Myocardial Myocyte Remodeling and Fibrosis in ... - Wiley Online Library

The Journal of Cardiometabolic Syndrome (ISSN 1524-6175) is published quarterly (March, June, Aug., Dec.) by Le Jacq, Three Parklands Drive, Darien, CT 06820-3652. Copyright ©2006 by Le Jacq, All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publishers. The opinions and ideas expressed in this publication are those of the authors and do not necessarily reflect those of the Editors or Publisher. For copies in excess of 25 or for commercial purposes, please contact Sarah Howell at [email protected] or 203.656.1711 x106.

CLINICAL APPLICATIONS OF BASIC RESEARCH

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Myocardial Myocyte Remodeling and Fibrosis in the Cardiometabolic Syndrome

T

he cardiometabolic syndrome (CMS), estimated to affect between 47 and 67 million people in the United States, is an international public health problem that is expected to worsen over the coming decades.1–3 The CMS is associated with increased cardiovascular disease (CVD) including coronary heart disease (CHD), congestive heart failure (CHF), and stroke.4,5 Myocardial cellular remodeling via the process of myocyte hypertrophy and extracellular matrix (ECM) remodeling fibrosis occurs in the CMS and is associated with left ventricular hypertrophy, which is known to be a strong risk marker for increased morbidity and mortality in the CMS.6 Diastolic dysfunction is an abnormality associated with impaired ventricular relaxation and an elevated end-diastolic pressure that frequently occurs in the CMS and in patients with obesity, impaired glucose tolerance (IGT), and overt type 2 diabetes mellitus (T2DM).4,7,8 These same cellular and ECM remodeling changes occur independently and synergistically with hypertension. ECM remodeling fibrosis also occurs as a consequence of CHD and CVD, including ischemic events of myocardial infarction and acute coronary syndromes. The overall morbidity and mortality in CHD–CVD is closely tied to the CMS and is known to be increased at least 3-fold in patients with the CMS.9 An interesting story emerges regarding the CMS and IGT. For example, nearly one half (46%) of patients with CHD will meet the criteria for the CMS, according to the Third Report of National Cholesterol Educational Program Adult Treatment Panel (NCEP ATP III).10 Additionally, one third of the patients with acute coronary syndromes will have overt T2DM and one third will have IGT when tested at 326

myocyte remodeling and fibrosis in the CMS ®

Myocardial cellular and extracellular matrix remodeling are important in the development of left ventricular hypertrophy and are essential for the adaptive and maladaptive changes associated with the cardiometabolic syndrome. This brief review of myocyte remodeling also presents preliminary observational findings regarding myocardial adaptive hypertrophy remodeling, including an increase in mitochondria and capillaries, convolutions and lengthening of intercalated discs, the addition of sarcomeres, thickened Z lines, and the novel presence of pericapillary fibrosis (in addition to perivascular arteriolar fibrosis). The 11-week-old TG(mREN-2)27 transgene rat model of tissue angiotensin II overexpression, which develops hypertension and insulin resistance, was chosen to examine both myocyte hypertrophy and extracellular matrix fibrosis. This review and the preliminary observational findings may provide the clinician and researcher a better understanding of remodeling changes in the myocardium and ultimately foster earlier recognition and therapeutic interventions. (JCMS. 2006;1:326–333) ©2006 Le Jacq Melvin R. Hayden, MD;1,2 Nazif Chowdhury, MD;1 Guru Govindarajan, MD;1,2,4,5 Poorna R. Karuparthi, MBBS;1 Javad Habibi, PhD;5 James R. Sowers, MD1,2,3,5 From the Department of Internal Medicine1 and the Divisions of Endocrinology, Diabetes and Metabolism,2 Medical Pharmacology and Physiology,3 and Cardiology,4 University of Missouri School of Medicine, Columbia, MO; and the Harry S. Truman VA Medical Center, Columbia, MO5 Address for correspondence: Melvin R. Hayden, MD, Research Professor, Department of Internal Medicine, University of Missouri School of Medicine, Health Sciences Center, MA410, DC043.00, Columbia, MO 65212 E-mail: [email protected] Manuscript received April 3, 2006; revised May 22, 2006; accepted June 19, 2006 discharge and again 3 months later— culminating in two thirds of patients with acute coronary syndromes/CHD having IGT.11 In another study of 3266 high-risk patients being scheduled for coronary angiography, one half of the patient population had unrecognized diabetes or IGT.12 Thus, each arm of the CMS (Figure 1) seems to be independently and synergistically involved with myocardial remodeling in CVD either in a direct or indirect capacity. This brief review describes: (1) the importance of microalbuminuria (MAU), a marker of endothelial dysfunction; (2) each of the 4 arms of the CMS (obesity, insulin resistance [IR]–hyperinsulinemia,

