Treatment With Dimethylthiourea Prevents Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction in Mice
Role of Oxidative Stress
Abstract—Oxidative stress might play an important role in the progression of left ventricular (LV) remodeling and failure that occur after myocardial infarction (MI). We determined whether reactive oxygen species (ROS) are increased in the LV remodeling and failure in experimental MI with the use of electron spin resonance spectroscopy and whether the long-term administration of dimethylthiourea (DMTU), hydroxyl radical (·OH) scavenger, could attenuate these changes. We studied 3 groups of mice: sham-operated (sham), MI, and MI animals that received DMTU (MI+DMTU). Drugs were administered to the animals daily via intraperitoneal injection for 4 weeks. ·OH was increased in the noninfarcted myocardium from MI animals, which was abolished in MI+DMTU. Fractional shortening was depressed by 65%, LV chamber diameter was increased by 53%, and the thickness of noninfarcted myocardium was increased by 37% in MI. MI+DMTU animals had significantly better LV contractile function and smaller increases in LV chamber size and hypertrophy than MI animals. Changes in myocyte cross-sectional area determined with LV mid–free wall specimens were concordant with the wall thickness data. Collagen volume fraction of the noninfarcted myocardium showed significant increases in the MI, which were also attenuated with DMTU. Myocardial matrix metalloproteinase-2 activity, measured with gelatin zymography, was increased with MI after 7 and 28 days, which was attenuated in MI+DMTU. Thus, the attenuation of increased myocardial ROS and metalloproteinase activity with DMTU may contribute, at least in part, to its beneficial effects on LV remodeling and failure. Therapies designed to interfere with oxidative stress might be beneficial to prevent myocardial failure.
Myocardial infarction (MI) frequently produces left ventricular (LV) dilatation and hypertrophy of the noninfarcted myocardium.1 These changes in LV geometry, referred to as remodeling, contribute to the development of depressed cardiac performance.2 Thus, surviving patients with MI are at an increased risk for occurrence of heart failure (HF), reinfarction, arrhythmia, and sudden cardiac death.2 LV remodeling is caused by the side-to-side slippage of cardiac myocytes at the infarcted region and the hypertrophic response of the noninfarcted myocytes, resulting in a progressive increase in the chamber diameter.3 The underlying mechanisms responsible for these processes have been attributed to hemodynamic stress as well as the activation of neurohumoral factors, including the renin-angiotensin system. However, the details of contributing factors in LV remodeling remain to be elucidated.
Reactive oxygen species (ROS) can produce myocardial contractile dysfunction and structural damage.4 There is growing evidence that ROS are increased in HF and may contribute to disease progression.5 6 Hill and Singal7 showed that antioxidant enzyme activities are decreased and that thiobarbituric acid reactive substances (TBARS) are increased in the failing myocardium due to MI. Further, recent study in isolated cardiac myocytes has shown that a subtle increase in ROS results in a phenotype characterized by hypertrophy and apoptosis,8 which play an important role in myocardial remodeling and failure.9 We recently demonstrated that ·OH is increased with the development of rapid pacing-induced HF with the use of electron spin resonance (ESR) spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO).6 In our studies, ·OH is generated from superoxide anion (·O2−) and H2O2 via metal-catalyzed Harber-Weiss reaction and Fenton reaction within the myocardial tissue. These observations have prompted the thought that oxidative stress can contribute to LV remodeling and HF after MI.
Several important questions remain to be answered. First, no direct evidence for the increased production of ROS has been obtained in post-MI hearts. Therefore, it should be determined whether our previous observations in rapid pacing–induced HF can be applicable to post-MI HF. Second, even though the direct demonstration of increased ROS in the failing myocardium should help to focus attention on the therapeutic value of antioxidants, it also raises the question of whether enhanced production of ROS is truly a mechanism of HF or merely a marker of the manifestation of the disease. Several previous studies have used antioxidant vitamins to address this question, in which, however, the changes of ROS production have not been rigorously examined concurrently with myocardial response.10 It is critically important to examine whether the administration of ROS scavengers can attenuate both ROS production and HF.
