AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H128-H136, 2006. First published September 9, 2005; doi:10.1152/ajpheart.00739.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/H128    most recent
00739.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by French, J. P.
Right arrow Articles by Powers, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by French, J. P.
Right arrow Articles by Powers, S. K.

Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition

Joel P. French, John C. Quindry, Darin J. Falk, Jessica L. Staib, Youngil Lee, Kevin K. W. Wang, and Scott K. Powers

Department of Applied Physiology and Kinesiology, Center for Exercise Science, and Departments of Psychiatry and Neuroscience, Center for Traumatic Brain Injury Studies, University of Florida, Gainesville, Florida

Submitted 13 July 2005 ; accepted in final form 7 September 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.

antioxidants; oxidative stress; cardioprotection

MYOCARDIAL ISCHEMIA-REPERFUSION (I/R) injury is a prevalent consequence of cardiovascular disease. Factors that contribute to myocardial I/R injury include oxidative stress due to the production of reactive oxygen species (ROS), disruption of Ca2+ homeostasis, activation of proteases, and neutrophil infiltration/activation (1, 10). Although each of these factors may contribute to cellular injury, growing evidence suggests that increased ROS production and cytosolic free Ca2+ overload, either independently or cooperatively, are major contributors to I/R-induced injury (1, 20, 22).

Increases in myocardial cytosolic Ca2+ levels have been observed during both ischemia and reperfusion (19, 30). In this regard, it has been hypothesized that one role of cytosolic Ca2+ in the pathogenesis of I/R-induced myocardial injury is through activation of the Ca2+-dependent protease calpain (11). Calpain exists in myocytes in two primary isoforms, micro (calpain I) and milli (calpain II), named for the respective amounts of Ca2+ required for their activation in vitro. Both calpain isoforms are activated by prolonged exposure to elevated cytosolic Ca2+ levels (11). Importantly, increased calpain activity can contribute to I/R-induced myocardial injury. Moreover, calpain's deleterious role in I/R injury is supported by strong evidence indicating that calpain inhibition significantly attenuates myocardial infarction (15, 33, 34, 38).

Recent work has linked I/R-induced ROS production to oxidative modification of Ca2+ handling proteins such as sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a (16, 28, 32). This is significant because damage to these proteins can lead to a disruption in cellular Ca2+ homeostasis. Additionally, antioxidant treatment significantly attenuates I/R-induced damage to Ca2+ handling proteins, maintaining Ca2+ homeostasis as well as contractile function (32). On the basis of these findings, there is evidence to suggest that an increase in endogenous antioxidants may provide cardioprotection, at least in part, via regulation of free cytosolic Ca2+ during I/R.

Muscular exercise (i.e., bouts of endurance) is a well-established means of inducing cardioprotection against I/R-induced injury. Specifically, work from numerous laboratories has consistently demonstrated exercise-induced cardioprotection against I/R insults of varying severities, ranging from minor injury to infarction (17, 18, 24, 31). Although the mechanism(s) responsible for exercise-induced cardioprotection is not fully understood, growing evidence suggests that endurance exercise may provide protection, at least in part, by upregulating endogenous antioxidants such as Mn-SOD (13, 25, 37). Moreover, antioxidant studies, using transgenic animals that overexpress antioxidants as well as exogenous antioxidants delivered to the heart, have provided additional evidence that antioxidants can protect the heart against I/R injury (6, 7, 12).

