FUNCTIONAL, STRUCTURAL AND MOLECULAR ALTERATIONS IN THE HEART AND KIDNEY ...

259 Pages·2016·6.83 MB·English

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FUNCTIONAL, STRUCTURAL AND MOLECULAR
ALTERATIONS IN THE HEART AND KIDNEY
DURING DIABETES MELLITUS

By

TEHREEM WAQAR

A thesis submitted in partial fulfilment for the requirements for the degree
of Doctor of Philosophy at the University of Central Lancashire

February 2016

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STUDENT DECLARATION FORM

Concurrent registration for two or more academic awards

I declare that while registered as a candidate for the research degree, I have not been a
registered candidate or enrolled student for another award of the University or other
academic or professional institution

Signature of Candidate _________________________________________

Type of Award ___________________________________________

School ___________________________________________

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DECLARATION I
DECLARATION

I declare that I have not been an enrolled or registered candidate at any other academic
or professional institution while being registered for the research degree at University of
Central Lancashire. Furthermore, no material contained in this thesis has been utilised
in any other submission for an academic award and is solely my own work.

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ABSTRACT II
ABSTRACT

Background: Diabetes mellitus (DM) is a major metabolic disorder leading to severe
long term complications including cardiomyopathy, nephropathy, retinopathy and
neuropathy that are common in type 1 DM (T1DM) and type 2 DM (T2DM).
Epidemiological studies have demonstrated a role of hyperglycaemia (HG) in eliciting
adverse cardiac and renal outcomes including heart failure (HF), diastolic and renal
dysfunction. This study investigated the effect of HG on left ventricle (LV) and kidney
structural remodelling, function and underlying molecular events associated with the
two organs over a period of 2 and 4 months compared to age-matched control.
Methods: Molecular mechanisms underlying HG-induced remodelling changes
including extracellular matrix (ECM) and myocyte apoptosis deposition, underlying
cytokine induction, recapitulation of foetal genes, and transcriptional alterations that
may influence the ECM and intracellular calcium [Ca2+] handling in the LV and kidney
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of T1DM as well as T2DM were examined in this study. LV and kidney isolations
following 2 and 4 months of the development of T1DM were used to assess the
remodelling changes and underlying transforming growth factor β1 (TGFβ1) activity,
gene expression profile of the ECM and calcium mediators using histological,
immunohistochemical and quantitative gene expression analyses compared to age-
matched Wistar control rats.

Results: The results show that T1DM over 4 months can elicit severe structural and
molecular changes in the LV and the kidney compared to 2 months of DM. The severity
of these changes was significantly less in respective healthy age-matched control
animals. The isolated ventricular cardiomyocytes from T1DM rats displayed altered
cellular calcium (Ca2+) homeostasis and [Ca2+]i translating to alterations in mRNA
abundance of key Ca2+ handling proteins, cardiac sarcoplasmic reticulum Ca2+ATPase
2a (SERCA2a), ryanodine receptor (RyR2), Na2+/Ca2+ exchanger, phospholamban (Plb),
L-type Ca2+ channel proteins (Cav1.2 and Cav1.3), calmodulin2 (Calm2) and
Ca2+/calmodulin-dependant protein kinase II delta (CaMK2d) were significantly
(p<0.05) altered in DM compared to age-matched control animals. The results showed
LV and kidney remodelling in the T1DM rats with increased ECM deposition that
translated into increased gene expressions of key components (collagen 1α, collagen 3α,
fibronectin and elastin) and modulators i.e. MMP2 and MMP9 and their tissue inhibitor
(TIMP4), connective tissue growth factor (CTGF), integrin 5α and connexin 43 (Cx43)
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ABSTRACT II
of the ECM. Molecular derangements underlying this phenotype included increased
TGFβ1 transcription and activity, recapitulation of foetal gene phenotype atrial
natriuretic peptide (ANP), brain natriuretic peptide (BNP) with marked hypertrophy,
underscored by caspase-3 mediated cell apoptosis. Electron microscopic analysis
revealed ultrastructural alterations in LV highlighted by increased mitochondrial
number and altered mitochondrial population, whereas the kidney presented with
increase glomerular basement membrane thickness in T1DM compared to controls.
These data clearly show that adult vs young adult, in combination with STZ-induced
T1DM, can elicit severe changes to both the heart and the kidney, respectively in
structural, functional and biochemical alterations. The final part of the study revealed
exercise training after 2-3 months may have beneficial effects in T2DM animals
compared to sedentary control rats. Ventricular myocyte and shortening were generally
well preserved despite alterations in mRNA gene expression encoding a variety of
cardiac muscle proteins in the exercised trained adult GK diabetic rat. LV remodelling
in GK rat presented with marked hypertrophy of cardiomyocytes and increased ECM
deposition that altogether translated into increased ECM components and regulators
which were reversed by exercise training.