Previous studies have reported increased myocardial metalloproteinase (MMP) activity in experimental models, including rapid pacing–induced HF11 and post-MI,12 13 as well as in human end-stage HF.14 Recently, an MMP inhibitor was shown to limit early LV dilatation in a murine model of MI.15 Because MMP can be activated by ROS in vivo,16 one proposed mechanism of LV remodeling is the activation of MMP secondary to increased ROS production. We hypothesized that ROS production and myocardial elaboration of MMP activation are interdependent and that the effects of ROS scavengers on LV remodeling are related, at least in part, to the modulation of this axis.
Accordingly, the first goal of the present study was to examine whether the production of ROS is increased in the remodeled LV after MI; the second goal was to determine whether chronic inhibition of ·OH production could inhibit the progression of LV remodeling and failure. A pharmacological intervention that can be used to prevent ·OH-mediated injury should ideally be capable of entering myocardial cells rapidly to encounter the generation of reactive oxygen metabolites. It should also maintain sufficient levels of tissue concentrations to afford protection against low levels of ·OH. However, most ROS scavengers have serum half-lives on the order of minutes and do not readily cross cell membranes. Dimethylthiourea (DMTU) is an agent that is highly diffusible, has a long half-life, and is effective in scavenging hydrogen peroxide (H2O2) and ·OH.17 Therefore, DMTU is expected to be an effective antioxidant, especially when administered in vivo.
In the present study, we created MI in mice by ligating the left anterior descending coronary artery, and we assessed the production of ·OH by measuring the rate of reduction of hydroxy-TEMPO in the myocardial tissue by using ESR spectroscopy. Further, we examined whether chronic in vivo administration of DMTU into MI animals can attenuate the LV remodeling and HF. We also examined myocardial MMP activity by using gelatin zymography.
Materials and Methods
The study was approved by our institutional animal research committee and conformed to the animal care guidelines of the American Physiological Society. MI was created in male CD-1 (Charles River) mice (6 to 8 weeks old, weight 30 to 40 g) by ligating the coronary artery according to the methods described by Michael et al.18
MI mice were randomly grouped to receive either saline (MI group) or DMTU (MI+DMTU group). DMTU (50 mg/kg in sterile saline) was administered daily via intraperitoneal injection beginning 6 hours after the creation of MI and throughout the study (4 weeks). This dose was chosen based on the previous studies of its efficacy.19 20
Echocardiography and Hemodynamic Evaluation
Serial echocardiographic measurements at baseline and 3, 7, 14, and 28 days after surgery were made in all groups of animals.21 After the echocardiographic measurements, LV pressure was measured according to the methods described by Williams et al.22 One subset of investigators (S.H. and N.S.), who were not informed of the experimental groups, performed in vivo LV function studies that included echocardiography and LV pressure measurements.
Experimental Protocol 1
Quantification of Myocardial ·OH by ESR Spectroscopy
We quantified ·OH in the noninfarcted LV myocardium according to the methods described previously.6
Experimental Protocol 2
LV Morphology and Morphometry
A separate group of animals, treated identically as in protocol 1, was used to evaluate the effects of chronic DMTU administration on LV chamber diameters. Infarct size in these hearts was determined according to the method described by Pfeffer et al.23
Myocyte Size and Collagen Volume Fraction
Myocyte cross-sectional area and collagen volume fraction were measured according to the methods described previously.24
Coronary artery ligation and a sham operation were performed in 84 and 32 mice, respectively. Eighty-one mice that survived 6 hours after coronary artery ligation were randomized to active treatment with DMTU (MI+DMTU; n=33) or no treatment (MI; n=48). During the follow-up period, 24 (30%) deaths occurred (6 mice receiving DMTU and 18 mice receiving no drugs; P=NS). All mice that died were confirmed to have MI on postmortem examination. All sham-operated animals survived until the end of the study period.
Serial 2-dimensional and M-mode echocardiography was performed in a group of sham-operated (n=10), MI (n=10), and MI+DMTU (n=8) animals. Figure 1⇓ demonstrates marked LV dilatation and contractile impairment in the MI mouse. These changes were attenuated in MI+DMTU mice. Figure 2⇓ shows that LV end-diastolic diameter increased and percent fractional shortening (FS) decreased by day 3 after ligation of coronary artery. They were significantly different from control values by days 3 to 28 of the control. DMTU significantly inhibited this LV diameter increase and percent FS decrease in MI as early as 3 days, which was maintained throughout the study period, indicating the persistent attenuation of LV dilatation and failure with DMTU from the early phase after MI.
