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Cardiac remodeling in pressure overload and myocardial infarction [Elektronische Ressource] : role of PKC_e63 and gp130 signaling pathways / von Praphulla Chandra Shukla

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Cardiac Remodeling in Pressure Overload and Myocardial Infarction: Role of PKC ε and gp130 Signaling Pathways Der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von M.Sc. Praphulla Chandra Shukla geboren am 19.01.1977 in Fatehpur, India 2006 Referent: Prof. Dr. Walter Müller Korreferent: Prof. Dr. Jörg Schmidtke thTag der Promotion: 7 December 2006 Key Words: Myokardinfarkt, Myokardhypertrophie, gp130 Myocardial Infarction, Myocardial Hypertrophy, gp130 INDEX SYNOPSIS..................................................................................................................................................................I LIST OF ABBREVIATIONS................................................................................................................................. V LIST OF FIGURES.............................................................................................................................................. VII LIST OF TABLES..................................................................................................................................................IX 1 INTRODUCTION (A) .............................................................................................................................. 1 1.

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Published 01 January 2006
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Cardiac Remodeling in Pressure Overload and Myocardial
Infarction: Role of PKC ε and gp130 Signaling Pathways


Der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
Doktor der Naturwissenschaften
(Dr. rer. nat.)
genehmigte Dissertation


von
M.Sc. Praphulla Chandra Shukla
geboren am 19.01.1977 in Fatehpur, India
2006

Referent: Prof. Dr. Walter Müller
Korreferent: Prof. Dr. Jörg Schmidtke
thTag der Promotion: 7 December 2006
Key Words: Myokardinfarkt, Myokardhypertrophie, gp130
Myocardial Infarction, Myocardial Hypertrophy, gp130




INDEX
SYNOPSIS..................................................................................................................................................................I
LIST OF ABBREVIATIONS................................................................................................................................. V
LIST OF FIGURES.............................................................................................................................................. VII
LIST OF TABLES..................................................................................................................................................IX
1 INTRODUCTION (A) .............................................................................................................................. 1
1.1 HYPERTROPHIC REMODELING OF THE HEART............................................................................................. 1
1.2 CELLULAR AND MOLECULAR RESPONSE OF CARDIOMYOCYTES TO BIOMECHANICAL STRETCH .............. 2
1.3 SIGNAL TRANSDUCTION IN CARDIAC HYPERTROPHY ................................................................................. 6
1.3.1 PKC and Cardiac Hypertrophy ......................................................................................................... 8
1.3.2 PKC Redundancy in Cardiac Hypertrophy..................................................................................... 10
1.4 PKCε KNOCKOUT MOUSE TO STUDY CARDIAC HYPERTROPHY............................................................... 11
2 AIM OF THE STUDY (A) ..................................................................................................................... 13
3 RESULTS (PART A) .............................................................................................................................. 14
3.1 CARDIAC PHENOTYPE OF PKCε KNOCKOUT MICE ................................................................................... 14
3.2 PRESSURE OVERLOAD TRIGGERED SIMILAR HYPERTROPHIC RESPONSES IN KNOCKOUT AND WILDTYPE
MICE........................................................................................................................................................... 16
3.3 PRESSURE OVERLOAD INCREASED COLLAGEN EXPRESSION IN KNOCKOUT MICE................................... 19
3.4 PKCε KNOCKOUT MICE SHOW DIASTOLIC DYSFUNCTION AFTER PRESSURE OVERLOAD....................... 21
3.5 UPREGULATION OF PKCδ IN PKCε KNOCKOUT MICE AFTER PRESSURE OVERLOAD .............................. 22
3.6 DIFFERENTIAL ACTIVATION OF MAPK SIGNALING PATHWAY IN KNOCKOUT MICE AFTER PRESSURE
OVERLOAD................................................................................................................................................. 23
3.7 APOPTOSIS IN PKCε KNOCKOUT AND WILDTYPE MICE AFTER PRESSURE OVERLOAD............................ 25
3.8 INDUCTION OF COL Iα1 EXPRESSION BY MECHANICAL STRETCH IN FIBROBLASTS................................. 25
4 DISCUSSION (PART A)........................................................................................................................ 28
5 INTRODUCTION (B) ............................................................................................................................ 31
5.1 THE PATHOPHYSIOLOGY OF MYOCARDIAL INFARCTION .......................................................................... 31
5.2 EXPERIMENTAL MI IN MICE AS A TOOL TO INVESTIGATE PATHOPHYSIOLOGICAL PROCESSES IN THE
INFARCTED HEART..................................................................................................................................... 32
5.3 PRO-INFLAMMATORY CYTOKINES IN THE INFARCTED AND THE FAILING HEART..................................... 33
5.3.1 Roles of the IL-6/gp130 Receptor System in Heart Failure............................................................ 34
5.4 THE MECHANISTIC INSIGHT INTO THE IL-6/GP130 SIGNALING CASCADE ................................................ 36
5.4.1 IL-6 Cytokine Family ....................................................................................................................... 37
5.4.2 The Structure of gp130 Receptor..................................................................................................... 38
5.4.3 Mutations in the gp130 Receptor..................................................................................................... 42
5.5 ROLE OF THE GP130 RECEPTOR SYSTEM IN THE ADULT HEART ............................................................... 46
5.6 ROLE OF SIGNALING PATHWAYS ACTIVATED BY THE GP130 RECEPTOR SYSTEM: MEK/ERK, JAK/STAT
AND PI3-K/AKT ......................................................................................................................................... 47

