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Development of a New Animal Model of Chronic Mitral Regurgitation in Rats Under Transesophageal Echocardiographic Guidance
Development of a New Animal Model of Chronic Mitral Regurgitation in Rats Under Transesophageal Echocardiographic Guidance Min Pu, MD, Zhaohui Gao, MD, Jianhua Li, PhD, Lawrence Sinoway, MD, and William R. Davidson Jr, MD, Hershey, Pennsylvania
Large animal models (dog and sheep) are often used for the investigation of the pathophysiology of chronic mitral regurgitation (MR). A major limitation of large animal models is cost. The aim of this study was to develop a new animal model of chronic MR. Left thoracotomy was performed in 34 rats. Under the guidance of transesophageal echocardiography, a fine needle was inserted into the left ventricle (LV) to damage the mitral leaflets and produce MR. Serial transthoracic echocardiography was per-
T
here are more than 5 million patients with moderate to severe valvular regurgitation in the United States.1 The Framingham Study noted that the prevalence of mitral regurgitation (MR) increased 1.3-fold with each decade of life.2 It is anticipated that the prevalence of MR will increase even further because of the rapidly growing aged population in the United States and worldwide. Although much progress has been made in the management of chronic MR,1,3 many aspects regarding pathophysiology and pharmacologic therapies of chronic MR are not fully understood. Animal models have been used in the study of the pathophysiology of MR and in the identification of potential surgical and/or medical therapies for chronic MR. Traditionally, dog or sheep MR models have been most commonly used.4-6 Although large animal models provide a reliable experimental model of chronic MR, a major limitation of these models is cost. Therefore, most previous studies used only 6 to 12 dogs or sheep.7-15 With limited numbers of animals, it is difficult to evaluate the effects of therapeutic interventions on the outcome From the Division of Cardiology, Milton S. Hershey Medical Center, Penn State College of Medicine. Supported in part by a grant-in-aid from the American Society of Echocardiography (M.P.), a Scientists Development Grant from the American Heart Association (M.P.), and National Institutes of Health R01 HL0060800 grant (L.S.). Reprint requests: Min Pu, MD, Division of Cardiology, H047, Penn State University, College of Medicine, Milton S. Hershey Medical Center, 500 University Dr, PO Box 850, Hershey, PA 17033 (E-mail: [email protected]). 0894-7317/$30.00 Copyright 2005 by the American Society of Echocardiography. doi:10.1016/j.echo.2004.10.005
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formed to assess LV remodeling and function. Left atrial and LV diameters were significantly larger, and LV fractional shortening was lower in the MR group than in the sham group. The 150-day survival was 59% in the MR group and 100% in the sham group (P < .01). This new animal model of chronic MR may be used in the study of the pathophysiology of chronic MR and pharmacologic therapies. (J Am Soc Echocardiogr 2005;18:468-74.)
(mortality/survival) in chronic MR. Therefore, development of a new cost-effective animal model of chronic MR is needed. Based on our pilot animal study16 and clinical experience, we hypothesized that a chronic MR model could be developed in rats. We chose rats for the development of a small animal model of chronic MR primarily for the following reasons: (1) rat models are commonly used in the experimental study of cardiovascular disease with an ample database that can serve as a pathophysiologic reference; (2) cardiovascular drugs (anigotensin-converting enzyme inhibiters, beta-blockers, and aldosterone receptor blockers) had first shown beneficial effects on left ventricular (LV) remodeling and function, and mortality in the rat models of myocardial infarction, hypertension, and congestive heart failure,17-20 before similar beneficial effects were validated in patients21-23–thus, a rat model of chronic MR could be particularly useful for the investigation of pharmacologic therapy for chronic MR; and (3) cellular and molecular biology studies (important for the investigation of fundamental pathophysiology of chronic MR) can be carried at a significantly lower cost in a rat model than dog or sheep models if experimental animals are required to be killed for ex vivo tissue and molecular analysis. Therefore, the aim of this experimental study was to develop and characterize a rat model of chronic MR. METHODS Animal Preparation The study protocol was approved by the institutional animal care and use committee. The study was performed
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Figure 1 Left ventricular (LV) long-axis view by transesophageal echocardiography. There was no mitral regurgitation (MR) before procedure (A). Severe MR was detected by color Doppler after MR procedure (B). LA, Left atrium.
Figure 2 Right pulmonary vein flow velocity recorded in rat before creation of mitral regurgitation (MR) (A) and after procedure (B). Pulmonary venous systolic flow was reversed after induction of severe MR (B).
