BASIC SCIENCE
Long-Term caspase inhibition ameliorates apoptosis, reduces myocardial troponin-I cleavage, protects left ventricular function, and attenuates remodeling in rats with myocardial infarction
Y. Chandrashekhar, MD*,*,
Soma Sen, MD*,
Ruth Anway, BA*,
Allan Shuros, BS* and
Inder Anand, FRCP, DPhil(Oxon), FACC*
* Division of Cardiology, VA Medical Center and University of Minnesota, Minneapolis, Minnesota, USA
Manuscript received June 19, 2003;
revised manuscript received September 15, 2003,
accepted September 15, 2003.
* Reprint requests and correspondence: Dr. Y. Chandrashekhar, Associate Professor of Medicine, University of Minnesota, Division of Cardiology IIIc, VAMC, 1 Veterans Drive, Minneapolis, Minnesota 55417, USA.
shekh003{at}tc.umn.edu
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Abstract
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OBJECTIVES: This study was designed to evaluate whether in vivo caspase inhibition can prevent myocardial contractile protein degradation, improve myocardial function, and attenuate ventricular remodeling.
BACKGROUND: Apoptosis is thought to play an important role in the development and progression of heart failure (HF) after a myocardial infarction (MI). However, it is not known whether inhibiting apoptosis can attenuate left ventricular (LV) remodeling and minimize systolic dysfunction.
METHODS: A 28-day infusion of caspase inhibitor (n = 12) or vehicle (n = 9) was administered to rats immediately after an anterior MI. In addition, five sham-operated rats given the caspase inhibitor were compared with 17 untreated sham-operated animals to study effects in non-MI rats. Left ventricular function, remodeling parameters, and hemodynamics were studied four weeks later. Myocardial caspase 3 activation and troponin-I contractile protein cleavage were studied in the non-infarct, remote LV myocardium using Western blots. Apoptosis was assessed using immunohistochemistry for activated caspase-positive cells as well as the TUNEL method. Collagen volume was estimated using morphometry.
RESULTS: Caspase inhibition reduced myocardial caspase 3 activation. This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus, and fewer apoptotic cardiomyocytes. Furthermore, caspase inhibition reduced LV-weight-to-body-weight ratio, decreased myocardial interstitial collagen deposition, attenuated LV remodeling, and better preserved LV systolic function after MI.
CONCLUSIONS: Caspase inhibition, started soon after MI and continued for four weeks, preserves myocardial contractile proteins, reduces systolic dysfunction, and attenuates ventricular remodeling. These findings may have important therapeutic implications in post-MI HF.
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Abbreviations and Acronyms
| | ANOVA | = analysis of variance | | DMSO | = dimethyl sulfoxide | | HF | = heart failure | | LV | = left ventricle/ventricular | | MI | = myocardial infarction |
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A million and a half patients suffer a myocardial infarction (MI) in the U.S. each year, and there are many times those numbers with a pre-existing MI. Some 15% to 25% of these patients develop heart failure (HF), which remains a progressive disease despite advances in pharmacologic therapy. Ischemic cardiomyopathy is thus a large and rapidly growing burden. Methods to reduce the occurrence of HF after MI are few and only modestly successful. The more effective ones, such as those that improve myocardial perfusion (e.g., thrombolytic therapy or primary coronary angioplasty), have their limitations, and the patient may be left with some degree of left ventricular (LV) damage (1). It is possible that a more direct approach, such as inhibiting cell death, could reduce the occurrence of HF and improve prognosis. Traditionally, necrosis has been regarded as the pathologic hallmark of myocardial cell death. However, there is accumulating evidence that apoptosis contributes significantly to myocardial cell death during and after MI (2–4). More importantly, apoptosis may also contribute to the development and progression of HF late after MI (5–7). Caspases play a crucial role in myocardial apoptosis (8). There are preliminary data that caspase inhibition can reduce apoptosis during ischemia and reperfusion with some beneficial results (9–11). However, there are no data yet to clarify whether inhibiting or attenuating apoptosis can reduce the occurrence of HF or impede its progression after MI. In this study, we show for the first time that long-term in vivo caspase inhibition protects against myocardial contractile protein cleavage and better preserves LV systolic function after MI. Furthermore, in vivo caspase inhibition also reduces non-infarct segment cardiomyocyte apoptosis and attenuates ventricular remodeling in rats with MI.