5.6.1 MEK/ERK Signaling ........................................................................................................................ 48
5.6.2 JAK/STAT Signaling......................................................................................................................... 49
5.6.3 PI3-K/Akt Signaling ......................................................................................................................... 49
6 AIM OF THE STUDY (B)...................................................................................................................... 51
7 RESULTS (PART B) .............................................................................................................................. 52
7.1 GENERATION OF CARDIOMYOCYTE SPECIFIC GP130 MUTANT MICE........................................................ 52
7.2 VERIFICATION OF CARDIOMYOCYTE SPECIFIC DELETION OF THE GP130 IN GP130-KO MICE.................. 56
7.3 CHARACTERIZATION OF THE CARDIAC PHENOTYPES OF GP130 MUTANT MICE AT BASELINE ................ 57
7.4 LIF INDUCES IMPAIRED DOWNSTREAM SIGNALING IN MICE HARBOURING CARDIOMYOCYTE SPECIFIC
MUTATIONS IN THE GP130 RECEPTOR ....................................................................................................... 57
7.5 CARDIOMYOCYTE SPECIFIC MUTATIONS IN THE GP130 RECEPTOR AFFECT THE ACTIVATION OF THE
MEK/ERK AND JAK/STAT SIGNALING................................................................................................... 59
7.6 INCREASED POST-MI MORTALITY IN GP130-KO AND MEK/ERK-KO BUT NOT IN JAK/STAT-KO MICE
............................................................................................................................................................... 61
7.7 LEFT VENTRICULAR FUNCTIONAL ANALYSIS ........................................................................................... 62
7.8 LEFT VENTRICULAR MORPHOMETRIC ANALYSIS...................................................................................... 63
7.8.1 Substantially Larger Infarct in gp130-KO and MEK/ERK-KO...................................................... 63
7.8.2 Gp130 Mutant Mice Show an Impaired Hypertrophic Response ................................................... 63
7.9 GENE EXPRESSION ANALYSIS IN GP130 MUTANT MICE POST-MI............................................................ 64
7.9.1 Reduction in Expression of skm- α-actin Gene ................................................................................ 64
7.9.2 Enhanced Induction of ANP Gene Expression in gp130 Mutant Mice........................................... 65
7.10 ENHANCED APOPTOSIS IN THE INFARCT BORDER ZONE OF GP130 MUTANT MICE .................................. 66
8 DISCUSSION (PART B)........................................................................................................................ 69
9 MATERIALS AND METHODS ...........................................................................................................74
A. CHEMICALS AND REAGENTS...................................................................................................................... 74
B. KITS............................................................................................................................................................ 75
C. ANTIBODIES ............................................................................................................................................... 75
D. INSTRUMENTS ............................................................................................................................................ 76
E. OTHER MATERIALS..................................................................................................................................... 76
9.1 MICE........................................................................................................................................................... 77
9.1.1 PKCε Knockout Mice....................................................................................................................... 77
9.1.2 Cardiomyocyte specific gp130-KO, MEK/ERK-KO and JAK/STAT-KO mice .............................. 78
9.2 GENOTYPING OF CARDIOMYOCYTE SPECIFIC GP130-KO, GP130-DEPENDENT MEK/ERK-KO AND
JAK/STAT-KO MICE................................................................................................................................80
9.3 TRANSVERSE AORTIC CONSTRICTION ....................................................................................................... 80
9.4 EXPERIMENTAL MYOCARDIAL INFARCTION.............................................................................................. 81
9.5 LEUKEMIA INHIBITORY FACTOR (LIF) INJECTION .................................................................................... 82
9.6 HEMODYNAMIC MEASUREMENTS (PRESSURE VOLUME ANALYSIS)......................................................... 82
9.7 IN SITU FIXATION........................................................................................................................................ 82

