HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/14/2718 01-0107fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ENGELHARDT, S. Articles by HEIN, L. Search for Related Content PubMed PubMed Citation Articles by ENGELHARDT, S. Articles by HEIN, L.

Early impairment of calcium handling and altered expression of junctin in hearts of mice overexpressing the ß₁-adrenergic receptor ¹

Institut für Pharmakologie und Toxikologie, Universität Würzburg, Germany; and
^* Institut für Pharmakologie und Toxikologie, Universität Münster, Germany

²Correspondence: Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany. E-mail: lohse{at}toxi.uni-wuerzburg.de

SPECIFIC AIMS

Chronic stimulation of cardiac ß₁-adrenergic receptors contributes to disease progression and mortality in patients and animal models of heart failure. To search for the mechanism of adrenergic impairment of cardiac function in vivo, we studied a transgenic mouse model with cardiac-specific overexpression of ß₁-adrenergic receptors.

PRINCIPAL FINDINGS

1. Progressive left ventricular fibrosis in ß₁-adrenergic receptor transgenic mice
Overexpression of ß₁-adrenergic receptors in the hearts of transgenic mice led to progressive left ventricular myocyte hypertrophy, and these mice developed overt heart failure at the age of 12 months. By staining cardiac sections with Sirius red, an age-dependent increase in collagen deposition was detected in the left ventricular wall of ß₁-transgenic mice but not in control littermates at 4 and 12 months of age (Fig. 1 ). However, in sections from 2-month-old transgenic and wild-type mice, no fibrotic changes were detected in hearts from either mouse line (Fig. 1a , 1d ).

View larger version (100K): [in this window] [in a new window]   Figure 1. Histological staining of connective tissue in myocardial sections. Transgenic mice with cardiac overexpression of the ß1-adrenergic receptor develop left ventricular fibrosis with increasing age. Histological sections of left ventricular myocardium were stained with Sirius red/picric acid. Connective tissue appears in red and myocytes are stained yellow. a–c) Sections from wild-type mice at 2, 4, and 12 months of age. d–f) Sections from ß1 receptor transgenic littermates at 2, 4, and 12 months of age.

2. Functional impairment precedes the development of structural alterations
To determine the time course of the hemodynamic impairment in ß₁ receptor transgenic mice, which ultimately results in cardiac contractile failure, we assessed in the left ventricle maximal rates of systolic pressure increase (dp/dt_max) and isovolumetric relaxation (dp/dt_min) by cardiac catheterization with a Millar microtip catheter. At 2 months of age, maximal contractility and rate of isovolumetric relaxation were increased in ß₁-transgenic mice compatible with a hyperfunctional ß₁-adrenergic receptor system. With increasing age, a progressive decline of left ventricular contractility and relaxation became apparent. The positive inotropic and positive lusitropic effects of transgenic ß₁ receptor overexpression were abolished in 4-month-old mice, and contractility and relaxation of ß₁-transgenic mice declined to 48% and 54% of the wild-type values, respectively, at 12 months of age. In the presence of the ß receptor antagonist propranolol, dp/dt_max and dp/dt_min were already significantly decreased in 2-month-old transgenic mice, indicating that enhanced adrenergic signaling in ß₁ receptor transgenic mice masks early contractile dysfunction. Similarly, in isolated electrically stimulated cardiac preparations (in the organ bath and thus in the absence of endogenous catecholamine release), slowed relaxation became evident at 2 months of age and more. These data clearly indicate that systolic and diastolic function in mice overexpressing ß₁-adrenergic receptors are already impaired (at 2 months of age) before fibrotic changes can be observed (starting at 4 months of age) in the left ventricle.