hypertension, and IGT–glucotoxicity); and (3) recent preliminary observational findings regarding myocardial remodeling in the TG(mREN-2)27 (Ren-2) rat model of tissue angiotensin II overexpression, hypertension, and IR.

MAU in CVD and the Myocardium

The clinical screening test for MAU is used to identify patients who are at increased risk for the development of CVD, stroke, and chronic kidney disease. MAU is strongly associated with the CMS. MAU is defined as 30–300 mg albumin/g creatinine. The American Diabetes Association (ADA) and National Kidney Foundation have JCMS fall 2006


The 4 Arms of the CMS and Myocardial Remodeling

I. Obesity. Adipose tissue is no longer regarded as a passive, space-filling tissue capable of storing triglyceride globules. Adipose tissue is now considered an endocrine organ. This has manifold implications for our understanding of the pathogenesis of chronic diseases and has even led to a new field of study— adipobiology.16 Obesity is an important determinant of CVD and remodeling.17 myocyte remodeling and fibrosis in the CMS ®

Myocardial remodeling

IGT +/- T2DM Prothrombotic ↑ PAI-1 ↑ Fibrinogen Hyperactive platelets Obesity epidemic is driving this construct

↑ FFA

Hypertension ROS O2–

IR

recently recommended measurement of albumin/creatinine ratio by dipstick method, due to its simplicity, in either a first morning voided specimen or a random spot urine collection. The ADA guidelines suggest that 2 of 3 tests for MAU must be positive in a 3–6-month period to identify those at risk for progressing to end-stage renal disease.13 It is important to note that transient elevations of MAU can be caused by exercise, urinary tract infections, hyperglycemia, febrile illness, severe hypertension, or heart failure, so abnormal results should be confirmed with retesting. MAU is associated with a systemic and glomerular endothelial dysfunction wherein the endothelium becomes more permeable to various proteins, including albumin and inflammatory cells, allowing for increased interaction with adhesion molecules and chemoattractants. Associated with the increased protein leak and inflammation in endothelial dysfunction is the uncoupling of the endothelial nitric oxide synthase (eNOS) enzyme and the decreased production of endothelial-derived nitric oxide (NO), with resultant oxidative stress at the endothelial–tissue interface (Figure 2). Once endothelial dysfunction is present, it can initiate and allow for accelerated atherosclerosis throughout the entire vascular system, resulting in a proinflammatory, pro-oxidative stress state and an increased risk of CVD and myocardial remodeling fibrosis.14,15 Therefore, the presence of MAU gives one a window to assess endothelial dysfunction in the complex signaling pathways involved in myocardial remodeling.

eNOS uncoupling ↑ROS

Microalbuminuria Endothelial dysfunction Uric Acid–XO hs CRP NAD(P)H oxidase ↑RAAS activity → Ang II

↓NO

Obesity

Hyperlipidemia Visceral obesity lipid triad

Compensatory Hyperinsulinemia Hyperamylinemia

Figure 1. Myocardial and extracellular matrix (ECM) remodeling fibrosis in cardiovascular disease and the cardiometabolic syndrome (CMS). Note the 4 arms of the CMS: (1) obesity; (2) insulin resistance (IR) with compensatory hyperinsulinemia and hyperamylinemia, renin-angiotensinaldosterone system (RAAS) activation, nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) oxidase enzyme activation, and reactive oxygen species (ROS); (3) hypertension; and (4) impaired glucose tolerance (IGT)–glucotoxicity (with or without overt type 2 diabetes mellitus (T2DM). PAI-1 indicates plasminogen activator inhibitor-1; FFA, free fatty acids; eNOS, endothelial NO synthase; XO, xanthine oxidase; and hsCRP, high-sensitivity C-reactive protein. Adapted with permission from Hayden et al.15