9.8 TRANSTHORACIC ECHOCARDIOGRAPHY.................................................................................................... 83
9.9 TISSUE DOPPLER IMAGING (TDI) .............................................................................................................. 83
9.10 MICROTOMY AND MORPHOMETRY............................................................................................................ 83
9.10.1 Haematoxylin-Eosin Staining .....................................................................................................84
9.10.2 Picro-Sirius Red Staining............................................................................................................ 84
9.11 TUNEL ASSAY.......................................................................................................................................... 85
9.12 CELL CULTURE AND MECHANICAL STRETCHING...................................................................................... 85
9.13 CARDIOMYOCYTE ISOLATION AND PCR ................................................................................................... 86
9.14 RNA ISOLATION AND REAL TIME PCR..................................................................................................... 86
9.15 NORTHERN BLOTTING ............................................................................................................................... 87
9.16 PROTEIN ASSAY AND IMMUNOBLOTTING .................................................................................................. 88
9.17 STATISTICAL ANALYSIS............................................................................................................................. 88
10 REFERENCES........................................................................................................................................ 89
11 CURRICULUM VITAE....................................................................................................................... 105
12 ACKNOWLEDGEMENT.................................................................................................................... 107
13 ERKLÄRUNG....................................................................................................................................... 108
Synopsis
Synopsis
Function of protein kinase C ε (PKC ε) and glycoprotein 130 (gp130) dependent signal
transduction in cardiac remodeling after pressure overload and myocardial infarction
(MI)
Part A: Role of PKC ε in the heart following pressure overload
Cardiac pressure overload, e.g. after aortic stenosis, leads to myocardial hypertrophy, which
correlates as a major risk factor with the development of heart insufficiency. Cardiomyocyte
specific overexpression of PKC ε causes concentric hypertrophy without cardiac dysfunction.
The aim of the study was the analysis of the role of PKC ε in the murine heart. PKC ε knockout
-/-mice with a systemic PKC ε deficiency (PKC ε ) showed no heart phenotype under basic
physiological conditions. Pressure overload after transverse aortic constriction (TAC), in
-/-PKC ε mice leads to pronounced interstitial fibrosis associated with increased expression of
collagen Iα1 and declined diastolic function in comparison to wildtype animals. Moreover, no
-/- differences were found in cardiomyocyte hypertrophy between PKC ε and wildtype mice on
morphometric or molecular level. These results suggested that PKC ε has important role in
cardiac remodeling, but not in the development of cardiac hypertrophy after pressure
overload.
Part B: Dissection of gp130 signaling pathways for cardioprotection and survival after
myocardial infarction
Left ventricular remodeling has a pivotal role in the development of chronic heart
insufficiency after MI associated with activation of neurohormonal system and increased
cytokine levels. Serum concentration of IL-6 is used as a relevant marker for the prognosis of
remodeling and survival for heart insufficient patients. IL-6 binding to the gp130 receptor
induces a signal transduction cascade, which is altered in patients with terminal heart
insufficiency. Heart specific gp130 knockout mice inhibit compensatory hypertrophy after
pressure overload and showed increased apoptosis and accelerated dilatation of left ventricle.
The aim of this study was to investigate the impact of gp130 on cardioprotection and
remodeling after MI. For this purpose, four different mutant mice were analyzed: (1) mice
i Synopsis
flox/floxwith cardiomyocyte specific gp130 knockout ( α−MHC-Cre; gp130 ; gp130-KO), (2)
mice with deletion of gp130-mediated activation of MEK/ERK signaling ( α−MHC-Cre;
flox/ERKgp130 ; MEK/ERK-KO), (3) mice with deletion of gp130-mediated activation of
flox/JAK-STATJAK/STAT signaling ( α−MHC-Cre; gp130 ; JAK/STAT-KO) and (4) mice with at
flox/floxleast two functional gp130 alleles (gp130 ; WT). All the mice displayed normal cardiac
