In den letzten Jahren wurden in mehreren Labors verschiedene Mausstämme mit Nullmutationen des fü... more In den letzten Jahren wurden in mehreren Labors verschiedene Mausstämme mit Nullmutationen des für einen myogenen Transkriptionsfaktor kodierenden Myf-Gens (Myogenic factor 5) generiert. Die Abwesenheit von Myf-5-Protein im sich entwickelnden Mausembryo führt zu einer Verzögerung der Myotombildung von 1,75 Tagen, bis die myogene Aktivität der einsetzenden MyoD-Expression benötigte Muskelvorläuferzellen determiniert. Die Muskulatur homozygoter Myf5-Mutanten entwickelt sich überraschenderweise normal. Die Abwesenheit des myogenen Myf5-Proteins führt unerwartet zum Fehlen distaler Rippen, in dessen Folge die Mäuse perinatal aufgrund mangelnder Respiration sterben. Um die Funktion von Myf-5 in adulten Mäusen zu untersuchen, wurde eine Mauslinie generiert, die mit Hilfe der Cre/loxP-Technologie ein konditional inaktivierbares Allel trägt (Myf-5loxP). Die kontrollierte Expression der Cre-Rekombinase mit dem Ziel der Inaktivierung des Myf5-Gens (Myf-5ΔloxP) wurde in 4 verschiedenen Ansätzen verfolgt. (1) Zeitliche Kontrolle der Exzision durch den induzierbaren Mx-Promotor. (2) Ausschließlich auf Skelettmuskulatur beschränkte Cre-Rekombinase-Expression durch den muskelspezifischen Muskel-Creatin-Kinase-Promotor. (3) Durch Adenovirus vermittelte Cre-Rekombinase-Expression, um unabhängig von verfügbaren Promotoren und ihren Expressionsprofilen zu sein. (4) Konstitutive Cre-Rekombinase Expression in einer Cre-deleter Mauslinie zwecks frühestmöglicher Myf5-Inaktivierung. Die Myf5-Exzision im Zygotenstadium sollte eine Phänokopie der alten Myf5-Allele darstellen. Homozygote Myf-5ΔloxP/ΔloxP Mutanten zeigen unerwartet keine Fehlentwicklung der Rippen. Trotz des völligen Fehlens der frühen Myotomentwicklung sind die Mutanten lebensfähig und fruchtbar, sie weisen keine erhöhte Sterblichkeit auf. Daraus kann geschlossen werden, dass der zuvor für Myf5 mutante Tiere beschriebene Rippenphänotyp durch Missregulation eines noch unbekannten Gens verursacht wird. Der Schwanz von Myf-5ΔloxP/ΔloxPMäusen weist mit einsetzender Adoleszenz [...]
Braun et al., 1992). Since the formation of the myotome Myf-5 Revisited: Loss of Early is depende... more Braun et al., 1992). Since the formation of the myotome Myf-5 Revisited: Loss of Early is dependent on Myf-5 and a number of potential induc-Myotome Formation Does Not Lead tive signals expressed in myotome like FGFs and PDGF are absent in Myf-5 mutant mice (Grass et al., 1996), to a Rib Phenotype in Homozygous this hypothesis appeared plausible and logical.
Ziel des Sonderforschungsbereichs (SFB) 1002 „Modulatorische Einheiten bei Herzinsuffizienz“ ist ... more Ziel des Sonderforschungsbereichs (SFB) 1002 „Modulatorische Einheiten bei Herzinsuffizienz“ ist es, neue Diagnose- und Therapieverfahren der Herzinsuffizienz zu identifizieren und bis zur Anwendung am Patienten zu entwickeln. Mit der Förderung des Deutschen Zentrums für Herz-Kreislauf-Forschung (DZHK) sollen schließlich „First-in-Man-Studien“ durchgeführt werden. Beispiele für den erfolgreichen Translationsprozess im SFB 1002 sind die klinischen Studien BioVAT-DZHK 20 „Safety and efficacy of induced pluripotent stem cell-derived engineered human myocardium as biological ventricular assist device in terminal heart failure“ (Herzpflaster) und REDUCE-MFA-DZHK 25 „Reduce myocardial fibrosis in aortic stenosis“ (Fibrosehemmung). Diese beiden Multicenterstudien basieren auf Grundlagenarbeiten des SFBs und befinden sich aktuell mit DZHK-Förderung in der Patientenrekrutierungsphase. Zur Target-Identifizierung im SFB werden sowohl die Herzinsuffizienzentwicklung als „transition to failure“ als auch der reversible Prozess „recovery from failure“ klinisch und experimentell analysiert. Im Vordergrund stehen hierbei funktionelle Mikrodomänen wie subzelluläre Kalziumfreisetzungseinheiten, Myozyten-Fibroblasten-Interaktionen und die Genregulation. Im Rahmen des SFBs wird darüber hinaus die TAVI-Bio‑/Datenbank aufgebaut. Hierbei werden Patienten, die sich einer „transcatheter aortic valve implantation“ (TAVI) unterziehen, intensiv phänotypisiert inklusive der Analyse von Blut und Myokardproben. Die klinischen Verläufe nach TAVI werden dann mit den hämodynamischen, bildgeberischen und molekularen Daten korreliert. Der SFB befindet sich in der dritten Förderperiode. Er wird flankiert von dem Internationalen Graduiertenkolleg IRTG 1816 „Phosphorylierungs- und Redox-vermittelte Signalmechanismen im kranken Herzen“.