Because recent evidence suggests the possibility of ROS-mediated disturbances in cytosolic Ca2+ homeostasis, it seems possible that exercise-induced increases in myocardial antioxidants can retard I/R-induced Ca2+ disturbances and, consequently, prevent or attenuate I/R-induced calpain activation. Therefore, this study addressed the question "Does endurance exercise training provide cardioprotection by diminishing I/R-induced calpain activation in the heart?" On the basis of work indicating that exercise protects against I/R-induced oxidative stress, we hypothesized that exercise training would attenuate I/R-induced calpain activation in the heart. Our results supported this hypothesis, and the objectives of our study were expanded to investigate the potential mechanisms responsible for this exercise-induced attenuation of calpain activation. Specifically, we determined whether exercise training decreased myocardial calpain levels and/or increased cardiac levels of calpain's endogenous inhibitor, calpastatin. Furthermore, we ascertained whether exercise was associated with a reduction in I/R-induced oxidative stress, as well as the preservation of intact SERCA2a and phospholamban (PLB) protein in the heart after I/R.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental design. These experiments were approved by the University of Florida Animal Care and Use Committee and followed guidelines established by the American Physiological Society for the use of animals in research. To investigate the relationship between exercise and I/R-induced calpain activation in the heart, adult male rats (6 mo old) were randomly assigned to one of seven experimental groups: nonperfused control (n = 11), nonperfused trained (n = 9), perfused control (n = 11), control I/R (n = 11), trained I/R (n = 12), calpain-inhibited I/R (n = 6), and vehicle I/R (n = 6). Nonperfused sedentary and exercise-trained animal groups were included to determine the effects of exercise training on the unstressed heart. Additionally, a sedentary perfused control group was included to serve as a control for any possible effects of perfusion before I/R. It should be noted that an exercise-trained, perfused group was not included in our experimental design because preliminary experiments in our laboratory revealed no differences in cardiac performance between trained and untrained rat hearts before ischemia. Throughout the experimental period, all animals were housed on a 12:12-h light-dark cycle and provided food (AIN93 diet) and water ad libitum.

Exercise training protocol. Animals assigned to the exercise-training groups were habituated to treadmill exercise for 5 consecutive days. This habituation period involved a gradual increase in running time beginning with 10 min/day and ending with 50 min/day. After 2 days of rest, the animals then performed 3 consecutive days of treadmill exercise for 60 min/day at 30 m/min, 0% grade (estimated work rate of 70% maximum O2 consumption) (9). All hearts were excised 24 h after the final exercise bout.

In vitro isolated, perfused working heart protocol. To investigate myocardial function before and after an I/R insult, we selected the in vitro isolated, perfused working heart model. This model is a highly reproducible preparation for examination of cardiac performance, as cardiac preload and afterload pressures are maintained constant. Furthermore, an advantage of the isolated, perfused working heart model vs. an in vivo I/R model is the elimination of the confounding influence of other organ systems, systemic circulation, and peripheral complications (14). Complete details of our isolated working heart preparation were described previously (17, 18, 26).

I/R protocol. Hearts were perfused with a modified Krebs-Henseleit perfusion buffer containing (in mM) 1.25 CaCl2, 130 NaCl, 5.4 KCl, 11 glucose, 0.5 MgCl2, 0.5 NaH2PO4, and 25 NaHCO3 and aerated with 95% O2-5% CO2. The simultaneous clamping of aortic and atrial lines induced global ischemia. After 30 min of perfusion during the preischemic protocol, ischemia was maintained for 25 min, followed by 45 min of reperfusion. During ischemia, the heart was enclosed in a sealed, water-jacketed chamber maintained at 37°C. After the ischemic period, the heart was switched to the retrograde perfusion mode for 10 min, followed by 10 min of assist mode (retrograde perfusion with the atrial cannula open) and 25 min of normal reperfusion.

Cardiac performance measurements. Cardiac performance measurements were made every 5 min before ischemia and during reperfusion. Measurements included timed collections of aortic flow (AF) during working heart mode and coronary flow (CF) during both working heart and retrograde perfusion modes. Cardiac output (CO) was defined as the sum of these flows (CO = AF + CF). Peak systolic pressure, diastolic pressure, and heart rate were measured via a pressure transducer (Harvard Instruments) connected to the aortic cannula. Data were recorded with a customized computer data-acquisition system.