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Materials and methods
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Animals and model.
This study was done in adult male Sprague-Dawley rats (200 to 250 g). Myocardial infarcts were produced as previously described (12). Briefly, rats were anesthetized (intraperitoneal sodium pentobarbital, 30 mg/kg), and the proximal left coronary artery was surgically occluded through a left thoracotomy. The suture was not tied in rats serving as controls (sham group). The same experienced surgical team, which has previously produced consistently sized infarcts, operated on all the animals. The protocol was approved by the institutional animal studies committee, and animals were used in accordance with the National Institutes of Health and institutional guidelines.
Caspase administration.
Caspase inhibitor (Z-Asp-2,6 DCBMk, a cell-permeable, broad-spectrum caspase inhibitor that does not affect other cysteine proteases, Alexis Biochemicals, San Diego, California) in dimethyl sulfoxide (DMSO) or saline + DMSO was administered via a peritoneal mini-pump in rats assigned to the caspase inhibitor (n = 12) or vehicle (n = 10, 9 rats completed the study) treatment groups, respectively. The surgical team was blinded to the contents of the pump. All rats in the caspase treatment group received a 2 mg bolus of the drug immediately before surgery (the control group received a bolus of vehicle), and an infusion (2 mg/day) was started just after coronary ligation. The infusion continued for 28 days after the surgery. This dose was obtained from previous studies (13) and our own pilot data. Hemodynamic and ventricular remodeling data from caspase inhibitor or vehicle treated rats were also compared with two additional separate groups of rats: untreated MI (rats not given a pump or saline, n = 15) and sham-operated rats (rats where a suture was passed under the left coronary artery but was not tied; n = 17).
Measurements.
All animals had the following measurements. Tissues for Western blots and caspase staining were obtained at one month after surgery from the non-infarct remote myocardium of the LV.
Caspase activation and troponin-I cleavage.
Caspase 3 activation and troponin I cleavage were assessed using Western blots. Tissue samples were homogenized in a Hepes buffer solution and a protease inhibitor cocktail (Sigma, St. Louis, Missouri). After determining protein concentrations, an equal amount of protein was added to each well. SDS-PAGE was performed and completed gels were transferred to nitrocellulose membrane in Towbin buffer. The gels were blocked in 10% powdered milk, 3% bovine serum albumin in TBS. Either rabbit anti-caspase-3 or goat anti-troponin-I or rabbit anti- -actin (Santa Cruz Biotechnology, Santa Cruz, California) was used as the primary antibody. The secondary antibodies were peroxidase-conjugated anti-rabbit and peroxidase-conjugated anti-goat (Sigma). The Westerns were developed using Supersignal West Pico chemiluminescence detection kit (Pierce, Rockford, Illinois). Blots were scanned, and image analysis was done using the NIH Image J program. Alpha-actin was used to normalize the test samples and serve as a loading control.
Cardiac apoptosis.
This was evaluated by using two separate techniques: 1) measuring the number of activated caspase-positive cells in myocardial sections stained with Apologix carboxyfluorescein caspase detection technique (Cell Technology, Minneapolis, Minnesota); and 2) TUNEL assay. The activated caspase detection technique has been clearly validated to show in situ apoptosis (14,15). Slides were labeled for activated caspases and counterstained with hematoxylin and eosin. Cells positive for activated caspase were counted in 30 random fields at 60x in each of the animals. A person blinded to therapy did the staining and counting. Even though the apoptosis-tagged (Apologix) slides had a reasonably clear definition of cell outline, a paired slide was also stained with hematoxylin and eosin to outline the myocyte. Myocyte origin of Apologix-positive apoptotic cells was confirmed by visualizing HE-stained paired slides under high power. TUNEL staining was done using the TACS-XL Blue Label kit (R and D Systems, Minneapolis, Minnesota). TUNEL-positive cells in 30 fields at 60x were counted in each animal. In view of the problems associated with using TUNEL to define apoptosis (16,17), the Apologix technique was used as the primary measure of apoptosis, and TUNEL provided secondary validation about the trends in apoptosis.