Conclusions: The present results have demonstrated that T1DM, if left untreated, can
lead to severe changes to both the heart and the kidney. These changes seem to occur at
structural and molecular levels leading to dysfunction of the heart and kidney and the
severity of the damage is enhanced over time. Data suggests that diabetic
cardiomyopathy (DCM) may have possible origins in pro-fibrotic and pro-hypertrophic
mechanisms. Moreover, this study demonstrates that physical exercise training
continues to be one of the most valuable forms of non-pharmacological therapy in DM.
Data concerning molecular signalling cascades and ECM phenotype is particularly
significant as targeting features of structural remodelling may delay onset and severity
of myocardial and renal complications.

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TABLE OF CONTENTS III
TABLE OF CONTENTS

DECLARATION----------------------------------------------------------------------I
ABSTRACT----------------------------------------------------------------------------II
TABLE OF CONTENTS------------------------------------------------------------III
LIST OF TABLES AND FIGURES-----------------------------------------------IV
ACKNOWLEDGEMENTS ---------------------------------------------------------V
ABBREVIATIONS--------------------------------------------------------------------VI

1. GENERAL INTRODUCTION
1.1 The functional anatomy of the heart -------------------------------------------------------1
1.1.2 The extracellular matrix ------------------------------------------------------------3
1.1.3 The cardiomyocyte ------------------------------------------------------------------7
1.1.4 Excitation contraction coupling (ECC) of the normal heart ------------------12
1.1.5 Mechanical events of the cardiac cycle------------------------------------------15

1.2 Heart failure-----------------------------------------------------------------------------------17
1.2.1 Pathophysiology of the failing heart ---------------------------------------------19
1.2.2 Cardiac structural remodelling----------------------------------------------------20
1.2.3 Molecular contributions in cardiac structural remodelling------------------- 23
1.2.4 TGFβ1 in cardiac remodelling------------------------------------------------25
1.2.5 Inflammation in HF and diabetes-------------------------------------------------27

1.3 Diabetes Mellitus-----------------------------------------------------------------------------29
1.3.1 Importance of studying diabetes mellitus -------------------------------------- 29
1.3.2 Diabetes mellitus classification --------------------------------------------------30
1.3.3 Cardiovascular disease in diabetes mellitus------------------------------------31
1.3.4 Diabetic cardiomyopathy in heart failure----------------------------------------32
1.3.5 Hyperglycemia and heart failure--------------------------------------------------34
1.3.6 Role of HG and its biochemical pathways in contractile dysfunction-------36

