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Models of cardiac hypertrophy and transition to heart failure
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 4, No. 4 2007
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Cardiovascular diseases
Models of cardiac hypertrophy and transition to heart failure Jeff M. Berry2, R. Haris Naseem2, Beverly A. Rothermel2, Joseph A. Hill1,2,3,* 1
Donald W. Reynolds Cardiovascular Clinical Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 3 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2
The prevalence of heart failure is increasing rapidly worldwide, and yet effective treatments remain elusive. Pathological remodeling of the ventricle – and
Section Editors: Rahul Kakkar and Richard T. Lee – Harvard Medical School, Brigham and Women’s Hospital, Cambridge, MA, USA
associated hypertrophic growth, fibrotic change, cavity dilatation and electrophysiological remodeling – is a significant contributor to the pathogenesis of this prevalent disorder. As a consequence, there is great interest in developing new therapies to target pathological remodeling of the heart with the intent to prevent, arrest, or possibly reverse the otherwise inexorable progression of disease. To tackle this problem, numerous models of disease have been developed, both in vivo and in vitro. Here, we review models of cardiac hypertrophy and failure, compare and contrast their strengths and limitations, and on occasion cite recent works where the use of these models has contributed to significant scientific advances.
Introduction The heart is capable of robust growth and remodeling in response to changes in workload. For example, diseaserelated stresses, such as unremitting hypertension or myocardial injury, trigger hypertrophic growth that confers markedly increased risk of functional decompensation (heart failure) and malignant rhythm disturbance. By contrast,
*Corresponding author: J.A. Hill ([email protected]) 1740-6757/$ ß 2007 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2007.06.003
growth of the heart in response to physiological demand (e.g. exercise or pregnancy) is adaptive and not associated with adverse sequelae. And the public health consequences of this cardiac remodeling are profound: congestive heart failure (CHF) contributes to over 300,000 deaths per year in the United States, and the prevalence of this disorder is rising rapidly [1]. Already a leading indication for hospitalization, CHF exacts a substantial financial toll on healthcare resources [1]. Heart failure is a syndrome that arises in the context of a multitude of disease states. Although there are common, overlapping aspects of the phenotype (e.g. elevated filling pressures, diminished forward flow), important differences exist across the spectrum of heart failure. Despite these substantial differences in the syndrome itself, treatment options are limited, and they are applied uniformly, irrespective of the functional phenotype, inciting stimulus, comorbid conditions, patient demographics, among others. A major focus in this field is to unveil disease-specific mechanisms that will lead to novel therapies to prevent, halt, or even reverse heart failure progression. Important new insights into the pathogenesis of this syndrome have been uncovered in recent years [2]. Among these insights is the emergence of left ventricular hypertrophy (LVH) as a major risk factor for development of heart failure [3]. Indeed, LVH is increasingly viewed as a crucial 197
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Figure 1. Paradigm of pathological remodeling of the ventricle. Moderate stress triggers a growth response leading to ventricular hypertrophy; this growth
step in the pathogenesis of many forms of heart failure [4,5] (Fig. 1). Numerous experimental models of heart failure exist, mimicking the vast array of clinical phenotypes. Here, we provide an overview of these models, comparing and contrasting their strengths and weaknesses (Table 1). Where appropriate, we cite recent examples where these models have been used to glean new insights into disease pathogenesis. We emphasize the role of hypertrophic remodeling, because this area of investigation has progressed rapidly in recent years, and several key insights have been uncovered that may emerge as clinically relevant therapeutic directions.
In vitro models of myocardial hypertrophy A truly ‘cardiac’ cell line does not exist. Thus, the great majority of cell culture-based studies have relied on neonatal cardiomyocytes in primary culture. These cells, typically isolated from rat, divide in culture approximately once and can be maintained in a healthy state for up to 1 week. To avoid overgrowth of co-purifying cardiac fibroblasts, differential-plating strategies are typically employed with mitosis inhibitors incorporated in the culture medium. It is likely that some of the divergent findings reported in the literature stem from the presence of variable fractions of co-cultured fibroblasts. 198
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Adult ventricular myocytes can be isolated and maintained for short periods of time in culture. Although this is suitable for certain types of investigation, such as whole cell electrophysiology studies, it is not optimal for biochemical or molecular biological approaches. For unknown reasons, adult mouse cardiac myocytes are particularly difficult to maintain in culture, although success has been reported [6]. However, even if successfully cultured, these cells begin to dedifferentiate over time and lose important aspects of the adult cardiomyocyte phenotype. The response of neonatal rat ventricular myocytes (NRVMs) to various neurohumoral signals has shed light on molecular mechanisms of myocardial hypertrophy and failure. For example, exposure to isoproterenol, a beta adrenergic agonist, triggers an increase in cardiomyocyte size and protein content with a gene expression profile similar to that observed with in vivo models of hypertrophy [7]. Phenylephrine, an alpha adrenergic agonist, and angiotensin II, a component of the renin–angiotensin–aldosterone system, each trigger growth of NRVMs in vitro and have been used to model hypertrophy [8,9]. The relationship between hypertrophic growth and contractile function has also been studied in vitro using NRVMs [9]. Contractility is assessed using digital imaging technologies to measure the amplitude and velocity of cell shortening
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Drug Discovery Today: Disease Models | Cardiovascular diseases
Table 1. Comparison of models of cardiac hypertrophy and failure Pros
In vitro models
In vivo models
In silico models
Cardiomyocytes and cardiac fibroblasts can be isolated and studied in isolation
Changes in molecular signaling and tissue structure can be examined in association with changes in cardiac function Stresses on the heart relevant to human disease can be modeled
One specific property of myocardial tissue can be tested while controlling all other properties Costs and ethical concerns of animal experimentation are avoided
Genetic constructs can be used to express specific proteins or measure promoter-driven gene expression Cons
Cardiomyocytes are difficult to isolate and maintain healthy in culture Results of NRVMs may not be relevant to in vivo models or human disease
Findings in animal models are not always relevant to human disease Ethical considerations when subjecting animals to cardiac stress
Model is a highly simplified representation of events occurring in the heart Hypotheses generated in silico must be tested experimentally
Best use of model
To characterize the molecular mechanisms of stress response signaling pathways To test the effects of endogenous or mutated proteins on gene expression
To understand the role of specific proteins in the development of hypertrophy and heart failure To test new therapeutic strategies for heart failure
When parameters are easily quantified and easily manipulated
How to get access to the model
Literature
Literature
Literature
Relevant patents
n/a
n/a
n/a
References
[6–12]
[15–20,22–28,31,32,34–36,40–49,51,52]
[65–68]
and relaxation. Intracellular calcium transients are estimated using a calcium-sensitive indicator and analyzing fluorescence emissions from selected individual myocytes. Fibroblasts comprise a substantial fraction of the cells in the heart. Despite this, relatively little is known regarding fibroblast function, proliferation, or collagen biosynthesis. Among their functions, fibroblasts secrete cytokines that influence the growth and function of neighboring cardiomyocytes. Fibroblasts also play a major role in disease-related remodeling of the extracellular matrix. For example, Jaffre et al. [10] have shown that treatment of murine cardiac fibroblasts in vitro with isoproterenol causes increased production of interleukin-6, tumor necrosis factora and interleukin-1b. Additional studies such as this may lead to novel therapies that specifically target the cardiac fibroblast. Alternative cell lines that are easier to maintain in culture have been used for experiments relating to cardiac hypertrophy. H9c2 myoblasts, derived from embryonic rat hearts, proliferate in culture and can be induced to differentiate into myotubes for study [11]. C2C12 myoblasts, a skeletal musclederived cell line, have also been used for molecular studies [12]. Whether results using these cell lines can be translated to the fully developed, adult myocardium is uncertain.