function and morphology up to the age of 3-4 months. Following experimental MI, gp130-KO
and MEK/ERK-KO mice showed significantly larger infarcts in comparison with WT. Post-
MI mortality was also higher in gp130-KO and MEK/ERK-KO mice in comparison with
JAK/STAT-KO and WT mice. Furthermore, analyzing mice with similar infarct sizes
revealed that, gp130-KO and MEK/ERK-KO has significantly diminished left ventricular
function. Interestingly, post-MI hypertrophy of the remote myocardium was blunted in
gp130-KO, while no hypertrophy in MEK/ERK-KO mice was detected.
In conclusion, these data show that after MI, gp130-mediated activation of MEK/ERK
signaling is essential for the cardioprotection, hypertrophy of the myocardium, cardiac
function and survival, and undermines the importance of gp130-mediated JAK/STAT
signaling in the heart.
ii Kurzzusammenfassung
Kurzzusammenfassung
Untersuchungen zur Funktion von Proteinkinase Cε (PKCε) und Glykoprotein 130
(gp130) -abhängiger Signaltransduktion im kardialen Remodelling nach
Druckbelastung und Myokardinfarkt
Teil A: Rolle von PKCε im druckbelasteten Myokard
Eine Druckbelastung des Myokards, z.B. durch eine erhöhte Nachlast bei Aortenstenose, führt
zu einer Myokardhypertrophie, welche ein wichtiger Risikofaktor für die Entwicklung einer
Herzinsuffizienz darstellt. Es ist bekannt, daß das Signalmolekül PKCε eine Rolle bei der
kardialen Hypertrophie spielt, da die Kardiomyozyten-spezifische Überexpression von PKCε
zu einer konzentrischen Myokardhypertrophie bei gleichzeitiger Funktionserhaltung führt. Da
Überexpressionsexperimente häufig unspezifische Phänotypen hervorrufen, wurde in dieser
Arbeit die Rolle von PKCε im Myocard in einer Maus mit einer systemischen PKCε-
-/- -/-Defizienz (PKCε ) untersucht. Die PKCε -Maus zeigt unter Basalbedingungen keine
phänotypischen Veränderungen. Die Druckbelastung nach einer transversen Aorta-
-/-Konstriktion führte in den PKCε -Mäusen zu einer deutlich stärkeren interstitiellen Fibrose,
die mit einer erhöhten Expression von Collagen Iα1 und einer verschlechterten diastolischen
Funktion im Vergleich zu Wildtyp-Mäusen assoziiert war. Es zeigten sich hingegen weder auf
morphometrischer noch auf molekularer Ebene Unterschiede in der Kardiomyozyten-
-/-Hypertrophie zwischen PKCε -Mäusen und Wildtyp-Tieren. Diese Ergebnisse zeigen, daß
PKCε ein wichtiger Faktor für das kardiale Remodelling, nicht aber für die Hypertrophie bei
Druckbelastung des Myokards darstellt.
Teil B: Diversifizierung des g130 Signalings für die Kardioprotektion und das
Überleben nach Myokardinfarkt
Das linksventrikuläre Remodeling nach Myokardinfarkt trägt entscheidend zur Entwicklung
einer chronischen Herzinsuffizienz bei. Neben der Aktivierung neurohumoraler Systeme sind
nach Infarkt auch Zytokinenspiegel erhöht. Es ist bekannt, daß die Serumkonzentration von
Interleukin 6 (IL-6) prognostisch für das Remodeling nach Infarkt und das Überleben von
herzinsuffizienten Patienten relevant ist. IL-6 induziert durch Bindung an die Rezeptor-
komponente gp130 eine spezifische Signalkaskasden, die in Patienten mit einer terminalen
iii Kurzzusammenfassung
Herzinsuffizienz deutliche Änderungen aufweisen. Außerdem war experimentell bekannt, daß
Druckbelastung durch Aortenstenose in Mäusen mit einem herzspezifischen gp130-Knockout
die kompensatorischen Hypertrophie unterbindet und zu einer hohe Apoptoserate sowie zu
einer frühen Dilatation führt.
In der vorliegenden Arbeit wurde die Rolle von gp130 für die Kardioprotektion und das
Remodelling nach Myokardinfarkt untersucht. Dazu wurden vier verschiedene
Mausgenotypen analysiert: Mäuse mit einem kardiomyozyten Knockout für gp130 ( α−MHC-
flox/floxCre; gp130 ; gp130-KO), Mäuse denen in Kardiomyozyten die gp130 vermittelte
flox/ERKAktivierung des MEK/ERK Signalweges fehlt ( α−MHC-Cre; gp130 ; MEK/ERK-KO),
Mäuse, denen in Kardiomyozyten die gp130 vermittelte Aktivierung des JAK/STAT
flox/JAK-STATSignalweges fehlt ( α−MHC-Cre; gp130 ; JAK/STAT-KO), sowie der
flox/floxkorrespondierende Wildtyp mit zwei funktionierenden gp130 Allelen (gp130 ; WT). Bis
im Alter von 3 bis 4 Monaten zeigten alle Genotypen eine normale Myokardfunktion und
Morphologie. Nach experimentellem Myokardinfarkt wiesen die gp130-KO Mäuse und die
MEK/ERK-KO Mäuse deutlich größere Infarkte und eine erhöhte Mortalität auf verglichen
mit den WT und den JAK/STAT-KO Mäusen. In ausgewählten Tieren mit vergleichbarer
Infarktgröße zeigten die gp130-KO und MEK/ERK-KO Mäuse zudem eine deutlich
schlechtere linksventrikuläre Funktion und eine reduzierte Hypertrophie des Restmyokard
auf. Die fehlende Hypertrophie war bei den MEK/ERK-KO Mäusen besonders ausgeprägt.
Zusammenfassend zeigen diese Daten, dass der gp130 vermittelten Aktivierung der
MEK/ERK Signalkaskade eine essentielle Funktion für die Kardioprotektion, die
Myokardhypertrophie, die kardiale Funktion und das Überleben nach Myokardinfarkt
zukommt, während die gp130 vermittelte Aktivierung des JAK/STAT Signalweges eher eine
untergeordnete Rolle spielt.