according to the guidelines of the American Physiological Society. A total of 34 Sprague-Dawley rats (180 g ⬃ 339) were used for the study. The rats were first anesthetized with 3% to 5% of isoflurane and then intubated. The lungs were ventilated using a respirator (Hallowell AWS, Piffsfield, Mass). Anesthesia was maintained by mixing isoflurane with oxygen (1%-2% isoflurane and 98%-99% oxygen). Heart rate and oxygen saturation were monitored using a respiratory gas monitor (Ohmeda 5250 RGM, Helsinki, Finland) throughout the study. Transesophageal Echocardiography Development of a rat model of chronic MR required a reliable method to assess the severity of MR and guide surgical creation of MR. Based on our clinical experience in the operating room,24-27 transesophageal echocardiography is one of the most useful tools for assessing MR intraoperatively. We performed transesophageal studies in rats using intracardiac echocardiography catheters (Acuson/Siemens Corp, Mountain View, Calif).16 This catheter is equipped with a 64-element phased-array transducer in a longitudinal plane on the tip with multiple imaging
frequencies from 5.5 to 10 MHz. The transducer produces M-mode, 2-dimensional, pulsed wave, and color Doppler images. The catheter tip can be flexed anteriorly, posteriorly, and laterally using the control knobs at the end of the catheter. The catheter was connected to a commercial echocardiograph (Sequoia 256, Acuson/Siemens Corp). In this study M-mode, 2-dimensional, color Doppler, and pulsed wave Doppler imaging was performed. The LV, left atrium (LA), mitral inflow, and pulmonary venous flow were examined. All images were stored digitally and on super-VHS tape. MR Operation After rats were anesthetized, an intracardiac echocardiographic catheter was inserted into the esophagus. A left thoracotomy was performed through the fifth or sixth intercostal space. A fine needle (0.36-mm diameter) was inserted into the LV through the LV apex under the guidance of transesophageal echocardiography. The needle was advanced into the mitral valve to damage mitral leaflets and create MR. The severity of MR was assessed using transesophageal echocardiography in real time. MR
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Figure 3 M-mode echocardiograms demonstrated significantly dilated left ventricle (LV) 12 weeks after creation of mitral regurgitation (MR). LV end-diastolic diameter (LVEDD) increased from 0.68 cm before MR procedure (A) to 1.3 cm 12 weeks after MR procedure (B).
was considered significant if a regurgitant jet area occupied more than 45% of the LA area (Figure 1), or reversed pulmonary venous flow pattern was detected (Figure 2), or both. The needle was then withdrawn and the chest was closed. In the sham group, a left thoracotomy was performed and the needle was inserted into the LV, but the mitral valve was not damaged and no MR was produced. The needle was then withdrawn and the chest closed. Transthoracic Echocardiography Transthoracic echocardiography (Acuson/Siemens Corp) was performed to assess LV remodeling and function. Rats were anesthetized with inhalation of 3% to 5% of isoflurane mixed with oxygen. No intubation was performed for transthoracic echocardiographic studies. Parasternal long- and short-axis views were recorded as previously described.28 M-mode image was obtained at the level of the chordae tendineae of the mitral valve under the guidance of 2-dimensional imaging. Transthoracic echocardiography was performed 1 week before operation and 1, 6, and 12 weeks after the surgical procedure in the MR and the sham groups. Data Analysis Semiquantitative assessment of MR. Color Doppler mapping of MR jets was used to semiquantitatively assess the severity of MR. MR jet areas and LA areas were measured in the LV long-axis view Figure 3. The ratio of MR jet area to the LA areas was calculated. Pulmonary venous flow was recorded. Pulmonary venous systolic flow reversal was used as a marker of severe MR (Figure 2, B). Assessment of LV remodeling. The LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and thicknesses of the interventricular septum and posterior wall were measured using M-mode echocardiography from the parasternal long-axis view (Figure 3). LV mass
was calculated using the following formula29: LV mass ⫽ 1.04 ⫻ [(LVEDD ⫹ posterior wall ⫹ interventricular septum)3 ⫺ end-diastolic diameter3]. The LV mass index was calculated as LV mass (in grams) divided by the rat’s body weight. Assessment of systolic function. LV fractional shortening was used for the assessment of LV systolic function. LV fractional shortening was calculated as [(LVEDD ⫺ LVESD)/LVEDD] ⫻ 100%. Measurement of LA dimension. The LA dimension was measured using M-mode echocardiography from the parasternal long-axis view. Costs Costs for rat, dog, and sheep models were analyzed according to standard charges from our institutional department of comparative medicine. Costs included direct costs for the purchase of animals and postoperative animal care. Statistical Analysis Values are expressed as mean ⫾ SD. Student unpaired t test was used to compare the means of continuous variables for the two independent groups. 2 Testing was used to examine difference in discrete variables. A P value ⬍ .05 was considered statistically significance. A KaplanMeier analysis was used to compare survival.
RESULTS MR Severity and Volume Overload Mean MR jet area was 20 ⫾ 4 mm2 with a ratio of MR jet area to the LA area of 71 ⫾ 11% in the MR group. There was no MR in the sham group. Mitral inflow
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Figure 4 Left atrial (LA) size was significant larger in mitral regurgitation (MR) group after creation of MR than in sham-operated group indicating significant volume overload of LA, although both groups had similar LA size before MR creation.
significantly increased immediately after the development of MR. This was reflected by a significantly increased mitral inflow peak velocity (94 ⫾ 16 cm/s vs 137 ⫾ 22 cm/s, P ⬍ .001) and an increased mitral inflow velocity-time integral (4.8 ⫾ 0.8 cm vs 6.4 ⫾ 0.9 cm, P ⬍ .01) in the rats with MR. There were no significant changes in the mitral inflow peak velocity (102 ⫾ 14 vs 106 ⫾ 11 cm/s, P ⬎ .05) or the velocity-time integral (4.8 ⫾ 0.8 vs 4.5 ⫾ 0.6 cm/s, P ⬎ .05) in the sham operated group. LA dimensions were significantly larger in the MR group than in the sham group after the MR operation (Figure 4). LV Remodeling One week after MR creation, LVEDD was mildly increased in the MR group. There was no significant change in LVESD 1 week after MR creation in either the MR group or the sham group. By the 12th week after the operation, however, both LVEDD and LVESD in the MR group were significantly larger than in the sham group (Figure 5). An increase in LVESD developed later than an increase in LVEDD. By the 12th week, the LV mass indexes were significantly higher in the MR group (2.72 ⫾ 0.78 g/kg) than in the sham group (1.98 ⫾ 0.26 g/kg, P ⬍ .01). LV Function LV fractional shortening initially increased at the first week after the operation in the MR group, indicating a hyperdynamic LV in the early stages of chronic MR. A decrease in LV fractional shortening was noted by the 12th week in the MR group, suggesting that LV systolic function was impaired because of long-term volume overload (Figure 6). There was no change in the LV fractional shortening in the sham group during the 12-week follow-up. Survival Six rats died within 6 days after operation in the MR group. No deaths were noted in the sham group with
Figure 5 Comparison of left ventricular (LV) end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) between mitral regurgitation (MR) group and sham-operated group. There were no significant differences in LVEDD and LVESD between MR and sham-operated groups before MR procedure. LVEDD was significantly larger in MR group than sham-operated group after creation of MR (A). However, LVESD was not significantly dilated until 6 weeks after MR procedure (B).