Visceral obesity is associated with increased sensitivity to lipolytic hormones and decreased sensitivity to insulin, resulting in increased free fatty acid synthesis and secretion into the portal system. This results not only in increasing systemic IR, but also increasing hepatic IR, with increased triglyceride synthesis and impaired first-pass insulin metabolism—supporting endogenous hyperinsulinemia.18 Additionally, as the National Health and Nutrition Examination Survey (NHANES) demonstrated, there is a linear relationship between increasing body mass index and systolic and diastolic blood pressure and pulse pressure.19 Obesity alone may account for essential hypertension in 65% of hypertensive women and 78% of hypertensive men, according to the Framingham Offspring Study.20 Adipocytes are responsible for the production of multiple adipocytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, IL-8, IL-18, leptin, resistin, plasminogen activator

inhibitor-1, and macrophage migration inhibition factor, and decreased production of the anti-inflammatory, antiatherosclerotic adiponectin.21 Tumor necrosis factor α produced by local macrophage-derived preadipocytes is of central importance in the inflammatory component of obesity via autocrine and paracrine mechanisms. Additionally, tumor necrosis factor α stimulates the production of IL-6 via an endocrine mechanism in hepatocytes responsible for the production of the acute-phase proteins (C-reactive protein, plasminogen activator inhibitor-1, and fibrinogen)—all of which suggest that obesity may be considered a chronic low-grade inflammatory and profibrotic disease state. This chronic state may relate to the causation and progression of hypertension, endothelial dysfunction, accelerated atherosclerosis, CVD, and myocardial fibrosis.14,15,17,22 Visceral obesity is associated with the lipid triad of: (1) elevated very-low-density lipoprotein cholesterol; (2) elevated small, JCMS fall 2006

327


Figure 2. Proposed signaling pathways in myocardial remodeling in the TG(mREN-2)27 (Ren2) rat model. The major players in these complex signaling pathways are reactive oxygen species (ROS), β protein kinase C β (PKC β), mitogen-activated protein kinase (MAPK), transforming growth factor (TGF) β-1, eNOS enzyme uncoupling, and matrix metalloproteinases (MMPs). Angiotensin II (Ang II)-induced TGF β via the At1 receptor (AT-1 R) plays a constant and important role in ECM remodeling fibrosis. AGE indicates advanced glycosylation end products; PLC, phospholipase C; DAG, 1,2-diacyl-sn-glycerol; PKC, protein kinase C; PKA, protein kinase A; NFkappaB, nuclear factor kappa B; TNF-α, tumor necrosis factor α; eNO, endothelial NO; TGF, transforming growth factor; PI3, phosphatidylinositol 3-kinase; Akt, protein kinase B; IR, insulin resistance; and MMPs, matrix metalloproteinases. A-FLIGHT-U is described in text.

dense, atherogenic low-density lipoprotein cholesterol; and (3) decreased high-density lipoprotein cholesterol. This triad is known to promote accelerated atherogenesis.14,15 Obesity in animal models (rabbits and dogs) and in humans has demonstrated the following associations: an increase in arterial pressure, heart rate, cardiac output, renal sympathetic activity, sodium balance, renal tubular reabsorption, and glomerular filtration rate.19 Obesity is associated with activation of the renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system, hyperinsulinemia/IR, dyslipidemia, dysglycemia, endothelial dysfunction, vascular disease, and hypertension, each of which independently and synergistically contributes to both functional and structural remodeling in the myocardium in CVD.14,15,18 Obesity is the driving force behind the CMS and is a major risk factor for CVD and myocardial remodeling, 328


acting through each of the component arms of the CMS (Figure 1). II. IR, Hyperinsulinemia, and Oxidative Stress. IR describes the condition of polygenetic and environmental (due to overnutrition and inadequate exercise) resistance to insulinmediated glucose uptake by peripheral cells (notably the skeletal muscle cells and hepatocytes). IR results in compensatory hypersecretion of the β-cellderived hormones (insulin, proinsulin, and amylin) that individually and synergistically activate a local tissue RAAS, resulting in the structural remodeling and dysfunction of the myocardium and increasing the number of angiotensin type 1 receptors.15,16,23 This compensatory hyperinsulinemia places patients at risk for CVD, stroke, and further myocardial remodeling.24,25 The World Health Organization (WHO) in 1999 and the NCEP ATP III in 2001 included the concept of the