Calpain inhibition. To determine the effects of I/R-induced calpain activation on myocardial function, calpain was inhibited (in vitro) with the selective inhibitor MDL 28170 or calpain inhibitor three (CI3; EMD Biosciences, La Jolla, CA). The efficacy of CI3 as a selective inhibition of calpain I and II has been well established (4, 5, 34, 36). The inhibitor was dissolved in DMSO and added to the perfusion buffer before heart perfusion at a concentration of 10 µM. In preliminary experiments, this concentration of CI3 was shown to inhibit calpain I and II without inhibiting the proteosome. In addition, because DMSO has antioxidant properties, a vehicle control group (vehicle I/R) was also included to demonstrate that our concentration of DMSO, without CI3, was not cardioprotective.

Tissue preparation. On the conclusion of nonperfusion, perfusion, or I/R treatments, the left ventricular free wall was immediately sectioned into four strips cut from base to apex. Before storage, heart sections were rinsed in a cold antioxidant buffer (in mM: 50 NaHPO4, 0.1 butylated hydroxytoluene, and 0.1 EDTA). These tissue sections were then rapidly frozen in liquid nitrogen and stored at –80°C until subsequent biochemical analysis.

I/R-induced lactate dehydrogenase release. Lactate dehydrogenase (LDH) activity in the coronary effluent was used as an indication of I/R-induced myocardial injury. LDH activity was measured in triplicate as described previously (8, 17, 18). Measurements were made in coronary effluent collected before and after ischemia. LDH activities were then normalized to heart wet weight and expressed as the percent difference between preischemic and postischemic values.

Biochemical analysis of endogenous antioxidant enzyme activity. To assess the effect of exercise training on myocardial antioxidant capacity, a section of left ventricular free wall from the nonperfused control and/or nonperfused trained groups was homogenized in cold 100 mM phosphate buffer with 0.5% bovine serum albumin (pH 7.4). Homogenates were centrifuged at 400 g for 10 min at 4°C. The resulting supernatant was used to determine protein content in addition to enzyme activities for SOD and catalase (Cat). Protein content was determined with the methods of Bradford (2). Total SOD activity as well as Mn-SOD and Cu/Zn-SOD activities and Cat activity were measured as described previously (17, 18). Both biochemical assays were performed on the same day at 25°C to avoid interassay variation. The coefficients of variation for SOD and Cat assays were 4% and 5%, respectively.

Western blot analysis of calpain cleavage products. To assess calpain activity, calpain-specific cleavage products of three proteins ({alpha}II-spectrin, talin, and calpastatin) were analyzed. Briefly, proteins were separated with standard SDS-PAGE techniques on a 4–20% polyacrylamide gel. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and exposed to a mouse monoclonal primary antibody to {alpha}II-spectrin (Biomol, Plymouth Meeting, PA), talin (Sigma, St. Louis, MO), or calpastatin (Triple Point Biologics, Forest Grove, OR). After washing, an anti-mouse or anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated secondary antibody was applied for chemiluminescence detection (Amersham, Piscataway, NJ). Both intact and calpain-cleaved fragments of each of the three proteins were analyzed with a Kodak imaging system (Kodak Image Station 440, Rochester, NY). The cleaved bands were then expressed as a percentage of the intact bands and finally normalized as a percentage of the nonperfused control group.

Western blot analysis of intact calpain, calpastatin, SERCA2a, and PLB. Briefly, proteins were separated with standard SDS-PAGE techniques on a 4–20% polyacrylamide gel. Proteins were then transferred to PVDF membranes and exposed to a mouse monoclonal primary antibody to calpain I, calpain II (Chemicon International, Temecula, CA), calpastatin (Triple Point Biologics), SERCA2a (Affinity Bioreragents, Golden, CO), or PLB (Upstate, Lake Placid, NY). After primary antibody exposure, an anti-mouse or anti-rabbit IgG-HRP-conjugated secondary antibody (Amersham) was applied for chemiluminescence detection. Each blot was analyzed with a Kodak imaging system (Kodak Image Station 440) and normalized to {beta}-actin to adjust for protein loading. Results were then expressed as a percentage of nonperfused control.

