Calpain inhibition preserves myocardial structure and function following myocardial infarction

Abstract

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca²⁺-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 ± 2% pre-MI to 30 ± 4% with MI only vs. 41 ± 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 ± 2 μl pre-MI to 73 ± 4 μl with MI only vs. 55 ± 4 μl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

several cardiovascular pathologies, including myocardial infarction (MI), are associated with left ventricular (LV) remodeling as defined by changes in LV geometry and structure, and concomitantly, LV pump function can be adversely affected post-MI (7, 34, 46). At the cellular level, both qualitative and quantitative changes in cardiomyocytes can also contribute to both compromised contractile function (9) and programmed cell death (21, 22, 30). One common mechanism for this maladaptive remodeling at the cardiomyocyte level is hyperactivation of cellular endopeptidases, such as members of the calpain (6, 15, 16, 58) and caspase families (5, 22, 26, 54). Activation of these proteases may result in the cleavage of several key cellular proteins that result in the loss of contractile proteins and thus function of cardiac muscle cells.

Although studies demonstrate activation of both calpain and caspases with cardiac disease states, our laboratory (30) recently demonstrated that calpain activation contributes substantially to programmed cell death in pressure-overloaded (PO) feline myocardium. Calpains are a family of Ca²⁺-dependent cysteine proteases. The predominant isoforms include μ-calpain (calpain-I), which requires a micromolar concentration of Ca²⁺; m-calpain (calpain-II), which requires a millimolar concentration of Ca²⁺; and the muscle-specific calpain-III (14, 18). Under physiological conditions, calpains contribute to protein processing and degradation, which are crucial for development as well as for normal tissue functions. However, both μ-calpain and m-calpain, which are highly expressed in the heart (32, 45), have been shown to increase activation under several pathological conditions. Our recent study in PO feline myocardium (30) revealed that inhibition of calpain with calpeptin, but not of caspases with Z-VD-fmk, prevented cardiomyocyte loss in PO myocardium. However, it remained unclear whether and to what degree calpain activation occurred in other forms of cardiac disease and whether calpain activation would contribute to a deterioration of LV pump function. Accordingly, calpain inhibition was utilized to determine the role of calpain activation in LV remodeling that occurs following MI. In the present study, we demonstrate that blunting MI-induced calpain activation in mice by calpeptin treatment reduces myocardial cell loss and improves ventricular function.

MATERIALS AND METHODS

Reagents.

Calpeptin was obtained from Calbiochem (San Diego, CA). The TdT-mediated dUTP nick-end labeling (TUNEL) staining kit was obtained from Chemicon (Temecula, CA). Antibodies against the following proteins were obtained from the indicated vendors: m-calpain (Chemicon, Temecula, CA), μ-calpain and α-actinin (Sigma Chemicals, St. Louis, MO), and fluorescence-labeled secondary antibodies (Molecular Probes, Carlsbad, CA). Calpain antibody for immunohistochemical studies (3) was kindly provided by Dr. Naren Banik.

Animal care.

All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council and were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.

Echocardiography.

Mice were initially anesthetized with 3–5% isoflurane vapor in an anesthesia chamber and then placed on a biofeedback warming station with nose cone anesthesia of 1.5–2.5% isoflurane, which was regulated to maintain physiological heart rate (521 ± 8 beats/min) while providing anesthesia (abolition of the toe pinch reflex). Ultrasound gel was placed on the chest, and echocardiography measurements were performed using a 40-MHz probe (Vevo770; Visualsonics). Two-dimensional and M-mode echo images were obtained in the parasternal short- and long-axis views. LV volumes were computed from the parasternal long-axis recordings using the “method of disks,” a modification of Simpson's algorithm (11, 31). For terminal studies, heart harvest was performed following this procedure. The entire echocardiography procedure took ∼20 min.

Mouse MI model.

For MI studies, coronary artery ligation was performed in C57BL/6 mice as described previously (10, 28, 33). Briefly, mice (n = 12) were anesthetized with 2% isoflurane and ventilated. A left-sided thoracotomy was performed, and the left lung was gently packed away using a saline-soaked sponge immediately upon accessing the thoracic space. After MI creation, the sponge was removed and excess fluid in the thorax evacuated carefully. When the thoracotomy was closed, the lungs were reinflated by transient occlusion of the outflow line.

