Infarction: Role of PKCε 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 Tag der Promotion: 7th 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 PKCεKNOCKOUT MOUSE TO STUDY CARDIAC HYPERTROPHY... 11
2 AIM OF THE STUDY (A) ... 13
3 RESULTS (PART A) ... 14
3.1 CARDIAC PHENOTYPE OF PKCεKNOCKOUT 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 PKCεKNOCKOUT MICE SHOW DIASTOLIC DYSFUNCTION AFTER PRESSURE OVERLOAD... 21
3.5 UPREGULATION OF PKCδ IN PKCεKNOCKOUT MICE AFTER PRESSURE OVERLOAD... 22
3.6 DIFFERENTIAL ACTIVATION OF MAPKSIGNALING PATHWAY IN KNOCKOUT MICE AFTER PRESSURE OVERLOAD... 23
3.7 APOPTOSIS IN PKCεKNOCKOUT AND WILDTYPE MICE AFTER PRESSURE OVERLOAD... 25
3.8 INDUCTION OF COL Iα1EXPRESSION 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/GP130SIGNALING 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 GP130RECEPTOR SYSTEM IN THE ADULT HEART... 46
5.6 ROLE OF SIGNALING PATHWAYS ACTIVATED BY THE GP130RECEPTOR 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 GP130MUTANT 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 GP130MUTANT MICE AT BASELINE... 57
7.4 LIFINDUCES IMPAIRED DOWNSTREAM SIGNALING IN MICE HARBOURING CARDIOMYOCYTE SPECIFIC MUTATIONS IN THE GP130RECEPTOR... 57
7.5 CARDIOMYOCYTE SPECIFIC MUTATIONS IN THE GP130 RECEPTOR AFFECT THE ACTIVATION OF THE MEK/ERK AND JAK/STATSIGNALING... 59
7.6 INCREASED POST-MIMORTALITY IN GP130-KO AND MEK/ERK-KO BUT NOT IN JAK/STAT-KOMICE ... 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 GP130MUTANT 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 GP130MUTANT 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-KOMICE... 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
9.8 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 TUNELASSAY... 85
9.12 CELL CULTURE AND MECHANICAL STRETCHING... 85
9.13 CARDIOMYOCYTE ISOLATION AND PCR ... 86
9.14 RNAISOLATION 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
Function of protein kinase Cε (PKCε) and glycoprotein 130 (gp130) dependent signal transduction in cardiac remodeling after pressure overload and myocardial infarction (MI)
Part A: Role of PKCε 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 PKCε causes concentric hypertrophy without cardiac dysfunction.
The aim of the study was the analysis of the role of PKCε in the murine heart. PKCε knockout mice with a systemic PKCε deficiency (PKCε-/-) showed no heart phenotype under basic physiological conditions. Pressure overload after transverse aortic constriction (TAC), in PKCε-/- 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 PKCε-/- and wildtype mice on morphometric or molecular level. These results suggested that PKCε 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
with cardiomyocyte specific gp130 knockout (α−MHC-Cre; gp130flox/flox; gp130-KO), (2) mice with deletion of gp130-mediated activation of MEK/ERK signaling (α−MHC-Cre;
gp130flox/ERK; MEK/ERK-KO), (3) mice with deletion of gp130-mediated activation of JAK/STAT signaling (α−MHC-Cre; gp130flox/JAK-STAT; JAK/STAT-KO) and (4) mice with at least two functional gp130 alleles (gp130flox/flox; 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.
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
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- Cre; gp130flox/flox; gp130-KO), Mäuse denen in Kardiomyozyten die gp130 vermittelte Aktivierung des MEK/ERK Signalweges fehlt (α−MHC-Cre; gp130flox/ERK; MEK/ERK-KO), Mäuse, denen in Kardiomyozyten die gp130 vermittelte Aktivierung des JAK/STAT Signalweges fehlt (α−MHC-Cre; gp130flox/JAK-STAT; JAK/STAT-KO), sowie der korrespondierende Wildtyp mit zwei funktionierenden gp130 Allelen (gp130flox/flox; 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.
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 GTP Guanosine Triphosphate
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
p90RSK p90 Ribosomal S6 Kinase PBS Phosphate Buffered Saline PI3-K Phosphatidylinositol 3-Kinase RACK Receptor for Activated C Kinase RIPA Radio Immunoprecipitation Assay RT-PCR Real Time Polymerase Chain Reaction SERCA Sarco-endoplasmic Reticulum Ca2+ -ATPase
SH Src Homology
SHP Src Homology Domain-2 Containing Tyrosine Phosphatase-1 Skm-α-actin Skeletal Muscle Alpha Actin
STAT Signal Transducers and Activators of Transcription TBST Tris Buffered Saline and Tween
TDI Tissue Doppler Imaging TGF Transforming Growth Factor
TIMP Tissue Inhibitor of Matrix Metalloproteinase TUNEL Terminal Transferase dUTP Nick End Labeling Tyk Tyrosine Kinase
α-MHC Alpha Myosin Heavy Chain β-MHC Beta Myosin Heavy Chain
List of Figures
Figure 1: Scheme of signal transduction pathways initiated by mechanical stretch in cardiomyocytes... 4
Figure 2: Roles of Ang II and other growth factors in stretch induced cardiac hypertrophy in vitro... 5
Figure 3: Mechanism of PKC activation... 10
Figure 4: Immunoblot showing the expression of PKCε in homozygous knockout, heterozygous knockout and wildtype mice... 15
Figure 5: H&E stained left ventricular sections of wildtype and knockout mice ... 15
Figure 6: H&E stained left ventricular sections of aortic banded mice ... 16
Figure 7: Real time analysis for expression of ANP and skm-α-actin... 17
Figure 8: Real time and immunoblot analysis for SERCA2a before and after banding... 18
Figure 9: Interstitial fibrosis in left ventricles of knockout mice after 4 weeks of transverse aortic banding ... 19
Figure 10: Immunoblot analysis for collagen Iα1 and collagen III before and after banding... 20
Figure 11: Real time analysis for the collagen Iα1 expression before and after banding ... 20