In vivo models of cardiac hypertrophy Despite the important insights gleaned from in vitro models, this field continues to rely heavily on animal models of cardiac hypertrophy and failure. Indeed, in several instances,
conclusions drawn from studies of NRVMs have not held up in vivo [13,14]. Animal models of cardiac hypertrophy and failure have been developed based on stress and growth signals, which are observed in human cardiac disease (Fig. 2).
Load-induced hypertrophy and failure: pressure overload Patients with hypertension or aortic stenosis (AS) develop LVH as a consequence of persistently increased afterload. The left ventricle, under these conditions, must generate a higher systolic blood pressure to eject an adequate stroke volume. This scenario is often referred to as pressure overload. For experimental purposes, an increase in afterload can be created by surgically banding the ascending, transverse, or descending aorta. This commonly used strategy has been utilized to develop numerous animal models of pressure-overload disease. Aortic banding in mice is a well-established model of cardiac hypertrophy and heart failure [15] (Fig. 3). Importantly, the resulting phenotype can vary significantly depending on the strain of mice and the surgical technique employed. For example, Rothermel et al. [16] showed that moderate aortic banding (inducing a trans-stenotic pressure gradient of 60–80 mmHg) in adult male C57BL6 mice produces ventricular hypertrophy with a normal ejection fraction; by contrast, slightly more severe banding leads to a dilated heart, depressed systolic function and clinical features of heart failure, including increased mortality. These two clinically relevant phenotypes – compensated versus www.drugdiscoverytoday.com
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Figure 2. Forms of cardiac stress that trigger hypertrophic remodeling. In vivo models of cardiac disease are generally developed by imposing a stress on
decompensated ventricular hypertrophy – are associated with differential activation of stress-response signaling pathways and calcium handling proteins [16]. In studies designed to evaluate antiremodeling therapy, it is crucial that the imposed hemodynamic load be equivalent across treatment groups. Unfortunately, there is no simple way to ensure that this has been accomplished. For example, trans-stenotic pressure gradients are a function of both the degree of vessel stenosis and the pressure generated by the LV; hence, they provide an indirect measure of arterial resistance. To infer that trans-stenotic pressure gradients are indicative of banding-induced stenosis, one must assume that cardiac output is unchanged by drug treatment (or genetic manipulation). As an analogy, to compare electrical resistance (R) in two circuits by measuring the voltage drop (V) across each circuit, one must assume that current (I) is the same in both circuits; V = IR. This important caveat is apparent to most clinicians, who recognize that the murmur of aortic stenosis increases as valve area declines; however, when the LV starts to fail, the aortic stenosis murmur diminishes. Thus, in order to ensure equivalence of experimentally imposed afterload, 200
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we recommend rigorous attention to study design: The surgeon must be blinded to the experimental arms, animals should be randomized between groups whenever possible, and large numbers of animals must be studied. A canine model of ventricular hypertrophy was used by Depre et al. [17] in a study that found evidence of increased proteasome activity in the hypertrophied left ventricle. In that study, puppies at age 8–10 weeks underwent aortic banding; as the animal grows, afterload gradually increases mimicking the effects of progressive hypertension or AS. By 1 year of age, a significant increase in LV systolic pressure and ventricular hypertrophy had developed. Moorjani et al. [18] developed a pressure-overload model in sheep using a Foley catheter balloon enclosed in a cuff around the aorta. The balloon was inflated every 2 weeks to steadily increase the severity of aortic constriction. These animals initially developed ventricular hypertrophy followed by a decline in fractional shortening on echocardiography. In this model, the development of heart failure is associated with an increase in caspase activity, a mediator of cardiomyocyte apoptosis.
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Load-induced hypertrophy and failure: volume overload
Figure 3. Schematic of surgically induced stenosis of the thoracic aorta. This model is often termed transverse aortic constriction
Pressure-overload stress has also been studied in the right ventricle. Cooper and co-workers have developed a model of myocardial hypertrophy in cats by banding the pulmonary artery. Using this model, Balasubramanian et al. [19] reported significant accumulation of polyubiquitinated cytoskeletal proteins during the early period of pressure-overload stress. Alternatives to aortic banding exist for modeling pressureoverload stress. Renal wrapping to produce persistent hypertension in dogs was first described by Page in 1939 [20]. This procedure involves wrapping the kidneys with cellophane or silk, which triggers progressive increases in blood pressure after several weeks. Munagala et al. [21] used this model recently to study diastolic dysfunction associated with LVH. The spontaneously hypertensive rat (SHR) gradually develops hypertension starting at a young age and has long been used to study load-induced cardiac remodeling. Rysa et al. [22] reported an increase in expression of genes encoding extracellular matrix proteins associated with the development of heart failure in these animals. The Dahl salt-sensitive rat is yet another model of pressure-overload hypertrophy and heart failure. When fed a high salt diet for 11 weeks, these animals develop significantly increased blood pressure. In a study by Iwanaga et al. [23], an increase in expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs was observed as hypertrophy progressed to heart failure.
Another pathological stress on the heart is persistently increased LV cavity volume. This so-called volume overload occurs in patients with chronic mitral regurgitation, aortic regurgitation, beriberi, arteriovenous malformations and certain forms of congenital heart disease. Under these conditions, stroke volume must increase to maintain forward cardiac output, either because of regurgitant or shunted blood flow. In this setting, preload increases, and over time the heart develops an increase in end-diastolic volume. A canine model of mitral regurgitation has been created using fluoroscopically guided catheterization to rupture the chordae tendineae. These animals develop an increase in both LV end-diastolic dimension and fractional shortening associated with enhanced norepinephrine release into the myocardial interstitial fluid [24]. Another way to model volume-overload hypertrophy is by creating an aortocaval shunt. This procedure is commonly performed in rats, and the hearts of these animals become significantly dilated over several months. Cantor et al. [25] used this model to compare the progression of volume-overload hypertrophy with a rat model of pressure-overload hypertrophy. A model of combined pressure and volume overload has been developed in rabbits wherein the aortic valve is ruptured and the abdominal aorta is subsequently constricted surgically. Using this model, Ai et al. [26] have provided evidence that calmodulin kinase II alters ryanodine receptor function and calcium handling in heart failure. Persistent tachycardia can trigger ventricular remodeling leading to heart failure. For example, patients with atrial fibrillation and a rapid ventricular response can manifest impaired ventricular function that can recover once heart rate is controlled again. Tachycardia-induced cardiomyopathy can be induced in dogs by implanting a pacemaker that rapidly paces the right ventricle for several weeks [27,28]. This model not only incorporates the stress of a rapid heart rate, but right ventricular pacing may also contribute to adverse remodeling by altering the sequence of ventricular depolarization. This would be consistent with clinical evidence that suggests that prolonged right ventricular pacing contributes to poor outcomes in patients with heart failure [29,30]. Myocardial infarction (MI) represents a unique stress on the heart, encompassing characteristics of each of the above stresses. The region of noninfarcted tissue experiences increased preload, increased afterload, hypertrophic growth and sometimes altered depolarization and dysynchrony. The mechanical load on this myocardial tissue varies depending on the location and size of the infarct. MI can be produced surgically by ligating a coronary artery, and this procedure has been performed in numerous species, including mice [31,32]. www.drugdiscoverytoday.com
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Neurohormonal stress Increases in sympathetic activity are routinely seen in heart failure and appear to contribute to deterioration of cardiac function [33]. Consistent with this notion, cardiac hypertrophy and heart failure can be modeled by activating adrenergic signaling pathways. Isoproterenol, when administered continuously using a subcutaneously implanted osmotic pump, causes an increase in cardiac mass in mice [34]. Other neurohumoral signaling pathways have also been studied, including the renin–angiotensin–aldosterone system, which plays a role in pathological ventricular remodeling leading to heart failure. Mice that are continuously administered angiotensin II develop increases in heart weight. Freund et al. [34] showed that transgenic mice expressing an inhibitor of nuclear factor-kappaB have an attenuated hypertrophic response to both isoproterenol and angiotensin II. Additional neurohormonal stress signals have been used to model cardiac hypertrophy in vivo. Growth hormone and insulin-like growth factor 1 stimulate ventricular hypertrophy in mice, although this growth response is distinct from that caused by pressure overload or MI [35] Similarly, thyroid hormone when given to rats causes significant cardiac hypertrophy associated with activation of the Akt signaling pathway [36]. These models of ventricular remodeling may be relevant to physiological growth of the heart (see below).
