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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0486fje. |
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Institute of Pharmacology and Toxicology,
* Department of Nuclear Medicine, the
Institute of Reproductive Medicine, University of Münster; and
Molecular Biology of the Cell I Division, German Cancer Research Center, Heidelberg, Germany
2Correspondence: Institute of Pharmacology and Toxicology, University of Münster, Domagkstr. 12, D-48149 Münster, Germany. E-mail: mullerf{at}uni-muenster.de
SPECIFIC AIMS
Transcriptional regulation mediated by cAMP response element and transcription factors of the CREB/CREM family represent an important mechanism of a cAMP-dependent gene control that may contribute to detrimental effects of chronic ß-adrenergic stimulation in the failing human heart. We addressed the specific cardiac role of transcription factor cAMP response element modulator (CREM) in mutant mice with complete inactivation of the CREM gene.
PRINCIPAL FINDINGS
1. Systolic and diastolic left ventricular dysfunction in CREM mutant mice
Cardiac function of CREM mutant mice (-/-) of 15–25 wk and of age-matched wild-type littermate controls (+/+) was studied in vivo by catheterization of the left ventricle and continuous recording of left ventricular pressure. Under basal conditions, CREM mutant mice showed depressed left ventricular contractile function (n=9 [+/+]-10 [-/-]; mean±SE) as assessed by maximal left ventricular pressure (LVPmax. -/-: 82±2 mm Hg*; +/+: 95±3 mm Hg; *P<0.05 vs.+/+) and the maximal positive first derivative of left ventricular pressure (dP/dtmax. -/-: 5969±349 mm Hg/s*; +/+: 7717±407 mm Hg/s; *P<0.05 vs.+/+) whereas heart rates were not different in both groups (-/-: 442±10 beats/min; +/+: 446±18 beats/min). CREM mutant mice displayed decreased maximal negative first derivative of left ventricular pressure (dP/dtmin. -/-: -6339±258 mm Hg/s*; +/+: -7784±345 mm Hg/s; *P<0.05 vs.+/+) and increased time to 90% relaxation (TTR90. -/-: 38.77±0.60 ms*; +/+: 35.23±0.92 ms; *P<0.05 vs.+/+) indicating impaired relaxation.
2. Reduced potency of ß1-adrenoreceptor agonist dobutamine in CREM mutant mice
Since reduced myocardial contraction and relaxation are associated with a diminished response to ß-adrenoreceptor stimulation in the human failing heart, we studied the effect of ß1-adrenoreceptor agonist dobutamine (2–400 µg/kg body weight) on hemodynamic parameters (Fig. 1
). Dobutamine had the same effect on heart rate in both groups. Although basal values were reduced, maximal effects on LVPmax, dP/dtmax and dP/dtmin were the same in CREM mutant mice compared with wild-type controls. This indicates that efficacy of ß-adrenoreceptor stimulation was not reduced in mice lacking CREM. However, half maximal effective doses (ED50) of dobutamine for effects on LVPmax and dP/dtmax were significantly higher in CREM mutant mice, suggesting decreased potency of dobutamine in regard to contractility-increasing effects (n=5 [-/-]-7 [+/+]; mean with 95% confidence interval. ED50,-LVPmax; -/-: 46 (13–77) ng/g*; +/+: 17 (5–29) ng/g. ED50, dP/dtmax; -/-: 57 (10–102) ng/g*; +/+: 15 (3–25) ng/g. *P < 0.05 vs. +/+).
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3. Decreased protein levels of sarcoplasmic Ca2+-ATPase (SERCA) in CREM mutant mice
The Ca2+-ATPase of the sarcoplasmic reticulum, SERCA, and its regulator protein phospholamban mediate the diastolic reuptake of Ca2+ into the sarcoplasmic reticulum and represent important regulators of cardiac contraction and relaxation. A reduced expression/function of SERCA is probably a major cause for myocardial systolic and diastolic dysfunction of the failing human heart. We therefore immunologically determined left ventricular protein levels of SERCA, phospholamban, calsequestrin, and junctin in the same CREM mutant and wild-type mice used for invasive assessment of left ventricular function (Fig. 2
). Whereas protein levels of phospholamban, calsequestrin, and junctin were not different in both groups, SERCA protein levels were 
42% lower in CREM mutant mice compared with wild-type controls (P<0.05). There was a moderate but significant down-regulation of ß1-adrenergic receptors in cardiac ventricular membranes of CREM mutant mice as measured by radioligand binding assays using the nonselective ß-adrenergic receptor ligand [125I]iodocyanopindolol (ICYP) in the absence and presence of a ß1-adrenergic receptor saturating concentration of the selective ß1-adrenergic receptor antagonist CGP20712A. The density of ß1-adrenergic receptors was diminished by 6 fmol/mg protein or 23% in CREM mutant mice compared with wild-type controls (n=5; P<0.05) that was also reflected by a significant decrease in total ICYP binding in CREM mutant mice.