Figure 12: Tissue Doppler imaging of left ventricular function... 21
Figure 13: Expression and activation of PKCα and PKCβII before and after transverse aortic banding... 22
Figure 14: Expression and activation of PKCδ in left ventricles before and after banding ... 23
Figure 15: Expression and activation of p38, JNK and ERK1/2 in left ventricles before and after banding ... 24
Figure 16: Myocardial apoptosis in wildtype and knockout mice at baseline and after transverse aortic banding25 Figure 17: Effect of biomechanical stretch on expression of collagen Iα1 in cultured fibroblasts isolated from wildtype and knockout mice... 26
Figure 18: Effect of specific inhibitors on collagen expression induced by biomechanical stretch ... 27
Figure 19a: IL-6 related cytokines and their receptor complexes ... 37
Figure 20: Activation of intracellular signaling... 41
Figure 21: Schematic representation of the gp130 receptor system... 42
Figure 22: Generation of the gp130∆STAT/∆STAT mice ... 44
Figure 23: Targeting strategy for the introduction of the gp130Y757F knock-in mutation... 45
Figure 24: Crossing scheme to generate mice harbouring cardiomyocyte specific mutations in the gp130 receptor using Cre-loxP system ... 52
Figure 25: Genotyping of gp130-KO, gp130-dependent MEK/ERK-KO and JAK/STAT-KO mice ... 54
Figure 26: Tissue and cell specificity and deletion efficiency of gp130 in gp130-KO mice ... 56
Figure 27: Analysis of the phosphorylation state of STAT3, ERK1/2 and Akt in WT and gp130 mutant mice after LIF injection... 58
Figure 28: Immunoblots showing the activation pattern of STAT3, ERK1/2 and Akt, 24 hours after MI ... 59
Figure 29: Immunoblots showing the activation of STAT3 and ERK1/2 MAP kinase, 14 days after MI ... 60
Figure 30: Kaplan-Meier curve for post-MI mortality of WT and gp130 mutant mice ... 61
Figure 31: Infarct size in WT, gp130-KO and MEK/ERK-KO, 14 days post-MI... 63
Figure 32: Analysis of mRNA expression of α-MHC in gp130 mutant mice after MI ... 65
Figure 33: Expression analysis of ANP in gp130 mutant and WT mice after MI... 66
Figure 34: TUNEL assay after 14 days of MI ... 67
Figure 35: Immunoblot analysis for cleaved caspase-3 in the border zone of gp130 mutant and WT mice ... 68
Figure 36: Transverse Aortic Constriction (TAC) of murine heart... 81
List of Tables
Table 1 Baseline parameters and dimensions of the hearts of wildtype and PKCε knockout mice………... 14 Table 2 Echocardiographic and morphometric parameters in wildtype and PKCε knockout mice
after banding... 17 Table 3 Diastolic function assessement by echocardiography and TDI at baseline and after
banding in wildtype and PKCε knockout mice………. 21 Table 4 Nomenclature and phenotype description of WT and gp130 mutant mice………... 53 Table 5 Frequencies of various genotypes after crossing, confirming Mendelian distribution of
gp130 mutant mice……… 55 Table 6 Baseline echocardiographic and morphometric parameters of the hearts of 12 weeks old
male WT and gp130 mutant mice………..…... 57 Table 7 LV hemodynamic analysis of WT and gp130 mutant mice after 14 days of
MI…..………..…….. 62 Table 8 Morphometric analysis of WT and gp130 mutant mice after 14 days of
MI………..……… 64 Table 9 Overview of experimental strategy used in PKCε knockout mice based banding
study………...……… 77 Table 10 Overview of plan showing male mice used in gp130 based MI study……….. 79
1 Introduction (A)
1.1 Hypertrophic Remodeling of the Heart
Heart failure is one of the major causes of death worldwide and is the reason for at least20 percent of all hospital admissions among elderly population (Jessup and Brozena 2003). The understanding of the associations and intricate relationships of cardiomyopathies with gene function is still far from completion. There are several different types of cardiomyopathies, caused by viral infections, abnormal gene function, pathophysiological responses, immune reactions and ischemia. Physiological and pathophysiological cardiac responses may converge and point towards a common fate of heart failure. These responses lead to adaptation of cardiac muscles and the process of adaptation is called cardiac remodeling, which is the fundamental response to adverse conditions in the heart and leads to growth of cardiomyocytes and their adaptation. The hypertrophic growth of cardiomyocytes is initiated by endocrine, paracrine and autocrine factors as well as mechanical stretch, that stimulate a wide range of membrane-bound receptors (Molkentin and Dorn 2001). It is an early event during the clinical course of heart failure and an important risk factor for subsequent cardiac morbidity and mortality.
In response to a variety of mechanical, hemodynamic, hormonal and pathologic stimuli, the heart adapts to increased demands for cardiac work by increasing muscle mass through the initiation of hypertrophic responses. At the cellular level, cardiomyocytes respond to biomechanical stress by initiating several different processes leading to hypertrophy.
Physiologic hypertrophy as seen in athletes is associated with proportional increase in the length and width of cardiomyocytes. In contrast, the assembly of contractile-protein units in series characterizes the eccentric hypertrophy that occurs in patients with dilated cardiomyopathy with a relatively greater increase in the length than width of myocytes.
Whereas, in pressure overload, new contractile-protein units are assembled in parallel, resulting in a relative increase in width of cardiomyocyte and therefore concentric hypertrophy (Hunter and Chien 1999).