acute mortality of 27% versus 0% (P ⬍ .01). Three rats died between 7 days and 150 days after the operation in the MR group with no death in the sham group. The 150-day survival was significantly lower in the MR group (59%) than in the sham group (100%) (P ⬍ .01) (Figure 7). Costs The table lists the estimated direct costs for a 150-day experiment in 34 rats in comparison with the estimated direct costs for 150-day experiment in 34 dogs or 34 sheep. Estimated direct costs were 42 times higher in the dog model and 48 times higher in the sheep model than in the rat model.
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Table Comparison of estimated direct costs among rat, dog, and sheep models of chronic mitral regurgitation with a 150-day follow-up Animals
Rat Dog Sheep
Costs for purchase of an animal ($)
Costs/day for postoperative care ($)
Total costs of 150 days for an animal ($)
Total costs for 34 animals ($)
22 800 650
0.10 5.0 7.5
37 1550 1775
1258 52,700 60,350
DISCUSSION MR Model Recent discoveries in genomics, and molecular and cellular biology, have led to the development and widespread use of small animal models of human diseases. Although large animal models have been used for the experimental study of chronic MR, there are few small animal models of chronic MR.30 In large animal models, acute or chronic MR is often produced by damaging the chordae tendineae using a biopsy catheter31-33 or by the ligation of the left circumflex coronary artery (ischemic MR).34-36 These large animal models have been used for the study of the pathophysiology of chronic MR and for evaluation of surgical and medical therapy for chronic MR.37 The high cost is a major limitation to these large animal models. The current study has established the first rat model of chronic MR. There are several advantages in this model: (1) costs for a rat model of chronic MR are significantly lower than that for large animal models– direct costs are $37 per rat as compared with $1550 per dog and $1775 per sheep for a 150-day follow-up study (Table); (2) LV remodeling, function, and hemodynamic changes caused by volume overload (chronic MR) can be assessed by noninvasive methods; (3) LV remodeling could be seen as early as the 6th week after MR procedure–this may be especially helpful for shortening the experimental cycle; and (4) a rat model can be particularly useful for the investigation of pharmacologic treatment of chronic MR–although the effects of pharmacologic therapy have been assessed using anatomic or functional surrogates (LV remodeling, function, and hemodynamic) invasively and noninvasively in large animal models of chronic MR,32,38 a mortality benefit could not be used as a primary end point for the assessment of the efficacy of medical therapy. This might be because high costs limited the numbers of experimental large animals (often ranging from 6-12 dogs or sheep) used in the previous studies. In the rat model of chronic MR, significant 150-day mortality was observed. Therefore, mortality (the hardest end point) can be used as a primary end point for the assessment of the effects of pharmacologic therapies for chronic MR.
LV Remodeling and Function in MR LV remodeling and function are most important factors determining prognosis for patients with severe chronic MR. The current study shows that LV remodeling in chronic MR has two stages. In the first stage (early) LV remodeling was mainly seen in diastole (diastolic remodeling) with an increase in the end-diastolic diameter. This early remodeling could be a compensatory response to volume overload posed by severe chronic MR with preserved LV systolic function (hyperdynamic stage). In the second stage, the LVESD increased as well (systolic remodeling stage), which was associated with impaired LV systolic function (decreased LV fractional shortening). The results of the current experimental study are consistent with clinical observations that LVESD is strongly associated with LV dysfunction in patients with chronic MR.39,40 The study suggests that treatment should target early LV remodeling in the early stages of chronic MR with the goal of preventing or delaying LV systolic remodeling. Study Limitations In large animal models of chronic MR, surgical repair or replacement of the mitral valve can be performed. Therefore, large animal models are important for the experimental investigation of surgical therapy for chronic MR. Currently, mitral valve operation cannot be performed in rats. Therefore, a rat model may not be used for the study of surgical therapy for chronic MR. In large animal models MR can be created using a biopsy catheter without an open-chest operation. In the current rat model of chronic MR, open-chest operation was performed. We were initially concerned that open-chest operation might impact rat survival, especially with a needle inserted into the LV temporarily. However, open-chest operation is routinely used for the rat model of myocardial infarction (coronary artery ligation) and is widely accepted in experimental studies. Furthermore, there was no death in the sham group, although there were the same number of needle insertions in LV in the sham-operated group, indicating that the rats tolerated the procedure well. Quantitative assessment of the severity of MR by the proximal isovelocity surface area method
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Figure 6 Dynamic changes in left ventricular (LV) systolic function. LV fractional shortening (LVFS) increased 1 week after creation of mitral regurgitation (MR), but decreased by 12th week in rats with chronic MR. There was no change in LVFS in sham group.