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the number of identified peptides and the analytical precision of quantitation. The new method was applied to study changes in protein expression in mouse hearts upon transverse aortic constriction, a model for cardiac stress.
Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hy... more Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. Methods and results Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P ¼ 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility. Conclusions Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.
This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differ... more This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differential pathways leading from a compensated challenged heart to either beneficial or maladaptive hypertrophic signaling. A model for adaptive hypertrophy (swimming) was compared to two models of maladaptive hypertrophy (transverse aortic constriction, TAC and myocardial infarction, MI) in wildtype (WT) and melusin overexpressing (OE) mice with special attention towards gender differences. Melusin, a muscle specific β1 integrin interacting protein, was shown to favorably influence heart failure after TAC. To achieve the highest possible standardization and homogeneity the interventions were carried out at a single center (1423 mice) and the harvested samples were distributed among the consortium members for in-depth analyses of signaling pathways, energy metabolism as well as characterization of the extracellular matrix. Swimming (n=391) and TAC (n=426) lead to comparable and significant degrees of hypertrophy (heart weight/body weight ratio +13.3% and +9.7%, respectively). While TAC caused a concentric hypertrophy (LVEDD −8.2%) swimming significantly increased LVEDD by 9.3%. At this compensated stage there were no significant morphometric or echocardiographic differences between WT and melusin OE mice. However, following MI (n=606), melusin OE mice showed reduced dilatation (LVEDD +49.8% wt vs. +29.0% melusin OE, n=136) and wall thinning (septum thickness −40.5% WT vs. −21.4% melusin OE). Gender separated analysis revealed a significant reduction in LVEDD only in the melusin OE males. A significantly improved survival rate was found for the melusin OE males (59% vs. 37%) whereas there was no significant effect on survival in females (82% vs. 76%). Analysis of shortening and calcium transients indicated a still compensated myocyte function on the single cell level for all three models, as intended. This is the first study that shows a prognostic effect of melusin overexpression in the setting of MI. Reduced mortality is associated with more favorable remodeling. Most interestingly, the effect is gender dependent. The present findings may be of future clinical relevance. Examinations of signaling pathways are currently being performed.
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the...
This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differ... more This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differential pathways leading from a compensated challenged heart to either beneficial or maladaptive hypertrophic signaling. A model for adaptive hypertrophy (swimming) was compared to two models of maladaptive hypertrophy (transverse aortic constriction, TAC and myocardial infarction, MI) in wildtype (WT) and melusin overexpressing (OE) mice with special attention towards gender differences. Melusin, a muscle specific β1 integrin interacting protein, was shown to favorably influence heart failure after TAC. To achieve the highest possible standardization and homogeneity the interventions were carried out at a single center (1423 mice) and the harvested samples were distributed among the consortium members for in-depth analyses of signaling pathways, energy metabolism as well as characterization of the extracellular matrix. Swimming (n=391) and TAC (n=426) lead to comparable and significant degrees of hypertrophy (heart weight/body weight ratio +13.3% and +9.7%, respectively). While TAC caused a concentric hypertrophy (LVEDD −8.2%) swimming significantly increased LVEDD by 9.3%. At this compensated stage there were no significant morphometric or echocardiographic differences between WT and melusin OE mice. However, following MI (n=606), melusin OE mice showed reduced dilatation (LVEDD +49.8% wt vs. +29.0% melusin OE, n=136) and wall thinning (septum thickness −40.5% WT vs. −21.4% melusin OE). Gender separated analysis revealed a significant reduction in LVEDD only in the melusin OE males. A significantly improved survival rate was found for the melusin OE males (59% vs. 37%) whereas there was no significant effect on survival in females (82% vs. 76%). Analysis of shortening and calcium transients indicated a still compensated myocyte function on the single cell level for all three models, as intended. This is the first study that shows a prognostic effect of melusin overexpression in the setting of MI. Reduced mortality is associated with more favorable remodeling. Most interestingly, the effect is gender dependent. The present findings may be of future clinical relevance. Examinations of signaling pathways are currently being performed.