"Hypertrophy" in literary terms means “growth in size”, and cardiac hypertrophy is an outcome of possible extrinsic factors such as arterial hypertension or valvular heart disease, and/or intrinsic as in familial hypertrophic cardiomyopathy. It eventually normalizes the
hypertrophy are an increase in size, enhanced protein synthesis, and a higher organization of sarcomere. These changes in cellular phenotype are preceded and accompanied by the re- entry of cells into the so-called fetal gene programme. The hypertrophic process is not entirely beneficial. This notion is further supported by observations in several clinical trials, such as the HOPE trial (Yusuf, Sleight et al. 2000). This study showed that inhibition or regression of cardiac hypertrophy by certain drugs, such as angiotensin-converting enzymes (ACE) inhibitors, lowers the risk for several endpoints, including progression to heart failure and death; whereas persistent cardiac hypertrophy, notwithstanding similar blood pressure changes, predicts an adverse outcome (Mathew, Sleight et al. 2001).
1.2 Cellular and Molecular Response of Cardiomyocytes to Biomechanical Stretch
In vivo external load plays a critical role in determining muscle mass and phenotype in both, cardiac and skeletal muscles (Morgan and Baker 1991). Cardiomyocytes have the ability to sense mechanical stretch and convert it into intracellular growth signals leading to hypertrophy. Cultured skeletal muscle cells grown on elastic substrate have been shown to undergo an increase in protein synthesis in response to static stretch of the substrate (Vandenburgh and Kaufman 1979). Similarly, stretching adult or neonatal cardiomyocytes cultured in serum-free media by 10 to 20% above resting length causes an increase in protein synthesis without cell division (Mann, Kent et al. 1989; Komuro, Kaida et al. 1990;
Sadoshima, Jahn et al. 1992). This response clearly shows that skeletal muscle cells and cardiomyocyte are able to sense external load in the absence of neuronal and hormonal factors. Mechanical stimuli also cause rapid change in gene expression (Komuro, Kaida et al.
1990; Sadoshima and Izumo 1995). Linear stretch of cardiomyocytes in vitro causes transcriptional activation of immediate-early genes. This is followed by an induction of fetal genes: atrial natriuretic peptide (ANP), skeletal muscle α-actin and β-myosin heavy chain (β- MHC) (Sadoshima, Jahn et al. 1992). The phenotypic features of stretched myocytes are very similar to those of pressure overload induced hypertrophy in vivo (Izumo, Nadal-Ginard et al.
1988). Analysis of signal transduction pathways shows that stretch activates multiple signal pathways (Vandenburgh 1992; Sadoshima and Izumo 1993). Reports indicate that mechanical stretch has a close relationship with autocrine/paracrine growth factors in the heart such as the cardiac renin-angiotensin system (RAS) (Baker, Booz et al. 1992; Sadoshima, Xu et al. 1993).
induced hypertrophy in vivo. Treatment of rats having aortic constriction with ACE inhibitor or Ang II type I receptor antagonists prevented left ventricular hypertrophy by pressure overload. These results are consistent with the involvement of renin-angiotensin system and its activation by hemodynamic loading in vivo. Its role in vitro as a critical mediator of stretch induced hypertrophy has also been shown in neonatal rat cardiomyocytes. Ang II receptor antagonists inhibit major markers of stretch induced hypertrophy i.e., c-fos, MAP kinase, ANP and skeletal muscle α-actin. Ang II causes an increase in protein synthesis in adult rat heart in vivo or in isolated perfused heart independent of blood pressure (Sadoshima and Izumo 1995).
The mechanism involved in sensing pressure overload or stretch, and translating it into hypertrophic response is not well defined. Integrins may play an important role in sensing and mediating stretch reponses in cardiomyocytes. Integrins are heterodimeric transmembrane receptors that couple the extracellular matrix (ECM) with the actin cytoskeleton. The cytoplasmic domain of integrins physically associates with cytoskeletal elements. For example, the cytoplasmic domain of the β-integrin interacts with actin and actin binding proteins such as talin, α-actinin and with the amino terminal domain of focal adhesion kinase (FAK). FAK further interacts with various signaling molecules such as PI3-Kinase, Src, Grb2, which subsequently activate downstream targets like MAP kinases, PKC and Rho/Cdc42 molecules. However, the full mechanism of mechanotransduction, the way cardiomyocytes sense mechanical stretch and convert it into intracellular growth signals, is still not elucidated.
Figure 1: Scheme of signal transduction pathways initiated by mechanical stretch in cardiomyocytes Arrows show the directionality of signaling, where “?” indicates conjecture. Abbreviations: AA, arachidonic acid; Ang II, angiotensin II; AT1-R, angiotensin II type 1 receptor; DAG, diacylglycerol; ER, endoplasmic reticulum; IE gene, immediate-early gene; IP3, inositol-1,4,5-triphosphate; PA, phosphatidic acid; PC, phosphatidylcholine; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; PIP2, phosphatidylinositol bisphosphate; PI3-K, phosphatidylinositol 3-kinase; SRE, serum response element; SRF, serum response factor; SA channel, stretch activated channel; TCF, ternary complex factor (Sadoshima and Izumo 1997).
Figure 2: Roles of Ang II and other growth factors in stretch induced cardiac hypertrophy in vitro Solid arrows indicate experimentally defined processes. Broken arrows indicate unidentified effect by stretch.
In general, mechanical stretch induced signal transduction is characterized by simultaneous activation of multiple second messengers. In cultured myocytes, stretch induces activation of PLC, PLD and PLA2; MAP kinases and their activators; c-Jun N-terminal kinases, PKC, phosphatidylinositol (PI) within 1 min. The activation of the phosphatidylinositol pathway leads to the formation of IP3 (Inositol-1,4,5-triphosphate) and DAG (Diacylglycerol). IP3 causes calcium release from intracellular calcium storage sites and DAG activates PKC, which plays an important role in stretch induced expression of immediate-early genes such as c-fos and Egr-1.