Cardiac protein carbonyl levels. Protein carbonyls are formed by a variety of oxidative mechanisms and are sensitive indexes of oxidative injury (3). Carbonyl formation was determined in left ventricle homogenates from all experimental groups with a sensitive and reliable commercially available ELISA (Zentech Technology, Dunedin, NZ).

Data analysis. To test our hypothesis, a one-way ANOVA was performed to assess I/R, calpain inhibition, and exercise training differences for the primary dependent measures. A Tukey post hoc test was used to determine group differences when indicated. Significance was established a priori at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal characteristics. The physical characteristics for the animals in all experimental groups are presented in Table 1. Although body mass differed among several of the experimental groups, heart weights and heart-to-body weight ratios were not significantly different between any of the experimental groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Animal characteristics

 
Myocardial performance and damage during I/R. The in vitro I/R protocol was successfully performed on 6–12 hearts from each experimental group. The preischemia and postreperfusion cardiac performance data are presented in Table 2. As expected, compared with preischemia, most indexes of cardiac performance were significantly depressed after I/R. It should be noted that postreperfusion cardiac output, heart rate, cardiac work, and rate-pressure product were preserved in exercise-trained and calpain-inhibited animals compared with sedentary controls. Cardiac work measurements for each group are also displayed, expressed as % recovery, in Fig. 1. Importantly, exercise-trained and calpain-inhibited hearts both recovered significantly better than control hearts in response to I/R. Additionally, LDH activity was measured in coronary effluent before and after ischemia to assess myocardial damage. These data, presented in Fig. 2, demonstrate an increase in LDH release during reperfusion after ischemia, which is attenuated by both exercise training and calpain inhibition. Combined with the measurements of cardiac work, these data indicate that both exercise and calpain inhibition provided cardioprotection against I/R injury.


View this table:
[in this window]
[in a new window]
  		Table 2. Cardiac performance data

 


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Mean % recovery of cardiac work [cardiac output (CO) x systolic blood pressure (SBP)]. Both exercise training and calpain inhibition significantly improved cardiac work after 25 min of ischemia. I/R, ischemia-reperfusion. Values are means ± SE. *Significantly different from control-I/R (P < 0.05).

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2. Effects of I/R injury, exercise and calpain inhibition on lactate dehydrogenase (LDH) release. LDH activity measurements were made in coronary effluent collected before and after ischemia. Activity was normalized to heart wet weight and expressed as % difference between preischemic and postischemic values. Importantly, LDH release was increased significantly after ischemic injury. However, both exercise training and calpain inhibition attenuated the I/R-induced increase in LDH release. *Significantly different from control-I/R (P < 0.05).

 
Calpain activity. In vivo calpain activity in the left ventricle was assessed via the proteolytic cleavage of three calpain substrates, talin, {alpha}II-spectrin, and calpastatin, illustrated in Figs. 3 –6. Compared with perfused control hearts, I/R induced a significant increase in calpain cleavage of all three substrates (Figs. 3– 5) as well as a decrease in intact calpastatin (Fig. 6). Additionally, the I/R-associated increase in calpain substrate cleavage was attenuated in exercised and calpain-inhibited hearts exposed to I/R. This indicates that our calpain inhibitor (CI3) successfully inhibited activation of calpain, and, similarly, exercise, through unknown mechanisms, also attenuated the I/R-induced increase in calpain activation. Collectively, these observations suggest that calpain regulation plays a critical role in exercise-induced cardioprotection.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. Analysis of calpain-cleaved talin. A: representative Western blot illustrating calpain-mediated cleavage of talin after experimental treatments. Intact talin (225 kDa) and breakdown product (BDP; 190 kDa) are as indicated. B: semiquantitative analysis of % calpain-cleaved talin protein present. Note that calpain activation was elevated with I/R (control-I/R and vehicle-I/R groups); however, both exercise (trained-IR) and calpain inhibition (inhibited-IR) attenuated the I/R-associated increase in calpain activation. Data are integrated from multiple Western blots. *Significantly different from nonperfused control (P < 0.05).