3. Impaired calcium handling of individual cardiomyocytes
To test whether overexpression of ß₁ receptors causes early dysfunction of the cardiac myocytes themselves in the absence of structural cardiac changes, intracellular Ca²⁺ transients were recorded in isolated cardiomyocytes in vitro. We focused on myocytes from 2-month-old mice, because in older mice concomitant fibrosis would make the interpretation of functional data more difficult. Ca²⁺ transients of isolated cardiomyocytes paced at 0.5 Hz were monitored with the Ca²⁺ indicator Fluo-3. Kinetic analysis revealed a marked prolongation of the Ca²⁺ transient in ß₁ receptor transgenic myocytes. The time to 50% decline of the Ca²⁺ transient was increased by 81% in transgenic mice compared with their wild-type littermates. Addition of 1 µM isoproterenol to the superfusate caused shortening of the Ca²⁺ transient in both wild-type and transgenic cardiomyocytes. However, isoproterenol could not normalize the impaired relaxation in ß₁-transgenic myocytes. These data clearly indicate that altered intracellular Ca²⁺ transients are responsible for early contractile dysfunction of ß₁ receptor transgenic mice and are observed before the appearance of interstitial fibrosis.

4. Decreased expression of the SR protein junctin
The altered Ca²⁺ transient of individual cardiomyocytes suggests that expression or function of sarcoplasmic reticulum (SR) proteins involved in Ca²⁺ release and storage may be altered in the ß₁-transgenic mice. Thus, we investigated the expression of SR Ca²⁺ ATPase (SERCA), phospholamban, calsequestrin, triadin-1, and junctin on mRNA and proteins levels. mRNA abundance was assessed by RNase protection assays. No significant alterations were detected in SERCA or phospholamban mRNA levels in ß₁-transgenic mice vs. nontransgenic littermates. At 2 months of age, expression of SERCA protein was increased by 29 ± 8% in ß₁-transgenic hearts. Phosphorylation of serine-16 in phospholamban was increased significantly in ß₁-transgenic animals, demonstrating that the transgenic ß₁ receptor was functionally active. By quantitative immunoblotting with monoclonal antibodies, we assessed the expression of the SR proteins calsequestrin, triadin-1, and junctin. Triadin and junctin are proteins that may play an important role in sarcoplasmic Ca²⁺ handling as they mediate binding of calsequestrin to the ryanodine receptor. Expression of calsequestrin and triadin was unaltered between wild-type and ß₁-transgenic mice. However, a significant decrease in the expression of junctin was detected in ß₁ receptor transgenic mice (Fig. 2 ). Analysis of the time course of junctin-expression revealed a significant reduction in junctin expression by 4 wk of age, with a progressive decline with time (Fig. 2B ). This change in the expression pattern of junctin closely paralleled the development of cardiomyocyte hypertrophy (cardiomyocyte cross-sectional area;+10±5.4% at 2 wk, n.s.;+48±9% at 4 wk, P<0.001;+60±9% at 8 wk of age, P<0.001; TG vs. WT).

View larger version (37K): [in this window] [in a new window]   Figure 2. Decreased cardiac expression of the SR protein junctin. A) A representative Western blot of left ventricular junctin from three wild-type and three ß1-transgenic mice at 2 months of age. B) Progressive decrease of the SR protein junctin in ß1 receptor transgenic mice with increasing age. Shown are means (±SE); n = 3–8; P < 0.01 for junctin transgene vs. wild-type.

CONCLUSIONS AND SIGNIFICANCE

Stimulation of cardiac ß-adrenergic receptors is the most potent mechanism to enhance myocardial contractility and relaxation. In chronic heart failure, overstimulation of the ß-adrenergic system occurs via increased levels of endogenously released catecholamines. This contributes to the progression of the disease, and chronic ß receptor blockade decreases the mortality of patients suffering from heart failure. Similarly, enhanced ß-adrenergic stimulation in transgenic mice with cardiac-specific overexpression of this receptor finally results in clinically overt heart failure. The mechanism underlying the detrimental effect of chronic adrenergic stimulation is unclear.

In the present study, we investigated the time course of the consequences of enhanced ß₁-adrenergic receptor signaling on contractile function in vivo and on the cardiomyocyte level. 1) Contractile dysfunction is visible at 2 months of age, and thus precedes the development of structural changes. 2) The impairment of contraction and relaxation parameters was paralleled by abnormal Ca²⁺ transients, which could be linked to decreased expression of junctin in 4-wk-old transgenic mice.