Studies aiming at elucidating the potential roles of signaling proteins that link hypertrophy and pressure overload are still going on. The most obvious and perhaps most difficult question in this whole picture of cardiac hypertrophy and mechanotransduction is: “what are the stretch receptors and how they are connected to intracellular signaling network facilitating hypertrophic responses?” PKC and its downstream molecules have been implicated in cardiac hypertrophy. Whether deletion of the PKCε gene in mice leads to a model in which stretch induced hypertrophy develops or not, has been answered in this study.
1.3 Signal Transduction in Cardiac Hypertrophy
The primary stimulus for cardiac hypertrophy is mechanical stress accompanied by neural or humoral factors. However, since hypertrophy can be achieved by hemodynamic load even after blockade of neural or humoral factors suggests mechanical stress itself is the primary factor (Cooper, Kent et al. 1985). Myocardial hypertrophy is mediated by several signal transduction pathways. Growth factors or hormones may be indirectly involved in load induced hypertrophy. Neurohormones such as epinephrine, norepinephrine, Ang II and aldosterone have been identified as the most important in stimulating stress mediated or reactive cardiac hypertrophy contributing to progression of heart failure. These are associated with heterotrimeric GTP-binding proteins and additionally, MAPKs, PKCs, calcineurin/NFAT, gp130, IGF-1, FGF-2 and TGFβ are also known to play a major role in cardiac hypertrophy (Frey and Olson 2003). Molecular signaling involved in hypertrophy is more complicated with many parallel and redundant transducer and effector pathways.
Adaptive cardiac growth occurs as a feature of normal postnatal cardiac development or as the physiological hypertrophy resulting from exercise. Maladaptive hypertrophy develops in response to excessive hemodynamic load. If the stimulus is not removed, it can lead to decompensated hypertrophy and heart failure. Physiological hypertrophy is largely mediated by signaling through the peptide growth factors: IGF-1 (Volterrani, Manelli et al. 2000; Neri Serneri, Boddi et al. 2001) and growth hormone (Tanaka, Ryoke et al. 1998). When IGF-1, insulin and other growth factors bind to its receptors, distal activation of Akt occurs via PI3- Kinase. It is known that PI3-K/Akt signaling transduces adaptive hypertrophy. Studies show that constitutive activation of PI3-K in cardiomyocytes does not cause maladaptive hypertrophy, whereas cardiac specific overexpression of constitutively active Akt stimulates heart growth that may (Shioi, McMullen et al. 2002) or may not (Condorelli, Drusco et al.
2002; Matsui, Li et al. 2002) culminate in left ventricular decompensation. Akt is a signaling cascade branch point. One branch leads to mTOR (mammalian target of rapamycin) and protein synthetic machinery, which is essential for all forms of hypertrophy. The other branch leads to glycogen synthase kinase-3 (GSK-3), which also regulates the general protein translational machinery along with specific transcription factor targets implicated in both normal and pathologic hypertrophy. Unlike Akt, GSK-3β was the first to be identified as a negative regulator of cardiac hypertrophy, which blocked cardiomyocyte hypertrophy in
response to ET-1 and PE. It negatively regulates both normal and pathologic hypertrophy of the heart.
PKCs were identified as mediators for Gq-stimulated hypertrophy. PKCε was pinpointed to be activated in maladaptive and stress-mediated hypertrophy. PKCs have long been implicated in cell proliferation, survival and apoptosis. There are at least 12 different isoforms of PKC, and in cardiac tissue the expression of PKC differs with species, cell type and developmental stage. Studies using various strategies such as; gene ablation, transgenic overexpression, expression of dominant negative form of an isoform or overexpression of activators of PKC showed the redundancy of PKCs in cardiac hypertrophy. Previously PKCε was believed to be maladaptive, however, later studies showed that activation of PKCε in cardiac hypertrophy is a compensatory event. Activation of PKC leads to the activation of downstream effectors such as MEKK1-4 and Raf, which further activate JNK1/2/3 and ERK1/2 MAP kinases. A very important difference in signaling pathways activated in adaptive and maladaptive hypertrophy is the involvement of different MAPKs. MAPKs are divided in the (i) extracellular-regulated kinases (ERKs), (ii) c-jun N-terminal kinases (JNKs), and (iii) p38 MAPKs. In maladaptive hypertrophy, there is strong recruitment of p38 MAPKs and JNKs whereas in adaptive cardiac hypertrophy their recruitment is very weak. MAPKs have been shown to be involved in all the form of hypertrophy but their individual role in different settings is still obscure. Studies show that in the heart ERK1 is the most highly expressed ERK (Ruwhof and van der Laarse 2000). ERK pathway can be stimulated by hormones via G protein coupled receptors such as ET-1 (Bogoyevitch, Glennon et al. 1994), Ang II (Sadoshima and Izumo 1995) and also by receptors with intrinsic tyrosine kinase activity such as IGF-1. Mechanical stretch has also been reported to activate ERK, Ras and p90RSK (Sadoshima and Izumo 1993; Yamazaki, Tobe et al. 1993; Kudoh, Komuro et al.
1998). Stretching myocytes causes activation of ERKs and subsequent increased expression of c-fos and skeletal α-actin, indicating involvement of ERKs in hypertrophy (Yazaki and Komuro 1992; Sadoshima and Izumo 1993). JNKs may also play a role in mechanical stretch induced hypertrophy via phosphorylation of c-Jun and (activating transcription factor-2) ATF2 (Clerk and Sugden 1997). Activation of JNKs is independent of secreted Ang II, extracellular Ca2+ and PKC. These MAPKs are also activated on cytokine stimulation.
Cytokines of the IL-6 family signal via gp130 receptor and binding of these cytokines leads to activation of JAK/STAT, MAPK and PI3-K pathways. It is known that inhibiting specific central signaling pathways can attenuate the hypertrophic response, contrastingly, if the
pathway is hyperactivated, it may not guarantee hypertrophy, thereby giving us cues about the complicated network existing underneath complex situations, having partly normal and partly pathological nature (Molkentin and Dorn 2001).