Figure 7 Kaplan-Meier plot of 150-day survival after development of mitral regurgitation (MR), which was lower in MR group (59%) than in sham group (100%) (P ⬍ .01).
may be difficult because of the rapid heart rate in rats (approximate 300-380/min). Therefore, this study used MR jet area, a ratio of MR jet area to LA area and a pulmonary venous systolic reversal as markers for the severity of MR. Our results showed significant LV remodeling, LA dilation, and a reduction in LV fractional shortening 12 weeks after MR creation, demonstrating that these techniques reliably identified significant MR, which produced volume overload. We thank Ms Valerie Kehoe for technical assistance in animal experiments.
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2. Singh JP, Evans JC, Levy D, Larson MG, Freed LA, Fuller DL, et al. Prevalence and clinical determinants of mitral, tricuspid, and aortic regurgitation (the Framingham heart study). Am J Cardiol 1999;83:897-902. 3. ACC/AHA guidelines for the management of patients with valvular heart disease. a report of the American College of Cardiology/American Heart Association task force on practice guidelines (committee on management of patients with valvular disease). J Am Coll Cardiol 1998;32:1486-588. 4. Nagatsu M, Ishihara K, Zile MR, Tsutsui H, Tagawa H, DeFreyte G, et al. The effects of complete versus incomplete mitral valve repair in experimental mitral regurgitation. J Thorac Cardiovasc Surg 1994;107:416-23. 5. Raman JS, Byrne MJ, Power JM, Alferness CA. Ventricular constraint in severe heart failure halts decline in cardiovascular function associated with experimental dilated cardiomyopathy. Ann Thorac Surg 2003;76:141-7. 6. Fukamachi K, Inoue M, Popovic ZB, Doi K, Schenk S, Nemeh H, et al. Off-pump mitral valve repair using the Coapsys device: a pilot study in a pacing-induced mitral regurgitation model. Ann Thorac Surg 2004;77:688-92. 7. Rankin JS, Nicholas LM, Kouchoukos NT. Experimental mitral regurgitation: effects on left ventricular function before and after elimination of chronic regurgitation in the dog. J Thorac Cardiovasc Surg 1975;70:478-88. 8. Carabello BA, Nakano K, Corin W, Biederman R, Spann JF Jr. Left ventricular function in experimental volume overload hypertrophy. Am J Physiol 1989;256:H974-81. 9. Yellin EL, Yoran C, Sonnenblick EH, Gabbay S, Frater RW. Dynamic changes in the canine mitral regurgitant orifice area during ventricular ejection. Circ Res 1979;45:677-83. 10. Tallaj J, Wei CC, Hankes GH, Holland M, Rynders P, Dillon AR, et al. Beta1-adrenergic receptor blockade attenuates angiotensin II-mediated catecholamine release into the cardiac interstitium in mitral regurgitation. Circulation 2003;108: 225-30. 11. Hoit BD, Jones M, Eidbo EE, Elias W, Sahn DJ. Sources of variability for Doppler color flow mapping of regurgitant jets in an animal model of mitral regurgitation. J Am Coll Cardiol 1989;13:1631-6. 12. Shiota T, Jones M, Teien DE, Yamada I, Passafini A, Ge S, et al. Dynamic change in mitral regurgitant orifice area: comparison of color Doppler echocardiographic and electromagnetic flowmeter-based methods in a chronic animal model. J Am Coll Cardiol 1995;26:528-36. 13. Gorman JH III, Gorman RC, Plappert T, Jackson BM, Hiramatsu Y, St John-Sutton MG, et al. Infarct size and location determine development of mitral regurgitation in the sheep model. J Thorac Cardiovasc Surg 1998;115:615-22. 14. Timek TA, Nielsen SL, Green GR, Dagum P, Bolger AF, Daughters GT, et al. Influence of anterior mitral leaflet second-order chordae on leaflet dynamics and valve competence. Ann Thorac Surg 2001;72:535-40. 15. Gregoric I, Conger JL, Reul H, Tamez D, Clubb FJ, Stainback RF, et al. Preclinical assessment of a trileaflet mechanical valve in the mitral position in a calf model. Ann Thorac Surg 2004;77:196-202. 16. Gao Z, Li J, Davidson WR Jr, Sinoway L, Pu M. Is comprehensive transesophageal Doppler echocardiographic study feasible in small animals [Abstract]? Circulation 2003;108:IV-2995. 17. Pfeffer JM, Pfeffer MA, Braunwald E. Hemodynamic benefits and prolonged survival with long-term captopril therapy in rats with myocardial infarction and heart failure. Circulation 1987;75:I149-55.