1.4 The Kidney------------------------------------------------------------------------------------39
1.4.1 Diabetic Nephropathy--------------------------------------------------------------42
1.4.2 Kidney and heart Failure----------------------------------------------------------44
1.5 Animal models used in this study ---------------------------------------------------------44
1.5.1 Aims and scope of the study-------------------------------------------------------47
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TABLE OF CONTENTS III
2. MATERIALS AND METHODS
2.1 Materials -----------------------------------------------------------------------------------49
2.2 Investigational design --------------------------------------------------------------------50
2.3 Experimental models---------------------------------------------------------------------50
2.3.1 Induction of diabetes------------------------------------------------------------50
2.3.2 GK model and exercise training-----------------------------------------------51
2.4 Sample collection-------------------------------------------------------------------------51
2.5 Histological studies-----------------------------------------------------------------------53
2.5.1 Immunohistochemistry---------------------------------------------------------54
2.5.2 Electron microscopy------------------------------------------------------------55
2.5.3 Immunofluorescence and confocal laser scanning microscopy-----------57

2.6 mRNA quantification by Quantitative Reverse transcriptase Polymerase chain
reaction…--------------------------------------------------------------------------------------59
2.6.1 mRNA isolation and generation of cDNA-----------------------------------59
2.6.2 Quantitative PCR ---------------------------------------------------------------61
2.6.3 Gene expression analyses------------------------------------------------------64
2.7 Functional studies in the STZ-induced type 1 diabetic rat and GK study
subgroups
2.7.1 Ventricular myocyte isolation -------------------------------------------------65
2.7.2 Measurement of shortening in ventricular myocytes-----------------------68
2.7.3 Measurement of intracellular calcium transients [Ca2+] -------------------68
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2.8 Transforming growth factor β1 Immunoassay----------------------------------------69
2.9 Statistical analysis ------------------------------------------------------------------------73

3. ASSESSMENT OF GENERAL CHARACTERISTICS, CARDIOMYOCYTE
CONTRACTILE FUNCTION AND CALCIUM HOMEOSTSTAIS IN THE STZ-
INDUCED TYPE 1 DIABETIC RAT
3.1 Abstract-------------------------------------------------------------------------------------74
3.2 Introduction--------------------------------------------------------------------------------75
3.3 Methods------------------------------------------------------------------------------------76
3.4 Results--------------------------------------------------------------------------------------77
3.5 Discussion----------------------------------------------------------------------------------83

4. STRUCTURAL AND MOLECULAR LEFT VENTRICLE REMODELLING
ASSOCIATED WITH HYPERGLYCAEMIA IN THE STZ-INDUCED TYPE 1
DIABETIC RAT
4.1 Abstract-------------------------------------------------------------------------------------88
4.2 Introduction--------------------------------------------------------------------------------89
4.3 Methods------------------------------------------------------------------------------------90
4.4 Results--------------------------------------------------------------------------------------91
4.5 Discussion--------------------------------------------------------------------------------109
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TABLE OF CONTENTS III
5. STRUCTURAL REMODELLING IN THE LEFT KIDNEY OF STZ-
INDUCED TYPE 1 DIABETIC RAT

5.1 Abstract-----------------------------------------------------------------------------------119
5.2 Introduction------------------------------------------------------------------------------120
5.3 Methods-----------------------------------------------------------------------------------121
5.4 Results------------------------------------------------------------------------------------122
5.5 Discussion -------------------------------------------------------------------------------137

6. A COMPARITIVE STUDY SHOWING THE STRUCTURAL AND
MOLECULAR CHANGES OBSERVED IN THE HEART AND KIDNEY IN
STZ-INDUCED TYPE 1 DIABETIC RAT

6.1 Introduction------------------------------------------------------------------------------146
6.2 Methods-----------------------------------------------------------------------------------147
6.3 Results------------------------------------------------------------------------------------148
6.4 Discussion--------------------------------------------------------------------------------160

7. EFFECT OF EXERCISE TRAINING ON LEFT VENTRICLE
CARDIOMYOCYTE CONTRATILE FUNCTION AND STRUCTURAL
REMODELLING IN THE GOTO-KAKIZAKI TYPE 2 DIABETIC RAT

7.1 Abstract-----------------------------------------------------------------------------------163
7.2 Introduction------------------------------------------------------------------------------164
7.3 Methods-----------------------------------------------------------------------------------165
7.4 Results------------------------------------------------------------------------------------166
7.5 Discussion--------------------------------------------------------------------------------181