Physiological hypertrophy Growth of the heart in response to the physiological demands of exercise is robust (up to 40–60% [37,38]) and yet is not associated with untoward sequelae. Mouse models of exercise-induced hypertrophy have proven valuable for dissecting molecular mechanisms of physiological and pathological ventricular remodeling. In many instances, the phosphoinositide-3-kinase (PI3K)/Akt pathway governs physiological growth of the heart [39]. DeBosch et al. [40] used a model of swimming-induced cardiac hypertrophy to show that Akt1 is necessary for physiological cardiac growth. Control mice subjected to forced swimming for 20 days developed significantly increased cardiac mass and cardiomyocyte size, whereas mice deficient in Akt1 showed no hypertrophy. It is important to recognize that forced exercise protocols use unpleasant stimuli to motivate animal activity, in contrast to voluntary exercise protocols, which are more physiological and less stressful. Voluntary wheel running for 5 weeks is associated with increased cardiac mass, although this hypertrophy appears to be more pronounced in female compared to male mice [41,42].
Genetic models of cardiac growth and failure A major objective in this field is to tease out specific signaling mechanisms that govern pathological and physiological cardiac remodeling. An integral part of this strategy is to utilize 202
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gain-of-function and loss-of-function strategies to decipher the role of specific signaling cascades [43]. For example, mice that overexpress the human beta1-adrenergic receptor specifically in the heart develop hypertrophy that progresses to heart failure with ventricular fibrosis [44]. Similarly, cardiacspecific overexpression of the angiotensin II type 1 receptor leads to cardiac hypertrophy and heart failure [45]. The cytoplasmic protein phosphatase calcineurin when expressed in cardiac myocytes is an especially potent inducer of pathological cardiac hypertrophy and failure [46]. Other recent examples include pathological remodeling triggered by expression in the heart of activated forms of CaMKII [47], MEK5 [48], or protein kinase D [49]. In addition to genetic activation of signaling pathways, cardiac hypertrophy can be triggered by mutations in genes coding for structural proteins and molecular elements of the sarcomere. For example, inherited forms of hypertrophic cardiomyopathy (HCM) in humans have been linked to numerous gene mutations [50], and many of these can be modeled in animals. Genes encoding components of the sarcomere including beta myosin heavy chain, troponin T, and myosin-binding protein C are among the most common to be implicated in the pathogenesis of HCM. Introduction of an Arg403Gln missense mutation in the murine alpha myosin heavy chain gene, a mutation associated with a severe form of HCM, leads to ventricular hypertrophy, cardiac dysfunction and arrhythmias [51]. Mice homozygous for a truncated form of myosin-binding protein C, similar to that observed in humans, develop myocardial hypertrophy and left ventricular dilation [52]. Although exceptions exist, a pattern has emerged where mutations of force-generating (e.g. sarcomeric) proteins trigger hypertrophic cardiomyopathy and mutations of structural (e.g. cytoskeletal) proteins induce dilated cardiomyopathy [50]. Although the primary impact of these mutations is probably an alteration of contractile efficiency, it is unknown how they influence the mechanotransduction machinery with which these proteins interconnect (reviewed in ref [53]).
Phenotypes of cardiac hypertrophy and heart failure Systolic heart failure Pathological cardiac signaling can lead to distinct patterns of morphological, functional and electrophysiological remodeling. Among the most common functional changes is impaired systolic contractile performance (systolic dysfunction), which can be assessed using a variety of invasive and noninvasive methods. A load-independent measure of LV contractility is obtained from the end-systolic pressurevolume relationship [54]. In the clinical setting, a reduced ejection fraction or an increase in LV volume is associated with adverse outcomes [55–57]. Development of systolic dysfunction, therefore, represents an important transition point in the progression of hypertrophic heart disease.
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Sadoshima et al. [58] showed that mitogen-activated protein kinase kinase (MEKK1) could counter the development of systolic dysfunction. After 2 weeks of aortic constriction, mice deficient in MEKK1 manifested increased mortality and decreased ejection fraction, while ventricular function remained stable in control mice. Okumura et al. [59] presented evidence that type 5 adenylyl cyclase (AC5) contributes to the transition to heart failure; 3 weeks after aortic constriction, wild-type mice manifested increases in LV size and reduction in ejection fraction, but AC5 knockout mice showed no such changes. Most et al. [31] showed that S100A1, a calciumbinding protein involved in excitation-contraction coupling, may protect the heart from systolic dysfunction following MI; S100A1 knockout mice subjected to coronary artery ligation manifested increased mortality and accelerated declines in systolic function compared to wild-type mice. Interestingly, transgenic mice that overexpress S100A1 showed blunted declines in systolic function and improved mortality [31].
Heart failure with normal ejection fraction Many patients experience heart failure symptoms despite apparently normal systolic function. In fact, a significant percentage of patients with CHF have a normal ejection fraction, and yet these patients are at increased risk of adverse events [56]. It is thought that diastolic dysfunction contributes to the transition from asymptomatic to symptomatic hypertrophic heart disease [60]. Elevated filling pressures, owing to impaired compliance of the left ventricle, are required to achieve an adequate preload and maintain cardiac output. These elevated pressures contribute to pulmonary congestion and CHF symptoms. Despite the prominence of heart failure with normal ejection fraction (HFNEF) in humans, development of a reliable animal model has been challenging. Munagala et al. [21] subjected dogs to renal wrapping to produce pressure overload, which resulted in left ventricular hypertrophy with preserved systolic function. These animals manifested prolonged LV relaxation times during diastole in association with increased ventricular fibrosis. Rysa et al. [22] examined spontaneously hypertensive rats at a stage where ventricular hypertrophy and diastolic dysfunction are present but ejection fraction is preserved; several genes were upregulated, most notably genes encoding extracellular matrix proteins. Studies in animals such as these are limited by the fact that one cannot assess symptoms, and signs of modest CHF (e.g. edema, pulmonary or hepatic congestion, lethargy) may be difficult to detect. Furthermore, accurate and precise measures of diastolic dysfunction are difficult, especially in small animals.
Ventricular fibrosis During pathological remodeling of the ventricle, disproportionate accumulation of fibrillar collagen occurs. This fibro-
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proliferative response contributes significantly to the development and worsening of both systolic and diastolic dysfunction and can lead to disruption of electrical wavefront activation and consequent re-entry. In general, cardiac extracellular matrix is regulated by a balance between production and degradation. Stressors that alter this delicate balance will lead to excessive ECM deposition resulting in myocardial dysfunction. Although the exact mechanisms by which excessive fibrosis leads to cardiac dysfunction are still unknown, drugs that block fibrogenesis would be of great clinical interest. Although models of pure myocardial fibrosis are more difficult to establish, given confounding abnormalities, examples exist of animal models where myocardial fibrosis is a dominant aspect of the phenotype. Probably the most studied and relevant animal models are those of pressureinduced hypertrophy and failure, where cardiac fibrosis and hypertrophy are classic features. (By contrast, cardiac fibrosis is not a prominent feature of exercise-induced hypertrophy.) Feutin-A knockout mice develop spontaneous dystrophic cardiac calcification and profound induction of fibroproliferative pathways [61]. Excessive myocardial fibrosis and diastolic dysfunction are characteristic abnormalities in this model. Interestingly, overexpression of activated protein kinase D in heart triggers profound failure with little fibrosis [62]. Perhaps even more compelling are data from animal models as well as clinical trials showing benefit in the progression of cardiac dysfunction from antifibrotic therapies. Examples of such therapies include antagonism of the renin–angiotensin–aldosterone axis with either ACE inhibitors, angiotensin receptor blockers, or mineralocorticoid receptor blockers [63]. Implantation of a left ventricular assist device (LVAD) has also been shown to improve myocardial function with regression of fibrosis [64].