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18. Sweet CS, Emmert SE, Stabilito II, Ribeiro LG. Increased survival in rats with congestive heart failure treated with enalapril. J Cardiovasc Pharmacol 1987;10:636-42. 19. Hu K, Gaudron P, Ertl G. Long-term effects of beta-adrenergic blocking agent treatment on hemodynamic function and left ventricular remodeling in rats with experimental myocardial infarction: importance of timing of treatment and infarct size. J Am Coll Cardiol 1998;31:692-700. 20. Smith EF III, Griswold DE, Hillegass LM, Slivjak MJ, Davis PA, DiMartino MJ. Cardioprotective effects of the vasodilator/beta-adrenoceptor blocker, carvedilol, in two models of myocardial infarction in the rat. Pharmacology 1992;44:297305. 21. Pfeffer MA, Braunwald E, Moye LA, Basta L, Brown EJ Jr, Cuddy TE, et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the survival and ventricular enlargement trial; the SAVE investigators. N Engl J Med 1992;327: 669-77. 22. Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, et al. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure: US carvedilol heart failure study group. N Engl J Med 1996;334:1349-55. 23. Hjalmarson A, Goldstein S, Fagerberg B, Wedel H, Waagstein F, Kjekshus J, et al. Effects of controlled-release metoprolol on total mortality, hospitalizations, and well-being in patients with heart failure: the metoprolol CR/XL randomized intervention trial in congestive heart failure (MERIT-HF); MERIT-HF study group. JAMA 2000;283:1295-302. 24. Pu M, Vandervoort PM, Griffin BP, Leung DY, Stewart WJ, Cosgrove DM, et al. Quantification of mitral regurgitation by the proximal convergence method using transesophageal echocardiography: clinical validation of a geometric correction for proximal flow constraint. Circulation 1995;92:2169-2. 25. Pu M, Griffin BP, Vandervoort PM, Leung DY, Cosgrove DM, Thomas JD. Intraoperative validation of mitral inflow determination by transesophageal echocardiography: comparison between single, biplane and thermodilution techniques. J Am Coll Cardiol 1995;26:1047-53. 26. Breburda CS, Griffin BP, Pu M, Rodriguez L, Cosgrove DM III, Thomas JD. Three-dimensional echocardiographic planimetry of maximal regurgitant orifice area in myxomatous mitral regurgitation: intraoperative comparison with proximal flow convergence. J Am Coll Cardiol 1998;32:432-7. 27. Pu M, Thomas JD, Vandervoort PM, Stewart WJ, Cosgrove DM, Griffin BP. Comparison of quantitative and semiquantitative methods for assessing mitral regurgitation by transesophageal echocardiography. J Am Cardiol 2001;87:66-70. 28. Pawlush DG, Moore RL, Musch TI, Davidson WR Jr. Echocardiographic evaluation of size, function, and mass of normal and hypertrophied rat ventricles. J Appl Physiol 1993;74: 2598-605.
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29. Litwin SE, Katz SE, Morgan JP, Douglas PS. Serial echocardiographic assessment of left ventricular geometry and function after large myocardial infarction in the rat. Circulation 1994;89:345-54. 30. Gunawardena S, Bravo E, Kappagoda CT. Effect of chronic mitral valve damage on activity of pulmonary rapidly adapting receptors in the rabbit. J Physiol 1998;511:79-88. 31. Zile MR, Tomita M, Ishihara K, Nakano K, Lindroth J, Spinale F, et al. Changes in diastolic function during development and correction of chronic LV volume overload produced by mitral regurgitation. Circulation 1993;97:1378-88. 32. Nagatsu M, Spinale FG, Koide M, Tagawa H, DeFreitas G, Cooper G, et al. Bradycardia and the role of beta-blockade in the amelioration of left ventricular dysfunction. Circulation 2000;101:653-9. 33. Dell’Italia LJ, Meng QC, Balcells E, Straeter-Knowlen IM, Hankes GH, Dillon R, et al. Increased ACE and chymase-like activity in cardiac tissue of dogs with chronic mitral regurgitation. Am J Physiol 1995;269:H2065-75. 34. Llaneras MR, Nance ML, Streicher JT, Lima JA, Savino JS, Bogen DK, et al. Large animal model of ischemic mitral regurgitation. Ann Thorac Surg 1994;57:432-9. 35. Otsuji Y, Handschumacher MD, Liel-Cohen N, Tanabe H, Jiang L, Schwammenthal E, et al. Mechanism of ischemic mitral regurgitation with segmental left ventricular dysfunction: three-dimensional echocardiographic studies in models of acute and chronic progressive regurgitation. J Am Coll Cardiol 2001;37:641-8. 36. Lai DT, Tibayan FA, Myrmel T, Timek TA, Dagum P, Daughters GT, et al. Mechanistic insights into posterior mitral leaflet inter-scallop malcoaptation during acute ischemic mitral regurgitation. Circulation 2002;106:I40-5. 37. Yun KL, Rayhill SC, Niczyporuk MA, Fann JI, Zipkin RE, Derby GC, et al. Mitral valve replacement in dilated canine hearts with chronic mitral regurgitation: importance of the mitral subvalvular apparatus. Circulation 1991;84: III112-24. 38. Nemoto S, Hamawaki M, De Freitas G, Carabello BA. Differential effects of the angiotensin-converting enzyme inhibitor lisinopril versus the beta-adrenergic receptor blocker atenolol on hemodynamics and left ventricular contractile function in experimental mitral regurgitation. J Am Coll Cardiol 2002; 40:149-54. 39. Matsumura T, Ohtaki E, Tanaka K, Misu K, Tobaru T, Asano R, et al. Echocardiographic prediction of left ventricular dysfunction after mitral valve repair for mitral regurgitation as an indicator to decide the optimal timing of repair. J Am Coll Cardiol 2003;42:458-63. 40. Tanaka K, Ohtaki E, Matsumura T, Misu K, Tohbaru T, Asano R, et al. Impact of a preoperative mitral regurgitation scoring system on outcome of surgical repair for mitral valve prolapse. Am J Cardiol 2003;92:1306-9.