Collagen distribution.
The percent of area occupied by collagen as opposed to non-collagen tissue was quantitated in sections stained with Masson's Trichrome using NIH J image densitometry (18). Collagen volume fraction was calculated as the sum of all collagen-positive areas divided by the sum of muscle areas and collagen areas. This approach estimates the proportion of myocardium occupied by fibrillar collagen and closely correlates with the hydroxyproline concentration of the tissue (19).
Hemodynamics, LV systolic function, and LV remodeling.
Left ventricular remodeling was evaluated using echocardiography at one and four weeks after surgery as previously described (12). Left ventricular systolic function was assessed by in vivo hemodynamics (done at 4 weeks after surgery) and by echocardiography (12). Ejection fraction was measured using Simpson's formula (20).
Statistical analysis.
Comparisons between normally distributed parameters were assessed with a t test. A non-parametric test (Mann-Whitney) was used for non-normally distributed between group data. Multiple groups (echocardiographic data) were compared using the analysis of variance (ANOVA) module (STATISTICA, Sysoft Inc., Tulsa, Oklahoma, 1994). Post hoc tests were performed using Bonferroni method when the ANOVA was significant. A p value < 0.05 was used for statistical significance. All results are expressed as mean ± SD.
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Results
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The caspase inhibitor was given to five sham-operated rats to see if the drug had any adverse effects on LV function or remodeling in non-MI rats. Echo indices in sham-operated animals given the drug were similar to sham-operated animals not given the drug (LV internal dimension [diastole]: 0.62 ± 0.03 vs. 0.69 ± 0.02; LV internal dimension [systolic]: 0.35 ± 0.03 vs. 0.34 ± 0.03 and EF: 81 ± 1.9 vs. 86 ± 2.3 in sham-operated rats with and without caspase inhibitor, respectively, all non-significant). Similarly, there were no differences in hemodynamic measurements between age-matched sham-operated animals treated with the caspase inhibitor and those not given the drug. In the MI group, although there was a 20% immediate surgical mortality, there were no study drug-related deaths. A total of 12 rats given the caspase inhibitor and 10 rats given vehicle alone survived the surgery. One rat in the saline group died at one week during the echocardiographic study. All the remaining rats (12 caspase inhibitor treated and 9 saline treated) lived until sacrifice and completed the study. The average infarct size, measured with morphometry, was comparable (34% ± 5% and 37% ± 6% of LV in vehicle- vs. caspase inhibitor treated rats) in both groups.
Caspase 3 activation.
Therapy with the caspase inhibitor significantly reduced caspase 3 activation, the main effector caspase, in the LV myocardium from rats with MI. In Western blots, caspase 3 cleavage was reduced by 55% in caspase inhibitor treated compared with vehicle treated rats (Fig. 1).
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Figure 1 (Top) Representative Western blot showing reduced caspase 3 and troponin-I cleavage in caspase inhibitor treated rats. Arrows identify the cleaved fragment. (Bottom) Mean density units normalized to actin (n = 8 each group) for caspase 3 and troponin-I cleaved fragments. Control = vehicle treated rats.
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Cardiac apoptosis.
Caspase 3 positivity predominantly involved the cardiomyocytes, as evidenced by double staining-activated Caspase 3 with either alpha sarcomeric actin or hematoxylin and eosin, where the caspase 3 staining was found within the outlines of apoptotic cardiomyocytes (Fig. 2A, panels A and B). Immunohistochemistry (Fig. 2A, panels C and D) also showed that caspase inhibition reduced myocyte apoptosis; there were 60% fewer caspase 3 positive cells in the remote LV myocardium of caspase inhibitor treated rats than in vehicle treated rats (Fig. 2B) (Mann-Whitney U test, p < 0.001). TUNEL (Fig. 2A, panels E and F) confirmed myocyte apoptosis and also showed that the caspase inhibitor reduced cardiomyocyte apoptosis.
