Deficiency of Capn4 Gene Inhibits Nuclear Factor-κB (NF-κB) Protein Signaling/Inflammation and Reduces Remodeling after Myocardial Infarction*

Background: The causal role of calpain in myocardial remodeling after infarction has not been addressed. Results: Deficiency of Capn4 inhibited NF-κB signaling and inflammation and reduced myocardial remodeling, dysfunction, and mortality after infarction. Conclusion: Calpain contributes to myocardial inflammation and remodeling after infarction. Significance: This study provides a significant insight into the function of calpain in post-myocardial infarction remodeling. Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses.


bition of NF-B activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of
Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IB degradation and NF-B activation, resulting in inhibition of inflammatory responses. 4 is the leading cause of death in most industrialized nations throughout the world. After MI, ischemia-induced myocardial cell death is followed by progressive remodeling of the heart (1). This well known sequence of events includes invasion of necrotic tissues by inflammatory cell infiltrates, activation and proliferation of resident cardiac fibroblasts, and breakdown of existing and generation of new extracellular matrix with scar formation, eventually leading to left ventricular (LV) dilatation and hypertrophy of the noninfarcted myocardium (2,3). Changes related to remodeling begin in the acute period of MI and tend to continue long after, leading to LV dysfunction and severe heart failure in later phases. However, the mechanisms of post-MI adverse remodeling and heart failure are only partially understood, and therapeutic interventions that aim to reduce adverse remodeling and prevent the progression to heart failure are relatively limited.

Myocardial infarction (MI)
Calpains are a family of calcium-dependent thiol proteases that participate in a wide variety of biological functions (4,5). Fifteen gene products of the calpain family are expressed in mammals. Among them, calpain-1 (-form) and calpain-2 (m-form) are ubiquitously expressed, and other calpain family members have more limited tissue distribution. The two most extensively studied isoforms, calpain-1 and calpain-2, are heterodimers, differing in their calcium requirement for activation (ϳ50 M for calpain-1 and ϳ1000 M for calpain-2) (6). They consist of a distinct large 80-kDa catalytic subunit encoded by the genes Capn1 and Capn2, respectively, and a common small 28-kDa regulatory subunit encoded by Capn4. The small subunit is indispensable for calpain-1 and calpain-2 stability and activity. Thus, Capn4 knock-out induces the impairment of calpain-1 and calpain-2 activity. Both calpain-1 and calpain-2 are tightly regulated by the intracellular concentration of free Ca 2ϩ and by its endogenous inhibitor calpastatin (6 -8). Calpain activity is increased in the infarcted heart and in myocardia of patients with heart failure (9,10). Pharmacologic inhibition of calpain reduces ischemic cardiac injury and preserves cardiac structure after acute MI (11)(12)(13)(14). A recent study showed that calpain-1 knock-out reduced whereas calpain-1 overexpression enhanced myocardial injury and dysfunction within 4 days after coronary occlusion (15). We and others have demonstrated that calpain-1 is important in cardiomyocyte apoptosis and cardiac proinflammatory responses under pathological conditions (16 -19). Both apoptosis and inflammation contribute to post-MI remodeling (20,21). Thus, calpain may be implicated in myocardial remodeling. Indeed, transgenic overexpression of calpain-1 is sufficient to induce dilated cardiomyopathy and heart failure (22). However, the role and mechanisms of calpain in myocardial remodeling after MI remain not fully understood.
The NF-B family plays an important role in inflammatory responses by promoting the expression of proinflammatory factors (23). Members of the NF-B family (p50, p52, p65, c-Rel, and Rel B) form homo or heterodimers (most commonly p50/ p65, p50/p50, or p65/p65) that are bound to inhibitory IB proteins in the cytosol (24). Degradation of IB frees NF-B dimmers and allows translocation of NF-B into the nucleus, where it can initiate transcription of target genes. Following MI, activation of NF-B contributes to maladaptive LV remodeling and functional deterioration by promoting inflammatory responses (25). Calpain activation has been demonstrated to induce IB degradation and NF-B activation (26,27). However, it has never been shown whether the calpain-mediated NF-B signaling is operative in the MI heart. In this study, we hypothesize that calpain activation induces IB degradation and NF-B activation, which mediate inflammatory responses in post-MI remodeling, and that cardiomyocyte-specific knock-out of Capn4 disrupts calpain-1 and calpain-2, inhibits cardiac inflammation, and reduces cardiac remodeling and dysfunction after MI.