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4. Absence of cardiac hypertrophy and unchanged mortality of CREM mutant mice
Besides changes in myocardial function and gene expression, human heart failure is characterized by ventricular hypertrophy at the organ and myocyte level and by poor prognosis of heart failure patients. Macroscopic and microscopic histopathological analysis did not reveal any striking differences in cardiac morphology of CREM mutant hearts; atrial and ventricular weights, absolute and indexed to the body weight, were not different in CREM mutant and wild-type control hearts. Mean survival times of 95 CREM mutant and 90 wild-type control mice were 504 days (95% confidence interval: 447–561 days) and 487 days (95% confidence interval: 400–574 days), respectively. This indicates that left ventricular dysfunction of CREM mutant mice was not associated with cardiac hypertrophy or increased mortality.
CONCLUSIONS AND SIGNIFICANCE
Mutant mice lacking transcription factor CREM resemble distinct characteristic properties of the failing human heart, i.e., reduced systolic and diastolic ventricular function, impaired response to ß-adrenergic stimulation, diminished expression of SERCA, and down-regulation of ß1-adrenergic receptors (Fig. 3
), whereas other features of heart failure (i.e., cardiac hypertrophy and increased mortality) were absent. Thus, results on CREM mutant mice suggest a specific cardiac role of CREM, providing evidence that CREM is essential for regular cardiac function maintaining expression of SERCA and of the ß1-adrenergic receptor.
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Congestive heart failure represents a leading cause of cardiovascular mortality in Western countries and is defined by the heart’s inability to adequately cover the organ’s blood demand. The failing heart displays characteristic functional defects, including impaired myocardial contraction and relaxation and diminished response to cAMP-elevating drugs, e.g., ß-adrenoreceptor agonists. Cardiac functional changes are explained by a changed program of myocardial gene expression implicating various cardiac regulatory proteins; numerous clinical and experimental data support the notion that chronic ß-adrenergic stimulation of the cAMP-dependent signaling pathway plays a central role in development and progression of heart failure and for altered gene regulation in the failing heart. However, underlying mechanisms controlling expression of functionally relevant cardiac genes on stimulation of the cAMP-dependent signaling pathway and PKA activation are largely unknown. The transcriptional regulation mediated by cAMP response element (CRE) and transcription factors of the CREB/CREM family represents an important mechanism of a cAMP-dependent expressional control; it has been hypothesized that this mechanism contributes to the altered cardiac gene regulation in heart failure and to detrimental effects of chronic ß-adrenergic stimulation. The cardiac phenotype of CREM mutant mice is remarkable in several aspects and provides the first evidence that CREM and the transcriptional regulation mediated by the CRE are of relevance in the heart. First, inactivation of the CREM gene led to impairment of cardiac systolic and diastolic function under basal conditions whereas ventricular function was normal on maximal ß-adrenergic stimulation. A similar observation was made in heterozygous SERCA2 gene knockout mice with comparable reduction of SERCA protein levels (60% of wild-type control). LVPmax, dP/dtmax and dP/dtmin were also decreased under basal conditions but LVPmax was not different from controls after maximal stimulation with dobutamine. This suggests that maximal ß-adrenoreceptor-stimulated contractile performance can be normal even in presence of reduced SERCA protein levels and that down-regulation of SERCA and subsequent reduction of the SERCA:phospholamban ratio are sufficient to explain impaired basal systolic and diastolic left ventricular function in CREM mutant mice. Second, whereas CREM mutant mice displayed several features of failing human hearts—reduced ventricular function, down-regulation of ß1-adrenoreceptor and of SERCA—cardiac hypertrophy and increase in mortality were absent in CREM mutant mice. This suggests a specific cardiac role of CREM as an important link between chronic ß-adrenergic stimulation and several distinct but not all detrimental effects of this stimulation on the heart. This phenotype is an argument against the possible concern that ablation of CREM might nonspecifically lead to cardiac hypertrophy and failure as observed as a common final stage of various human cardiac diseases.
Two important questions arise from the results on CREM mutant mice: 1) Which other cardiac genes beside ß1-adrenoreceptor and SERCA are regulated by CREM. 2) How do CREM and CRE-mediated transcriptional regulation contribute to detrimental cardiac effects of chronic ß-adrenergic stimulation in the failing human heart? Recent experiments on transiently transfected chick cardiac myocytes revealed that CRE-mediated transcriptional activation is desensitized on prolonged ß-adrenergic stimulation in the heart. This led to the hypothesis that transcriptional regulation mediated by the CRE is altered in the failing heart. Thus, detailed investigation of CREB/CREM-transcription factors and of CREB/CREM-regulated target genes, e.g., by the use of cDNA array technologies, during development and progression of human heart failure will be an important subject for further investigation. This knowledge might be of great interest for understanding the pathophysiology of heart failure with the potential to develop new therapeutic approaches aiming on unwanted expressional changes on chronic ß-adrenergic stimulation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0486fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0486fje 
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) mice. The highest dose of dobutamine (400 µg/kg) was only tolerated by mutant mice whereas control mice responded with arrhythmia. Note comparable effects of dobutamine on heart rate (upper left panel) and equal maximal effects of dobutamine on maximal LV pressure (lower left panel), dP/dtmax (upper right panel) and dP/dtmin (lower right panel) in both groups. *Significant differences (P<0.05) between both groups; bpm, beats/min.
) and 10 wild-type control hearts (
), data presented in relative PhosphorImager intensity units (mean of wild-type signals set to 100%). Note down-regulation of SERCA in mutant mice to 58% of wild-type controls. *Significant differences (P<0.05) between both groups.