1.3.1 PKC and Cardiac Hypertrophy
The PKC isoforms are a family of ubiquitous lipid-binding serine-threonine kinases, acting downstream of virtually all membrane-associated signal transduction pathways (Nishizuka 1986). They are predominantly activated by Gq/G11-coupled receptors. Multiple studies have implicated the various PKC isoforms in the pathogenesis of cardiac hypertrophy. PKC family consists of more than 10 isoenzymes encoded by different genes; exhibiting distinct patterns of tissue-specific expression and agonist-mediated activation. Based on enzymatic properties, PKC isoforms are classified into three families; (i) conventional or calcium dependent (cPKC:
PKCα, PKCβ1, PKCβ2 and PKCγ), (ii) novel or calcium independent (nPKC: PKCδ, PKCε, PKCη and PKCθ) and (iii) atypical (aPKC: PKCζ, PKCλ and PKCτ) (Puceat and Vassort 1996; Newton 1997). The third family of PKC, also called atypical PKCs, is activated by lipids other than diacylglycerol. Thus, these three families of PKCs comprise many sub- families, each of which may contain multiple isoforms derived from distinct genetic loci. An important feature of PKC isoforms is that, when activated, they translocate to distinct sub- cellular sites (Molkentin and Dorn 2001). Alpha adrenergic receptor stimulation of cultured rat cardiomyocytes is associated with translocation of PKCβ1 from cytosol to nucleus, PKCβII from fibrillar structures to perinuclear space and sarcolemma, PKCε from nucleus and cytosol to myofibrils and PKCδ to the perinuclear region (Molkentin and Dorn 2001).
The differential sub-cellular compartmentalization of activated PKC isoforms implies distinct substrate and therefore unique cellular function for each isoform (Hug and Sarre 1993). The mechanism of sub-cellular translocation and activation of PKC isoforms involves binding to anchoring proteins termed RACKs (receptors for activated C kinases) (Mochly-Rosen 1995).
Each PKC isoform or group of related isoforms binds to a specific RACK for activation.
Activated PKCs are known to modulate transcription factors such as c-jun, c-fos and STATs, voltage dependent calcium channels and myofilament proteins such as troponin-I and -T.
Inactive PKC exists in a conformation where the active sites for substrates are not exposed. In the presence of phospholipids or calcium (depending upon the PKC isoform), it changes the conformation and exposes the substrate and RACK-binding sites, facilitating activation. By
interfering RACK-binding via inhibiting or promoting peptides, PKC inactivation or activation, respectively, is possible (Ron and Mochly-Rosen 1995; Johnson, Gray et al. 1996;
Gray, Karliner et al. 1997).
In cardiac tissue, PKC enzymatic activity is increased after ischemia and acute or chronic pressure overload, where it is postulated to mediate ischemic pre-conditioning and transduce hypertrophic signaling, respectively (Molkentin and Dorn 2001). Pressure overload or mechanical stretch rapidly induces growth factors like Ang II and ET-1. Multiple studies implicate the various PKC isoforms in the pathogenesis of cardiac hypertrophy. Transgenic overexpression of PKCβ is sufficient to elicit cardiac hypertrophy and sudden death (Bowman, Steinberg et al. 1997), but not required for cardiac hypertrophy after pressure overload, whereas, PKCα is both required and sufficient for cardiomyocyte hypertrophy in vitro (Braz, Bueno et al. 2002). Phorbol esters such as PMA (Phorbol-12-myristate-13- acetate) activate PKC resulting in myocyte hypertrophy (Frey and Olson 2003).
The mechanisms of activation of PKC isoforms in cardiomyocytes have been largely pioneered by Mochly-Rosen. Accordingly, PKCε V1 fragment (144 amino acids) or the eight- amino-acid PKCε RACK-binding site peptide was shown to attenuate PMA induced or norepinephrine-dependent negative chronotropy, and prevented ischemic preconditioning in cultured neonatal cardiomyocytes (Johnson, Gray et al. 1996; Gray, Karliner et al. 1997).
Furthermore, in cardiomyocytes specific activation of PKCε with octapeptide pseudo-RACK peptide protected cardiomyocytes from ischemic damage (Dorn, Souroujon et al. 1999). It has been observed that PKCε is selectively translocated from cytosol to the particulate fractions in cardiomyocytes during acute or chronic pressure overload (Gu and Bishop 1994; Paul, Ball et al. 1997; De Windt, Lim et al. 2000), and also after Ang II stimulation. Many reports have shown the existence of several associations between PKC activity and different pathological cardiac responses, suggesting that PKC signaling contributes mechanistically to these events, however, gain- and loss-of-function studies are necessary for causality to be established.
Figure 3: Mechanism of PKC activation
The pseudo-RACK-binding site normally holds PKC factors in an inactive conformation unless stimulated. Once stimulated by diacylglycerol or calcium, PKC changes conformation, allowing interaction with RACK domain- containing proteins and exposure of the active site (Molkentin and Dorn 2001).
Mochly-Rosen and Dorn have used transgenic techniques to express PKCε-activating and - inhibiting peptides in the mouse heart, where it was shown that increasing basal translocation of PKCε by about 20% was enough to exert a strong protective effect on cardiac contractile function and integrity of myocytes in isolated hearts subjected to ischemia and reperfusion (Mochly-Rosen, Wu et al. 2000; Inagaki, Hahn et al. 2003). Later, it was reported that PKCε activation causes a physiological form of hypertrophy, whereas, inhibition (by inhibiting translocation of PKCε using RACK-binding peptide; εV1) resulted in the opposite response, i.e., thinning of ventricular walls and heart failure due to dilated cardiomyopathy (Mochly- Rosen, Wu et al. 2000).