metabolic syndrome/CMS in the cholesterol guidelines to emphasize their concern regarding the obesity and diabetes epidemics and to promote weight reduction and increased physical activity. Bringing the metabolic syndrome/ CMS into prominence has enhanced the recognition by health care providers of the increased risk of T2DM (7–28fold), CVD and stroke (3-fold), and myocardial remodeling.15 The WHO diagnostic criteria specify IGT or IR (T2DM) as an essential component of the CMS; MAU is included as an additional criterion.22 MAU is an independent risk factor for CVD26,27 and is associated with IR and CKD.28,29 Endothelial dysfunction is strongly associated with MAU and myocardial remodeling fibrosis (Figure 2). The strong association of underlying oxidative stress, largely due to an uncoupling of eNOS, vascular nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) oxidase, xanthine oxidase, reactive oxygen species (ROS), reactive nitrogen species, and the associated depletion of cellular and extracellular antioxidants, is the result of multiple metabolic toxicities.15 Currently, the presence of MAU is not a requirement for clinical diagnosis of the CMS, but it has been demonstrated to be an independent variable and is included in the WHO component guidelines.30 The multiple metabolic toxicities (angiotensin II and amylin; free fatty acids; lipotoxicity; insulin toxicity; glucotoxicity; hypertension toxicity; triglyceride toxicity and uric acid toxicity [A-FLIGHT-U acronym15]) result in the functional uncoupling of eNOS, with the endothelium becoming a net producer of superoxide, an ROS, instead of the protective antioxidant, anti-inflammatory constitutive endothelial NO.15 The uncoupling of eNOS at the endothelial–tissue interface may be responsible for the initiation of perivascular fibrosis and interstitial ECM remodeling fibrosis in the endomysium.4,14 Research with the Ren-2 rat model of tissue angiotensin II overexpression and hypertension has demonstrated IR as early as 9 weeks of age. Additionally, it has been shown that insulin levels are JCMS fall 2006


elevated in the fasting state, as well as during intraperitoneal glucose tolerance testing, in the Ren-2 model compared with the Sprague-Dawley control rat.31 III. Hypertension. There is a significant association between hypertension and the development of diastolic and systolic dysfunction and CHF.32 Hypertension is strongly related to the development of left ventricular hypertrophy and diastolic dysfunction with impaired ventricular relaxation. This impaired relaxation is due to a stiffened myocardium, the result of an accumulation of ECM (primarily types I and III collagen and fibronectin).4,33 In considering the appropriate research model to study cellular/myocyte adaptive remodeling and early interstitial remodeling fibrosis to elucidate the relationship between hypertension and diastolic dysfunction, we chose the male Ren-2 model of spontaneous hypertension (mean systolic blood pressure of 199±4 mm Hg) at 11 weeks of age. This model also develops IR at 9–11 weeks of age via an increase in the local tissue expression of RAAS and angiotensin II due to manipulation of a single gene transfection. 4,31,33–36 This model of hypertension has permitted novel observations of myocardial remodeling on the cellular level, as described below.

SDC A

B

Figure 3. Mitochondria biogenesis in the TG(mREN-2)27 (Ren-2) myocardium. (A) the Sprague-Dawley control (SCD) myocardium at 11 weeks of age; (B) Ren-2 model of hypertension. Note the increased numbers of interdigitating mitochondria, representing adaptive cellular remodeling and mitochondria biogenesis. SDC

A

CMS. It consists of fibronectin and types I and III collagen matrix accumulation in the endomysium and perimysium of the myocardium, resulting in a stiffened, concentrically remodeled myocardium with impaired relaxation and diastolic dysfunction. Additionally, there is a specific perivascular fibrosis of the arterioles within the myocardium, detectable with light microscopy in the Ren-2 model (Figure 6). These observational findings have been reported by others, and the view that this is a form of perivascular remodeling is now accepted.4,33–36

ECM Remodeling Fibrosis and Perivascular Fibrosis. ECM fibrosis is prevalent in hypertension and in the

Novel Pericapillary Fibrosis. In addition to perivascular fibrosis in the Ren-2 model, we have been able to demonstrate a

®

Ren-2 B B

Figure 4. Z-line thickening in myocyte hypertrophy and convoluted intercalated discs. Note the Z-line thickening (arrowheads) of the TG(mREN-2)27 (Ren-2) model in (B) compared with the Z lines in (A) of the Sprague-Dawley control (SCD) at 11 weeks. Also note the increased convolutions of the intercalated disk (arrows) in (B) of the Ren-2 vs the intercalated disc in (A) of the controls. The increased convolutions also increase the length of the intercalated discs. Original magnification ×10,000.