Large animal models (dog and sheep) are often used for the investigation of the pathophysiology of chronic mitral regurgitation (MR). A major limitation of large animal models is cost. The aim of this study was to develop a new animal model of chronic MR. Left thoracotomy was performed in 34 rats. Under the guidance of transesophageal echocardiography, a fine needle was inserted into the left ventricle (LV) to damage the mitral leaflets and produce MR. Serial transthoracic echocardiography was per-
T
here are more than 5 million patients with moderate to severe valvular regurgitation in the United States.1 The Framingham Study noted that the prevalence of mitral regurgitation (MR) increased 1.3-fold with each decade of life.2 It is anticipated that the prevalence of MR will increase even further because of the rapidly growing aged population in the United States and worldwide. Although much progress has been made in the management of chronic MR,1,3 many aspects regarding pathophysiology and pharmacologic therapies of chronic MR are not fully understood. Animal models have been used in the study of the pathophysiology of MR and in the identification of potential surgical and/or medical therapies for chronic MR. Traditionally, dog or sheep MR models have been most commonly used.4-6 Although large animal models provide a reliable experimental model of chronic MR, a major limitation of these models is cost. Therefore, most previous studies used only 6 to 12 dogs or sheep.7-15 With limited numbers of animals, it is difficult to evaluate the effects of therapeutic interventions on the outcome From the Division of Cardiology, Milton S. Hershey Medical Center, Penn State College of Medicine. Supported in part by a grant-in-aid from the American Society of Echocardiography (M.P.), a Scientists Development Grant from the American Heart Association (M.P.), and National Institutes of Health R01 HL0060800 grant (L.S.). Reprint requests: Min Pu, MD, Division of Cardiology, H047, Penn State University, College of Medicine, Milton S. Hershey Medical Center, 500 University Dr, PO Box 850, Hershey, PA 17033 (E-mail: [email protected]). 0894-7317/$30.00 Copyright 2005 by the American Society of Echocardiography. doi:10.1016/j.echo.2004.10.005
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formed to assess LV remodeling and function. Left atrial and LV diameters were significantly larger, and LV fractional shortening was lower in the MR group than in the sham group. The 150-day survival was 59% in the MR group and 100% in the sham group (P < .01). This new animal model of chronic MR may be used in the study of the pathophysiology of chronic MR and pharmacologic therapies. (J Am Soc Echocardiogr 2005;18:468-74.)
(mortality/survival) in chronic MR. Therefore, development of a new cost-effective animal model of chronic MR is needed. Based on our pilot animal study16 and clinical experience, we hypothesized that a chronic MR model could be developed in rats. We chose rats for the development of a small animal model of chronic MR primarily for the following reasons: (1) rat models are commonly used in the experimental study of cardiovascular disease with an ample database that can serve as a pathophysiologic reference; (2) cardiovascular drugs (anigotensin-converting enzyme inhibiters, beta-blockers, and aldosterone receptor blockers) had first shown beneficial effects on left ventricular (LV) remodeling and function, and mortality in the rat models of myocardial infarction, hypertension, and congestive heart failure,17-20 before similar beneficial effects were validated in patients21-23–thus, a rat model of chronic MR could be particularly useful for the investigation of pharmacologic therapy for chronic MR; and (3) cellular and molecular biology studies (important for the investigation of fundamental pathophysiology of chronic MR) can be carried at a significantly lower cost in a rat model than dog or sheep models if experimental animals are required to be killed for ex vivo tissue and molecular analysis. Therefore, the aim of this experimental study was to develop and characterize a rat model of chronic MR. METHODS Animal Preparation The study protocol was approved by the institutional animal care and use committee. The study was performed
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Figure 1 Left ventricular (LV) long-axis view by transesophageal echocardiography. There was no mitral regurgitation (MR) before procedure (A). Severe MR was detected by color Doppler after MR procedure (B). LA, Left atrium.
Figure 2 Right pulmonary vein flow velocity recorded in rat before creation of mitral regurgitation (MR) (A) and after procedure (B). Pulmonary venous systolic flow was reversed after induction of severe MR (B).
according to the guidelines of the American Physiological Society. A total of 34 Sprague-Dawley rats (180 g ⬃ 339) were used for the study. The rats were first anesthetized with 3% to 5% of isoflurane and then intubated. The lungs were ventilated using a respirator (Hallowell AWS, Piffsfield, Mass). Anesthesia was maintained by mixing isoflurane with oxygen (1%-2% isoflurane and 98%-99% oxygen). Heart rate and oxygen saturation were monitored using a respiratory gas monitor (Ohmeda 5250 RGM, Helsinki, Finland) throughout the study. Transesophageal Echocardiography Development of a rat model of chronic MR required a reliable method to assess the severity of MR and guide surgical creation of MR. Based on our clinical experience in the operating room,24-27 transesophageal echocardiography is one of the most useful tools for assessing MR intraoperatively. We performed transesophageal studies in rats using intracardiac echocardiography catheters (Acuson/Siemens Corp, Mountain View, Calif).16 This catheter is equipped with a 64-element phased-array transducer in a longitudinal plane on the tip with multiple imaging
frequencies from 5.5 to 10 MHz. The transducer produces M-mode, 2-dimensional, pulsed wave, and color Doppler images. The catheter tip can be flexed anteriorly, posteriorly, and laterally using the control knobs at the end of the catheter. The catheter was connected to a commercial echocardiograph (Sequoia 256, Acuson/Siemens Corp). In this study M-mode, 2-dimensional, color Doppler, and pulsed wave Doppler imaging was performed. The LV, left atrium (LA), mitral inflow, and pulmonary venous flow were examined. All images were stored digitally and on super-VHS tape. MR Operation After rats were anesthetized, an intracardiac echocardiographic catheter was inserted into the esophagus. A left thoracotomy was performed through the fifth or sixth intercostal space. A fine needle (0.36-mm diameter) was inserted into the LV through the LV apex under the guidance of transesophageal echocardiography. The needle was advanced into the mitral valve to damage mitral leaflets and create MR. The severity of MR was assessed using transesophageal echocardiography in real time. MR
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Figure 3 M-mode echocardiograms demonstrated significantly dilated left ventricle (LV) 12 weeks after creation of mitral regurgitation (MR). LV end-diastolic diameter (LVEDD) increased from 0.68 cm before MR procedure (A) to 1.3 cm 12 weeks after MR procedure (B).