 


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 6. Analysis of intact calpastatin protein. A: representative Western blot illustrating calpastatin protein from experimental groups. B: semiquantitative analysis of calpastatin protein present. Note that I/R resulted in a significant decrease in intact calpastatin protein. *Significantly different from nonperfused control (P < 0.05). Data are integrated from multiple Western blots.

 


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 5. Analysis of calpain-cleaved calpastatin. A: representative Western blot illustrating cleavage of calpastatin after experimental treatments. B: semiquantitative analysis of % cleaved calpastatin protein present. Note that calpastatin cleavage was elevated with I/R (control-I/R and vehicle-I/R groups); however, both exercise (trained-I/R) and calpain inhibition (inhibited-I/R) attenuated the I/R-associated increase in calpastatin cleavage. Data are integrated from multiple Western blots. *Significantly different from nonperfused control (P < 0.05).

 
Calpain and calpastatin protein levels. Intact calpastatin, calpain II, and calpain I protein levels were measured via Western blot analysis. The results, displayed in Figs. 6–8, reveal that neither calpain nor calpastatin protein contents were altered with exercise training before I/R. These data indicate that exercise does not regulate calpain activity through alterations in protein levels of calpain or its inhibitor calpastatin.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 8. Analysis of calpain I protein. A: representative Western blot illustrating calpain I protein from all experimental groups. B: semiquantitative analysis of calpain II protein present. Note that calpain I protein was not altered in any of the experimental groups. Data are integrated from multiple Western blots.

 
Antioxidant enzyme activities. The activities of key antioxidant enzymes were determined in hearts from sham-treated animals and are reported in Table 3. Endurance exercise training resulted in a significant increase in myocardial Mn-SOD and Cat activities.


View this table:
[in this window]
[in a new window]
  		Table 3. Myocardial antioxidant enzyme activity

 
Oxidative stress. Oxidative stress was assessed through the measurement of protein carbonyl formation. There was a significant increase in myocardial carbonyl levels after I/R, which was attenuated in exercise-trained animals (Fig. 9).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 9. Effects of I/R injury and exercise on the formation of protein carbonyls. An increase in oxidative stress, measured by protein carbonyl formation, was observed after I/R. Importantly, exercise training significantly attenuated oxidative stress after I/R. Values are means ± SE. *Significantly different from perfused control (P < 0.05).

 
SERCA2a and PLB protein. Intact SERCA2a and PLB protein were measured by Western blotting. The results indicate a significant decrease in both proteins resulting from I/R, which was attenuated by both exercise training and calpain inhibition (Figs. 10 and 11).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 10. Analysis of intact sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a protein. A: representative Western blot illustrating intact SERCA2a protein. B: semiquantitative analysis of intact SERCA2a protein present. Note that I/R was associated with a decrease in intact SERCA2a protein (control-I/R and vehicle-I/R groups); however, both exercise training and calpain inhibition maintained SERCA2a protein after I/R. Data are integrated from multiple Western blots. *Significantly different from perfused control (P < 0.05); {bullet}significantly different from nonperfused control (P < 0.05).