In silico models Numerical models that reliably simulate ventricular hypertrophy progressing to heart failure do not exist, which is a reflection of the complexity of the disease process. Success has been achieved, however, in developing numerical models of the cardiac action potential and disease-associated electrical remodeling. For example, a model developed by Luo and Rudy [65,66] which describes the ventricular cardiomyocyte action potential as a function of individual ionic currents has been used extensively. Nattel and colleagues have used mathematical models that simulate the electrical properties of canine atrial tissue to evaluate propensity to sustain fibrillatory activity [67]. Wiegerinck et al. [68] used a computer simulation of strands of ventricular myocytes to test the effect of increased cardiomyocyte size on conduction velocity. Their data correlated with measurements obtained from an in vivo model of heart failure. www.drugdiscoverytoday.com
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Model comparison In vitro studies utilizing cardiomyocytes in culture have proven useful for testing specific molecular pathways in the pathogenesis of cardiac hypertrophy and failure. On occasion, however, results derived from studies of cultured myocytes have not been consistent with observations made in vivo. Uncoupling of adjacent cardiomyocytes, separation from the extracellular matrix, variable extent of fibroblast contamination and nutrient deprivation may each contribute to divergent findings. Furthermore, neonatal cardiomyocytes are immature, and signaling pathways in these cells are expected to differ from those of adult cardiomyocytes. Mouse models of cardiac hypertrophy have provided a wealth of evidence regarding mechanisms of ventricular remodeling. This is due, in large part, to the strength of transgenic and gene knockout technologies to define the expression and function of specific proteins. In addition, mice are relatively inexpensive and widely used, allowing numerous investigators to study similar models. By contrast, the small size of mice is a limiting factor. Measurement of cardiac function can be challenging, necessitating small instrumentation techniques with limited precision. Animal restraint or anesthesia is frequently required, which may further confound physiological measurements. Accurate and precise measurements of cardiac function are feasible in large animal models of cardiac disease. Also, it is also possible to study the effects of cardiac stress on particular regions of the heart. However, use of dog or pig models is expensive and requires considerable expertise for development and handling. In addition, transgenic technologies, which have proven so useful with mice and rats, are not as well established in large animals.
Model translation to humans Findings made with animal models must be interpreted with caution before applying them to human disease. For example, as compared with the human heart, the mouse heart is significantly smaller and beats 10 times faster, and resulting differences in cardiac structure and response to stress are anticipated. These differences are exemplified by the shape of the action potential of a ventricular myocyte: The human action potential includes a long plateau phase which is absent in the mouse. Aortic banding induces abrupt elevations in ventricular wall stress, analogous to that seen in the spared regions of infarcted LV. These models are less applicable to hypertension or aortic stenosis, however, where ventricular pressures typically rise gradually and myocyte hypertrophy precedes failure. Furthermore, most studies focus on rodents whose physical activity is limited by their being maintained in cages. Despite these limitations, these models serve as a useful platform for defining the role of specific pathways and 204
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mechanisms in the pathogenesis of load-induced heart disease.
Conclusions Enormous strides have been made in recent years in defining the fundamental biology of cardiac hypertrophy and failure. Numerous models have been developed to accomplish this important objective, each with attendant strengths and weaknesses. It is likely that additional work in this field will lead to insights that benefit patients with heart disease.
Acknowledgements This work was supported by grants from the Donald W. Reynolds Cardiovascular Clinical Research Center, NIH (HL-075173, HL-006296, HL-080144, HL-072016) and AHA (0640084N, 0655202Y).
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Depre, C. et al. (2006) Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy. Circulation 114, 1821–1828 Moorjani, N. et al. (2006) Activation of apoptotic caspase cascade during the transition to pressure overload-induced heart failure. J. Am. Coll. Cardiol. 48, 1451–1458 Balasubramanian, S. et al. (2006) Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases. J. Mol. Cell Cardiol. 41, 669–679 Page, I.H. (1939) A method for producing persistent hypertension by cellophane. Science 89, 273–274 Munagala, V.K. et al. (2005) Ventricular structure and function in aged dogs with renal hypertension: a model of experimental diastolic heart failure. Circulation 111, 1128–1135 Rysa, J. et al. (2005) Distinct upregulation of extracellular matrix genes in transition from hypertrophy to hypertensive heart failure. Hypertension 45, 927–933 Iwanaga, Y. et al. (2002) Excessive activation of matrix metalloproteinases coincides with left ventricular remodeling during transition from hypertrophy to heart failure in hypertensive rats. J. Am. Coll. Cardiol. 39, 1384–1391 Hankes, G.H. et al. (2006) Beta1-adrenoceptor blockade mitigates excessive norepinephrine release into cardiac interstitium in mitral regurgitation in dog. Am. J. Physiol. Heart Circ. Physiol. 291, H147–H151 Cantor, E.J. et al. (2005) A comparative serial echocardiographic analysis of cardiac structure and function in rats subjected to pressure or volume overload. J. Mol. Cell Cardiol. 38, 777–786 Ai, X. et al. (2005) Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ. Res. 97, 1314–1322 Akar, F.G. et al. (2005) Molecular mechanisms underlying K+ current downregulation in canine tachycardia-induced heart failure. Am. J. Physiol. Heart Circ. Physiol. 288, H2887–H2896 Poelzing, S. and Rosenbaum, D.S. (2004) Altered connexin43 expression produces arrhythmia substrate in heart failure. Am. J. Physiol. Heart Circ. Physiol. 287, H1762–H1770 Wilkoff, B.L. et al. (2002) Dual-chamber pacing or ventricular backup pacing in patients with an implantable defibrillator: the Dual Chamber and VVI Implantable Defibrillator (DAVID) Trial. JAMA 288, 3115–3123 Sharma, A.D. et al. (2005) Percent right ventricular pacing predicts outcomes in the DAVID trial. Heart Rhythm 2, 830–834 Most, P. et al. (2006) Cardiac S100A1 protein levels determine contractile performance and propensity toward heart failure after myocardial infarction. Circulation 114, 1258–1268 Curcio, A. et al. (2006) Competitive displacement of phosphoinositide 3kinase from beta-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling. Am. J. Physiol. Heart Circ. Physiol. 291, H1754–H1760 Port, J.D. and Bristow, M.R. (2001) Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure. J. Mol. Cell Cardiol. 33, 887–905 Freund, C. et al. (2005) Requirement of nuclear factor-kappaB in angiotensin II- and isoproterenol-induced cardiac hypertrophy in vivo. Circulation 111, 2319–2325 Wilkins, B.J. et al. (2004) Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ. Res. 94, 110–118 Degens, H. et al. (2003) Functional and metabolic adaptation of the heart to prolonged thyroid hormone treatment. Am. J. Physiol. Heart Circ. Physiol. 284, H108–H115 Milliken, M.C. et al. (1988) Left ventricular mass as determined by magnetic resonance imaging in male endurance athletes. Am. J. Cardiol. 62, 301–305 Fagard, R. (2003) Athlete’s heart. Heart 89, 1455–1461
