The Senescence-Accelerated Mouse (SAM): A Murine Model of Age-Associated Diastolic Dysfunction Open Access

Reed, Alana Leigh (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/br86b432b?locale=en
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Abstract

Diastolic heart failure, a form of heart failure in which patients exhibit signs and symptoms of heart failure despite the maintenance of a normal ejection fraction, is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness of the left ventricle. The goal of this dissertation project has been to use a murine model of spontaneous aging, the senescence-accelerated mouse (SAM) model as a model for the investigation of the mechanisms contributing to age-associated diastolic dysfunction. This model is comprised of senescence-prone (SAMP8) mice and senescence-resistant (SAMR1) controls. Using echocardiography and invasive hemodynamics, it was found that by 6 months of age, SAMP8 mice exhibit diastolic dysfunction. Since diastolic dysfunction is characterized by impaired relaxation and left ventricular stiffening, fibrosis was measured. SAMP8 mice showed increased deposition of fibrotic tissue in the interstitial and perivascular areas of the myocardium compared to SAMR1 controls at 6 months of age. Furthermore, gene expression of collagen 1A1, collagen 3A, and fibronectin was increased. Expression of the pro-fibrotic cytokines transforming growth factor-beta (TGF-ß ) and connective tissue growth factor was increased in the hearts of 6-month-old SAMP8 mice. Cardiac fibroblasts isolated from SAMP8 mice exhibited a decrease in collagen 3A, which is a more elastic form of collagen, in response to TGF- ß , suggesting that perhaps the loss of the more elastic isoform of collagen could promote stiffening of the left ventricle. Next, the role of oxidative stress was examined. Increased oxidative stress was found in the blood and vasculature of SAMP8 mice; however, no increase in myocardial superoxide was observed. Increased gene expression of the NADPH oxidases Nox2 and Nox4 as well as antioxidants was observed in the hearts of 6-month-old SAMP8 mice. It is possible that in the heart, antioxidant upregulation at least partially compensates for increased ROS resulting from increased expression of NADPH oxidases. In this dissertation, the SAM model has been established as a model of age-associated diastolic dysfunction, and cardiac fibrosis has been demonstrated as a mechanism of diastolic dysfunction in this complicated pathology.

Table of Contents

TABLE OF CONTENTS

Chapter 1: General Introduction (page 1)

Aging and cardiovascular disease (page 2)

1.1 Aging: demographics and lifespan (page 2)

1.2 Mechanisms of aging of mammalian cells (page 3)

· Mitochondrial ROS production (page 3)

· Telomere shortening (page 5)

· Senescence-associated signaling pathways (page 6)

· Impact of aging on stem cells and renewal (page 8)

1.3 Cardiovascular aging and disease (page 9)

· Vascular and arterial aging (page 9)

· Cardiac aging (page 10)

· Cellular and molecular mechanisms driving the cardiac aging process (page 14)

Diastolic heart failure and diastolic dysfunction (page 16)

1.4 Characteristics and clinical perspective (page 16)

· Heart failure patient characteristics (page 16)

· Diagnosis and treatment (page 18)

· Diastolic dysfunction (page 20)

1.5 Mechanisms of development (page 21)

· Myofilaments and myofilament proteins (page 22)

· Cardiac myocytes (page 23)

· Fibrosis (page 24)

o Characteristics of fibrosis (page 24)

o Mechanisms of fibrosis (page 25)

§ The role of TGF-ß (page 25)

§ The role of CTGF (page 27)

§ The role of the ECM (page 27)

· The role of fibrosis in age-associated heart failure (page 28)

· Cardiac and extra-cardiac effects (page 30)

1.6 Animal models of diastolic dysfunction (page 31)

The senescence-accelerated mouse (SAM) model (page 39)

1.7 Development and characterization of the SAM model (page 39)

1.8 Oxidative stress in the SAM model (page 41)

1.9 Cardiovascular diseases in the SAM model (page 44)

Objectives of this dissertation (page 47)

Chapter 2: The SAM model is a model of age-related diastolic dysfunction (page 48)

2.1 Introduction (page 49)

2.2 Methods (page 51)

· Animal maintenance (page 51)

· RNA isolation and quantitative real-time PCR (qRT-PCR) (page 51)

· Assessment of cardiac dimensions and diastolic function using echocardiography (page 51)

· Assessment of cardiac function using invasive hemodynamics (page 52)

· Measurement of sarcomere length shortening and relengthening (page 53)

· Acquisition of blood pressure data using telemetry (page 53)

· Measurement of lung weight and right/left ventricle weight ratio (page 54)

· Measurement of right ventricular systolic pressure (RVSP) using a pressure-transducing catheter (page 54)

· Statistical analysis (page 54)

2.3 Experimental results (page 56)

· SAMP8 mice show accelerated senescence at 6 months of age (page 56)