Aims Melusin is a muscle-specific chaperone protein whose expression is required for a compensato... more Aims Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. Methods and results Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein e...
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the...
In den letzten Jahren wurden in mehreren Labors verschiedene Mausstämme mit Nullmutationen des fü... more In den letzten Jahren wurden in mehreren Labors verschiedene Mausstämme mit Nullmutationen des für einen myogenen Transkriptionsfaktor kodierenden Myf-Gens (Myogenic factor 5) generiert. Die Abwesenheit von Myf-5-Protein im sich entwickelnden Mausembryo führt zu einer Verzögerung der Myotombildung von 1,75 Tagen, bis die myogene Aktivität der einsetzenden MyoD-Expression benötigte Muskelvorläuferzellen determiniert. Die Muskulatur homozygoter Myf5-Mutanten entwickelt sich überraschenderweise normal. Die Abwesenheit des myogenen Myf5-Proteins führt unerwartet zum Fehlen distaler Rippen, in dessen Folge die Mäuse perinatal aufgrund mangelnder Respiration sterben. Um die Funktion von Myf-5 in adulten Mäusen zu untersuchen, wurde eine Mauslinie generiert, die mit Hilfe der Cre/loxP-Technologie ein konditional inaktivierbares Allel trägt (Myf-5loxP). Die kontrollierte Expression der Cre-Rekombinase mit dem Ziel der Inaktivierung des Myf5-Gens (Myf-5ΔloxP) wurde in 4 verschiedenen Ansätzen verfolgt. (1) Zeitliche Kontrolle der Exzision durch den induzierbaren Mx-Promotor. (2) Ausschließlich auf Skelettmuskulatur beschränkte Cre-Rekombinase-Expression durch den muskelspezifischen Muskel-Creatin-Kinase-Promotor. (3) Durch Adenovirus vermittelte Cre-Rekombinase-Expression, um unabhängig von verfügbaren Promotoren und ihren Expressionsprofilen zu sein. (4) Konstitutive Cre-Rekombinase Expression in einer Cre-deleter Mauslinie zwecks frühestmöglicher Myf5-Inaktivierung. Die Myf5-Exzision im Zygotenstadium sollte eine Phänokopie der alten Myf5-Allele darstellen. Homozygote Myf-5ΔloxP/ΔloxP Mutanten zeigen unerwartet keine Fehlentwicklung der Rippen. Trotz des völligen Fehlens der frühen Myotomentwicklung sind die Mutanten lebensfähig und fruchtbar, sie weisen keine erhöhte Sterblichkeit auf. Daraus kann geschlossen werden, dass der zuvor für Myf5 mutante Tiere beschriebene Rippenphänotyp durch Missregulation eines noch unbekannten Gens verursacht wird. Der Schwanz von Myf-5ΔloxP/ΔloxPMäusen weist mit einsetzender Adoleszenz [...]
Braun et al., 1992). Since the formation of the myotome Myf-5 Revisited: Loss of Early is depende... more Braun et al., 1992). Since the formation of the myotome Myf-5 Revisited: Loss of Early is dependent on Myf-5 and a number of potential induc-Myotome Formation Does Not Lead tive signals expressed in myotome like FGFs and PDGF are absent in Myf-5 mutant mice (Grass et al., 1996), to a Rib Phenotype in Homozygous this hypothesis appeared plausible and logical.