1.3.2 PKC Redundancy in Cardiac Hypertrophy
The family of PKC is divided in many sub-families. A rational view about the roles of protein kinases is that each of the isoforms has a distinct role in cellular regulation. The alternate view is redundancy, which leads to the formation of cobweb like complicated network inside the cell. It is quite well known that in cardiac hypertrophy PKCs are involved but differentiation of individual role of each isoform has been a difficult task for many years.
Now, one thing has become clear and unequivocal, PKC isoforms have propensity for shared activation stimuli and common substrate specificities. Altering one PKC isoform almost invariably has consequences for the activation /expression/ localization of other family
(Steinberg and Sussman 2005). Cardiomyocytes co-express multiple PKC isoforms (Sabri and Steinberg 2003), and each isoform mediates specific effects in the heart. Their activity depends on expression level, localization within the cell and phosphorylation state (Malhotra, Kang et al. 2001). Several reports have shown that a particular PKC isoform, when activated or overexpressed causes hypertrophy e.g., PKCβ overexpression leads to hypertrophy of the heart (Wakasaki, Koya et al. 1997; Takeishi, Chu et al. 1998), while systemic deletion of the same has no visible effects (Roman, Geenen et al. 2001). Similarly, studies using adenovirus based transfection and inhibition in cardiomyocytes revealed that, PKCα was sufficient to induce cell hypertrophy and inhibition of only PKCα isoform inhibited agonist mediated hypertrophy (Braz, Bueno et al. 2002). However, an in vivo analysis of PKCα effects in the mouse heart utilizing gene ablation and transgenic overexpression revealed no effect of PKCα overexpression on cardiac growth and no effect of PKCα inhibition on the hypertrophic response to pressure overload (Braz, Gregory et al. 2004). Instead, ablation of PKCα improved contractility, while overexpression diminished it. Furthermore, a study in the hypertensive rats showed differential roles of PKCs in the cardiac hypertrophy (Johnsen, Kacimi et al. 2005). Thus these types of ambiguities in PKCs roles lead us to speculate the redundancy amongst different PKC isoforms and coordinated orchestra played by them.
1.4 PKCε Knockout Mouse to Study Cardiac Hypertrophy
The mouse represents an appropriate model to study the function of genes and proteins, which have been implicated in human pathophysiological conditions. There are mice available, in which, specific PKC isoforms are overexpressed or knocked-out. These mice have provided an insight into the mechanisms of cardiac hypertrophy and involvement of protein kinases. In mice, cardiac hypertrophy has been extensively studied, either by the use of drugs such as Ang II and phenylephrine, which induces cardiac hypertrophy by activation of genes involved in protein biosynthesis, activation of pro-hypertrophic factors (Calcineurin, STAT and MAP kinases) (Mascareno, Dhar et al. 1998; Kodama, Fukuda et al. 2000; Booz, Day et al. 2002) or using interventional methods like transverse aortic constriction (TAC or Banding). Mochly- Rosen has shown that activation of PKCε signaling stimulates physiological growth of cardiomyocytes and is compensatory in nature, whereas postnatally inhibiting translocation of PKCε leads to lethal dilated cardiomyopathy (Wu, Toyokawa et al. 2000). Therefore, we focused on PKCε and studied whether PKCε is important for the development of cardiac hypertrophy in the situation of pressure overload. For this purpose PKCε-/- mice of C57BL/6
× 129O1a background were used. These were kindly provided by Dr. M. Leitges (Department of Experimental Endocrinology, Max-Planck-Institute, Hannover, Germany). In brief, E14 ES cells (129/O1a) were used for targeting experiment following the standard procedures of the gene-targeting approach in mouse. Homologue-recombined ES cell clones were then introduced by injection into C57BL/6 blastocysts. The possible germ line transmission of injected ES cells was identified by crossing the observed chimeric males to C57BL/6 females and subsequently the presence of agouti coat color in the F1 progeny. F1 heterozygote breeding gave rise to homozygous animals that were finally used in this study and corresponded to the hybrid (C57BL/6 × 129O1a) background. Although adult mice have a heart rate of about 600 beats per minute and an aorta that is 1.0 - 1.2 mm in diameter, they are a valid model system to study both pressure overload induced hypertrophy and heart failure.
One of the most commonly used surgical intervention for pressure overload induced hypertrophy is coarctation of the ascending aorta i.e. aortic banding (Figure 36). This system has been very well characterized and proven to be highly reproducible with a low mortality rate of 10-20% or less in experienced hands. Aortic banding in mice is an excellent model system to evaluate the process of development of left ventricular hypertrophy in response to hemodynamic stress.
2 Aim of the Study (A)
The aim of the first part of my thesis was to evaluate the role of PKCε in cardiac hypertrophy and remodeling following pressure overload.
More specifically, I evaluated the cardiac phenotype of mice with a systemic knockout for PKCε in reponse to transverse aortic constriction.
3 Results (Part A)
3.1 Cardiac Phenotype of PKCε Knockout Mice
The primary goal of this part of the work was to determine the role of PKCε in the heart after pressure overload. To examine this, systemic knockout mice were chosen.
Systemic knockout (PKCε-KO) mice were born in the expected Mendelian ratio and did not display any morphological abnormalities in the heart. These mice had normal heart weight, body weight and heart weight to body weight ratio compared with the wildtype (WT) mice.
They showed normal survival and no signs of any functional impairment. Echocardiographic analysis showed normal cardiac function (Table 1).