MI was induced by ligating the left coronary artery with an 8.0 ethilon suture (Ethicon, VP-72-28086). MI was confirmed by LV blanching and ST segment elevation on the electrocardiogram. For groups with calpeptin treatment alone, nonoperated control mice were given subcutaneous injections of calpeptin (0.5 mg·kg·⁻¹day⁻¹). For calpeptin treatment in the MI group, a similar protocol was adopted where the first intravenous delivery of calpeptin was done 15 min before MI induction. Once the MI was confirmed, the thoracotomy was repaired. The mice were given buprenorphine (0.05 mg/kg) by subcutaneous injection and placed in a 37°C incubator with room air supplemented with oxygen. They were monitored closely until ambulatory, at which time they were returned to their cages and monitored daily. For mice randomized to the calpeptin group, subcutaneous injections of calpeptin (0.5 mg·kg·⁻¹day⁻¹) were given once per day for the next three days, followed by a terminal echocardiographic analysis (as described above) and euthanization for immunohistochemical and biochemical analyses on day 4 post-MI.

After terminal echocardiography procedures were completed, while mice were under full surgical anesthesia, a midline sternotomy was performed, the heart and great vessels were removed, and the LV was quickly processed for performance of biochemical and histological studies. Since extracellular matrix changes in the 4-day infarcted heart are minimal and determination of infarct size by collagen content was difficult, hematoxylin and eosin (H&E) staining was performed to detect the MI region. To obtain the percentage of MI area, we measured both MI area and remote area in H&E images using NIH ImageJ and then performed calculations using the formula %MI = [MI area/(remote area + MI area)] × 100, as detailed previously (57).

Confocal imaging.

Fresh LV tissue samples were embedded in tissue freezing medium [optimal cutting temperature (OCT) compound], and 15-μm-thick cryosections were prepared using a Leica cryomicrotome for immunohistochemistry. The sections were placed on slides, fixed in 10% neutral buffered formalin, and then processed for H&E staining. For immunostaining, sections were fixed in 2% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 10% normal donkey serum for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: mouse anti-α-actinin (1:200) and rabbit anti-calpain antibody (1:100). After sections were washed with PBS, Alexa647-labeled anti-mouse IgG and Alexa568-labeled anti-rabbit IgG were used as secondary antibodies and incubated for 2 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (Molecular Probes). Apoptotic cell death was detected by TUNEL staining as per the manufacturer's protocol (Chemicon). The fluorescence staining was visualized under laser scanning confocal microscopy (IX71; Olympus Optical, Tokyo, Japan). For quantitation of TUNEL-positive cells, ∼1,200 nuclei were counted from multiple sections from 4 separate mice, chosen randomly for each group (control, MI remote, MI border zone, calpeptin-treated MI remote, and calpeptin-treated MI border zone), and the average percentage of TUNEL-positive nuclei was calculated.

Western blotting.

Triton X-100-soluble proteins were extracted from LV samples and processed with SDS sample buffer as described previously (30). Proteins in SDS sample buffer were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h using 1% BSA and 5% milk in TBST (10 mM Tris, 0.1 M NaCl, and 0.1% Tween 20, pH 7.4). Blots were incubated with primary antibodies in TBST buffer overnight at 4°C, washed five times, each for 5 min with TBST, and then incubated with horseradish peroxidase-conjugated secondary antibody in TBST for 1 h at room temperature. After five washes, each for 5 min with TBST, the proteins were detected by enhanced chemiluminescence.

Statistical analysis.

For echocardiographic measurements, LV geometry and function were compared between control and control + calpeptin groups and between the MI-only and MI + calpeptin groups by using two-way analysis of variance (ANOVA), where the main parameters were time (pre- vs. post-MI) and treatment (no treatment vs. calpeptin). After the ANOVA was performed, post hoc pairwise comparisons were performed for the MI-only and MI + calpeptin groups using Bonferroni-adjusted t-tests. Changes in end-diastolic volume (EDV) and ejection fraction (EF) from baseline (pre-MI) values at 4-days post-MI were computed as the percent change from respective individual baseline values and compared by subjecting these computations to a t-test. For Western blot quantitation of calpain isoforms and cleaved caspase-3, densitometry was performed using the NIH Image J program. For control samples, the levels of calpain isoform in each experiment were assigned a value of 1, and the relative value for each group was then calculated. Differences between groups were compared using one-way ANOVA followed by Tukey's test. Statistical analyses were performed using the STATA statistical software package (Statacorp, College Station, TX). Results are means ± SE. Values of P < 0.05 were considered to be statistically significant.