Ziel des Sonderforschungsbereichs (SFB) 1002 „Modulatorische Einheiten bei Herzinsuffizienz“ ist ... more Ziel des Sonderforschungsbereichs (SFB) 1002 „Modulatorische Einheiten bei Herzinsuffizienz“ ist es, neue Diagnose- und Therapieverfahren der Herzinsuffizienz zu identifizieren und bis zur Anwendung am Patienten zu entwickeln. Mit der Förderung des Deutschen Zentrums für Herz-Kreislauf-Forschung (DZHK) sollen schließlich „First-in-Man-Studien“ durchgeführt werden. Beispiele für den erfolgreichen Translationsprozess im SFB 1002 sind die klinischen Studien BioVAT-DZHK 20 „Safety and efficacy of induced pluripotent stem cell-derived engineered human myocardium as biological ventricular assist device in terminal heart failure“ (Herzpflaster) und REDUCE-MFA-DZHK 25 „Reduce myocardial fibrosis in aortic stenosis“ (Fibrosehemmung). Diese beiden Multicenterstudien basieren auf Grundlagenarbeiten des SFBs und befinden sich aktuell mit DZHK-Förderung in der Patientenrekrutierungsphase. Zur Target-Identifizierung im SFB werden sowohl die Herzinsuffizienzentwicklung als „transition to failure“ als auch der reversible Prozess „recovery from failure“ klinisch und experimentell analysiert. Im Vordergrund stehen hierbei funktionelle Mikrodomänen wie subzelluläre Kalziumfreisetzungseinheiten, Myozyten-Fibroblasten-Interaktionen und die Genregulation. Im Rahmen des SFBs wird darüber hinaus die TAVI-Bio‑/Datenbank aufgebaut. Hierbei werden Patienten, die sich einer „transcatheter aortic valve implantation“ (TAVI) unterziehen, intensiv phänotypisiert inklusive der Analyse von Blut und Myokardproben. Die klinischen Verläufe nach TAVI werden dann mit den hämodynamischen, bildgeberischen und molekularen Daten korreliert. Der SFB befindet sich in der dritten Förderperiode. Er wird flankiert von dem Internationalen Graduiertenkolleg IRTG 1816 „Phosphorylierungs- und Redox-vermittelte Signalmechanismen im kranken Herzen“.
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the number of identified peptides and the analytical precision of quantitation. The new method was applied to study changes in protein expression in mouse hearts upon transverse aortic constriction, a model for cardiac stress.
Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hy... more Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. Methods and results Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P ¼ 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein expression in the infarcted area. At 2 weeks after MI, melusin overexpression conferred a favourable adaptive remodelling characterized by reduced left ventricle dilatation and better preserved contractility in the presence of a comparable degree of hypertrophy. Adaptive remodelling in melusin TG mice was characterized by reduced apoptosis and fibrosis as well as increased cardiomyocyte contractility. Conclusions Consistent with its function as a chaperone protein, melusin overexpression exerts a dual protective action following MI reducing an array of maladaptive processes. In the early phase after MI, reduced inflammation and myocyte remodelling protect against cardiac rupture. Chronically, reduced myocyte loss and matrix remodelling, with preserved myocyte contractility, confer adaptive LV remodelling.
This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differ... more This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differential pathways leading from a compensated challenged heart to either beneficial or maladaptive hypertrophic signaling. A model for adaptive hypertrophy (swimming) was compared to two models of maladaptive hypertrophy (transverse aortic constriction, TAC and myocardial infarction, MI) in wildtype (WT) and melusin overexpressing (OE) mice with special attention towards gender differences. Melusin, a muscle specific β1 integrin interacting protein, was shown to favorably influence heart failure after TAC. To achieve the highest possible standardization and homogeneity the interventions were carried out at a single center (1423 mice) and the harvested samples were distributed among the consortium members for in-depth analyses of signaling pathways, energy metabolism as well as characterization of the extracellular matrix. Swimming (n=391) and TAC (n=426) lead to comparable and significant degrees of hypertrophy (heart weight/body weight ratio +13.3% and +9.7%, respectively). While TAC caused a concentric hypertrophy (LVEDD −8.2%) swimming significantly increased LVEDD by 9.3%. At this compensated stage there were no significant morphometric or echocardiographic differences between WT and melusin OE mice. However, following MI (n=606), melusin OE mice showed reduced dilatation (LVEDD +49.8% wt vs. +29.0% melusin OE, n=136) and wall thinning (septum thickness −40.5% WT vs. −21.4% melusin OE). Gender separated analysis revealed a significant reduction in LVEDD only in the melusin OE males. A significantly improved survival rate was found for the melusin OE males (59% vs. 37%) whereas there was no significant effect on survival in females (82% vs. 76%). Analysis of shortening and calcium transients indicated a still compensated myocyte function on the single cell level for all three models, as intended. This is the first study that shows a prognostic effect of melusin overexpression in the setting of MI. Reduced mortality is associated with more favorable remodeling. Most interestingly, the effect is gender dependent. The present findings may be of future clinical relevance. Examinations of signaling pathways are currently being performed.
