A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.
Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.
Heart failure is a complex clinical syndrome, the symptoms of which include fatigue, dyspnea, exercise intolerance, and fluid retention in peripheral tissues. It is the leading cause of death in developed countries1. Aside from inherited cardiomyopathy caused by mutations in sarcomere proteins or ion channels2, myocardial dysfunction can be caused by a variety of medical conditions, including hypertension, valvular heart diseases, obesity, and diabetes3. Changes in myocardial structure, electrical conduction, and energy metabolism lead to inadequate cardiac pumping capacity to meet circulatory demands, which ultimately results in heart failure3,4. Investigating the mechanisms underlying heart failure, therefore, is critical in the field of cardiovascular research. Identifying molecular mechanisms leading to heart failure progression can eventually aid in the discovery of novel therapeutic targets or useful biomarkers1. It is therefore important to develop heart failure animal models that share key clinical features with heart failure in humans5.
Cardiac hypertrophy and remodeling plays a critical role in the development of heart failure. Hypertensive heart disease is the key contributing factor of cardiac hypertrophy and the maladaptive remodeling seen in human patients1. To mimic these human conditions, animal models are often established through surgical procedures. In particular, the transverse or abdominal aorta can be constricted to increase the resistance against the left ventricle, which ultimately leads to a pressure overload in the heart. This phenomenon usually results in cardiac hypertrophy, a physiological compensation of the cardiomyocytes to meet the functional demand of the cardiovascular system. However, the functional demand overrides the normal physiological compensatory mechanisms, leading to cardiac fibrosis and contractile impairment. Transverse aortic constriction (TAC) surgery often involves complicated procedures, including thoracotomy, mechanical ventilation, and separation of the thymus and fat tissue from the aortic arch. In contrast, abdominal aortic constriction requires simpler experimental techniques6-8. The abdominal aorta, between the left and right renal arteries, is constricted during the surgery. Cardiac hypertrophy and remodeling can be observed several weeks after the abdominal aortic constriction surgery6-8; they produce robust hypertensive heart disease similar to that generated by the transverse aortic constriction surgery9,10. Here, we describe a protocol to conduct abdominal aortic constriction in rats using an efficient, highly-reproducible, and minimally-invasive method. The abdominal aorta adjacent to the renal arteries is constricted by a 0.72 mm loop formed by a 4-0 silk thread. Ten weeks after the surgery, cardiac hypertrophy and remodeling can be observed. The rat model of abdominal aortic constriction-induced cardiac hypertrophy provides a platform for studying disease mechanisms and pathophysiology, as well as the development of potential therapeutics.
All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). This protocol was approved by and in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee at National Taiwan University.
1. Animal Surgery
2. Tissue and Blood Sample Collection
3. Tissue Fibrosis Quantification
4. Blood Troponin Quantification
10 weeks after the abdominal aortic constriction surgery, the resulting cardiac pathology was analyzed. The cardiac histology was measured by calculating the ratio of the heart weight to the body weight and by detecting the amount of collagen in the heart. Cardiac injury was confirmed by measuring plasma cardiac troponin concentration.
As shown in Figure 1A, the cardiac size was enlarged after abdominal aortic constriction surgery, as demonstrated by a higher heart-weight-to-body-weight ratio (Figure 1B), an indicator of cardiac hypertrophy. By using picrosirius red staining, fibrotic myocardium stained with an increased collagen content (red) can be distinguished from normal areas (yellow, Figure 2). Cardiac fibrosis was increased after abdominal aortic constriction surgery (Figure 2B) as compared to controls (Figure 2A). The results correlate with plasma troponin concentrations (Figure 3). An increased troponin concentration indicates that cardiac remodeling and injury during pressure overload has occurred. Taken together, abdominal aortic constriction results in evident cardiac injury, marked cardiac hypertrophy, and tissue remodeling.
Figure 1: Cardiac Hypertrophy during Pressure Overload. (A) Representative heart and (B) heart-weight-to-body-weight ratios are shown 10 weeks after abdominal aortic constriction surgery. Data represent the mean ± SEM of six independent experiments performed. Differences between groups were assessed by Student's t-test. *p <0.05 versus control. Please click here to view a larger version of this figure.
Figure 2: Cardiac Fibrosis during Pressure Overload.
Picrosirius red staining revealed increased collagen expression. (A) Representative picrosirius red staining (Scale bar = 200 µm) and (B) percentage of fibrotic area 10 weeks after abdominal aortic constriction surgery. Data represent the mean ± SEM of six independent experiments performed. Differences between groups were assessed by Student's t-test. *p <0.05 versus control. Please click here to view a larger version of this figure.
Figure 3: Cardiac Injury during Pressure Overload.
Blood plasma troponin concentrations were measured 10 weeks after abdominal aortic constriction surgery. Data represent the mean ± SEM of six independent experiments performed. Differences between groups were assessed by Student's t-test. *p <0.05 versus control. Please click here to view a larger version of this figure.