RESULTS

The goal of the present study was to determine whether calpeptin treatment preserves ventricular geometry and function following MI. First, however, the effect of calpain on LV pump function and geometry was analyzed by echocardiography in non-MI (control) mice. Calpeptin treatment alone for 4 days did not exhibit any significant changes in either EDV (46.29 ± 2.46 μl at baseline vs. 45.15 ± 2.88 μl after calpeptin treatment) or EF (62.24 ± 1.49% at baseline vs. 61.73 ± 1.64% after calpeptin treatment). Next, changes in LV geometry and function were analyzed by echocardiography of MI and calpeptin-treated MI mice at baseline and after 4 days of MI (Fig. 1A). For these studies, only 12 mice, including those used for 4 days of MI induction, underwent surgery, and all of them with and without calpeptin treatment survived through the experimental period. Compared with pre-MI values, 4-day MI mice exhibited a significant increase in EDV (Fig. 1B), and this increase was significantly blunted with calpeptin treatment. Concomitantly, EF was reduced at 4 days post-MI, consistent with previous reports (13). However, this reduction in EF was significantly attenuated in the calpeptin-treated mice (Fig. 1C). Together these data clearly demonstrate that 4-day calpeptin treatment does not affect ventricular geometry and function of normal mice but has a significant effect in MI mice, where it preserves LV geometry and attenuates the reduction of LV pump performance.

Fig. 1.

Inhibition of calpain improves cardiac function in 4-day postmyocardial infarction (MI) mice. A: echocardiograph showing the effect of calpeptin treatment in reducing the chamber dilation in 4-day post-MI mice. Echo images are end-diastolic frames from the long-axis view (LV, left ventricle; LA, left atrium; Ao, aorta). Scale bar, 2 mm. B and C: end-diastolic volume (EDV; B) and percent ejection fraction (EF; C) are shown for MI and calpeptin-treated MI mice. Calpep, calpeptin. Data are means ± SE (no. of animals in each experiment = 6). *P < 0.05 vs. MI only. #P < 0.05 vs. pre-MI.

Since calpain activation is linked to programmed cell death (38, 47, 56), we next examined whether calpain activation occurs in the infarcted myocardium, whether such activation is accompanied by programmed cell death, and whether inhibition of calpain activity with calpeptin can block these changes. For this, we performed histochemical analyses to detect both TUNEL-positive nuclei and calpain enrichment in the MI border zone compared with that in control groups with and without calpeptin treatment. Our recent study in PO myocardium demonstrates a strong correlation among calpain enrichment in cardiomyocytes, increased calpain activity, decreased levels of calpastatin (an endogenous inhibitor of calpain), and TUNEL-positive cardiomyocytes (30). Therefore, we next analyzed changes in calpain activation and programmed cell death in MI mice (n = 4). Compared with the control or control + calpeptin group that exhibited neither calpain enrichment nor TUNEL-positive nuclei, mice with MI alone showed (Fig. 2A) a robust enrichment of calpain (stained red) in the border zone, which was absent in the remote area. Furthermore, a substantial number of TUNEL-positive cells were observed in the border zone but not in the remote area, and many of these dying cells could be cardiomyocytes based on the α-actinin staining. Calpeptin treatment blunted both calpain enrichment and TUNEL reactivity in the MI border zone. As with the MI-alone group, the remote region in the calpeptin-treated animal did not stain for either calpain or TUNEL. Quantitation of TUNEL-positive cells in the border zone of the MI only group was 6.9 ± 1.2% (Fig. 2B), which was effectively prevented by calpeptin treatment. We also extended our analyses to the infarcted area (data not shown): mice with MI alone showed a patchy loss of cardiomyocytes as evidenced by the reduced level of sarcomeric α-actinin, whereas in the calpeptin-treated MI mice, this loss was found to be substantially reduced.

Fig. 2.

Calpeptin treatment reduces calpain activation and cell death in MI mice. A: left, hematoxylin and eosin-stained whole LV section is represented for comparing the anatomical locations, namely, the infarct, border, and remote zones. Nonoperated control animals with and without calpeptin treatment served as controls. Right, tissue sections were stained for calpain (red), TdT-mediated dUTP nick-end labeling (TUNEL; green), α-actinin (blue), and nucleus (purple). Scale bar, 10 μm. B: percentage of TUNEL-positive cells was quantitated in control and MI border zone with and without calpeptin treatment. Approximately 1,200 nuclei were counted from multiple sections from 4 separate mice in each group. *P < 0.05 compared with untreated control. #P < 0.05 compared with untreated MI border zone.

