franz,
Günter Breithardt,
Edward Carmeliet,
Michael Schäfers,
Sebastian K G Maier,
Peter Carmeliet,
Paulus Kirchhof
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ABSTRACT: Clinical observations in patients with long QT syndrome carrying sodium channel mutations (LQT3) suggest that bradycardia caused by parasympathetic stimulation may provoke torsades de pointes (TdP). Beta-adrenoceptor blockers appear less effective in LQT3 than in other forms of the disease.
We studied effects of autonomic modulation on arrhythmias in vivo and in vitro and quantified sympathetic innervation by autoradiography in heterozygous mice with a knock-in deletion (DeltaKPQ) in the Scn5a gene coding for the cardiac sodium channel and increased late sodium current (LQT3 mice). Cholinergic stimulation by carbachol provoked bigemini and TdP in freely roaming LQT3 mice. No arrhythmias were provoked by physical stress, mental stress, isoproterenol, or atropine. In isolated, beating hearts, carbachol did not prolong action potentials per se, but caused bradycardia and rate-dependent action potential prolongation. The muscarinic inhibitor AFDX116 prevented effects of carbachol on heart rate and arrhythmias. beta-Adrenoceptor stimulation suppressed arrhythmias, shortened rate-corrected action potential duration, increased rate, and minimized difference in late sodium current between genotypes. Beta-adrenoceptor density was reduced in LQT3 hearts. Acute beta-adrenoceptor blockade by esmolol, propranolol or chronic propranolol in vivo did not suppress arrhythmias. Chronic flecainide pre-treatment prevented arrhythmias (all P < 0.05).
Cholinergic stimulation provokes arrhythmias in this model of LQT3 by triggering bradycardia. beta-Adrenoceptor density is reduced, and beta-adrenoceptor blockade does not prevent arrhythmias. Sodium channel blockade and beta-adrenoceptor stimulation suppress arrhythmias by shortening repolarization and minimizing difference in late sodium current.
Cardiovascular research 07/2010; 87(1):60-72. · 5.80 Impact Factor
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Uwe Kirchhefer,
Diana Wehrmeister,
Alex V Postma,
Gottfried Pohlentz,
Michael Mormann,
Dana Kucerova,
Frank U Müller,
Wilhelm Schmitz,
Eric Schulze-Bahr,
Arthur A Wilde,
Joachim Neumann
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ABSTRACT: Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQ(K206N) were investigated in comparison to the wild-type form of CASQ2 (CSQ(WT)) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca(2+) binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQ(K206N)-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca(2+) load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [(3)H]ryanodine binding was increased in CSQ(K206N)-expressing myocytes, suggesting a higher opening state of the SR Ca(2+) release channel. Myocytes with expression of CSQ(K206N) showed a higher rate of spontaneous SR Ca(2+) releases under basal conditions and after beta-adrenergic stimulation. We conclude that CSQ(K206N) caused a reduced Ca(2+) binding leading to an abnormal regulation of intracellular Ca(2+) in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca(2+) transients in CSQ(K206N)-expressing myocytes.
Journal of Molecular and Cellular Cardiology 03/2010; 49(1):95-105. · 5.17 Impact Factor
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ABSTRACT: Mono-unsaturated fatty acids (MUFAs) like oleic acid have been shown to cause apoptosis of cultured endothelial cells by activating protein phosphatase type 2C alpha and beta (PP2C). The question arises whether damage of endothelial or other cells could be observed in intact animals fed with a trioleate-enriched diet.
Dunkin-Hartley guinea pigs were fed with a trioleate-enriched diet for 5 months. Advanced atherosclerotic changes of the aorta and the coronary arteries could not be seen but the arteries appeared in a pre-atherosclerotic stage of vascular remodelling. However, the weight and size of the hearts were lower than in controls and the number of apoptotic myocytes increased in the hearts of trioleate-fed animals. To confirm the idea that oleic acid may have caused this apoptosis by activation of PP2C, cultured cardiomyocytes from guinea pigs and mice were treated with various lipids. It was demonstrable that oleic acid dose-dependently caused apoptosis of cardiomyocytes from both species, yet, similar to previous experiments with cultured neurons and endothelial cells, stearic acid, elaidic acid and oleic acid methylester did not. The apoptotic effect caused by oleic acid was diminished when PP2C alpha and beta were downregulated by siRNA showing that PP2C was causally involved in apoptosis caused by oleic acid.
The glycerol trioleate diet given to guinea pigs for 5 months did not cause marked atherosclerosis but clearly damaged the hearts by activating PP2C alpha and beta. The diet used with 24% (wt/wt) glycerol trioleate is not comparable to human diets. The detrimental role of MUFAs for guinea pig heart tissue in vivo is shown for the first time. Whether it is true for humans remains to be shown.