Hypertensive heart disease, a major health problem that contributes greatly to morbidity and mortality, can lead to cardiac hypertrophy and heart failure5. The pathogenesis and progression of hypertensive heart disease in humans is complex, so an appropriate animal model is critical to investigate the underlying mechanisms and to test novel therapeutics that aim to improve cardiac structure and function5. The abdominal aortic constriction model, which simulates chronic heart disease, is an effective method for cardiovascular research. The abdominal aorta adjacent to the renal arteries is constricted to induce cardiac remodeling, which eventually leads to cardiac injury. The extent of cardiac remodeling is evaluated by calculating the ratio of heart weight to body weight, performing picrosirius staining for the measurement of collagen expression, and conducting an ELISA-based method for the detection of plasma cardiac troponin levels. The abdominal aortic constriction method allows more reproducible results simulating cardiac remodeling in rat models.
Surgical models of heart disease are advantageous for closely mimicking the pathophysiology of hypertension and aortic stenosis1. Most of the currently-available surgical techniques to induce cardiac hypertrophy are conducted through transverse aortic constriction12,13, which is a common experimental procedure used to create a pressure overload. The sudden onset of hypertension that is achieved causes an approximately 50% increase in left ventricle mass within 2 weeks5, making the model an excellent choice to examine the molecular mechanisms leading to cardiac hypertrophy. However, transverse aortic constriction requires complex procedures and a high level of surgical skill. The stress associated with open-chest surgery and mechanical ventilation results in high surgical mortality. Moreover, the acute onset of severe hypertension, characteristic of this model, lacks direct clinical relevance5. In contrast, abdominal aortic constriction is less technically demanding. The onset and progression of cardiac hypertrophy is gradual, making this model clinically relevant to hypertensive cardiac diseases1,3. In addition, renal hypoperfusion by abdominal aortic constriction consequently activates the renin-angiotensin system, and therefore, the same surgical technique can be used as an animal model of kidney hypoperfusion injury15.
After surgery, cardiac pathology, including the appearance of fibrosis in the heart and changes in cardiac function, develops. In the early stages of hypertensive heart disease, myocardial contractility is enhanced by cardiac hypertrophy to compensate for the pressure overload10. In the later stages of hypertensive heart disease, cardiac function decompensates with fibrosis, which leads to heart failure10. We did not elaborate on the functional measurements of the heart, including pressure-volume loop analysis16 and echocardiography11. These approaches are invasive or non-invasive methods that are useful for understanding the changes in cardiac function. The time period after surgery can be varied to produce different degrees of cardiac remodeling. The longer the aorta is constricted, the greater the extent of cardiac dysfunction as a result of remodeling. The picrosirius red staining to measure fibrotic area and the ELISA measurements of plasma troponin levels are useful for the assessment of the degree of cardiac remodeling in order to set the endpoints after the surgery.
A key aspect of the surgery to induce cardiac hypertrophy is the clear identification and constriction of the abdominal aorta. Precision in the placement of the constriction site improves consistency of hypertrophy induction time, since variation in constriction placement may affect the length of time required for hypertrophy induction. The closer the stricture is to the heart, the shorter the induction time needed, although the harder it is to isolate the aorta. The abdominal aorta, between the origins of the right and left renal arteries, is a suitable site for performing constriction. The use of a rat model for the abdominal aortic constriction method has great advantages for the study of cardiac hypertrophy and heart failure. A limitation of this approach is that the surgical incision leads to tissue damage and to the secretion of inflammatory cytokines, which are different from the human cardiac hypertrophy and heart failure resulting from hypertension. Furthermore, the use of anesthetics and analgesics should be cautious, since some of these agents are reported to offer cardioprotective effects17.
The method described here provides a simple and effective approach to produce cardiac remodeling and injury in rats. Our technique is easy to perform, and the results are robust and reproducible. Once our surgical approach is mastered, this procedure will prove to be a useful platform for the investigation of disease mechanisms and the development of therapeutics in cardiac hypertrophy and remodeling.
The authors have nothing to disclose.
The authors’ work was supported by a grant from Ministry of Science and Technology (MOST 103-2320-B-002-068-MY2), the National Health Research Institute (NHRI-EX104-10418SC), and National Taiwan University (NTU 104R4000).
22-Gauge syringe needle | BD Biosciences | 309572 | |
EDTA Blood Collection Tubes | BD Biosciences | REF365974 | |
4-0 silk suture | Sharpoint™ Products | DC-2515N | |
6-0 silk suture | Sharpoint™ Products | DC-2150N | |
Pentobarbital | Sigma Aldrich | 1507002 | |
Paraformaldehyde | Sigma Aldrich | 441244 | |
Acetaminophen | Sigma Aldrich | A7085 | |
Picrosirius red solution | Abcam | ab150681 | |
Cardiac troponin kit | Abcam | ab200016 | |
Imagequant | Molecular Dynamics | ||
Langendorff | ADInstruments | ML870B2 |