EXPERIMENTAL PROCEDURES
Animals and Cardiomyocyte Culture-This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication Number 85-23). All experimental procedures were approved by the Animal Use Subcommittee at the University of Western Ontario. Mice bearing the targeted Capn4 PZ allele containing loxP sites flanking essential coding exons were generated as described previously (28). Transgenic mice with cardiomyocyte-specific expression of Cre recombinase under the control of ␣-myosin heavy chain (␣-MHC) (Tg-Cre) were generously provided by Dr. Dale E. Abel (University of Utah) (29). Mice with cardiomyocyte-specific disruption of Capn4 (Capn4-ko) were generated by crossing floxed Capn4 PZ mice with transgenic mice overexpressing Cre under the control of the ␣-MHC promoter as we recently described (30). All of the mice used in this study, including controls, were littermates of the same generation. Capn4 PZ/PZ mice were used as wild-type control for Capn4-ko group. Adult mouse ventricle cardiomyocytes were isolated and cultured as described (17).
Myocardial Infarction Model-Under anesthesia with ketamine (100 mg/kg)/xylazine (5 mg/kg, intraperitoneally), adult male mice (about 2 months old) including Capn4-ko (49 mice), Capn4 PZ/PZ (50 mice), and Tg-Cre (7 mice) were subjected to left coronary artery ligation as described previously (31,32). Sham-operated animals (10 Capn4-ko and 10 Capn4 PZ/PZ mice) underwent the same surgical procedure without left coronary artery ligation. After surgery, buprenorphine (0.05 mg/kg per 6 h, subcutaneously) were given for 48 h. Animals were subjected to the following experiments at 24 h, 7 days, and 30 days after MI. For survival study, a separate set of adult Capn4 PZ/PZ and Capn4-ko mice received left coronary artery ligation surgery. Twenty-four hours after surgery, surviving mice including 61 Capn4 PZ/PZ and 27 Capn4-ko mice were monitored and mortality was recorded for 30 days.
Hemodynamic Measurements-Mice were anesthetized with ketamine (100 mg/kg)/xylazine (5 mg/kg) and ventilated. The chest was opened, and a Scisense mouse pressure volume catheter (FTE-1212B-4518, 1.2French) was directly inserted into the LV via the apex to measure LV pressures and volumes as recently described (30).
Histological Analysis-The collagen contents and cardiomyocyte cross-sectional area in noninfarcted areas were assessed as described in our recent study (30).
Myocardial Inflammation-As an index of polymorphonuclear neutrophil infiltration, myeloperoxidase activity in the myocardium was determined by measuring hydrogen peroxide-dependent oxidation of 3,3Ј, 5,5Ј-tetramethylbenzidine as described previously (33). To quantify the mast cells in the heart, cardiac tissue sections were stained with 0.02% toluidine blue in 0.25% acetic acid (pH 2.0 -2.5). The number of mast cells in each heart section was evaluated (31).
To determine monocyte/macrophage infiltration, 4-m sections were cut from cardiac frozen tissue samples embedded in Tissue-Tek OCT gel (Sakura Finetek), mounted on gelatincoated glass microscope slides. Monocytes/macrophages were detected with a biotin-conjugated rat anti-mouse Mac-1 mAb (Cedarlane Laboratories) and then stained by a standard indirect avidin-biotin immunoperoxidase method using an Elite VECTASTAIN ABC kit (Vector Laboratories) as described previously (34). In addition, CD68 mRNA expression was determined as an additional marker of monocytes/macrophages.
Calpain Activity-Calpain activities were determined in noninfarcted areas as described previously (30).
Apoptosis-Apoptosis was determined by assessing caspase-3 activity and TUNEL staining positive cells as described previously (35).
NF-B Activity-NF-B activity was determined in noninfarcted areas by using ELISA-based transcription factor filter plate assay kits according to the manufacturer's instructions (Signosis, Inc.) (30).
Statistical Analysis-All data were presented as mean Ϯ S.D. Analysis of variance followed by Newman-Keuls test was performed for multigroup comparisons. Survival curves were created by the method of Kaplan and Meier, and compared by log-rank test. A value of p Ͻ 0.05 was considered statistically significant.