was considered significant if a regurgitant jet area occupied more than 45% of the LA area (Figure 1), or reversed pulmonary venous flow pattern was detected (Figure 2), or both. The needle was then withdrawn and the chest was closed. In the sham group, a left thoracotomy was performed and the needle was inserted into the LV, but the mitral valve was not damaged and no MR was produced. The needle was then withdrawn and the chest closed. Transthoracic Echocardiography Transthoracic echocardiography (Acuson/Siemens Corp) was performed to assess LV remodeling and function. Rats were anesthetized with inhalation of 3% to 5% of isoflurane mixed with oxygen. No intubation was performed for transthoracic echocardiographic studies. Parasternal long- and short-axis views were recorded as previously described.28 M-mode image was obtained at the level of the chordae tendineae of the mitral valve under the guidance of 2-dimensional imaging. Transthoracic echocardiography was performed 1 week before operation and 1, 6, and 12 weeks after the surgical procedure in the MR and the sham groups. Data Analysis Semiquantitative assessment of MR. Color Doppler mapping of MR jets was used to semiquantitatively assess the severity of MR. MR jet areas and LA areas were measured in the LV long-axis view Figure 3. The ratio of MR jet area to the LA areas was calculated. Pulmonary venous flow was recorded. Pulmonary venous systolic flow reversal was used as a marker of severe MR (Figure 2, B). Assessment of LV remodeling. The LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and thicknesses of the interventricular septum and posterior wall were measured using M-mode echocardiography from the parasternal long-axis view (Figure 3). LV mass
was calculated using the following formula29: LV mass ⫽ 1.04 ⫻ [(LVEDD ⫹ posterior wall ⫹ interventricular septum)3 ⫺ end-diastolic diameter3]. The LV mass index was calculated as LV mass (in grams) divided by the rat’s body weight. Assessment of systolic function. LV fractional shortening was used for the assessment of LV systolic function. LV fractional shortening was calculated as [(LVEDD ⫺ LVESD)/LVEDD] ⫻ 100%. Measurement of LA dimension. The LA dimension was measured using M-mode echocardiography from the parasternal long-axis view. Costs Costs for rat, dog, and sheep models were analyzed according to standard charges from our institutional department of comparative medicine. Costs included direct costs for the purchase of animals and postoperative animal care. Statistical Analysis Values are expressed as mean ⫾ SD. Student unpaired t test was used to compare the means of continuous variables for the two independent groups. 2 Testing was used to examine difference in discrete variables. A P value ⬍ .05 was considered statistically significance. A KaplanMeier analysis was used to compare survival.
RESULTS MR Severity and Volume Overload Mean MR jet area was 20 ⫾ 4 mm2 with a ratio of MR jet area to the LA area of 71 ⫾ 11% in the MR group. There was no MR in the sham group. Mitral inflow
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Figure 4 Left atrial (LA) size was significant larger in mitral regurgitation (MR) group after creation of MR than in sham-operated group indicating significant volume overload of LA, although both groups had similar LA size before MR creation.
significantly increased immediately after the development of MR. This was reflected by a significantly increased mitral inflow peak velocity (94 ⫾ 16 cm/s vs 137 ⫾ 22 cm/s, P ⬍ .001) and an increased mitral inflow velocity-time integral (4.8 ⫾ 0.8 cm vs 6.4 ⫾ 0.9 cm, P ⬍ .01) in the rats with MR. There were no significant changes in the mitral inflow peak velocity (102 ⫾ 14 vs 106 ⫾ 11 cm/s, P ⬎ .05) or the velocity-time integral (4.8 ⫾ 0.8 vs 4.5 ⫾ 0.6 cm/s, P ⬎ .05) in the sham operated group. LA dimensions were significantly larger in the MR group than in the sham group after the MR operation (Figure 4). LV Remodeling One week after MR creation, LVEDD was mildly increased in the MR group. There was no significant change in LVESD 1 week after MR creation in either the MR group or the sham group. By the 12th week after the operation, however, both LVEDD and LVESD in the MR group were significantly larger than in the sham group (Figure 5). An increase in LVESD developed later than an increase in LVEDD. By the 12th week, the LV mass indexes were significantly higher in the MR group (2.72 ⫾ 0.78 g/kg) than in the sham group (1.98 ⫾ 0.26 g/kg, P ⬍ .01). LV Function LV fractional shortening initially increased at the first week after the operation in the MR group, indicating a hyperdynamic LV in the early stages of chronic MR. A decrease in LV fractional shortening was noted by the 12th week in the MR group, suggesting that LV systolic function was impaired because of long-term volume overload (Figure 6). There was no change in the LV fractional shortening in the sham group during the 12-week follow-up. Survival Six rats died within 6 days after operation in the MR group. No deaths were noted in the sham group with
Figure 5 Comparison of left ventricular (LV) end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) between mitral regurgitation (MR) group and sham-operated group. There were no significant differences in LVEDD and LVESD between MR and sham-operated groups before MR procedure. LVEDD was significantly larger in MR group than sham-operated group after creation of MR (A). However, LVESD was not significantly dilated until 6 weeks after MR procedure (B).
acute mortality of 27% versus 0% (P ⬍ .01). Three rats died between 7 days and 150 days after the operation in the MR group with no death in the sham group. The 150-day survival was significantly lower in the MR group (59%) than in the sham group (100%) (P ⬍ .01) (Figure 7). Costs The table lists the estimated direct costs for a 150-day experiment in 34 rats in comparison with the estimated direct costs for 150-day experiment in 34 dogs or 34 sheep. Estimated direct costs were 42 times higher in the dog model and 48 times higher in the sheep model than in the rat model.