Abnormal cardiomyocyte Ca²⁺ transients are the earliest functional abnormality that might account for the cardiac phenotype of ß₁ receptor transgenic mice. Among the potential biochemical alterations that might cause changes in calcium transients, we observed a modest increase of SERCA2a protein in the hypertrophic phase before the onset of clinical heart failure, which agrees with data from hypertrophic human hearts. Phospholamban, the SR inhibitor of SERCA, did not show expression changes in ß₁ receptor transgenic mice. Recently, additional key regulators of SR calcium handling—triadin and junctin—have been identified. Triadin-1 and junctin are homologous proteins of the sarcoplasmic reticulum, each projecting a highly charged carboxyl terminus into the junctional SR lumen. The physiological functions of these proteins are still largely unknown. Their role in human heart failure has not yet been characterized, but data from in vitro studies suggest a role for junctin in cardiomyocyte calcium handling. In our transgenic model, expression of triadin was unchanged whereas junctin was decreased by 45% at 2 months of age. This is the first time that altered expression of junctin has been implied in the development of hypertrophy and heart failure. The question arises whether the changes observed in junctin expression are secondary to cardiac hypertrophy and failure or whether they contribute to the development of the phenotype in these mice. Experiments on SHR rats demonstrate that cardiac hypertrophy per se does not lead to alterations in the expression of SR proteins. Most notably, triadin and junctin levels were not altered in SHR rats despite cardiac hypertrophy and increased relaxation times. These findings suggest that altered junctin expression is caused by ß₁ receptor signaling rather than by cardiac hypertrophy in this mouse model. Thus, altered junctin expression might be a mechanism specifically involved in adrenergically induced heart failure.

The development of hypertrophy and fibrosis with overexpression of the ß₁-adrenergic receptor is clearly different from what has been observed in mice with overexpression of the ß₂ subtype under the same promoter. At comparable levels of cardiac receptor overexpression, ß₂ receptor transgenic mice do not develop significant fibrosis. ß₂-Transgenic mice display markedly enhanced contractility and heart rate without developing hypertrophy or heart failure. At very high levels of overexpression of the ß₂-adrenergic receptor, cardiac hypertrophy and fibrosis may develop. Taking these results into account and given the comparably modest increase in basal heart rate and contractility in the ß₁ receptor transgenic mice, we suggest that ß₁-adrenergic receptor-specific signaling rather than enhanced myocardial contractility is responsible for the observed cardiomyopathic changes in ß₁ receptor transgenic mice. Several differences between the actions of ß₁ and ß₂ subtypes have been described, including differential effects on cardiomyocyte apoptosis, coupling to G_s and G_i proteins and Ca²⁺ handling. We did not observe a significant change in the expression of phospholamban, which is in contrast to findings in ß₂-transgenic mice. It remains to be determined which of the described signaling differences are responsible for the different phenotype of ß₁ and ß₂ receptor-overexpressing mice.

In conclusion, our experiments show that cardiac overexpression of ß₁-adrenergic receptors results in early contractile dysfunction with impaired calcium transients on the cardiomyocyte level. Decreased expression of a recently described key regulator of SR-calcium (junctin) might be involved in the observed alterations. Future studies will have to address the role of this protein in human heart failure.

View larger version (95K): [in this window] [in a new window]   Figure 3. The detrimental effect of chronic ß-adrenergic activation involves down-regulation of junctin. Acute stimulation of cardiac ß1-adrenergic receptors leads to an increase in the amplitude and shortening of cardiomyocyte calcium transients via phosphorylation of phospholamban and opening of sarcolemmal calcium channels (left part of the diagram, depicted in blue). During chronic stimulation of cardiac ß1-adrenergic receptors alterations in cardiomyocyte calcium transients occur (right part of the diagram, depicted in red). Expression of junctin, a key regulator of cardiomyocyte calcium handling, is reduced by 50% and the calcium transient is greatly prolonged. The impairment of cardiomyocyte calcium handling is believed to represent a critical step in the development of cardiac hypertrophy, cell death, and ultimately heart failure.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0107fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0107fje

This article has been cited by other articles:

				U. Gergs, T. Berndt, J. Buskase, L. R. Jones, U. Kirchhefer, F. U. Muller, K.-D. Schluter, W. Schmitz, and J. Neumann On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H728 - H734. [Abstract] [Full Text] [PDF]

				V. Regitz-Zagrosek, B. Hocher, M. Bettmann, M. Brede, K. Hadamek, C. Gerstner, H. B. Lehmkuhl, R. Hetzer, and L. Hein {alpha}2C-Adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomyopathy Eur. Heart J., February 2, 2006; 27(4): 454 - 459. [Abstract] [Full Text] [PDF]