The summarized data for echocardiographic measurements at baseline and after 4 weeks are presented in Table 1 online (data supplement available at http://www.circresaha.org). In comparison with sham-operated animals, MI animals showed a 38% decrease (P<0.01) in the thickness of the infarcted region and a 37% (P<0.05) increase in the thickness of the noninfarcted region. DMTU significantly attenuated the hypertrophy of the noninfarcted myocardium (P<0.01) but did not affect the thickness of the infarcted portion.
Hemodynamics and Organ Weights
Hemodynamic measurements could be obtained in a group of MI (n=8), MI+DMTU (n=8), and sham-operated (n=7) animals (Table 2 online; data supplement available at http://www.circresaha.org). They had similar body weights (P=NS). The MI mice tended to exhibit lower aortic blood pressures than the sham group, which, however, did not reach statistical significance (P=0.06). LV end-diastolic pressure was significantly elevated and LV +dP/dt was depressed in the MI group (P<0.01 for both), which was attenuated with DMTU. Coincident with an increased LV end-diastolic pressure, the ratio of lung weight to body weight was significantly increased in the MI group versus the sham group (9.1±1.5 versus 4.6±0.1 g/kg, P<0.01), which was also attenuated with DMTU treatment (5.7±0.4 g/kg, P<0.01 versus MI).
ROS in the Noninfarcted LV
ESR signals of hydroxy-TEMPO reduced more rapidly in the presence of homogenates from post-MI hearts compared with sham (Figure 3A⇓). There was a linear relation in the semilogarithmic plot of peak signal intensity versus time, indicating the first-order kinetics of the signal decay (Figure 3B⇓). The rate constant of signal decay was significantly (P<0.01) larger in MI than that in sham (Figure 3C⇓) animals. DMTU (50 mmol/L) added to the reaction mixture completely abolished an increase of signal decay in MI, indicating that ROS indeed contributed to the increase of signal decay rate in MI. Catalase (50 U/mL) plus superoxide dismutase (SOD; 50 U/mL) also attenuated an increase in signal decay rate, which implies the contribution of ·O2− to the production of ·OH.
In the MI+DMTU group, the “DMTU-inhibitable” rate of signal decay was normalized, which provided evidence that the chronic in vivo administration of ·OH scavenger DMTU into MI animals completely prevented the production of ·OH (Figure 4⇓).
LV Morphology and Morphometry
Figure 5⇓ shows the transverse LV sections (midcavity) stained with Masson’s trichrome. The sections obtained from the MI mouse revealed an anteroapical infarct that extends into the anterolateral wall (Figure 5B⇓). The interventricular septum was generally spared. Infarct size was estimated to establish whether the scarred myocardium was comparable between MI and MI+DMTU and to provide a basis of comparison. Two MI and 2 MI+DMTU mice with an infarct size of <40% were excluded from the analysis of LV morphology. Infarct size was identical between MI (59±3%, range 42% to 70%; n=12) and MI+DMTU (56±2%, range 49% to 66%; n=11) animals. Figure 6⇓ illustrates MI size and LV chamber diameter in all groups. Consistent with echocardiographic data (Table 1 online; data supplement available at http://www.circresaha.org), MI+DMTU animals had significantly smaller LV chamber diameters and volume than MI animals.
Myocyte cross-sectional area was increased in MI, which was significantly attenuated with DMTU treatment (Figure 7⇓). These results are concordant with LV wall thickness data obtained from echocardiography (Table 1 online; data supplement available at http://www.circresaha.org). Collagen volume fraction was also increased in MI, which was inhibited with DMTU treatment (Figure 7⇓).
Myocardial MMP Activity
Figure 8⇓ shows representative gelatin zymography of the LV from sham, MI, and MI+DMTU mice. There was minimal MMP activity in the sham group. MI after 7 days markedly enhanced MMP-2 (62- and 58-kDa gelatinases) activity in the noninfarcted LV. The increase in MMP-2 activity persisted at day 28 after MI.12 13 There was a modest but significant increase in MMP-1 activity (54 kDa) in MI at both days 7 and 28.12 13 With DMTU, there was significant attenuation of MMP activities at 7 and 28 days after the induction of MI (Figure 8⇓).