· SAMP8 mice develop diastolic dysfunction by 6 months of age (page 58)

o Echocardiography (page 58)

o Invasive hemodynamics (page 62)

· SAMP8 mice do not show differences in myocyte contraction or relaxation at 6 months of age (page 64)

· SAMP8 mice do not show differences in blood pressure from 3 to 6 months of age (page 66)

· Body and metabolic characteristics of SAM mice (page 68)

2.4 Discussion (page 71)

Chapter 3: Diastolic dysfunction is associated with fibrosis in the SAM model (page 74)

3.1 Introduction (page 75)

3.2 Methods (page 77)

· Histology (page 77)

· RNA isolation and qRT-PCR (page 77)

· Western blot analysis (page 78)

· TGF-β enzyme-linked immunosorbent assay (ELISA) (page 79)

· Cardiac fibroblast isolation and culture (page 79)

· MTT cell proliferation assay (page 80)

· Amplex® Red H2O2 assay (page 81)

· TGF-β stimulation, RNA isolation, and qRT-PCR (page 81)

· Statistical analysis (page 83)

3.3 Experimental results (page 84)

· SAMP8 mice show evidence of myocardial fibrosis by 6 months of age (page 84)

· Cardiac fibrosis in SAMP8 mice is associated with increased expression of pro-fibrotic cytokines (page 88)

· Assessment of TGF-β (page 90)

· Cardiac fibroblasts from 6-month-old SAMP8 mice show no differences in cell proliferation or H2O2 production (page 92)

· Gene expression of fibrosis markers in cardiac fibroblasts (page 95)

· Response to cardiac fibroblasts to TGF-β stimulation (page 95)

3.4 Discussion (page 99)

Chapter 4: The role of oxidative stress in the SAM model (page 102)

4.1 Introduction (page 103)

4.2 Methods (page 105)

· High-performance liquid chromatography (HPLC) detection of reduced and oxidized forms of plasma GSH and cysteine (page 105)

· ESR detection of O2•- (page 105)

· HPLC detection of intracellular O2•- with dihydroethidium (DHE) (page 106)

· HPLC detection of cardiac biopterin content (page 106)

· RNA isolation and qRT-PCR (page 107)

4.3 Experimental results (page 109)

· SAMP8 mice show evidence of altered systemic redox states independent of changes in cardiac redox states at 6 months of age (page 109)

· SAMP8 mice demonstrate vascular, but not myocardial, oxidative stress at 6 months of age (page 111)

· SAMP8 mice do not show differences in myocardial biopterin content at 6 months of age (page 111)

· SAMP8 mice exhibit increased myocardial gene expression of oxidative stress-associated genes at 6 months of age (page 115)

4.4 Discussion (page 118)

Chapter 5: Discussion (page 121)

References (page 137)


INDEX OF FIGURES

Figure 1.1 Age-associated changes in the heart (page 13)

Figure 2.1 Gene expression of p19 (page 57)

Figure 2.2 Functional analysis of isolated cardiomyocytes (page 65)

Figure 2.3 Blood pressure in SAM mice (page 67)

Figure 2.4 Pulmonary measurements in SAM mice (page 70)

Figure 3.1 Histological analysis of cardiac collagen deposition (page 86)

Figure 3.2 Gene expression of cardiac ECM components (page 87)

Figure 3.3 Gene expression of pro-fibrotic cytokines and protein expression of alpha-SMA (page 89)

Figure 3.4 Quantification of TGF-β1 by ELISA (page 91)

Figure 3.5 MTT assay for cardiac fibroblast proliferation (page 93)

Figure 3.6 Amplex ® Red assay of cardiac fibroblast H2O2 production (page 94)

Figure 3.7 Response of isolated cardiac fibroblasts to treatment with TGF-β (page 97)

Figure 3.8 Dose-dependence of the treatment of isolated cardiac fibroblasts with TGF-β (page 98)

Figure 4.1 Analysis of plasma redox states (page 110)

Figure 4.2 Measurement of vascular oxidative stress (page 112)

Figure 4.3 Measurement of myocardial oxidative stress (page 113)

Figure 4.4 Measurement of myocardial biopterin isoforms (page 114)

Figure 4.5 Gene expression of NADPH oxidases (page 116)

Figure 4.6 Gene expression of antioxidant enzymes (page 117)

INDEX OF TABLES

Table 2.1 Echocardiographic comparison of SAMR1 and SAMP8 mice at 3 and 6 months of age (page 60)

Table 2.2 Echocardiographic comparison of SAMR1 and SAMP8 mice at 12 months of age (page 61)

Table 2.3 Invasive hemodynamic comparison of SAMR1 and SAMP8 mice at 6 months of age (page 63)

Table 2.4 Metabolic profile of SAM mice at 6 months of age (page 69)

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