We next analyzed whether the increased levels of calpain enrichment and TUNEL reactivity correlate with infarct size, LV geometry, or function. In the MI-only group, the mean infarct size was 39.5 ± 3.2%, which was significantly reduced to 25.4 ± 2.6% (P < 0.05) in calpeptin-treated MI mice. The increased infarct size in the MI-only group exhibited a positive correlation with the increased levels of both calpain enrichment (Fig. 3A) and TUNEL reactivity (Fig. 3B). Importantly, in the calpeptin-treated MI mice, the reduced levels of infarct size by calpeptin correlate very well with the reduction in both calpain enrichment (Fig. 3A) and TUNEL reactivity (Fig. 3B). Similarly, positive correlations between EDV and calpain enrichment (Fig. 3C) and between EDV and TUNEL (Fig. 3D) and negative correlations between EF and calpain enrichment (Fig. 3E) and between EF and TUNEL (Fig. 3F) were also observed.

Fig. 3.

Infarct size and EDV exhibit positive correlation whereas EF shows negative correlation with calpain enrichment and TUNEL reactivity in MI border zone. Infarct size in MI-only (•) and MI + calpeptin (▪) groups was calculated as described in materials and methods. Graphs A and B show positive correlations between MI area and the number of calpain-enriched cells (r = 0.92) and the number of TUNEL-positive cells (r = 0.89), respectively. Similarly, graphs C and D show positive correlations between EDV and the number of calpain-enriched cells (r = 0.74) and the number of TUNEL-positive cells (r = 0.63, respectively). Graphs E and F show negative correlation between EF and the number of calpain-enriched cells (r = −0.55) and the number of TUNEL-positive cells (r = −0.64), respectively.

Finally, the abundance of calpain isoforms and caspase-3 was analyzed by Western blotting to determine whether there were any differential changes in MI. Analyses of both μ- and m-calpain isoforms with specific antibodies showed the basal level of 80-kDa protein bands (Fig. 4). In control mice, calpeptin treatment caused a significant increase in μ-calpain, although m-calpain remained unaltered. To study changes in calpain levels in MI mice, we used the border zone because it showed a substantial staining of calpain enrichment and TUNEL staining (Fig. 2A). Compared with the basal levels in control mice, analysis in the border zone showed a substantial decrease in μ-calpain level, whereas the band corresponding to m-calpain was significantly increased (Fig. 4). Calpeptin treatment significantly reversed this trend, and the levels of both these isoforms were similar to that of calpeptin-treated control mice. The presence of cleaved (active) caspase-3 (19 kDa), another marker for programmed cell death, was also increased in MI mice, and calpeptin treatment significantly blocked the generation of the cleaved caspase-3.

Fig. 4.

Calpeptin blocks MI-induced changes in calpain isoforms. A: Triton X-100-soluble samples were prepared from control LV and MI border zone of mice treated with or without calpeptin. Protein sample prepared in SDS sample buffer were resolved by polyacrylamide gel electrophoresis and Western blotted with anti-μ- and anti-m-calpain antibodies and anti-active caspase-3 antibody. Adjusting GAPDH levels normalized protein loading between samples. Molecular mass (MW) in kDa corresponding to each protein band is also indicated at right. B– D: changes in calpain isoform and caspase-3 levels were assessed by densitometry using at least 3 independent experiments. The value for control (Cont) in each experiment was arbitrarily assigned a value of 1, and changes in all other treatment conditions were then calculated for statistical analysis as detailed in materials and methods. Summary data are presented as histograms for μ-calpain (B), m-calpain (C), and cleaved (active) caspase-3 (D). Values are means ± SE. *P < 0.05 compared with untreated control. #P < 0.05 compared with untreated MI border zone.