Deletion of Capn4 Improves Cardiac Function after MI-
Consistent with the previous studies (9, 10), the mRNA levels of Capn1, Capn2, Capn4, and calpain enzymatic activities were elevated in the noninfarcted areas of Capn4 PZ/PZ hearts at 7 and 30 days after MI when compared with their sham group (supplemental sFig. 1, A-D). To investigate the role of calpain-1 and calpain-2, we used cardiomyocyte-specific Capn4 knock-out mice because Capn4 is required for calpain-1 and calpain-2 activities. We first confirmed that Capn4 deletion resulted in a robust reduction of calpain-1 and calpain-2 protein (supplemental sFig. 1E) and disruption of calpain-1 and calpain-2 activities in myocardial tissues from Capn4-ko when compared with Capn4 PZ/PZ hearts (supplemental sFig. 1F). We then induced MI on Capn4-ko mice and their wild-type littermates (Capn4 PZ/PZ ). To evaluate the effect of Capn4 knock-out on cardiac function, LV hemodynamic parameters were analyzed 7 and 30 days after MI. There was no significant difference in the heart rate between Capn4-ko and Capn4 PZ/PZ sham and MI mice (supplemental Table 1). Seven days after MI, cardiac function of both Capn4-ko and Capn4 PZ/PZ MI mice as determined by maximal positive and minimal negative first derivative of LV pressure (ϩdP/dt max and ϪdP/dt min ) and ejection fraction value was significantly decreased when compared with sham groups (p Ͻ 0.05, Fig. 1, A-C). However, LV ϩdP/dt max , ϪdP/ dt min , and ejection fraction value were greater in Capn4-ko versus Capn4 PZ/PZ MI mice (Fig. 1, A-C). Thirty days after surgery, LV ϩdP/dt max , ϪdP/dt min , and ejection fraction value were significantly decreased in both Capn4-ko and Capn4 PZ/PZ animals when compared with their sham groups (Fig. 1, D-F). However, cardiac-specific deletion of Capn4 relatively improved cardiac function (Fig. 1, D-F). To exclude potential effect of Cre transgene, we induced MI on Capn4 PZ/PZ and Tg-Cre mice and analyzed cardiac function 7 days after MI. There was no difference between Capn4 PZ/PZ and Tg-Cre mice (supplemental sFig. 2, A and B).
To further assess cardiac function, echocardiography was performed 30 days after MI. The systolic function index percentage of fractional shortening showed a significant decrease in Capn4-ko and Capn4 PZ/PZ MI mice when compared with their sham animals. However, cardiac-specific deletion of Capn4 exhibited relative improvement of cardiac function in Capn4-ko MI mice ( Fig. 1G and supplemental Table 2). Consistently, the ratios of lung-to-body weight (supplemental sFig. 3) and dry-to-wet lung weight (Fig. 1H) indexes of lung congestion were increased in Capn4 PZ/PZ MI mice when compared with sham animals but decreased in Capn4-ko MI mice. As a consequence, the post-MI mortality was decreased in Capn4-ko when compared with Capn4 PZ/PZ mice (p Ͻ 0.05, Fig. 1I). Taken together, targeted disruption of calpain attenuates myocardial dysfunction and improves survival after MI.
Capn4 Knock-out Prevents LV Dilation and Limits Infarct Expansion Post-MI-Thirty days after MI, echocardiographic analysis revealed that LV end-diastolic and end-systolic diameters were increased in Capn4-ko and Capn4 PZ/PZ MI groups when compared with their shams, indicative of LV dilation and remodeling. However, these changes were significantly attenuated in Capn4-ko MI mice when compared with their wild-type MI groups (supplemental Table 2). These results suggest that disruption of calpain prevents LV dilation after MI. Hemodynamic analysis supported the inhibitory effects of Capn4 knock-out on the enlargement of LV chamber in MI mice. The LV end-diastolic volume and end-systolic volume were significantly reduced in Capn4-ko MI mice when compared with Capn4 PZ/PZ MI group (supplemental Table 1).
To substantiate the role of calpain in LV remodeling after MI, we analyzed infarct size, scar area, LV inner perimeter, and LV cavity size. Initial myocardial injury was not affected by Capn4 knock-out as 2,3,5-triphenyltetrazolium chloride (TTC) staining showed that infarct size did not differ between Capn4-ko and Capn4 PZ/PZ groups at 24 h post-MI (Fig. 2, A-C). Seven days after MI, we assessed scar size at the level of the papillary muscles. The ratio of scar/cross-sectional area was smaller in Capn4-ko MI when compared with Capn4 PZ/PZ MI mice (Fig. 2,  D and E). In parallel with this alteration, deficiency of Capn4 reduced the ratio of inner scar length to inner perimeter and ratio of outer scar length to outer perimeter of LV chamber at the papillary level after MI (Fig. 2, F and G), supporting the limitation of LV infarct expansion in Capn4-ko MI when compared with Capn4 PZ/PZ MI animals. Consistently, there was no difference of scar/cross-sectional area between Capn4 PZ/PZ and Tg-Cre MI mice (supplemental sFig. 2C). These results suggest that inhibition of calpain prevents infarct expansion after MI. Similarly, LV cavity size was preserved in Capn4-ko when compared with Capn4 PZ/PZ mice at 30 days after MI (Fig.  2H).