8. GENERAL DISCUSSION

8.1 Discussion--------------------------------------------------------------------------------188
8.2 Limitation of the study------------------------------------------------------------------192
8.3 Conclusion -------------------------------------------------------------------------------194
8.3 Scope for Future studies ---------------------------------------------------------------197

BIBLIOGRAPHY------------------------------------------------------------------------------VII
COMMUNICATIONS-------------------------------------------------------------------------IX
PUBLICATIONS--------------------------------------------------------------------------------IX

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LIST OF TABLES AND FIGURES IV
LIST OF TABLES

CHAPTER 2
Table 2.1: Commercially available primers used in the study
CHAPTER 3
Table 3.1: Heart- General characteristics of rats at 2 and 4 months post STZ-
administration
CHAPTER 5
Table 5.1: Kidney- General characteristics of rats at 2 and 4 months post STZ-
administration
CHAPTER 7
Table 7.1: General Glucometry and gravimetry characteristics

LIST OF FIGURES

CHAPTER 1
Figure 1.1: The mammalian heart
Figure 1.2: Components of the extracellular membrane
Figure 1.3: Cellular crosstalk paradigms in the heart
Figure 1.4: Functional anatomy of ventricular cardiomyocytes
Figure 1.5: Diagram showing a typical action potential of a ventricular myocyte
Figure 1.6: Diagram showing the processes involved in cardiac muscle contraction
Figure 1.7: Schematic representation of Ca2+ regulation in cardiomyocytes
Figure 1.8: Mechanical events of the cardiac cycle
Figure 1.9: Heart failure pathogenesis
Figure 1.10: A schematic representation highlighting the role of hyperglycaemia and its
biochemical pathways
Figure 1.11: Anatomy of the human kidney
Figure 1.12: Anatomy of the Nephron

CHAPTER 2
Figure 2.1: Representative validation plots of RTqPCR methodology
Figure 2.2: A photograph showing isolation apparatus for ventricular myocyte isolation
Figure 2.3: A photograph showing an isolated rat heart on the cannula of Langendorff
apparatus.
Figure 2.3: A typical TGFβ standard calibration curve
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LIST OF TABLES AND FIGURES IV
CHAPTER 3
Figure 3.1: Ventricular cardiomyocyte contractility
Figure 3.2: Contractility in ventricular cardiomyocytes
Figure 3.3: Fast time base recordings of representative Ca2+ transient
Figure 3.4: [Ca2+] transient kinetics in ventricular cardiomyocytes
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CHAPTER 4
Figure 4.1: Left ventricle histology
Figure 4.2: Myocyte diameter
Figure 4.3: Investigations of ECM deposition in LV myocardium
Figure 4.4: Myocyte apoptosis
Figure 4.5: Myocyte ultrastructure and quantitative investigations
Figure 4.6: ECM components gene expression
Figure 4.7: ECM regulatory components gene expression
Figure 4.8: ANP and BNP gene expression
Figure 4.9: [Ca2+] gene expression
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Figure 4.10: TGFβ1 protein level and gene expression profile

CHAPTER 5
Figure 5.1: Kidney histology
Figure 5.2: Investigations of glomerular basement membrane
Figure 5.3: Investigations of ECM deposition in the kidneys
Figure 5.4: Kidney apoptosis
Figure 5.5: Kidney ultrastructure
Figure 5.6: ECM components gene expression
Figure 5.7: ECM regulatory components gene expression
Figure 5.8: Hypertrophy biomarkers ANP and BNP gene expression
Figure 5.9: TGFβ1 protein level and gene expression profile

CHAPTER 6
Figure 6.1: Comparison of the organ specific remodelling changes observed in heart
and kidney at 2 and 4 months of STZ-treatment
Figure 6.2: Comparison of the Organ specific effects on TGFβ1 concentration in heart
and kidney at 2 and 4 months after STZ-treatment
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