PLoS ONE 01/2010; 5(3):e9561. · 4.09 Impact Factor
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Alexander P Schwoerer,
Christiane Neuber,
Ariane Schmechel,
Ivan Melnychenko,
Giulia Mearini,
Peter Boknik, Uwe Kirchhefer,
Wilhelm Schmitz,
Heimo Ehmke,
Thomas Eschenhagen,
Ali El-Armouche
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ABSTRACT: Mechanical unloading of failing hearts by left ventricular (LV) assist devices is regularly used as a bridge to transplantation and may lead to symptomatic improvement. The latter has been associated with altered phosphorylation of cardiac regulatory proteins, but the underlying mechanisms remained unknown. Here, we tested whether cardiac unloading alters protein phosphorylation by affecting the corresponding kinase-phosphatase balance. Cardiac unloading and reduction in LV mass were induced by heterotopic heart transplantation in rats for two weeks (n=8). Native in situ hearts from the recipient animals were used as controls (n=8). The steady-state protein kinase A (PKA) and/or Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) phosphorylation levels of phospholamban (PLB, Ser(16) and Thr(17)) and troponin I (TnI, Ser(23/24)) were decreased by 40-60% in unloaded hearts. Consistently, in these hearts PKA activity was decreased by approximately 80% and the activity of protein phosphatase 1 and 2A was increased by 50% and 90%, respectively. In contrast, CaMKII activity was approximately 60% higher, which may serve as a partial compensation. These data indicate that unloading shifts the kinase-phosphatase balance towards net dephosphorylation of PLB and TnI. This shift may also contribute to the reduction in phosphorylation levels of cardiac phosphoproteins observed in diseased human hearts after LVAD.
Journal of Molecular and Cellular Cardiology 10/2008; 45(6):846-52. · 5.17 Impact Factor
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Nicole Brüchert,
Nirmala Mavila,
Peter Boknik,
Hideo A Baba,
Larissa Fabritz,
Ulrich Gergs, Uwe Kirchhefer,
Paulus Kirchhof,
Marek Matus,
Wilhelm Schmitz,
Anna A DePaoli-Roach,
Joachim Neumann
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ABSTRACT: Cardiac-specific overexpression of the catalytic subunit of protein phosphatase type 1 (PP1) in mice results in hypertrophy, depressed contractility, propensity to heart failure, and premature death. To further address the role of PP1 in heart function, PP1 mice were crossed with mice that overexpress a functional COOH-terminally truncated form of PP1 inhibitor-2 (I-2(140)). Protein phosphatase activity was increased in PP1 mice but was normalized in double transgenic (DT) mice. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were reduced in catheterized PP1 mice but normalized in DT mice. Similar contractile abnormalities were observed in isolated, perfused work-performing hearts and in whole animals by means of echocardiography. The increased absolute and relative heart weights observed in PP1 mice were normalized in DT mice. Histological analyses indicated that PP1 mice had significant cardiac fibrosis, which was absent in DT mice. Furthermore, PP1 mice exhibited an age-dependent increase in mortality, which was abrogated in DT mice. These results indicate that I-2 overexpression prevents the detrimental effects of PP1 overexpression in the heart and further underscore the fundamental role of PP1 in cardiac function. Therefore, PP1 inhibitors such as I-2 could offer new therapeutic options to ameliorate the deleterious effects of heart failure.
AJP Heart and Circulatory Physiology 09/2008; 295(4):H1539-46. · 3.71 Impact Factor
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Stephanie Grote-Wessels,
Hideo A Baba,
Peter Boknik,
Ali El-Armouche,
Larissa Fabritz,
Hans-Jörg Gillmann,
Dana Kucerova,
Marek Matus,
Frank U Müller,
Joachim Neumann,
Martina Schmitz,
Frank Stümpel,
Gregor Theilmeier,
Jeremias Wohlschlaeger,
Wilhelm Schmitz, Uwe Kirchhefer
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ABSTRACT: The progression of human heart failure is associated with increased protein phosphatase 1 (PP1) activity, which leads to a higher dephosphorylation of cardiac regulatory proteins such as phospholamban. In this study, we tested the hypothesis whether the inhibitor-2 (I-2) of PP1 can mediate cardiac protection by inhibition of PP1 activity.