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Figure 2 (A) Immunostaining for activated caspases in cardiomyocytes. Representative sections of left ventricular myocardium (200 to 400x). The left panel shows Apologix carboxyfluorescein activated caspase stain (white spots in panel A) and the same field stained with alpha sarcomeric actin (B) below. Most of the apoptotic cells were myocytes. One fibroblast with apoptosis is also shown (arrowhead in panels A and B) and has a different morphology. The next two panels show the effect of caspase inhibitors (caspase Rx) on number of apoptotic cells measured with Apologix staining (seen as large white bright spots in panels C and D) or TUNEL (greenish color in panels E and F). There were fewer (panels C and E) activated caspase-positive cells (panel C) or TUNEL-positive cells (panel E) in the myocardium of animals treated with caspase inhibitors than in the myocardium of animals with vehicle treatment (no caspase Rx, panels D and F). (B) Mean data for the number of apoptotic cardiomyocytes from rats with myocardial infarction treated or not treated with the caspase inhibitor (n = 8 each group).
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Troponin-I cleavage.
Caspase activation cleaves contractile proteins, and this may have implications for contractile dysfunction. Caspase inhibition reduced troponin I cleavage 51% compared with vehicle treatment (Fig. 1).
Hemodynamics.
In vivo LV end-diastolic pressure (8 ± 0.9 vs. 13 ± 1.6 mm Hg, p < 0.05) was lower, whereas the LV developed pressure trended to be higher (117 ± 7 vs. 100 ± 4 mm Hg, p = 0.04) in caspase inhibitor treated rats than in vehicle treated rats.
LV systolic function.
Consistent with the reduced myocardial troponin I cleavage seen in Figure 1, the caspase inhibitor treatment significantly attenuated the decline in systolic function seen after MI (Figs. 3 and 4). Ejection fraction (39% and 46% greater at 1 and 4 weeks after MI, p < 0.05) and fractional shortening (55% and 45% greater at 1 and 4 weeks after MI, p < 0.05) were better preserved (Fig. 3) and LV end-systolic dimensions were 26% smaller in caspase inhibitor treated animals than in either vehicle treated or saline treated animals with MI (Fig. 4, lower panel). Vehicle treated animals were no different from animals with MI not given any infusion. Left ventricular function, however, remained worse in caspase-inhibited animals than in sham-operated animals. The mean arterial pressure was similar in caspase inhibitor and vehicle treated animals (93 ± 2.6 vs. 100 ± 1.7 mm Hg, p = ns), suggesting that the differences in systolic function were not due to a vasodilator effect.
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Figure 3 Systolic function was better preserved both at one week and four weeks after myocardial infarction (MI) in caspase inhibitor (CI) treated (MI + CI, n = 12) rats than in vehicle (dimethyl sulfoxide) treated (MI + DMSO, n = 9) rats or MI rats without any pump (untreated MI, n = 15). Systolic function still remained worse than in sham-operated animals (n = 17). EF = ejection fraction; FS = fractional shortening.
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Figure 4 Left ventricular (LV) remodeling and function were improved in rats with myocardial infarction (MI) given a caspase inhibitor (MI + CI, n = 12) for four weeks compared with vehicle (dimethyl sulfoxide) treated (MI + DMSO, n = 9) rats or MI rats without any pump (untreated MI, n = 15). Left ventricular remodeling still remained worse than in sham-operated animals (n = 17).
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Myocardial interstitial collagen.
The extent of myocardial fibrosis in the vehicle treated animals was similar to that described by other investigators in rats with moderately large MIs (21). However, there was a 46% reduction in the percentage of LV myocardium occupied by collagen in the caspase inhibitor treated compared with the vehicle treated rats (8% ± 0.7% vs. 15% ± 1.7%, p < 0.05).