Deficiency of Capn4 Attenuates LV Hypertrophy following MI-The ratio of heart weight to body weight, a measure of cardiac hypertrophy, was similar between sham groups but was significantly increased in Capn4 PZ/PZ MI mice. However, this ratio was significantly decreased in Capn4-ko MI when compared with Capn4 PZ/PZ MI group (supplemental sFig. 4). MI-induced cardiac hypertrophy was further studied at the cellular level by histological analysis. The myocyte cross-sectional area was similar between sham groups but was significantly increased in the Capn4 PZ/PZ MI mice, indicative of hypertrophy. However, the Capn4-ko MI mice showed smaller myocyte cross-sectional areas when compared with those of Capn4 PZ/PZ MI mice (Fig. 3A). Furthermore, gene expression measurements of Capn4 PZ/PZ MI hearts showed up-regulation of both atrial natriuretic peptide and ␤-MHC mRNA (Fig. 3, B and C), providing further evidence of a hypertrophic phenotype. The mRNA levels of both genes were significantly reduced in Capn4-ko MI hearts (Fig. 3, B and C). There were no differences in hypertrophic gene expression when comparing Capn4-ko and Capn4 PZ/PZ sham hearts (Fig. 3, B and C). These results demonstrate that disruption of calpain prevents myocardial hypertrophy after MI.
Lack of Capn4 in Cardiomyocytes Prevents Apoptosis following MI-Seven days after MI, apoptosis was determined by caspase-3 activity and in situ TUNEL staining. Both TUNELpositive cells (Fig. 3, D and E) and caspase-3 activity (Fig. 3F) were significantly increased in the noninfarcted area of Capn4 PZ/PZ MI mice when compared with their sham group, whereas these indexes were significantly decreased in  AUGUST 10, 2012 • VOLUME 287 • NUMBER 33

D-F).
Post-MI Myocardial Fibrosis Is Reduced in Capn4-ko Hearts-To assess fibrosis, myocardial tissues were stained with picrosirius red to highlight collagen deposition. Ratios of collagen area to total area were measured in noninfarcted areas. MI increased collagen deposition in all mice; however, cardiac-specific deletion of Capn4 reduced collagen deposition (Fig. 4, A  and B). In line with altered collagen deposition, the mRNA levels of collagens I and III were decreased in Capn4-ko MI when compared with Capn4 PZ/PZ MI hearts (Fig. 4, C and D).
Capn4 Knock-out Prevents IB Degradation and Inhibits NF-B Activation in the MI Heart-To gain insights into the downstream signaling, we focused on NF-B pathway because IB has been a target of calpain (26,27). A significant reduction of IB protein was observed in the Capn4 PZ/PZ MI heart; however, its level was restored in the heart of Capn4-ko mice after MI (Fig. 5A). This result suggests that calpain mediates IB degradation in the MI heart. Degradation of IB frees NF-B dimers, which in turn translocate to the nucleus and promote transcription. Consistently, the protein levels of p65 protein and NF-B activity in the nucleus were significantly increased in the Capn4 PZ/PZ MI heart but reduced in the Capn4-ko MI heart (Fig. 5, B and C). In line with the alteration of the NF-B signaling, NF-B-targeted cytokines, such as TNF-␣ and Mcp-1 expression, were significantly elevated in the Capn4 PZ/PZ MI heart. However, their mRNA levels were much lower in the heart of Capn4-ko when compared with Capn4 PZ/PZ MI mice (Fig. 5, D and E).