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Shiojima, I. and Walsh, K. (2006) Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway. Genes Dev. 20, 3347–3365 DeBosch, B. et al. (2006) Akt1 is required for physiological cardiac growth. Circulation 113, 2097–2104 De Bono, J.P. et al. (2006) Novel quantitative phenotypes of exercise training in mouse models. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R926–R934 Konhilas, J.P. et al. (2004) Sex modifies exercise and cardiac adaptation in mice. Am. J. Physiol. Heart Circ. Physiol. 287, H2768–H2776 Yutzey, K.E. and Robbins, J. (2007) Principles of genetic murine models for cardiac disease. Circulation 115, 792–799 Seeland, U. et al. (2007) Interstitial remodeling in beta1-adrenergic receptor transgenic mice. Basic Res. Cardiol. 102, 183–193 Zhai, P. et al. (2006) An angiotensin II type 1 receptor mutant lacking epidermal growth factor receptor transactivation does not induce angiotensin II-mediated cardiac hypertrophy. Circ. Res. 99, 528–536 Molkentin, J.D. et al. (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228 Zhang, T. et al. (2003) The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ. Res. 92, 912–919 Nicol, R.L. et al. (2001) Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J. 20, 2757–2767 Harrison, B.C. et al. (2006) Regulation of cardiac stress signaling by protein kinase d1. Mol. Cell Biol. 26, 3875–3888 Ahmad, F. et al. (2005) The genetic basis for cardiac remodeling. Annu. Rev. Genomics Hum. Genet 6, 185–216 Wolf, C.M. et al. (2005) Somatic events modify hypertrophic cardiomyopathy pathology and link hypertrophy to arrhythmia. Proc. Natl. Acad. Sci. U. S. A. 102, 18123–18128 Song, Q. et al. (2003) Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J. Clin. Invest 111, 859–867 Sadoshima, J. and Izumo, S. (1997) The cellular and molecular response of cardiac myocytes to mechanical stress. Annu. Rev. Physiol. 59, 551–571 Suga, H. et al. (1973) Load independence of the instantaneous pressurevolume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circ. Res. 32, 314–322 Vasan, R.S. et al. (1997) Left ventricular dilatation and the risk of congestive heart failure in people without myocardial infarction. N. Engl. J. Med. 336, 1350–1355 Vasan, R.S. et al. (1999) Congestive heart failure in subjects with normal versus reduced left ventricular ejection fraction: prevalence and mortality in a population-based cohort. J. Am. Coll. Cardiol. 33, 1948–1955 Wang, T.J. et al. (2003) Natural history of asymptomatic left ventricular systolic dysfunction in the community. Circulation 108, 977–982 Sadoshima, J. et al. (2002) The MEKK1-JNK pathway plays a protective role in pressure overload but does not mediate cardiac hypertrophy. J. Clin. Invest. 110, 271–279 Okumura, S. et al. (2003) Disruption of type 5 adenylyl cyclase gene preserves cardiac function against pressure overload. Proc. Natl. Acad. Sci. U. S. A. 100, 9986–9990 Zile, M.R. et al. (2004) Diastolic heart failure–abnormalities in active relaxation and passive stiffness of the left ventricle. N. Engl. J. Med. 350, 1953–1959 Merx, M.W. et al. (2005) Myocardial stiffness, cardiac remodeling and diastolic dysfunction in calcification-prone fetuin-A-deficient mice. J. Am. Soc. Nephrol. 16, 3357–3364 Vega, R.B. et al. (2004) Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5. Mol. Cell Biol. 24, 8374–8385 Brown, R.D. et al. (2005) The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev. Pharmacol. Toxicol. 45, 657–687
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DRUG DISCOVERY
TODAY
DISEASE
MODELS
Vol. 4, No. 4 2007
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
Cardiovascular diseases
Models of cardiac hypertrophy and transition to heart failure Jeff M. Berry2, R. Haris Naseem2, Beverly A. Rothermel2, Joseph A. Hill1,2,3,* 1
Donald W. Reynolds Cardiovascular Clinical Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 3 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 2
The prevalence of heart failure is increasing rapidly worldwide, and yet effective treatments remain elusive. Pathological remodeling of the ventricle – and
Section Editors: Rahul Kakkar and Richard T. Lee – Harvard Medical School, Brigham and Women’s Hospital, Cambridge, MA, USA
associated hypertrophic growth, fibrotic change, cavity dilatation and electrophysiological remodeling – is a significant contributor to the pathogenesis of this prevalent disorder. As a consequence, there is great interest in developing new therapies to target pathological remodeling of the heart with the intent to prevent, arrest, or possibly reverse the otherwise inexorable progression of disease. To tackle this problem, numerous models of disease have been developed, both in vivo and in vitro. Here, we review models of cardiac hypertrophy and failure, compare and contrast their strengths and limitations, and on occasion cite recent works where the use of these models has contributed to significant scientific advances.
Introduction The heart is capable of robust growth and remodeling in response to changes in workload. For example, diseaserelated stresses, such as unremitting hypertension or myocardial injury, trigger hypertrophic growth that confers markedly increased risk of functional decompensation (heart failure) and malignant rhythm disturbance. By contrast,
*Corresponding author: J.A. Hill ([email protected]) 1740-6757/$ ß 2007 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmod.2007.06.003
growth of the heart in response to physiological demand (e.g. exercise or pregnancy) is adaptive and not associated with adverse sequelae. And the public health consequences of this cardiac remodeling are profound: congestive heart failure (CHF) contributes to over 300,000 deaths per year in the United States, and the prevalence of this disorder is rising rapidly [1]. Already a leading indication for hospitalization, CHF exacts a substantial financial toll on healthcare resources [1]. Heart failure is a syndrome that arises in the context of a multitude of disease states. Although there are common, overlapping aspects of the phenotype (e.g. elevated filling pressures, diminished forward flow), important differences exist across the spectrum of heart failure. Despite these substantial differences in the syndrome itself, treatment options are limited, and they are applied uniformly, irrespective of the functional phenotype, inciting stimulus, comorbid conditions, patient demographics, among others. A major focus in this field is to unveil disease-specific mechanisms that will lead to novel therapies to prevent, halt, or even reverse heart failure progression. Important new insights into the pathogenesis of this syndrome have been uncovered in recent years [2]. Among these insights is the emergence of left ventricular hypertrophy (LVH) as a major risk factor for development of heart failure [3]. Indeed, LVH is increasingly viewed as a crucial 197
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Figure 1. Paradigm of pathological remodeling of the ventricle. Moderate stress triggers a growth response leading to ventricular hypertrophy; this growth
step in the pathogenesis of many forms of heart failure [4,5] (Fig. 1). Numerous experimental models of heart failure exist, mimicking the vast array of clinical phenotypes. Here, we provide an overview of these models, comparing and contrasting their strengths and weaknesses (Table 1). Where appropriate, we cite recent examples where these models have been used to glean new insights into disease pathogenesis. We emphasize the role of hypertrophic remodeling, because this area of investigation has progressed rapidly in recent years, and several key insights have been uncovered that may emerge as clinically relevant therapeutic directions.