Myocyte Cellular Remodeling. Preliminary transmission electron microscopy observational findings of myocyte adaptive cellular remodeling hypertrophy in the Ren-2 model include the following: (1) increased numbers of mitochondria that are more globular, compared with an oval or elongated morphology in other organs (Figure 3); (2) increased endomysial capillaries representing angiogenesis (observational, no images); (3) thickened Z lines (Figure 4); (4) increased convolutions of intercalated discs (Figure 4); and (5) additions to existing sarcomeres and the building of new sarcomeres (Figure 5).

myocyte remodeling and fibrosis in the CMS

Ren-2

previously unreported pericapillary fibrosis in this model of hypertension and IR (Figure 7). A similar pericapillary fibrosis has also been found in other end organs in the Ren-2 and Zucker obese model of the CMS, namely the pancreatic islet, kidney, and liver.15,37 Pericapillary fibrosis seems to be a unifying structural change in each of the end organs involved in the CMS. It represents a structural endothelial–tissue uncoupling that is associated with the functional eNOS enzyme uncoupling discussed earlier. We hypothesize that pericapillary and perivascular fibrosis may be a direct result of eNOS uncoupling and generation of a local vascular ROS (superoxide/peroxynitrite), together with the action of vascular membranous JCMS fall 2006

329


X

ER M

M

M

X

mm

Figure 5. Sarcomere biogenesis in the TG(mREN-2)27 (Ren-2) myocardium. This image captures what may be the biogenesis of sarcomeres in the adaptive, hypertrophic myocardium. Note the immature mitochondria (M) without distinct cristae compared with the mature mitochondria (mm) on the right side of this image. Also note the endoplasmic reticulum (ER) on the right indicating active remodeling. One seldom observes this prominent ER in the healthy control myocardium. Also note that some Z lines seem to traverse open spaces. X indicates incomplete sarcomere biogenesis.

NAD(P)H oxidase also generating local vascular ROS due to angiotensin II in end organs involved in the CMS. Oxidative stress and reduced myocardial NO are known to induce matrix metalloproteinase activity associated with ECM accumulation in the interstitium and perivascular areas of the myocardium.4,15,37 The interstitial remodeling fibrosis in the Ren-2 model seems to originate in the pericapillary areas at the site of endothelial dysfunction and oxidative stress (possibly due to ROS generation, as described above), and then extend in a fanning-out mechanism to involve the interstitial endomysial space, and eventually the perimysial space, resulting in an endothelial–myocyte structural uncoupling. This new observation may be the earliest ECM remodeling fibrotic change found in the myocardium of the Ren-2 model. While complex, most of the possible signaling pathways involved are presented in Figure 2. IV. Impaired Glucose Tolerance. IGT and glucotoxicity, the fourth arm of the CMS, also play a role in myocardial remodeling 330


in the CMS. Overt T2DM is a component of the CMS, but it is not a prerequisite for the diagnosis of the CMS. IGT results in glucotoxicity of the endothelial vascular wall and assumes a driving role in the activation of a local RAAS in endorgan tissues (including the myocardium) once the β cells have failed in their role of compensatory hypersecretion of insulin, proinsulin, and amylin.23 Glucotoxicity is believed to interfere with myocardial function by promoting increased synthesis of cardiac fibroblast protein, collagen, and angiotensin II receptor type 1 messenger RNA. Glucotoxicity may therefore be considered profibrotic in the myocardium, mirroring its effects in the kidney.4,37,38 Additionally, glucotoxicity contributes to advanced glycation end products, which in turn contribute to collagen crosslinking, resulting in resistance to enzymatic proteolysis and degradation, thus further promoting a stiffened myocardium via another mechanism in addition to ECM accumulation. Fasting blood glucose is known to be higher in the Ren-2 model compared with the Sprague-Dawley control at 9

weeks of age (9.0±1.3 mmol/L vs 6.5±0.4 mmol/L; P