DISCUSSION

Activation of both calpain (6, 15, 16, 58) and caspases (5, 22, 26, 54) has been reported to be associated with adverse myocardial remodeling in animal models of cardiac disease. In this context, a recent study (30) from this laboratory using a feline model of PO-induced cardiac hypertrophy demonstrated a transient activation of both calpain and caspase-3 that was accompanied by both the cleavage of cytoskeletal proteins and a substantial increase in TUNEL-positive cardiomyocytes. Importantly, inhibition of calpain with calpeptin, but not of caspases with Z-VD-fmk, prevented cardiomyocyte death and its associated changes, suggesting calpain could be a potential therapeutic target to both preserve cytoskeletal structure and prevent loss of cardiomyocytes in hypertrophying myocardium (30). Therefore, our primary goal in the present study was to establish that calpain activation occurs in other models of LV remodeling and/or failure and that calpeptin treatment could be used to improve ventricular function under such conditions. We tested this idea using a mouse MI model. Our studies with MI demonstrate that calpeptin attenuates both chamber dilation and reduction of LV pump function and that these observations are accompanied by reduced calpain- and caspase-3-associated changes and cardiomyocyte loss in the border zone.

Similar to our earlier studies that showed calpeptin treatment could block programmed cell death of cardiomyocytes in PO myocardium (30), the present study supports the idea that calpeptin treatment may prevent cardiomyocyte loss during LV remodeling and/or failure. The cleavage of intracellular target proteins by calpain may contribute to both programmed cell death and contractile dysfunction as part of pathological remodeling. In the heart, numerous studies have indicated that increased calpain activity might be a contributing factor to various pathological states, including MI, hypertensive cardiomyopathy, and ischemia-reperfusion injury (1, 23, 24, 37, 39, 44, 47). In the case of MI, the levels of both μ- and m-calpain isoforms were shown to increase in rats following left ventricular coronary artery ligation for 2 wk, and cardiac failure then developed by 8 wk (47) with profound cardiac remodeling and concomitant calpain activity. In the present study, measuring calpain isoforms levels in the border zone of 4-day MI mice indicated that m-calpain is primarily increased, whereas the level of μ-calpain is significantly decreased (Fig. 4). Previous studies (18) demonstrated that a process of self-cleavage of calpain is involved in the calpain activation in addition to the calpain-mediated cleavage of the endogenous inhibitor calpastatin. Therefore, although the μ-calpain level was decreased in the MI border zone, it was indicated that this isoform undergoes cleavage for its activation. Our immunohistochemical studies showed increased enrichment of calpain in cardiomyocytes of MI border zone (Fig. 2A), which could be partly due to the increased m-calpain level (Fig. 4). In MI mice, both the increased level of m-calpain and enrichment of total calpain, as analyzed by Western blotting and confocal immunostaining, respectively, were restored to basal conditions following calpeptin treatment. In the case of μ-calpain, calpeptin treatment not only blocked the cleavage of this isoform in the MI border zone but also caused it to increase above the basal level that was comparable to its level in calpeptin-treated control mice. These findings suggest that calpeptin treatment either blocks degradation and/or increases de novo synthesis of μ-calpain in the remote and border zones following MI. However, the Western blot observation on the increase in μ-calpain levels during calpeptin treatment of control (Fig. 4) is not reflected in the immunostaining analyses, where a corresponding increase in calpain enrichment was absent (Fig. 2A). A recent study (40) showed that in postinfarct heart, m-calpain activity level was higher in the beginning (3 days following an MI) and μ-calpain was higher in 2-wk post-MI heart. Overall, all these studies indicate that calpain enrichment observed in the border zone of 4-day MI mice might be primarily contributed by m-calpain, although the possible contribution by the cleaved (active) form of μ-calpain could not be ruled out from these studies.

Two potential mechanisms could be proposed for calpain activation observed in the border zone of MI mice. First, during the development of ischemic heart disease, there is associated endoplasmic reticulum (ER) stress (2). It must be recognized that LV remodeling post-MI is a heterogeneous process where the LV wall becomes thinner at the MI region while the region remote from the site of MI undergoes hypertrophy (34). Cardiac hypertrophy with accelerated protein synthesis is also often accompanied by ER stress (50). Several studies have demonstrated that excessive calcium release under ER stress conditions causes the activation of both calpain and its downstream targets, such as caspase-12 (36, 37, 41, 48, 49), leading to programmed cell death. Second, calpain activation might be mediated through α₂-integrin activation (4, 42, 51, 52). In this scenario, extracellular matrix degradation may lead to α₂β₁-integrin activation, which has been shown to result in calpain activation. This pathway is particularly intriguing, since activation of matrix metalloproteinases observed in both PO myocardium and the postinfarct heart can result in the generation of cleaved collagen products (12, 20). Whether α₂-integrin activation under these conditions contributes to calpain activation in this murine MI model remains to be established.