				G. Feriotto, A. Finotti, P. Volpe, S. Treves, S. Ferrari, C. Angelelli, F. Zorzato, and R. Gambari Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human A{beta}H-J-J Locus, Encoding Aspartyl-{beta}-Hydroxylase, Junctin, and Junctate Mol. Cell. Biol., April 15, 2005; 25(8): 3261 - 3275. [Abstract] [Full Text] [PDF]

				D. Terentyev, S. E. Cala, T. D. Houle, S. Viatchenko-Karpinski, I. Gyorke, R. Terentyeva, S. C. Williams, and S. Gyorke Triadin Overexpression Stimulates Excitation-Contraction Coupling and Increases Predisposition to Cellular Arrhythmia in Cardiac Myocytes Circ. Res., April 1, 2005; 96(6): 651 - 658. [Abstract] [Full Text] [PDF]

				U. Kirchhefer, H. A. Baba, G. Hanske, L. R. Jones, P. Kirchhof, W. Schmitz, and J. Neumann Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2216 - H2225. [Abstract] [Full Text] [PDF]

				L. Barki-Harrington, C. Perrino, and H. A Rockman Network integration of the adrenergic system in cardiac hypertrophy Cardiovasc Res, August 15, 2004; 63(3): 391 - 402. [Abstract] [Full Text] [PDF]

				X.-J. Du Gender modulates cardiac phenotype development in genetically modified mice Cardiovasc Res, August 15, 2004; 63(3): 510 - 519. [Abstract] [Full Text] [PDF]

				S. Engelhardt, L. Hein, V. Dyachenkow, E. G. Kranias, G. Isenberg, and M. J. Lohse Altered Calcium Handling Is Critically Involved in the Cardiotoxic Effects of Chronic {beta}-Adrenergic Stimulation Circulation, March 9, 2004; 109(9): 1154 - 1160. [Abstract] [Full Text] [PDF]

				G. Chu, G. F. Egnaczyk, W. Zhao, S.-H. Jo, G.-C. Fan, J. E. Maggio, R.-P. Xiao, and E. G. Kranias Phosphoproteome Analysis of Cardiomyocytes Subjected to {beta}-Adrenergic Stimulation: Identification and Characterization of a Cardiac Heat Shock Protein p20 Circ. Res., February 6, 2004; 94(2): 184 - 193. [Abstract] [Full Text] [PDF]

				M. J. Lohse, S. Engelhardt, and T. Eschenhagen What Is the Role of {beta}-Adrenergic Signaling in Heart Failure? Circ. Res., November 14, 2003; 93(10): 896 - 906. [Abstract] [Full Text] [PDF]

				C. E Huggins, A. A Domenighetti, T. Pedrazzini, S. Pepe, and L. M. Delbridge Elevated intracardiac angiotensin II leads to cardiac hypertrophy and mechanical dysfunction in normotensive mice Journal of Renin-Angiotensin-Aldosterone System, September 1, 2003; 4(3): 186 - 190. [Abstract] [PDF]

				M. Brede, F. Wiesmann, R. Jahns, K. Hadamek, C. Arnolt, S. Neubauer, M. J. Lohse, and L. Hein Feedback Inhibition of Catecholamine Release by Two Different {alpha}2-Adrenoceptor Subtypes Prevents Progression of Heart Failure Circulation, November 5, 2002; 106(19): 2491 - 2496. [Abstract] [Full Text] [PDF]

				S. Engelhardt, L. Hein, U. Keller, K. Klambt, and M. J. Lohse Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in {beta}1-Adrenergic Receptor Transgenic Mice Circ. Res., April 19, 2002; 90(7): 814 - 819. [Abstract] [Full Text] [PDF]

				S. Engelhardt, L. Hein, U. Keller, K. Klambt, and M. J. Lohse Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in {beta}1-Adrenergic Receptor Transgenic Mice Circ. Res., April 19, 2002; 90(7): 814 - 819. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/14/2718 01-0107fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ENGELHARDT, S. Articles by HEIN, L. Search for Related Content PubMed PubMed Citation Articles by ENGELHARDT, S. Articles by HEIN, L.