To examine whether effect of Capn4 deletion on proinflammatory response was restricted to cardiomyocytes, we isolated adult cardiomyocytes, blood mononuclear cells, and peritoneal macrophages from Capn4-ko and Capn4 PZ/PZ mice and exposed them to lipopolysaccharides (LPS) for 2 h. LPS-in-FIGURE 2. Infarct size and LV cavity size after MI. A, representative TTC staining showing no changes in infarct size between Capn4 pz/pz and Capn4-ko mice 24 h after MI. After perfusion with Evans blue dye, the hearts were excised, and the left ventricle was sectioned into six slices for TTC staining of infarct areas. B and C, quantifications of the ratios of ischemic/risk area and ischemia/total area. Data are mean Ϯ S.D., n ϭ 5. D, representative photomicrographs of hematoxylin and eosin-stained sections at the papillary muscle level in Capn4 pz/pz (WT) and Capn4-ko (KO) mice 7 and 30 days after MI (magnification ϫ2). E-G, quantification of scar size and ratios of inner (outer) infarct to total inner (outer) perimeter at the papillary muscle level 7 days after MI. H, LV cavity size was determined at the papillary muscle level 30 days after MI. Data are mean Ϯ S.D., n ϭ 6 -7. *, p Ͻ 0.05 versus WT or sham group; #, p Ͻ 0.05 versus MI in WT mice.
duced TNF-␣ mRNA was significantly reduced in Capn4-ko when compared with Capn4 PZ/PZ cardiomyocytes (supplemental sFig. 5A). In contrast, no difference of TNF-␣ expression was observed in either blood mononuclear cells or peritoneal macrophages from Capn4-ko versus Capn4 PZ/PZ mice (supplemental sFig.5, B and C). These results confirm that deletion of Capn4 affects cardiomyocytes but not inflammatory cells in Capn4-ko mice in terms of proinflammatory response.
The Inflammatory Response following MI Is Decreased in Capn4-ko Mice-NF-B is a ubiquitous transcription factor that controls the expression of genes involved in inflammatory responses (23). Having shown that calpain activates NF-B signaling and promotes proinflammatory factors (TNF-␣ and Mcp-1) in the MI heart, we hypothesized that calpain is responsible for cardiac inflammatory responses, which significantly contribute to cardiac remodeling (21). To test this hypothesis, we measured inflammatory cell infiltration in peri-infarct and infarct areas. As shown in Fig. 6, the infil-tration of inflammatory cells including neutrophils, macrophages, and mast cells was increased in the Capn4 PZ/PZ MI heart and decreased in the heart of Capn4-ko mice (Fig. 6,  A-D). These data suggest an important role of calpain in cardiac inflammation following MI.

DISCUSSION
Calpain has been implicated in post-MI myocardial injury (11)(12)(13). The present study employed cardiomyocyte-specific Capn4 knock-out mice to investigate the role of calpain-1 and calpain-2 in LV remodeling and myocardial dysfunction following MI. We demonstrated that inhibition of calpain-1 and calpain-2 by targeted deletion of Capn4 in cardiomyocytes alleviated post-MI myocardial dysfunction. The improvement of post-MI myocardial function by Capn4 knock-out correlated with a significant attenuation of adverse LV remodeling and a reduction in mortality. The attenuation of myocardial remodeling in Capn4-ko mice was associated with restoration of IB and inhibition of NF-B signaling, leading to suppression of inflammatory responses in the MI heart. Thus, our study has provided direct evidences supporting an important role of calpain-1 and calpain-2 in LV remodeling and myocardial dysfunction after MI and demonstrates that the role of calpain may be mediated through the NF-B signaling and inflammatory responses.
In the present study, cardiomyocyte-specific Capn4 knockout did not influence the initial myocardial injury because myocardial infarct size was comparable after 24 h of MI. This is attributable to the permanent nature of coronary artery ligation. Therefore, this model was used to assess the influence of calpain on adverse myocardial remodeling and dysfunction after MI. Targeted deletion of Capn4 resulted in a significant attenuation of LV remodeling, both of infarct and of remote area, and functional improvement. LV end-diastolic volume and end-systolic volume were markedly reduced in Capn4-ko mice at 7 and 30 days after MI. The infarct area of Capn4 mice was smaller when compared with Capn4 PZ/PZ mice, making it less susceptible to the impact of mechanical loading forces that drive the remodeling process. In addition, Capn4 deletion decreased heart mass, cardiomyocyte hypertrophy, and myocardial fibrosis after MI. Although our hemodynamic measurement was unable to obtain the exact LV end-diastolic pressure due to directly inserting a catheter into the LV chamber via the apex, which can be considered as a limitation of this study, the differences in cardiac functional parameters from both hemodynamic measurement and echocardiography between Capn4-ko and Capn4 PZ/PZ mice were remarkable 7 and 30 days after coronary artery ligation. These protections observed on Capn4-ko MI mice are not due to Cre overexpression because Cre transgene alone did not affect both cardiac function and infarct expansion, which is consistent with a recent study (36). Thus, Capn4 deletion provides a protective effect on both early LV remodeling and late adverse LV remodeling following MI.