LV remodeling.
Caspase inhibition attenuated LV remodeling, as assessed by a number of measures. Heart weight was increased in both groups as expected after MI. However, heart weight and heart-weight-to-body-weight ratio were significantly lower in the caspase inhibitor than in the vehicle treated group (3.17 ± 0.14 vs. 4.7 ± 0.3, p = 0.028). The body weights were not significantly different (336 ± 3 vs. 342 ± 2 g, p = ns, in caspase inhibitor vs. vehicle treated animals). Echocardiographic LV end-diastolic dimensions were lower in the caspase inhibitor treated animals (Fig. 4, upper panel). These differences persisted even when normalized to body weight. Echocardiographic LV mass tended to be lower in the caspase inhibitor treated animals (0.57 ± 0.04 g vs. 0.64 ± 0.04 g), once again suggesting reduced remodeling, but this was not statistically significant.
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Discussion
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We have shown for the first time that a four-week period of in vivo caspase inhibition, starting immediately after MI, reduced myocardial caspase 3 activation, is a crucial step in mediating apoptosis (8). This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus. Furthermore, caspase inhibition modestly reduced ventricular fibrosis, attenuated ventricular remodeling, and better preserved LV systolic function.
Cell loss and progression to HF after MI.
The syndrome of congestive HF is characterized by a relentless progression and high mortality. Although the exact mechanisms responsible for progression are not totally clear, it appears that myocardial structural remodeling is an important predictor of adverse prognosis in HF. Remodeling after MI is a progressive, often self-perpetuating process, and cell loss through apoptosis may be an important component in its genesis (5–7). Increased wall stress and neurohormonal/cytokine activation are known to adversely influence remodeling. There is evidence that these triggers can also mediate apoptosis. Apoptotic cell loss may contribute to ventricular dilation and increased wall stress, which may further perpetuate HF (5,22). Thus, interruption of apoptosis may have a therapeutic potential.
Apoptosis in MI and HF.
Apoptosis has been shown to be prominent in both animal (2,3) and human (4) MI. The amount of apoptosis is variable and time dependent. There is evidence for an increase in many of the apoptosis intermediates, including a co-localization of caspase 3 and apoptosis in the ischemic heart (8,23). Apoptosis is also found in the chronically failing heart (6,7), albeit in a lesser amount, and a progressive loss of myocytes through apoptosis has been thought to be important for progression in this syndrome (5). Some investigators have also found chronic ongoing apoptosis in the hibernating heart, although this is controversial (24).
Caspases and HF.
Caspases, which are activated after MI and in various stages of HF, are the key effector molecules for apoptosis. Cellular caspases exist as inactive precursors and need proteolytic cleavage for activation. They seem to be a crucial step in myocardial apoptosis after MI (8). Caspase activation co-localizes to apoptotic areas and precedes DNA degradation and the development of apoptotic morphology (23). Furthermore, caspase can also influence the contractile machinery of myocytes through cleavage of troponin (25,26). This can result in contractile dysfunction. Indeed, over-expression of caspase 3 induces contractile dysfunction in mice (27). However, caspase inhibition has not been studied in depth for modifying the course of the failing heart.
Caspase inhibition in hf and mi.
Although caspase inhibitors have been tested in animal models of cardiac and non-cardiac ischemia, there are no published studies that have evaluated chronic caspase inhibition in the failing heart. A few studies report the short-term effects of caspase inhibition in MI (11,28). Caspase inhibition reduces apoptosis in experimental models of ischemia-reperfusion, but the degree of infarct size reduction is controversial (10,11). None of these studies has evaluated longer-term effects of reducing apoptosis. Our data is perhaps the first demonstration that long-term caspase inhibition reduces cardiomyocyte apoptosis in the non-infarct myocardium, attenuates systolic dysfunction, and reduces ventricular remodeling after MI.