The present study demonstrates an increase of ROS in the noninfarcted myocardium, in association with LV remodeling (ie, the chamber dilatation and hypertrophy of the noninfarcted region). Despite the equal infarct size, chronic administration of DMTU into the MI animals results in a tendency toward normalization of LV size and contractile function. The changes in LV structure after DMTU treatment are associated with reduced myocyte hypertrophy and interstitial fibrosis, suggesting that they are responsible, at least in part, for post-MI LV remodeling and reverse remodeling with DMTU.
Increased ROS in the Post-MI Heart
The ESR method for the measurement of ROS in the myocardial tissue used in the present study has been well validated in our previous studies.5 6 This can provide a direct method to quantify the generation of ROS within biological tissue.6 We extended our earlier observations in rapid ventricular pacing–induced HF by showing that the production of ·OH was increased in the myocardium with remodeling and dysfunction after MI. These results also confirm and extend previous observations that lipid peroxidation products measured as TBARS are increased in post-MI hearts.7 In addition, the present study demonstrates that the addition of catalase plus SOD into the reaction mixture significantly attenuates the increase of signal decay (Figure 3C⇑). These findings indicate that as was shown in our previous study,6 ·OH can be secondarily produced from ·O2− through the electron exchange between ·O2− and H2O2 via Harber-Weiss reaction and Fenton reaction.25
·O2− can be produced intracellularly through electron leakage from mitochondria during oxidative phosphorylation and through the activation of several cellular enzymes, including NADPH oxidase, xanthine oxidase, and nitric oxide synthase.8 We previously used ESR with the ·O2− spin trap, 5,5′-dimethyl-1-pyrroline-N-oxide, to show that formation of ·O2− was increased in submitochondrial particles from rapid pacing–induced HF.5 Even though the source of ROS was not determined in the present study due to the limited amount of available myocardial tissue, it is well expected that the same mechanism might be operative in this model.
Role of DMTU Administration on Post-MI Remodeling and Failure
Previous studies have shown that ROS play an important role in the pathogenesis of myocardial ischemia-reperfusion,26 doxorubicin-induced cardiomyopathy,27 and HF.5 6 ROS have been implicated as an important contributing factor in LV remodeling after MI.7 However, whether and to what degree ROS inhibition can attenuate the LV remodeling process remain unexplored. This question is also important to establish a cause-and-effect relationship between oxidative stress and HF. We observed that DMTU prevented LV dilatation and hypertrophy of noninfarcted LV in association with contractile dysfunction. The beneficial effects of DMTU were not due to its MI size-sparing effect because the administration was started 6 hours after coronary artery ligation and the resultant MI size was not affected with DMTU (Figure 6A⇑). DMTU has been shown to readily enter myocardium, to have a long serum half-life of 43 hours, and to have no cardiac toxicity in intact animals.28 These properties are thought to be effective in accomplishing meaningful reduction in myocardial damage. Therefore, the present study provided clear evidence that DMTU can modulate the myocardial response to infarction and that increased ROS are responsible, at least in part, for remodeling and failure.
Serial echocardiographic measurements demonstrated that the beneficial effects of DMTU on LV remodeling could be identified at the early phase of its process (Figure 2⇑), indicating that ROS are involved in the early remodeling after MI. Nevertheless, we could not exclude the possibility that ROS are also involved in the late phase. To clarify the mechanisms of DMTU-induced effects, it is necessary to determine whether the protective ability of DMTU is due to its capability to chelate metals with the use of desferrioxamine. However, the chelation of iron with desferrioxamine produces ferrioxamine, and the iron in ferrioxamine can be a redox reactant. In fact, desferrioxamine has been shown to have a biphasic antioxidant/pro-oxidant behavior29 and to amplify oxidative damage through the generation of ·OH.30 Future studies that confirm whether the same effects could be observed with the use of other antioxidants are warranted.
Dhalla et al10 have shown that the transition from hypertrophy to failure could be prevented with the antioxidant vitamin E in the guinea pig model of ascending aortic constriction. In addition, probucol has been shown to exert protective effects against adriamycin-induced cardiomyopathy.27 The present study extends the previous observation by demonstrating substantial reductions in LV dilation and hypertrophy after DMTU treatment in association with the improvement in contractile function. The changes in LV structure were associated with reduced myocyte hypertrophy and interstitial fibrosis, suggesting that they are responsible, at least in part, for post-MI LV remodeling and reverse remodeling with DMTU.