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the...
This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differ... more This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differential pathways leading from a compensated challenged heart to either beneficial or maladaptive hypertrophic signaling. A model for adaptive hypertrophy (swimming) was compared to two models of maladaptive hypertrophy (transverse aortic constriction, TAC and myocardial infarction, MI) in wildtype (WT) and melusin overexpressing (OE) mice with special attention towards gender differences. Melusin, a muscle specific β1 integrin interacting protein, was shown to favorably influence heart failure after TAC. To achieve the highest possible standardization and homogeneity the interventions were carried out at a single center (1423 mice) and the harvested samples were distributed among the consortium members for in-depth analyses of signaling pathways, energy metabolism as well as characterization of the extracellular matrix. Swimming (n=391) and TAC (n=426) lead to comparable and significant degrees of hypertrophy (heart weight/body weight ratio +13.3% and +9.7%, respectively). While TAC caused a concentric hypertrophy (LVEDD −8.2%) swimming significantly increased LVEDD by 9.3%. At this compensated stage there were no significant morphometric or echocardiographic differences between WT and melusin OE mice. However, following MI (n=606), melusin OE mice showed reduced dilatation (LVEDD +49.8% wt vs. +29.0% melusin OE, n=136) and wall thinning (septum thickness −40.5% WT vs. −21.4% melusin OE). Gender separated analysis revealed a significant reduction in LVEDD only in the melusin OE males. A significantly improved survival rate was found for the melusin OE males (59% vs. 37%) whereas there was no significant effect on survival in females (82% vs. 76%). Analysis of shortening and calcium transients indicated a still compensated myocyte function on the single cell level for all three models, as intended. This is the first study that shows a prognostic effect of melusin overexpression in the setting of MI. Reduced mortality is associated with more favorable remodeling. Most interestingly, the effect is gender dependent. The present findings may be of future clinical relevance. Examinations of signaling pathways are currently being performed.
Aims Melusin is a muscle-specific chaperone protein whose expression is required for a compensato... more Aims Melusin is a muscle-specific chaperone protein whose expression is required for a compensatory hypertrophy response to pressure overload. Here, we evaluated the consequences of melusin overexpression in the setting of myocardial infarction (MI) using a comprehensive multicentre approach. Methods and results Mice overexpressing melusin in the heart (TG) and wild-type controls (WT) were subjected to permanent LAD ligation and both the acute response (Day 3) and subsequent remodelling (2 weeks) were examined. Mortality in wild-type mice was significant between Days 3 and 7, primarily due to cardiac rupture, but melusin's overexpression strongly reduced mortality (43.2% in wild-type vs. 27.3% in melusin-TG, P = 0.005). At Day 3 after MI, a time point preceding the mortality peak, TG hearts had increased heat shock protein 70 expression, increased ERK1/2 signalling, reduced cardiomyocyte hyper-contractility and inflammatory cell infiltrates, and increased matricellular protein e...
The development of quantitative techniques in mass spectrometry has generated the ability to syst... more The development of quantitative techniques in mass spectrometry has generated the ability to systematically monitor protein expression. Isobaric tags for relative and absolute quantification (iTRAQ) have become a widely used tool for the quantification of proteins. However, application of iTRAQ methodology using ion traps and hybrid mass spectrometers containing an ion trap such as the LTQ-Orbitrap was not possible until the development of pulsed Q dissociation (PQD) and higher energy C-trap dissociation (HCD). Both methods allow iTRAQ-based quantification on an LTQ-Orbitrap but are less suited for protein identification at a proteomic scale than the commonly used collisional induced dissociation (CID) fragmentation. We developed an analytical strategy combining the advantages of CID and HCD, allowing sensitive and accurate protein identification and quantitation at the same time. In a direct comparison, the novel method outperformed PQD and HCD regarding its limit of detection, the...
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Papers by Axel Kaul