Parameter WT (Baseline) KO (Baseline)
Heart weight (mg) 114 ± 17 116 ± 27
HW/BW (mg/g) 4.6 ± 0.6 4.7 ± 0.7
LV/BW (mg/g) 3.5 ± 0.5 3.5 ± 0.7
Age (weeks) 12 ± 2 12 ± 2
SBP (mmHg) 110 ± 14 115 ± 10
LVEDD (mm) 4.2 ± 0.1 4.2 ± 0.1
LVESD (mm) 2.9 ± 0.1 2.8 ± 0.1
LVAWD (mm) 0.70 ± 0.03 0.70 ± 0.04
LVPWD (mm) 0.50 ± 0.03 0.60 ± 0.03
FS (%) 32.0 ± 1.5 34.0 ± 2.1
CSA (µm2) 144.5 ± 7.7 140.7 ± 14.2
Table 1: Table shows the baseline parameters and dimensions of the heart of wildtype and PKC knockout mice.
HW/BW, heart weight to body weight ratio; LV/BW, left ventricle weight to body weight ratio; SBP, systolic blood pressure; LVEDD, left ventricle end-diastolic diameter; LVESD, left ventricle end-systolic diameter;
LVAWD, left ventricle anterior wall dimension; LVPWD, left ventricle posterior wall dimension; FS, fractional shortening; CSA, septal myocyte cross sectional area.
The next objective was to check the expression of PKCε in homozygous knockout, heterozygous knockout and wildtype mice. Expression of PKCε in the heart was absent in mutant mice, whereas heterozygous (PKCε+/-) mice expressed half of the amount of protein compared with the wildtype mice (Figure 4).
PKCε
Figure 4: Immunoblot showing the expression of PKCε in homozygous knockout, heterozygous knockout and wildtype mice
Tissues were harvested at the age of 12±2 weeks of age and SDS-PAGE was performed. The blots were probed with antibodies against PKCε. Wildtype has maximum band density indicating normal amount of PKCε, heterozygous mice showed diminished amounts whereas, no signals were observed in homozygous knockout mice.
Morphometric analysis of in situ fixed, H&E-stained heart sections showed normal and similar cardiac dimensions and cardiomyocyte cross sectional area in both knockout and wildtype mice (Table 1 and Figure 5).
Figure 5: H&E stained left ventricular sections of wildtype and knockout mice
Male mice were sacrificed without any intervention at the age of 12±2 weeks and in situ fixed. Left ventricular sections were stained with haematoxylin-eosin. No differences were seen concerning septum thickness, LV inner and outer diameters and cross sectional area of myocytes amongst two groups. Bar = 1mm
Homozygous Heterozygous WT
WT KO
WT KO
3.2 Pressure Overload Triggered Similar Hypertrophic Responses in Knockout and Wildtype Mice
Transverse aortic constriction produces pressure overload due to stenosis or narrowing of the aorta. This consequently increases the workload on the cardiac muscles thereby triggering them to grow.
After 4 weeks of aortic constriction, knockout and wildtype mice showed a similar rise in pre- stenotic blood pressure as measured by MILLAR pressure volume catheter transducer and a similar amount of myocardial hypertrophy assessed by left ventricular weight to body weight ratio, echocardiography and morphometric measurements (Table 2 and Figure 6).
Conventional cardiac hypertrophy markers such as atrial natriuretic peptide (ANP) and skeletal muscle α-actin (skm-α-actin) were also increased significantly in left ventricles of both, knockout and wildtype mice compared with the non-banded mice (Figure 7). Within banded groups the induction of ANP was also comparable (ns). Sarco-endoplasmic reticulum Ca2+ATPase2a (SERCA2a), which is an important regulator of calcium signaling in myocytes and linked to the progression of cardiac hypertrophy, was unchanged between knockout and wildtype mice as assessed by mRNA, and protein levels were also unaffected after banding (Figure 8).
Figure 6: H&E stained left ventricular sections of aortic banded mice
Banding was performed on 12±2 weeks old male mice and kept under observation for 4 weeks. After 4 weeks the hearts were in situ fixed and from paraffin embedded tissue serial sections were obtained. No differences were observed concerning hypertrophic growth in wildtype and knockout mice. Bar = 1mm.
WT KO
WT KO
Parameter WT-TAC KO-TAC
Heart Weight (mg) 233 ± 38* 189 ± 31*
Systolic Blood Pressure (mmHg) 168 ± 12* 178 ± 9*
HV/BW (mg/g) 6.2 ± 1.2* 6.1 ± 1.3*
LV/BW (mg/g) 5.7 ± 0.9* 5.4 ± 0.8*
LVEDD (mm) 4.5 ± 0.2 4.4 ± 0.2
LVESD (mm) 3.1 ± 0.2 3.0 ± 0.1
LVAWD (mm) 1.00 ± 0.05 1.10 ± 0.10
LVPWD (mm) 0.80 ± 0.08* 0.90 ± 0.13*
CSA (µm2) 476.7 ± 27.3* 517.1 ± 65.5*
Table 2: Echocardiographic and morphometric parameters in wildtype and PKCε knockout mice after transverse aortic banding. Mice were banded and kept for 4 weeks and later they underwent echocardiographic measurements and/or in situ fixed for morphometric analysis. *P<0.05 compared with respective baseline mice (Table 1).
A B
Atrial Natriuretic Peptide (ANP) Skm-α-actin
Figure 7: Real time analysis for expression of ANP and skm-α-actin
Bar graphs depict gene expression as assessed by RT-PCR for (A) ANP and (B) skeletal muscle α-actin, 4 weeks after aortic banding. Both ANP and skm-α-actin were upregulated after 4 weeks of banding in similar fashion in wildtype and knockout mice.
* P<0.05
SERCA2a
Figure 8: Real time and immunoblot analysis for SERCA2a before and after banding
Bar graphs depicts gene expression as assessed by RT-PCR for Sarco-endoplasmic reticulum Ca2+ATPase2a (SERCA2a), 4 weeks after aortic banding. Immunoblot analysis for the same is shown on the right side. For loading controls the blots were probed with antibodies against sarcomeric actin. No differences were observed in the expression of SERCA2a in the myocardium, before and after banding in wildtype and knockout mice.
relative expressionrelative expression
3.3 Pressure Overload Increased Collagen Expression in Knockout Mice
Interstitial matrix deposition, as analyzed by three independent experiments, was found to be higher in knockout mice after pressure overload. Picro-Sirius Red staining of the paraffin sections showed increased deposition of collagen I and collagen III in the ventricles of knockout mice compared with the wildtype mice after pressure overload (Figure 9).