The present study demonstrated that the cleaved (active) caspase-3 product was increased in the border zone, but this effect was reduced with calpeptin treatment. These findings suggest that calpain functions upstream of caspase-3 activation during post-MI remodeling. This observation is similar to our recent study (30), where ventricular PO caused the activation of both calpain and caspase-3, and treatment with calpeptin abolished both their activation and prevented myocardial cell death. Previous studies show a calpain-mediated mechanism for the activation of caspase-3 in Jurkat T cells that could be prevented by calpeptin treatment (25). TUNEL reactivity and caspase-3 activation are primarily used as markers of apoptosis, although they often overlap with necrotic cell death. Therefore, it is still possible that part of the cell death observed in the border zone occurs via necrosis. Nevertheless, calpain activation that controls multiple processes, including caspase-3 activation, might be a critical target to prevent both types of cell death in the infarcted heart.

Calpain activation results in the cleavage of a number of specific cardiomyocyte proteins that might lead to subcellular remodeling and contractile dysfunction (44). Calpain-mediated proteolysis of contractile proteins, such as troponins, has been demonstrated in stunned myocardium (17). Often, calpain activation and myofibrillar protein cleavage are followed by the activation of apoptotic pathways, suggesting that contractile dysfunction of cardiomyocytes occurs before subsequent cell death (8). Other targets of calpain include several regulatory proteins of the ER (43), sarcolemmal proteins (23), proteins of the focal adhesion complex (42), and cytoskeletal proteins (30, 53). Thus the process of programmed cell death may be anticipated to cause detrimental effects with respect to contractile function in two ways. First, since the cell death process is mediated through the activation of proteases, such as calpain and caspases that cleave both structural and contractile proteins, the loss of contractility of these cells might precede their death. Second, a decrease in the number of cardiomyocytes in the heart will have an impact on the remaining viable myocardium by increasing their workload; this in turn can contribute to pathological changes in the residual myocardium, thus leading to an overall loss of ventricular function. Therefore, both acute loss of cardiomyocytes in the infarcted area and the subsequent progressive loss of cardiomyocytes in the border zone as part of the remodeling process could contribute to LV pump dysfunction (27). Previous studies have demonstrated that programmed cell death in the postinfarct residual viable myocardium correlate with worsening heart failure (29). Therefore, one potential mechanism for the preserved ventricular geometry and function that we observed in calpeptin-treated MI mice could be through the prevention of calpain-mediated cardiomyocyte death.

A recent study demonstrated that ischemic preconditioning attenuates calpain activation during reperfusion and that the associated recovery of Na⁺-K⁺-ATPase activity contributes to ischemic preconditioning (19). In this context, isoflurane, which is used as anesthesia, has been shown to have a cardioprotective effect (35). However, such an effect (if any) in the present study by isoflurane does not appear to be adequate, since both ventricular dilation and loss of EF could still be observed 4 days after MI where calpeptin treatment significantly blunted these changes. Importantly, it must be noted that a similar anesthetic regiment using isoflurane was used in both MI groups; therefore, the observed effect of calpeptin on LV remodeling post-MI occurred over and above any putative preconditioning effect from the use of isoflurane.

Together, results of the present study demonstrate that calpain activation and the subsequent cleavage of intracellular target proteins post-MI may contribute to programmed cell death as part of pathological remodeling, leading to contractile dysfunction. Calpeptin prevents these key maladaptive changes. However, it must be recognized that in the present study, calpeptin treatment was initiated before the induction of MI, and its protective effect needs to be evaluated following an MI to establish a clinically relevant cohort. Moreover, whether calpeptin treatment can provide similar beneficial effect with respect to LV remodeling over longer post-MI durations remains to be established. Nevertheless, past studies have provided evidence of a causal relationship between programmed cell death and loss of ventricular pump function (55). The present work builds on these past reports by demonstrating that even short-term inhibition of calpain activation attenuates adverse LV remodeling post-MI by controlling programmed cell death and suggests calpain as a potential therapeutic target in treating MI patients.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant PPG HL-48788, a Merit Award from the Research Service of the Department of Veterans Affairs, and NHLBI Grant R01 HL-092124 (to D. Kuppuswamy).