In vitro models of myocardial hypertrophy A truly ‘cardiac’ cell line does not exist. Thus, the great majority of cell culture-based studies have relied on neonatal cardiomyocytes in primary culture. These cells, typically isolated from rat, divide in culture approximately once and can be maintained in a healthy state for up to 1 week. To avoid overgrowth of co-purifying cardiac fibroblasts, differential-plating strategies are typically employed with mitosis inhibitors incorporated in the culture medium. It is likely that some of the divergent findings reported in the literature stem from the presence of variable fractions of co-cultured fibroblasts. 198
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Adult ventricular myocytes can be isolated and maintained for short periods of time in culture. Although this is suitable for certain types of investigation, such as whole cell electrophysiology studies, it is not optimal for biochemical or molecular biological approaches. For unknown reasons, adult mouse cardiac myocytes are particularly difficult to maintain in culture, although success has been reported [6]. However, even if successfully cultured, these cells begin to dedifferentiate over time and lose important aspects of the adult cardiomyocyte phenotype. The response of neonatal rat ventricular myocytes (NRVMs) to various neurohumoral signals has shed light on molecular mechanisms of myocardial hypertrophy and failure. For example, exposure to isoproterenol, a beta adrenergic agonist, triggers an increase in cardiomyocyte size and protein content with a gene expression profile similar to that observed with in vivo models of hypertrophy [7]. Phenylephrine, an alpha adrenergic agonist, and angiotensin II, a component of the renin–angiotensin–aldosterone system, each trigger growth of NRVMs in vitro and have been used to model hypertrophy [8,9]. The relationship between hypertrophic growth and contractile function has also been studied in vitro using NRVMs [9]. Contractility is assessed using digital imaging technologies to measure the amplitude and velocity of cell shortening
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Table 1. Comparison of models of cardiac hypertrophy and failure Pros
In vitro models
In vivo models
In silico models
Cardiomyocytes and cardiac fibroblasts can be isolated and studied in isolation
Changes in molecular signaling and tissue structure can be examined in association with changes in cardiac function Stresses on the heart relevant to human disease can be modeled
One specific property of myocardial tissue can be tested while controlling all other properties Costs and ethical concerns of animal experimentation are avoided
Genetic constructs can be used to express specific proteins or measure promoter-driven gene expression Cons
Cardiomyocytes are difficult to isolate and maintain healthy in culture Results of NRVMs may not be relevant to in vivo models or human disease
Findings in animal models are not always relevant to human disease Ethical considerations when subjecting animals to cardiac stress
Model is a highly simplified representation of events occurring in the heart Hypotheses generated in silico must be tested experimentally
Best use of model
To characterize the molecular mechanisms of stress response signaling pathways To test the effects of endogenous or mutated proteins on gene expression
To understand the role of specific proteins in the development of hypertrophy and heart failure To test new therapeutic strategies for heart failure
When parameters are easily quantified and easily manipulated
How to get access to the model
Literature
Literature
Literature
Relevant patents
n/a
n/a
n/a
References
[6–12]
[15–20,22–28,31,32,34–36,40–49,51,52]
[65–68]
and relaxation. Intracellular calcium transients are estimated using a calcium-sensitive indicator and analyzing fluorescence emissions from selected individual myocytes. Fibroblasts comprise a substantial fraction of the cells in the heart. Despite this, relatively little is known regarding fibroblast function, proliferation, or collagen biosynthesis. Among their functions, fibroblasts secrete cytokines that influence the growth and function of neighboring cardiomyocytes. Fibroblasts also play a major role in disease-related remodeling of the extracellular matrix. For example, Jaffre et al. [10] have shown that treatment of murine cardiac fibroblasts in vitro with isoproterenol causes increased production of interleukin-6, tumor necrosis factora and interleukin-1b. Additional studies such as this may lead to novel therapies that specifically target the cardiac fibroblast. Alternative cell lines that are easier to maintain in culture have been used for experiments relating to cardiac hypertrophy. H9c2 myoblasts, derived from embryonic rat hearts, proliferate in culture and can be induced to differentiate into myotubes for study [11]. C2C12 myoblasts, a skeletal musclederived cell line, have also been used for molecular studies [12]. Whether results using these cell lines can be translated to the fully developed, adult myocardium is uncertain.
In vivo models of cardiac hypertrophy Despite the important insights gleaned from in vitro models, this field continues to rely heavily on animal models of cardiac hypertrophy and failure. Indeed, in several instances,
conclusions drawn from studies of NRVMs have not held up in vivo [13,14]. Animal models of cardiac hypertrophy and failure have been developed based on stress and growth signals, which are observed in human cardiac disease (Fig. 2).
Load-induced hypertrophy and failure: pressure overload Patients with hypertension or aortic stenosis (AS) develop LVH as a consequence of persistently increased afterload. The left ventricle, under these conditions, must generate a higher systolic blood pressure to eject an adequate stroke volume. This scenario is often referred to as pressure overload. For experimental purposes, an increase in afterload can be created by surgically banding the ascending, transverse, or descending aorta. This commonly used strategy has been utilized to develop numerous animal models of pressure-overload disease. Aortic banding in mice is a well-established model of cardiac hypertrophy and heart failure [15] (Fig. 3). Importantly, the resulting phenotype can vary significantly depending on the strain of mice and the surgical technique employed. For example, Rothermel et al. [16] showed that moderate aortic banding (inducing a trans-stenotic pressure gradient of 60–80 mmHg) in adult male C57BL6 mice produces ventricular hypertrophy with a normal ejection fraction; by contrast, slightly more severe banding leads to a dilated heart, depressed systolic function and clinical features of heart failure, including increased mortality. These two clinically relevant phenotypes – compensated versus www.drugdiscoverytoday.com
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Figure 2. Forms of cardiac stress that trigger hypertrophic remodeling. In vivo models of cardiac disease are generally developed by imposing a stress on
decompensated ventricular hypertrophy – are associated with differential activation of stress-response signaling pathways and calcium handling proteins [16]. In studies designed to evaluate antiremodeling therapy, it is crucial that the imposed hemodynamic load be equivalent across treatment groups. Unfortunately, there is no simple way to ensure that this has been accomplished. For example, trans-stenotic pressure gradients are a function of both the degree of vessel stenosis and the pressure generated by the LV; hence, they provide an indirect measure of arterial resistance. To infer that trans-stenotic pressure gradients are indicative of banding-induced stenosis, one must assume that cardiac output is unchanged by drug treatment (or genetic manipulation). As an analogy, to compare electrical resistance (R) in two circuits by measuring the voltage drop (V) across each circuit, one must assume that current (I) is the same in both circuits; V = IR. This important caveat is apparent to most clinicians, who recognize that the murmur of aortic stenosis increases as valve area declines; however, when the LV starts to fail, the aortic stenosis murmur diminishes. Thus, in order to ensure equivalence of experimentally imposed afterload, 200
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we recommend rigorous attention to study design: The surgeon must be blinded to the experimental arms, animals should be randomized between groups whenever possible, and large numbers of animals must be studied. A canine model of ventricular hypertrophy was used by Depre et al. [17] in a study that found evidence of increased proteasome activity in the hypertrophied left ventricle. In that study, puppies at age 8–10 weeks underwent aortic banding; as the animal grows, afterload gradually increases mimicking the effects of progressive hypertension or AS. By 1 year of age, a significant increase in LV systolic pressure and ventricular hypertrophy had developed. Moorjani et al. [18] developed a pressure-overload model in sheep using a Foley catheter balloon enclosed in a cuff around the aorta. The balloon was inflated every 2 weeks to steadily increase the severity of aortic constriction. These animals initially developed ventricular hypertrophy followed by a decline in fractional shortening on echocardiography. In this model, the development of heart failure is associated with an increase in caspase activity, a mediator of cardiomyocyte apoptosis.