We induced pressure overload by transverse aortic constriction (TAC) for 28 days in transgenic (TG) mice with heart-directed overexpression of a constitutively active form of I-2 (TG(TAC)) and wild-type littermates (WT(TAC)). Both groups were compared with sham-operated mice. TAC treatment resulted in comparable ventricular hypertrophy in both groups. However, TG(TAC) exhibited a higher atrial mass and an enhanced ventricular mRNA expression of beta-myosin heavy chain. The increased afterload was associated with the development of focal fibrosis in TG. Consistent with signs of overt heart failure, fractional shortening and diastolic function were impaired in TG(TAC) as revealed by Doppler echocardiography. The contractility was reduced in catheterized banded TG mice, which is in line with a depressed shortening of isolated myocytes. This is due to profoundly abnormal cytosolic Ca(2+) transients and a reduced stimulation of phosphorylation of phospholamban (PLB)(Ser16) after TAC in TG mice. Moreover, administration of isoproterenol was followed by a blunted contractile response in isolated myocytes of TG(TAC) mice.
These results suggest that cardiac-specific overexpression of a constitutively active form of I-2 is deleterious for cardiac function under conditions of pressure overload. Thus, the long-term inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure.
Cardiovascular Research 06/2008; 79(3):464-71. · 6.06 Impact Factor
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ABSTRACT: 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.
AJP Heart and Circulatory Physiology 12/2007; 293(5):H3165-74. · 3.71 Impact Factor
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ABSTRACT: Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca(2+) release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca(2+) sensitivity of the Ca(2+) release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca(2+) transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca(2+) homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca(2+) transients showed a decreased maximum Ca(2+) amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca(2+) homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.
AJP Heart and Circulatory Physiology 07/2007; 293(1):H728-34. · 3.71 Impact Factor
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Paulus Kirchhof,
Jan Klimas,
Larissa Fabritz,
Melanie Zwiener,
Larry R Jones,
Michael Schäfers,
Sven Hermann,
Peter Boknik,
Wilhelm Schmitz,
Günter Breithardt, Uwe Kirchhefer,
Joachim Neumann
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ABSTRACT: Reduced function of the cardiac ryanodine receptor or calsequestrin causes catecholaminergic ventricular tachycardia (VT). These proteins regulate sarcoplasmic Ca(2+) release in close conjunction with two accessory proteins, triadin and junctin. Based on data from cardiomyocytes, we hypothesized that enhanced triadin expression could cause VT. We assessed arrhythmias and electrophysiological changes in vivo and in the beating heart in mice expressing junctin, triadin, or both proteins (TRDxJCN), and measured calcium transients in isolated ventricular cardiomyocytes. TRDxJCN mice were studied to compensate the down-regulation of junctin expression in triadin-expressing mice. Exercise or stress provoked repetitive VT in freely roaming TRDxJCN mice whenever heart rate increased above approximately 600 bpm (p<0.05 vs. the three other genotypes). TRDxJCN mice expressed total triadin 2.9-fold (p<0.05) and total junctin not different to wildtype (p=ns). Left ventricular systolic function was not different between lineages. beta-adrenoreceptor stimulation (orciprenaline 1.7 microM) provoked early-coupled ventricular ectopy and repetitive VT in isolated, Langendorff-perfused TRDxJCN hearts (p<0.05). Under conditions associated with VT (high pacing rate, catecholamine stimulation), action potential duration was shorter in TRDxJCN with VT than in the other genotypes and shorter than in TRDxJCN hearts without VT (p<0.05). Ca(2+) transient duration was prolonged in Indo1-loaded TRDxJCN cardiomyocytes under VT-provoking conditions. Action potential prolongation by mexiletine (2 microM or 4 microM) or clarithromycine (150 microM) suppressed VT. Expression of triadin provokes stress- and tachycardia-related ventricular arrhythmias in mice. An imbalance between prolonged intracellular calcium release and shortening of the ventricular action potential may contribute to genesis of arrhythmias in this model.
Journal of Molecular and Cellular Cardiology 05/2007; 42(5):962-71. · 5.17 Impact Factor
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ABSTRACT: In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).
Cell Calcium 03/2006; 39(2):131-42. · 3.77 Impact Factor
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Uwe Kirchhefer,
Hideo A Baba,
Peter Bokník,
Kristine M Breeden,
Nirmala Mavila,
Nicole Brüchert,
Isabel Justus,
Marek Matus,
Wilhelm Schmitz,
Anna A Depaoli-Roach,
Joachim Neumann
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ABSTRACT: Protein phosphatase 1 (PP1) has been implicated in the control of cardiac function. Cardiac specific overexpression of the catalytic subunit, PP1c, results in hypertrophy and depressed contractility.