iv List of Abbreviations
List of Abbreviations
BSA Bovine Serum Albumin
CSA Cross Sectional Area
DAPI 4,6-diamidino-2-phenylindole
DMEM Dulbecco’s Modified Eagle Medium
ECL Enhanced Chemiluminescence
Egr Epidermal Growth Factor Receptor
ERK Extracellular Signal Regulated Kinase
ES Cells Embryonic Stem Cells
ET-1 Endothelin-1
FAK Focal Adhesion Kinase
bFGF Basic Fibroblast Growth Factor
FITC Fluorescein Isothiocynate
GAPDH Glyceraldehyde-3-phosphate Dehydrogenase
GH Growth Hormone
gp130 Glycoprotein 130
GPCR G-protein Coupled Receptors
Guanosine Triphosphate GTP
HOPE Heart Outcomes Prevention Evaluation Study
Hr Hour
IGF Insulin Like Growth Factor
JAK Janus Kinase
JNK c-jun N-Terminal Kinase
kDa Kilo Dalton
LV Left Ventricle
MAPK Mitogen Activated Protein Kinase
MEK MAP Kinase Kinase
MI Myocardial Infarction
Min Minute
MKP MAPK Phosphatase
MMP Matrix Metalloproteinase
NFAT Nuclear Factor of Activated T-cells
v