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Load-induced hypertrophy and failure: volume overload
Figure 3. Schematic of surgically induced stenosis of the thoracic aorta. This model is often termed transverse aortic constriction
Pressure-overload stress has also been studied in the right ventricle. Cooper and co-workers have developed a model of myocardial hypertrophy in cats by banding the pulmonary artery. Using this model, Balasubramanian et al. [19] reported significant accumulation of polyubiquitinated cytoskeletal proteins during the early period of pressure-overload stress. Alternatives to aortic banding exist for modeling pressureoverload stress. Renal wrapping to produce persistent hypertension in dogs was first described by Page in 1939 [20]. This procedure involves wrapping the kidneys with cellophane or silk, which triggers progressive increases in blood pressure after several weeks. Munagala et al. [21] used this model recently to study diastolic dysfunction associated with LVH. The spontaneously hypertensive rat (SHR) gradually develops hypertension starting at a young age and has long been used to study load-induced cardiac remodeling. Rysa et al. [22] reported an increase in expression of genes encoding extracellular matrix proteins associated with the development of heart failure in these animals. The Dahl salt-sensitive rat is yet another model of pressure-overload hypertrophy and heart failure. When fed a high salt diet for 11 weeks, these animals develop significantly increased blood pressure. In a study by Iwanaga et al. [23], an increase in expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs was observed as hypertrophy progressed to heart failure.
Another pathological stress on the heart is persistently increased LV cavity volume. This so-called volume overload occurs in patients with chronic mitral regurgitation, aortic regurgitation, beriberi, arteriovenous malformations and certain forms of congenital heart disease. Under these conditions, stroke volume must increase to maintain forward cardiac output, either because of regurgitant or shunted blood flow. In this setting, preload increases, and over time the heart develops an increase in end-diastolic volume. A canine model of mitral regurgitation has been created using fluoroscopically guided catheterization to rupture the chordae tendineae. These animals develop an increase in both LV end-diastolic dimension and fractional shortening associated with enhanced norepinephrine release into the myocardial interstitial fluid [24]. Another way to model volume-overload hypertrophy is by creating an aortocaval shunt. This procedure is commonly performed in rats, and the hearts of these animals become significantly dilated over several months. Cantor et al. [25] used this model to compare the progression of volume-overload hypertrophy with a rat model of pressure-overload hypertrophy. A model of combined pressure and volume overload has been developed in rabbits wherein the aortic valve is ruptured and the abdominal aorta is subsequently constricted surgically. Using this model, Ai et al. [26] have provided evidence that calmodulin kinase II alters ryanodine receptor function and calcium handling in heart failure. Persistent tachycardia can trigger ventricular remodeling leading to heart failure. For example, patients with atrial fibrillation and a rapid ventricular response can manifest impaired ventricular function that can recover once heart rate is controlled again. Tachycardia-induced cardiomyopathy can be induced in dogs by implanting a pacemaker that rapidly paces the right ventricle for several weeks [27,28]. This model not only incorporates the stress of a rapid heart rate, but right ventricular pacing may also contribute to adverse remodeling by altering the sequence of ventricular depolarization. This would be consistent with clinical evidence that suggests that prolonged right ventricular pacing contributes to poor outcomes in patients with heart failure [29,30]. Myocardial infarction (MI) represents a unique stress on the heart, encompassing characteristics of each of the above stresses. The region of noninfarcted tissue experiences increased preload, increased afterload, hypertrophic growth and sometimes altered depolarization and dysynchrony. The mechanical load on this myocardial tissue varies depending on the location and size of the infarct. MI can be produced surgically by ligating a coronary artery, and this procedure has been performed in numerous species, including mice [31,32]. www.drugdiscoverytoday.com
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Neurohormonal stress Increases in sympathetic activity are routinely seen in heart failure and appear to contribute to deterioration of cardiac function [33]. Consistent with this notion, cardiac hypertrophy and heart failure can be modeled by activating adrenergic signaling pathways. Isoproterenol, when administered continuously using a subcutaneously implanted osmotic pump, causes an increase in cardiac mass in mice [34]. Other neurohumoral signaling pathways have also been studied, including the renin–angiotensin–aldosterone system, which plays a role in pathological ventricular remodeling leading to heart failure. Mice that are continuously administered angiotensin II develop increases in heart weight. Freund et al. [34] showed that transgenic mice expressing an inhibitor of nuclear factor-kappaB have an attenuated hypertrophic response to both isoproterenol and angiotensin II. Additional neurohormonal stress signals have been used to model cardiac hypertrophy in vivo. Growth hormone and insulin-like growth factor 1 stimulate ventricular hypertrophy in mice, although this growth response is distinct from that caused by pressure overload or MI [35] Similarly, thyroid hormone when given to rats causes significant cardiac hypertrophy associated with activation of the Akt signaling pathway [36]. These models of ventricular remodeling may be relevant to physiological growth of the heart (see below).
Physiological hypertrophy Growth of the heart in response to the physiological demands of exercise is robust (up to 40–60% [37,38]) and yet is not associated with untoward sequelae. Mouse models of exercise-induced hypertrophy have proven valuable for dissecting molecular mechanisms of physiological and pathological ventricular remodeling. In many instances, the phosphoinositide-3-kinase (PI3K)/Akt pathway governs physiological growth of the heart [39]. DeBosch et al. [40] used a model of swimming-induced cardiac hypertrophy to show that Akt1 is necessary for physiological cardiac growth. Control mice subjected to forced swimming for 20 days developed significantly increased cardiac mass and cardiomyocyte size, whereas mice deficient in Akt1 showed no hypertrophy. It is important to recognize that forced exercise protocols use unpleasant stimuli to motivate animal activity, in contrast to voluntary exercise protocols, which are more physiological and less stressful. Voluntary wheel running for 5 weeks is associated with increased cardiac mass, although this hypertrophy appears to be more pronounced in female compared to male mice [41,42].
Genetic models of cardiac growth and failure A major objective in this field is to tease out specific signaling mechanisms that govern pathological and physiological cardiac remodeling. An integral part of this strategy is to utilize 202
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gain-of-function and loss-of-function strategies to decipher the role of specific signaling cascades [43]. For example, mice that overexpress the human beta1-adrenergic receptor specifically in the heart develop hypertrophy that progresses to heart failure with ventricular fibrosis [44]. Similarly, cardiacspecific overexpression of the angiotensin II type 1 receptor leads to cardiac hypertrophy and heart failure [45]. The cytoplasmic protein phosphatase calcineurin when expressed in cardiac myocytes is an especially potent inducer of pathological cardiac hypertrophy and failure [46]. Other recent examples include pathological remodeling triggered by expression in the heart of activated forms of CaMKII [47], MEK5 [48], or protein kinase D [49]. In addition to genetic activation of signaling pathways, cardiac hypertrophy can be triggered by mutations in genes coding for structural proteins and molecular elements of the sarcomere. For example, inherited forms of hypertrophic cardiomyopathy (HCM) in humans have been linked to numerous gene mutations [50], and many of these can be modeled in animals. Genes encoding components of the sarcomere including beta myosin heavy chain, troponin T, and myosin-binding protein C are among the most common to be implicated in the pathogenesis of HCM. Introduction of an Arg403Gln missense mutation in the murine alpha myosin heavy chain gene, a mutation associated with a severe form of HCM, leads to ventricular hypertrophy, cardiac dysfunction and arrhythmias [51]. Mice homozygous for a truncated form of myosin-binding protein C, similar to that observed in humans, develop myocardial hypertrophy and left ventricular dilation [52]. Although exceptions exist, a pattern has emerged where mutations of force-generating (e.g. sarcomeric) proteins trigger hypertrophic cardiomyopathy and mutations of structural (e.g. cytoskeletal) proteins induce dilated cardiomyopathy [50]. Although the primary impact of these mutations is probably an alteration of contractile efficiency, it is unknown how they influence the mechanotransduction machinery with which these proteins interconnect (reviewed in ref [53]).
Phenotypes of cardiac hypertrophy and heart failure Systolic heart failure Pathological cardiac signaling can lead to distinct patterns of morphological, functional and electrophysiological remodeling. Among the most common functional changes is impaired systolic contractile performance (systolic dysfunction), which can be assessed using a variety of invasive and noninvasive methods. A load-independent measure of LV contractility is obtained from the end-systolic pressurevolume relationship [54]. In the clinical setting, a reduced ejection fraction or an increase in LV volume is associated with adverse outcomes [55–57]. Development of systolic dysfunction, therefore, represents an important transition point in the progression of hypertrophic heart disease.