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Table Comparison of estimated direct costs among rat, dog, and sheep models of chronic mitral regurgitation with a 150-day follow-up Animals
Rat Dog Sheep
Costs for purchase of an animal ($)
Costs/day for postoperative care ($)
Total costs of 150 days for an animal ($)
Total costs for 34 animals ($)
22 800 650
0.10 5.0 7.5
37 1550 1775
1258 52,700 60,350
DISCUSSION MR Model Recent discoveries in genomics, and molecular and cellular biology, have led to the development and widespread use of small animal models of human diseases. Although large animal models have been used for the experimental study of chronic MR, there are few small animal models of chronic MR.30 In large animal models, acute or chronic MR is often produced by damaging the chordae tendineae using a biopsy catheter31-33 or by the ligation of the left circumflex coronary artery (ischemic MR).34-36 These large animal models have been used for the study of the pathophysiology of chronic MR and for evaluation of surgical and medical therapy for chronic MR.37 The high cost is a major limitation to these large animal models. The current study has established the first rat model of chronic MR. There are several advantages in this model: (1) costs for a rat model of chronic MR are significantly lower than that for large animal models– direct costs are $37 per rat as compared with $1550 per dog and $1775 per sheep for a 150-day follow-up study (Table); (2) LV remodeling, function, and hemodynamic changes caused by volume overload (chronic MR) can be assessed by noninvasive methods; (3) LV remodeling could be seen as early as the 6th week after MR procedure–this may be especially helpful for shortening the experimental cycle; and (4) a rat model can be particularly useful for the investigation of pharmacologic treatment of chronic MR–although the effects of pharmacologic therapy have been assessed using anatomic or functional surrogates (LV remodeling, function, and hemodynamic) invasively and noninvasively in large animal models of chronic MR,32,38 a mortality benefit could not be used as a primary end point for the assessment of the efficacy of medical therapy. This might be because high costs limited the numbers of experimental large animals (often ranging from 6-12 dogs or sheep) used in the previous studies. In the rat model of chronic MR, significant 150-day mortality was observed. Therefore, mortality (the hardest end point) can be used as a primary end point for the assessment of the effects of pharmacologic therapies for chronic MR.
LV Remodeling and Function in MR LV remodeling and function are most important factors determining prognosis for patients with severe chronic MR. The current study shows that LV remodeling in chronic MR has two stages. In the first stage (early) LV remodeling was mainly seen in diastole (diastolic remodeling) with an increase in the end-diastolic diameter. This early remodeling could be a compensatory response to volume overload posed by severe chronic MR with preserved LV systolic function (hyperdynamic stage). In the second stage, the LVESD increased as well (systolic remodeling stage), which was associated with impaired LV systolic function (decreased LV fractional shortening). The results of the current experimental study are consistent with clinical observations that LVESD is strongly associated with LV dysfunction in patients with chronic MR.39,40 The study suggests that treatment should target early LV remodeling in the early stages of chronic MR with the goal of preventing or delaying LV systolic remodeling. Study Limitations In large animal models of chronic MR, surgical repair or replacement of the mitral valve can be performed. Therefore, large animal models are important for the experimental investigation of surgical therapy for chronic MR. Currently, mitral valve operation cannot be performed in rats. Therefore, a rat model may not be used for the study of surgical therapy for chronic MR. In large animal models MR can be created using a biopsy catheter without an open-chest operation. In the current rat model of chronic MR, open-chest operation was performed. We were initially concerned that open-chest operation might impact rat survival, especially with a needle inserted into the LV temporarily. However, open-chest operation is routinely used for the rat model of myocardial infarction (coronary artery ligation) and is widely accepted in experimental studies. Furthermore, there was no death in the sham group, although there were the same number of needle insertions in LV in the sham-operated group, indicating that the rats tolerated the procedure well. Quantitative assessment of the severity of MR by the proximal isovelocity surface area method
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Figure 6 Dynamic changes in left ventricular (LV) systolic function. LV fractional shortening (LVFS) increased 1 week after creation of mitral regurgitation (MR), but decreased by 12th week in rats with chronic MR. There was no change in LVFS in sham group.
Figure 7 Kaplan-Meier plot of 150-day survival after development of mitral regurgitation (MR), which was lower in MR group (59%) than in sham group (100%) (P ⬍ .01).
may be difficult because of the rapid heart rate in rats (approximate 300-380/min). Therefore, this study used MR jet area, a ratio of MR jet area to LA area and a pulmonary venous systolic reversal as markers for the severity of MR. Our results showed significant LV remodeling, LA dilation, and a reduction in LV fractional shortening 12 weeks after MR creation, demonstrating that these techniques reliably identified significant MR, which produced volume overload. We thank Ms Valerie Kehoe for technical assistance in animal experiments.
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