The present results suggest that increased myocardial ROS could contribute to the activation of MMP and thus to the development of LV remodeling after MI. It has been reported that MMPs are increased in the noninfarcted myocardium obtained from a rat model of MI.13 Further, an MMP inhibitor has been shown to limit the chamber dilatation in a murine model of MI.15 Sustained MMP activation might therefore influence the structural properties of the myocardium by providing an abnormal extracellular environment with which the myocytes interact.31 Importantly, the present study has demonstrated that DMTU inhibits the activation of MMP in association with the development of LV remodeling. These data raise the interesting possibility that increased ROS after MI can be a stimulus for myocardial MMP activation, which might play an important role in the development of HF. Although this concept warrants further exploration, there is recent evidence for the activation of vascular MMP by ROS in vivo.16 In addition, the beneficial effects of DMTU on LV failure may be related to its antiedema action because small increases in interstitial water content can greatly increase LV chamber stiffness.32
Moreover, ROS have direct effects on cellular structure and function and may be integral signaling intermediates in myocardial remodeling.33 Higher levels of ROS are known to cause direct damage to proteins and lipids, leading to myocyte death through necrosis or apoptosis.4 Further, more powerful pro-oxidant peroxynitrite can be produced, particularly when both ·O2− and nitric oxide are present.34 A recent study by Siwik et al8 demonstrated that a subtle increase in ROS caused by partial inhibition of SOD results in a phenotype characterized by hypertrophy and apoptosis in isolated cardiac myocytes, which appears to be present in the noninfarcted myocardium. ROS of mitochondrial origin may be particularly prone to trigger apoptosis.35
The demonstration of a preventive effect of DMTU on HF implies that antioxidants could be of clinical relevance. Recently, carvedilol, a β-blocker with antioxidant activity, has been shown to be beneficial in the treatment of patients with congestive HF.36 One must take into account that this drug possesses additional properties, including α-adrenoceptor–blocking action and antiproliferative activities. However, as in the settings of ischemia-reperfusion, one might assume that the beneficial effects of carvedilol on HF are also mediated, at least in part, through the prevention of ROS-induced damage.
There are several limitations that should be acknowledged in this study. First, because the hemodynamic profile of DMTU is incompletely described, we could not exclude the possibility that the beneficial effects of DMTU might be due to hypothetical favorable hemodynamic effects on systemic and coronary circulation. However, this possibility might be less likely because arterial pressure and heart rate were not influenced with DMTU (Table 2 online; data supplement available at http://www.circresaha.org). Second, we administered DMTU into the animals for 4 weeks to allow sufficient time to identify changes in LV morphology, because this study was designed to address the hypothesis that ROS inhibition could attenuate long-term LV remodeling. Thus, the present study does not preclude an additional effect if DMTU was initiated at the time of coronary ligation or the effects on coronary occlusion with reperfusion. Further studies in genetically altered mice and other models will also improve understanding of the role of oxidative stress in LV remodeling. Third, in the present study, we examined the amount of ROS at the time point after the development of LV remodeling. Significant changes in ROS may also occur at the onset and during the development of remodeling. Further studies that focus in the temporal changes of oxidative stress may be necessary.
·OH radicals were increased within the post-MI hearts, which might be involved in myocardial remodeling and failure. Therapies designed to interfere with oxidative stress could be beneficial to prevent the progression of HF.
This work was supported in part by grants 07266220, 07670789, 08258221, and 09670724 from the Ministry of Education, Science and Culture.
- Received March 21, 2000.
- Revision received July 5, 2000.
- Accepted July 10, 2000.
- © 2000 American Heart Association, Inc.
Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald E. Progressive ventricular remodeling in rat with myocardial infarction. Am J Physiol. 1991;260:H1406–H1414.
Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation. 1990;81:1161–1172.
Olivetti G, Capasso JM, Sonnenblick EH, Anversa P. Side-to-side slippage of myocytes participates in ventricular wall remodeling acutely after myocardial infarction in rats. Circ Res. 1990;67:23–34.
Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL. Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes. J Biol Chem. 1991;266:2354–2361.
Ide T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N, Uchida K, Arimura K, Egashira K, Takeshita A. Mitochondrial electron transport complex I is the potential source of oxygen free radicals in the failing myocardium. Circ Res. 1999;85:357–363.
Ide T, Tsutsui H, Kinugawa S, Suematsu N, Hayashidani S, Ichikawa K, Utsumi H, Machida Y, Egashira K, Takeshita A. Direct evidence for increased hydroxyl radicals originated from superoxide in the failing myocardium. Circ Res. 2000;86:152–157.