Left ventricular remodeling is an active process that contributes to physiological deterioration after MI. Cardiomyocyte drop-out due to apoptosis is a defining cellular event related to ventricular remodeling and development of heart failure (37,38). Continuing cardiomyocyte apoptosis increases infarct expansion, which occurs after MI, leading to compensative and maladaptive hypertrophy, and loss of cardiomyocytes is replaced by fibrosis as cardiomyocytes are unable to proliferate and the generation of new cardiomyocytes is largely limited (37). Calpain is an important contributor to apoptosis in car- diomyocytes (18). In support of this view, deficiency of Capn4 reduced cardiac apoptosis in Capn4-ko mice following MI. Thus, inhibition of cardiomyocyte apoptosis by disruption of calpain may be one of the important mechanisms for reduction of post-MI remodeling in Capn4-ko mice.
Calpain activation has been suggested to induce degradation of myocardial Ca 2ϩ -handling proteins (39) and to cleave protein kinase C-␣ (15), leading to Ca 2ϩ deregulation and protein kinase C-␣ activation. In addition, calpain cleaves myofibrillar proteins such as troponin T, troponin I, titin, and desmin in cardiomyocytes (40,41). Ca 2ϩ deregulation, protein kinase C signaling, and degradation of myofibrillar proteins occur in ischemic hearts and play an important role in myocardial remodeling and dysfunction. The present study provides an additional mechanism that calpain activation induces IB protein degradation, leading to NF-B activation in the MI heart. It is well known that the NF-B signaling is important in promot-ing the expression of proinflammatory factors, which mediate inflammatory responses (26,27). In line with the alteration of IB protein and NF-B activity, the present study demonstrated that the expression of TNF-␣ and Mcp-1 was significantly attenuated in Capn4-ko hearts, which correlated with a reduction of infiltration of inflammatory cells in the MI heart. Thus, it is postulated that after MI, activation of calpain induces IB degradation and subsequent NF-B activation, resulting in the expression of proinflammatory cytokines such as TNF-␣ and Mcp-1 in cardiomyocytes, which promote the infiltration of inflammatory cells, contributing to myocardial remodeling (21). In addition, the proinflammatory cytokines released from cardiomyocytes not only act as an autocrine signaling to induce cardiomyocyte apoptosis and hypertrophy, but also as a paracrine signaling to promote fibrosis through fibroblasts after MI (42). As such, cardiac-specific deletion of Capn4, via prevention of IB degradation and NF-B activation, inhibits inflamma- FIGURE 5. Effects of Capn4 knock-out on IB degradation and NF-B signaling after MI. A, the upper two panels are representative Western blots for IB protein from 3-4 out of 6 -8 different hearts in each group. The lower panel is the quantification of IB protein relative to GAPDH. B, the upper panel is a representative Western blot for p65 protein in the nucleus from 2 out of 6 different hearts in each group, and the lower panel is the quantification of p65 protein relative to histone 2A (H2A). C, NF-B activity was measured in Sham and MI Capn4 pz/pz and Capn4-ko hearts. D and E, the mRNA levels of TNF-␣ (D) and Mcp-1 (E) were measured in noninfarcted areas 7 days after MI. Data are mean Ϯ S.D., n ϭ 6 -8. *, p Ͻ 0.05 versus sham group; #, p Ͻ 0.05 versus MI in Capn4 pz/pz mice. tory responses and blocks both autocrine and paracrine signalings induced by local proinflammatory cytokines from cardiomyocytes, leading to a reduction in myocardial remodeling and an improvement of myocardial function in the MI heart. Given the critical role of NF-B signaling in cardiac remodeling (25,43), IB degradation and consequent NF-B activation may represent an important mechanism for calpain-mediated remodeling after MI. It is worthwhile to mention that calpain from cardiac fibroblasts may also contribute to post-MI myocardial remodeling because our recent study has revealed an important role of calpain in proliferation of cardiac fibroblasts (30).
In summary, cardiomyocyte-specific deletion of Capn4 markedly reduces the protein levels of calpain-1 and calpain-2 and impairs the activities of both calpains in the heart. Calpain induces the NF-B signaling, inflammatory response, and apoptosis following MI, and thus, targeted deletion of Capn4 results in attenuation of LV remodeling and improvement of cardiac function, leading to a reduction in mortality. This study provides a significant insight into the function of calpain in post-MI myocardial remodeling and healing.