To further address the role of PP1, transgenic mice (TG) were generated that overexpress in heart a functional COOH-terminally truncated form (amino acids 1-140) of the PP1 inhibitor-2 (I-2(140)).
The TG hearts show increased levels of I-2(140) mRNA as well as protein and activity. No increase in absolute or relative heart weight was observed, nor any changes in gross pathology or increase in morbidity or mortality in the TG mice. Immunohistochemical and biochemical analyses revealed that expression of the I-2(140) protein is confined to cardiomyocytes where it is mainly localized in the cytosol. The total protein phosphatase (PP) activity was reduced by 80% in TG hearts as compared to wild-type littermates (WT). The PP1c mRNA level was the same in TG and WT, while the protein level was increased by approximately 7-fold in TG animals. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were increased by 32% and 40%, respectively, in the intact catheterized TG mice compared to WT. However, the maximal contractile response to beta-adrenergic agonists was comparable in hearts from TG and WT mice. In isolated cardiomyocytes of TG mice, Ca2+transient amplitude was increased by 50% under basal conditions and by 60% upon rapid caffeine application. The phospholamban (PLB) protein level was unchanged whereas the basal phosphorylation of PLB at Ser(16) was significantly increased in TG hearts.
These results indicate that I-2(140) overexpression results in decreased PP1 activity and enhanced contractility in the heart, underscoring the fundamental role of PP1 in cardiac function.
Cardiovascular Research 11/2005; 68(1):98-108. · 6.06 Impact Factor
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Frank U Müller,
Geertje Lewin,
Hideo A Baba,
Peter Bokník,
Larissa Fabritz, Uwe Kirchhefer,
Paulus Kirchhof,
Karin Loser,
Marek Matus,
Joachim Neumann,
Burkhard Riemann,
Wilhelm Schmitz
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ABSTRACT: The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic beta-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-IbDeltaC-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca(2+)-ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of beta(1)-adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-IbDeltaC-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.
Journal of Biological Chemistry 03/2005; 280(8):6906-14. · 4.77 Impact Factor
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ABSTRACT: Junctin is a transmembrane protein of the cardiac junctional sarcoplasmic reticulum (SR) that binds to the ryanodine receptor, calsequestrin, and triadin 1. This quaternary protein complex is thought to facilitate SR Ca2+ release. To improve our understanding of the contribution of junctin to the regulation of SR function, we examined the age-dependent effects of junctin overexpression in the atrium of 3-, 6-, and 18-wk-old transgenic mice. The ratio of atrial weight and body weight was unchanged between junctin-overexpressing (JCN) and wild-type (WT) mice at all ages investigated (n=6-8). The protein expression of triadin 1 was decreased starting in 3-wk-old JCN atria (by 69%), whereas the expression of the ryanodine receptor was diminished in 6- (by 48%) and 18-wk-old (by 57%) JCN atria compared with age-matched WT atria. Force of contraction was decreased by 35% in 18-wk-old JCN compared with age-matched WT left atrial muscle strips, which was accompanied by a prolonged time of relaxation (48.1 +/- 0.9 vs. 44.2 +/- 0.8 ms, respectively, n=6-8, P <0.05). The spontaneous beating rate of isolated right atria was higher in 18-wk-old JCN mice compared with age-matched WT mice (389 +/- 10 vs. 357 +/- 6 beats/min, respectively, n=6-8, P <0.05). Heart rate was lower by 9% in telemetric ECG recordings in 18-wk-old JCN mice during stress tests. Three-week-old JCN atria exhibited a higher potentiation of force of contraction at rest pauses of 30 s (by 13%) and of 300 s (by 35%), suggesting increased SR Ca2+ content. This was consistent with the higher force of contraction in 3-wk-old JCN atria (by 29%) compared with age-matched WT atria (by 10%) under the administration of caffeine. We conclude that in 3-wk-old atria, junctin overexpression was associated with a reduced expression of triadin 1 resulting in a higher SR Ca2+ load without changes in contractility or heart rate. In 6-wk-old JCN atria, the compensatory downregulation of the ryanodine receptor may offset the effects of junctin overexpression. Finally, the progressive decrease in ryanodine receptor density may contribute to the decreased atrial contractility and lower heart rate during stress in 18-wk-old JCN mice.
AJP Heart and Circulatory Physiology 11/2004; 287(5):H2216-25. · 3.71 Impact Factor
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ABSTRACT: 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.
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.
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 beta-adrenergic receptor (beta-AR) stimulation in TG hearts was associated with an increased protein expression of beta-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 beta-AR stimulation.
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 beta-adrenergic agonists in TG mice.
Cardiovascular Research 05/2004; 62(1):122-34. · 6.06 Impact Factor