Hill MF, Singal PK. Right and left myocardial antioxidant responses during heart failure subsequent to myocardial infarction. Circulation. 1997;96:2414–2420.
Siwik DA, Tzortzis JD, Pimental DR, Chang DL, Pagano PJ, Singh K, Sawyer DB, Colucci WS. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apoptosis in neonatal rat cardiac myocytes in vitro. Circ Res. 1999;85:147–153.
Sawyer DB, Colucci WS. Mitochondrial oxidative stress in heart failure: “oxygen wastage” revisited. Circ Res. 2000;86:119–120.
Spinale FG, Coker ML, Thomas CV, Walker JD, Mukherjee R, Hebbar L. Time-dependent changes in matrix metalloproteinase activity and expression during the progression of congestive heart failure: relation to ventricular and myocyte function. Circ Res. 1998;82:482–495.
Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ, Spinale FG. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation. 1998;97:1708–1715.
Rohde LE, Ducharme A, Arroyo LH, Aikawa M, Sukhova GH, Lopez-Anaya A, McClure KF, Mitchell PG, Libby P, Lee RT. Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation. 1999;99:3063–3070.
Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Galis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro: implications for atherosclerotic plaque stability. J Clin Invest. 1996;98:2572–2579.
Fox RB. Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest. 1984;74:1456–1464.
Michael LH, Entman ML, Hartley CJ, Youker KA, Zhu J, Hall SR, Hawkins HK, Berens K, Ballantyne CM. Myocardial ischemia and reperfusion: a murine model. Am J Physiol. 1995;269:H2147–H2154.
Mayhan WG, Patel KP. Treatment with dimethylthiourea prevents impaired dilatation of the basilar artery during diabetes mellitus. Am J Physiol. 1998;274:H1895–H1901.
Patten RD, Aronovitz MJ, Deras-Mejia L, Pandian NG, Hanak GG, Smith JJ, Mendelsohn ME, Konstam MA. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol. 1998;274:H1812–H1820.
Williams RV, Lorenz JN, Witt SA, Hellard DT, Khoury PR, Kimball TR. End-systolic stress-velocity and pressure-dimension relationships by transthoracic echocardiography in mice. Am J Physiol. 1998;274:H1828–H1835.
Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, Braunwald E. Myocardial infarct size and ventricular function in rats. Circ Res. 1979;44:503–512.
Namba T, Tsutsui H, Tagawa H, Takahashi M, Saito K, Kozai T, Usui M, Imanaka-Yoshida K, Imaizumi T, Takeshita A. Regulation of fibrillar collagen gene expression and protein accumulation in volume-overloaded cardiac hypertrophy. Circulation. 1997;95:2448–2454.
Repine JE, Pfenninger OW, Talmage DW, Berger EM, Pettijohn DE. Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical. Proc Natl Acad Sci U S A. 1981;78:1001–1003.
Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A. 1987;84:1404–1407.
Siveski-Iliskovic N, Hill M, Chow DA, Singal PK. Probucol protects against adriamycin cardiomyopathy without interfering with its antitumor effect. Circulation. 1995;91:10–15.
Carrea FP, Lesnefsky EJ, Repine JE, Shikes RH, Horwitz LD. Reduction of canine myocardial infarct size by a diffusible reactive oxygen metabolite scavenger: efficacy of dimethylthiourea given at the onset of reperfusion. Circ Res. 1991;68:1652–1659.
Senzaki H, Paolocci N, Gluzband YA, Lindsey ML, Janicki JS, Crow MT, Kass DA. β-Blockade prevents sustained metalloproteinase activation and diastolic stiffening induced by angiotensin II combined with evolving cardiac dysfunction. Circ Res. 2000;86:807–815.
Laine GA, Allen SJ. Left ventricular myocardial edema: lymph flow, interstitial fibrosis, and cardiac function. Circ Res. 1991;68:1713–1721.
Yamamoto S, Tsutsui H, Tagawa H, Saito K, Takahashi M, Tada H, Yamamoto M, Katoh M, Egashira K, Takeshita A. Role of myocyte nitric oxide in β-adrenergic hyporesponsiveness in heart failure. Circulation. 1997;95:1111–1114.
Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998;281:1309–1312.