Immunoblots probed with antibodies against collagen Iα1 and collagen III showed increase in expression of both col Iα1 and col III in knockout compared with the wildtype mice after pressure overload (Figure 10). Another approach to confirm our finding was real time PCR for col Iα1, which also confirmed that col Iα1 was upregulated at transcriptional level in knockout mice after pressure overload (Figure 11).
Figure 9: Interstitial fibrosis in left ventricles of knockout mice after 4 weeks of transverse aortic banding Left panels show bright field photomicrograph of Picro-Sirius Red stained LV sections of wildtype compared with the knockout mice. The collagen appears red in bright field after Picro-Sirius Red staining. Right panels show the same in polarized light, where collagen III is green and collagen I is yellow. Bar graphs summarizing the quantification of interstitial fibrosis from wildtype (n = 5) and knockout (n = 5) at baseline and 4 weeks of transverse aortic banding in percent of circumferential area.
* P<0.05
Figure 10: Immunoblot analysis for collagen Iα1 and collagen III before and after banding
Bar graphs showing quantification of immunoblots for the expression of col Iα1 and col III in wildtype and knockout mice left ventricles (n = 4 for each group). Bands were quantified densitometrically and plotted after normalization with GAPDH. Knockout mice show significant increase in expression of both, collagen Iα1 and collagen III after 4 weeks of aortic banding.
Figure 11: Real time analysis for the collagen Iα1 expression before and after banding
Bar graph depicts the real time PCR expression analysis for increase of collagen Iα1 mRNA in left ventricles of wildtype and knockout mice (n = 4 to 6 in each group). GAPDH was taken for normalization of gene expression.
Knockout mice showed increased amounts of mRNA for collagen Iα 1 after 4 weeks of aortic banding.
* P<0.05
* P<0.05
3.4 PKCε Knockout Mice Show Diastolic Dysfunction after Pressure Overload
Non-invasive investigation of the left ventricular function done by echocardiography showed that knockout and wildtype mice have similar and preserved systolic function after 4 weeks of pressure overload (Table 3). However, the blood flow pattern at mitral valve, as revealed by the tissue Doppler imaging (TDI) in terms of Ea/Aa ratio (passive to active flow ratio) were altered and significantly reduced in knockout mice compared with the wildtype mice after aortic banding (Figure 12), thereby indicating the development of diastolic dysfunction in knockout mice.
Parameter WT (Baseline) KO (Baseline) WT-TAC (n=6) KO-TAC
E (m/s) 1.4 ± 0.3 1.1 ± 0.1 1.3 ± 0.4 1.2 ± 0.4
A (m/s) 0.5 ± 0.2 0.5 ± 0.2 0.5 ± 0.1 0.8 ± 0.2 *
E/A 2.6 ± 0.2 2.3 ± 0.1 2.5 ± 0.3 1.6 ± 0.2 *§
Ea (m/s) 3.1 ± 0.3 2.7 ± 0.3 3.9 ± 0.1 2.3 ± 0.1 * Aa (m/s) 1.5 ± 0.2 1.3 ± 0.1 2.0 ± 0.4 2.3 ± 0.1 *
Ea/Aa 2.2 ± 0.1 2.1 ± 0.3 2.0 ± 0.2 1.0 ± 0.2 *§
Table 3: Table shows diastolic function assessed by echocardiography and TDI (Tissue Doppler Imaging) at baseline and after 4 weeks of transverse aortic banding in wildtype and knockout mice. E and A denotes velocity of blood flow measured by echocardiography at mitral valve in early diastole and atrial contraction, respectively.
Ea and Aa are early diastolic and late diastolic blood flow velocities, respectively, measured by TD at mitral annulus.*P<0.05 baseline vs banded. §P<0.05 KO-TAC vs WT-TAC.
Figure 12: Tissue Doppler imaging of left ventricular function
Representative photographs of Tissue Doppler Imaging (TDI) at the posterior left ventricular wall showing diastolic dysfunction in knockout mice compared with the wildtype after 4 weeks of aortic banding. Ea indicates tissue imaging of early diastolic myocardial velocity and Aa is TDI of late diastolic myocardial velocity.
3.5 Upregulation of PKCδ in PKCε Knockout Mice after Pressure Overload
PKC isoforms are divided into classical, novel, typical and atypical isoforms. In the heart, the main PKC isoforms expressed are PKCα, PKCβII, PKCδ and PKCε. It is plausible to investigate the level of expression of other isoforms in PKCε knockout mice after pressure overload. In order to test whether the lack of PKCε modifies or alters the expression of other PKC isoforms, we analyzed protein levels and activation patterns of PKCα, PKCβII and PKCδ in left ventricles of knockout and wildtype mice. Levels of PKCα and PKCβII isoforms were not different in wildtype and knockout mice before and after aortic banding (Figure 13), but the levels of total PKCδ and phospho-PKCδ (Thr-505 phosphorylation) were increased to the same extent (two-fold) in knockout mice compared with the the wildtype after aortic banding (Figure 14), this indicates that higher activation may be because of increased total content of PKCδ in the tissue.
Figure 13: Expression and activation of PKCα and PKCβII before and after transverse aortic banding Blots were first probed with antibodies against activated forms of PKCα and PKCβII. Blots were stripped and re-probed for non-phosphorylated forms. Bands were measured densitometrically and graphs were plotted. There were no differences among non-phosphorylated and phosphorylated forms of PKCα and PKCβII proteins, before and after aortic banding.