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Sadoshima et al. [58] showed that mitogen-activated protein kinase kinase (MEKK1) could counter the development of systolic dysfunction. After 2 weeks of aortic constriction, mice deficient in MEKK1 manifested increased mortality and decreased ejection fraction, while ventricular function remained stable in control mice. Okumura et al. [59] presented evidence that type 5 adenylyl cyclase (AC5) contributes to the transition to heart failure; 3 weeks after aortic constriction, wild-type mice manifested increases in LV size and reduction in ejection fraction, but AC5 knockout mice showed no such changes. Most et al. [31] showed that S100A1, a calciumbinding protein involved in excitation-contraction coupling, may protect the heart from systolic dysfunction following MI; S100A1 knockout mice subjected to coronary artery ligation manifested increased mortality and accelerated declines in systolic function compared to wild-type mice. Interestingly, transgenic mice that overexpress S100A1 showed blunted declines in systolic function and improved mortality [31].
Heart failure with normal ejection fraction Many patients experience heart failure symptoms despite apparently normal systolic function. In fact, a significant percentage of patients with CHF have a normal ejection fraction, and yet these patients are at increased risk of adverse events [56]. It is thought that diastolic dysfunction contributes to the transition from asymptomatic to symptomatic hypertrophic heart disease [60]. Elevated filling pressures, owing to impaired compliance of the left ventricle, are required to achieve an adequate preload and maintain cardiac output. These elevated pressures contribute to pulmonary congestion and CHF symptoms. Despite the prominence of heart failure with normal ejection fraction (HFNEF) in humans, development of a reliable animal model has been challenging. Munagala et al. [21] subjected dogs to renal wrapping to produce pressure overload, which resulted in left ventricular hypertrophy with preserved systolic function. These animals manifested prolonged LV relaxation times during diastole in association with increased ventricular fibrosis. Rysa et al. [22] examined spontaneously hypertensive rats at a stage where ventricular hypertrophy and diastolic dysfunction are present but ejection fraction is preserved; several genes were upregulated, most notably genes encoding extracellular matrix proteins. Studies in animals such as these are limited by the fact that one cannot assess symptoms, and signs of modest CHF (e.g. edema, pulmonary or hepatic congestion, lethargy) may be difficult to detect. Furthermore, accurate and precise measures of diastolic dysfunction are difficult, especially in small animals.
Ventricular fibrosis During pathological remodeling of the ventricle, disproportionate accumulation of fibrillar collagen occurs. This fibro-
Drug Discovery Today: Disease Models | Cardiovascular diseases
proliferative response contributes significantly to the development and worsening of both systolic and diastolic dysfunction and can lead to disruption of electrical wavefront activation and consequent re-entry. In general, cardiac extracellular matrix is regulated by a balance between production and degradation. Stressors that alter this delicate balance will lead to excessive ECM deposition resulting in myocardial dysfunction. Although the exact mechanisms by which excessive fibrosis leads to cardiac dysfunction are still unknown, drugs that block fibrogenesis would be of great clinical interest. Although models of pure myocardial fibrosis are more difficult to establish, given confounding abnormalities, examples exist of animal models where myocardial fibrosis is a dominant aspect of the phenotype. Probably the most studied and relevant animal models are those of pressureinduced hypertrophy and failure, where cardiac fibrosis and hypertrophy are classic features. (By contrast, cardiac fibrosis is not a prominent feature of exercise-induced hypertrophy.) Feutin-A knockout mice develop spontaneous dystrophic cardiac calcification and profound induction of fibroproliferative pathways [61]. Excessive myocardial fibrosis and diastolic dysfunction are characteristic abnormalities in this model. Interestingly, overexpression of activated protein kinase D in heart triggers profound failure with little fibrosis [62]. Perhaps even more compelling are data from animal models as well as clinical trials showing benefit in the progression of cardiac dysfunction from antifibrotic therapies. Examples of such therapies include antagonism of the renin–angiotensin–aldosterone axis with either ACE inhibitors, angiotensin receptor blockers, or mineralocorticoid receptor blockers [63]. Implantation of a left ventricular assist device (LVAD) has also been shown to improve myocardial function with regression of fibrosis [64].
In silico models Numerical models that reliably simulate ventricular hypertrophy progressing to heart failure do not exist, which is a reflection of the complexity of the disease process. Success has been achieved, however, in developing numerical models of the cardiac action potential and disease-associated electrical remodeling. For example, a model developed by Luo and Rudy [65,66] which describes the ventricular cardiomyocyte action potential as a function of individual ionic currents has been used extensively. Nattel and colleagues have used mathematical models that simulate the electrical properties of canine atrial tissue to evaluate propensity to sustain fibrillatory activity [67]. Wiegerinck et al. [68] used a computer simulation of strands of ventricular myocytes to test the effect of increased cardiomyocyte size on conduction velocity. Their data correlated with measurements obtained from an in vivo model of heart failure. www.drugdiscoverytoday.com
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Model comparison In vitro studies utilizing cardiomyocytes in culture have proven useful for testing specific molecular pathways in the pathogenesis of cardiac hypertrophy and failure. On occasion, however, results derived from studies of cultured myocytes have not been consistent with observations made in vivo. Uncoupling of adjacent cardiomyocytes, separation from the extracellular matrix, variable extent of fibroblast contamination and nutrient deprivation may each contribute to divergent findings. Furthermore, neonatal cardiomyocytes are immature, and signaling pathways in these cells are expected to differ from those of adult cardiomyocytes. Mouse models of cardiac hypertrophy have provided a wealth of evidence regarding mechanisms of ventricular remodeling. This is due, in large part, to the strength of transgenic and gene knockout technologies to define the expression and function of specific proteins. In addition, mice are relatively inexpensive and widely used, allowing numerous investigators to study similar models. By contrast, the small size of mice is a limiting factor. Measurement of cardiac function can be challenging, necessitating small instrumentation techniques with limited precision. Animal restraint or anesthesia is frequently required, which may further confound physiological measurements. Accurate and precise measurements of cardiac function are feasible in large animal models of cardiac disease. Also, it is also possible to study the effects of cardiac stress on particular regions of the heart. However, use of dog or pig models is expensive and requires considerable expertise for development and handling. In addition, transgenic technologies, which have proven so useful with mice and rats, are not as well established in large animals.
Model translation to humans Findings made with animal models must be interpreted with caution before applying them to human disease. For example, as compared with the human heart, the mouse heart is significantly smaller and beats 10 times faster, and resulting differences in cardiac structure and response to stress are anticipated. These differences are exemplified by the shape of the action potential of a ventricular myocyte: The human action potential includes a long plateau phase which is absent in the mouse. Aortic banding induces abrupt elevations in ventricular wall stress, analogous to that seen in the spared regions of infarcted LV. These models are less applicable to hypertension or aortic stenosis, however, where ventricular pressures typically rise gradually and myocyte hypertrophy precedes failure. Furthermore, most studies focus on rodents whose physical activity is limited by their being maintained in cages. Despite these limitations, these models serve as a useful platform for defining the role of specific pathways and 204
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mechanisms in the pathogenesis of load-induced heart disease.
Conclusions Enormous strides have been made in recent years in defining the fundamental biology of cardiac hypertrophy and failure. Numerous models have been developed to accomplish this important objective, each with attendant strengths and weaknesses. It is likely that additional work in this field will lead to insights that benefit patients with heart disease.
Acknowledgements This work was supported by grants from the Donald W. Reynolds Cardiovascular Clinical Research Center, NIH (HL-075173, HL-006296, HL-080144, HL-072016) and AHA (0640084N, 0655202Y).
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