Cellular targets for improving ventricular remodeling.
It is interesting that caspase inhibition, apart from attenuating ventricular dilation, also reduced myocardial fibrosis. Caspase inhibition may have affected a variety of cells in the remodeling myocardium, including cardiomyocytes, fibroblasts, endothelial cells, and even the inflammatory cell infiltrate. Although anti-apoptotic effects on each of these cell populations are likely to have complex and possibly discordant effects on ventricular remodeling, the net effect in our study was an attenuated LV remodeling. At the time we measured apoptosis (4 weeks after MI), most of the apoptotic cells appeared to be cardiomyocytes. This is consistent with data in other models of HF (29) and even in hearts with risk factors for myocardial ischemia but no failure (30).
There are no studies in literature to clarify how anti-apoptotic therapy might affect various components of ventricular remodeling. Our study also does not allow us to determine how caspase inhibition therapy affected other cell populations involved in ventricular remodeling and their interactions in the remodeling process. Although reduction in myocyte apoptosis could explain attenuated ventricular remodeling, it is not clear whether blocking myofibroblast apoptosis would be beneficial or harmful. Inhibiting myofibroblast apoptosis could make the fragile granulation tissue survive longer (31) and might make the scar tissue stronger. This might prevent excessive ventricular dilation, and indeed, there is very recent evidence (32) supporting this using ischemia-reperfusion—a model that, however, is significantly different from our model. Anti-apoptotic effects on fibroblasts early after MI have been suggested to retard ventricular remodeling (33). Secondly, fibroblast death affects the interconnection between myocytes, which would be expected to increase myocyte slippage, enhance mural thinning and chamber dilation, and increase wall stress (29,33). All of these features worsen remodeling and could be attenuated if anti-apoptotic therapy prolonged myofibroblast survival. This is supported by studies showing that inhibiting collagen degradation attenuates LV remodeling (34) and enhancing collagen degradation hastens the development of HF (35). Thus the attenuated remodeling seen in our model could be the result of beneficial effects on both myocyte and non-myocyte components.
The reduction in non-infarct myocardial fibrosis that we found at four weeks after MI in the caspase inhibitor group may not, at first glance, be consistent with reduced fibroblast or myofibroblast apoptosis. However, the initial stimulus to myofibroblast proliferation and increased fibrosis may arise from increased regional wall stress or stretch and neurohormonal activation (36). The latter would be attenuated if anti-apoptotic therapy reduced cardiomyocyte loss. Moreover, anti-apoptotic effects on myofibroblasts, in the critical initial days after MI, could have protected against ventricular dilation and wall stress and thus reduced the stimulus for fibrosis. Whereas our studies concentrated on the net effects of anti-apoptotic therapy and were not designed to tease out these individual elements, one could speculate that reduced myocyte apoptosis (and possibly reduced myofibroblast apoptosis in the early post-MI period) might have reduced the stimuli for fibrous tissue proliferation.
Study limitations.
There are several limitations of this study. Measurements including those for apoptosis were made at four weeks after MI. Even though we did find significant anti-apoptotic effects, earlier measurements might have shown a larger difference in apoptosis. Caspases also have beneficial housekeeping actions, and caspase inhibition may result in unwanted consequences. These were not evaluated in this study. Caspase inhibition may not block all pro-apoptotic stimuli, and combined strategies, attacking multiple pathways, may be more effective but remain subject to future studies. We chose the dose from previous studies in literature (13), but it is possible that a higher dose may have had a greater effect on remodeling. Finally, it is uncertain whether remodeling would worsen once caspase inhibition is terminated. This is the subject of a more long-term investigation.
Conclusions.
We conclude that long-term caspase inhibition, started in the peri-MI period and continued for four weeks, significantly inhibited non-infarct myocardial caspase 3 activation and contractile protein cleavage in rats with a large anterior wall MI. This was accompanied by better preserved LV function and attenuated LV remodeling. Caspase inhibition might have a therapeutic role in improving the natural history of MI.
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Abstract
Materials and methods