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 11. Analysis of intact phospholamban (PLB) protein. A: representative Western blot illustrating PLB protein. B: semiquantitative analysis of PLB protein present. Note that I/R was associated with a decrease in PLB protein (control-I/R and vehicle-I/R groups); however, both exercise training and calpain inhibition maintained PLB protein after I/R. Data are integrated from multiple Western blots. *Significantly different from perfused control (P < 0.05); {bullet}significantly different from nonperfused control (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
These experiments tested the hypothesis that endurance exercise training would provide cardioprotection against I/R-induced injury, at least in part, by attenuating the I/R-associated increase in myocardial calpain activity. Our findings clearly supported this hypothesis. Moreover, our results suggest that the mechanism explaining the exercise-induced reduction in I/R-induced calpain activation in the heart was not due to diminished calpain protein levels or increased levels of the endogenous calpain inhibitor calpastatin. However, we did observe an increase in the I/R-induced degradation of the Ca2+-ATPase SERCA2a along with the Ca2+ regulatory protein PLB, which was prevented by exercise training. Collectively, these findings advance our understanding of the mechanism(s) responsible for both I/R-induced injury and cardioprotection and are discussed in the following paragraphs.

Calpain inhibition attenuates I/R-induced contractile dysfunction. It is well established that myocardial I/R results in elevated cytosolic levels of Ca2+ that lead to the activation of the Ca2+-regulated protease calpain (1, 5, 11, 15, 32, 34, 38). This is significant because calpain activation promotes the degradation of key cardiac proteins, leading to myocardial contractile dysfunction and cell death. Indeed, activation of calpain can injure cardiac myocytes via several different pathways. For example, calpains cleave several structural proteins leading to the release of myofilaments, facilitating their degradation by the proteosome (11, 21, 27, 35, 36). Moreover, calpains may contribute to apoptosis, through cleavage of Bid, mediating cytochrome c release from the mitochondria (5, 11). Also, calpains increase the expression of cell adhesion molecules, leading to an increase in neutrophil-mediated oxidative damage (23, 29). Each of these pathways has been shown to significantly contribute to I/R-associated injury.

Our findings confirmed the damaging impact of calpain activation on I/R-induced cardiac injury as indicated by the observation that a selective calpain inhibitor (MDL 28170) provided cardioprotection against I/R-induced injury (Fig. 1). These results provide physiological support for the notion that calpain activation plays a significant role in I/R-induced myocardial injury and are consistent with previous work on this topic (15, 33, 34, 38).

Exercise retards I/R-induced calpain activation. Although it is well established that exercise provides protection against I/R-induced cardiac injury, the mechanism(s) responsible for this form of cardioprotection remains unknown. We postulated that exercise-induced cardioprotection against I/R injury could be due, in part, to diminished calpain activation during the I/R insult. Our calpain activity data (Figs. 3–6) were consistent with this hypothesis. Therefore, we also investigated several potential mechanisms responsible for exercise-induced protection against I/R-induced calpain activation. Theoretically, exercise-induced protection against I/R-induced calpain activation could result from one or more of the following possibilities: 1) decreased cardiac levels of calpain I or calpain II, 2) increased cellular levels of the calpain inhibitor calpastatin, and/or 3) improved maintenance of Ca2+ homeostasis in the heart during I/R.

The ratio of calpain to calpastatin in cells is physiologically important because this ratio greatly influences the ability of Ca2+ to activate calpain. That is, an increased calpain-to-calpastatin ratio would favor calpain activation, whereas a decreased calpain-to-calpastatin ratio would favor calpain inhibition. Although it is possible that exercise training could decrease I/R-induced calpain activation by promoting a decrease in cardiac levels of calpain and/or an increase in calpastatin, our results indicate that exercise did not alter the levels of calpain (I and II) or calpastatin in the unstressed heart (Figs. 6–8). Therefore, by process of elimination, we hypothesize that exercise diminishes I/R-induced calpain activation in the heart by improving maintenance of Ca2+ homeostasis. This postulate is supported by the knowledge that calpain is activated by prolonged exposure to elevated Ca2+ levels (11); hence, improved maintenance of myocyte Ca2+ homeostasis would minimize I/R-induced calpain activation. The question remains, however: How does exercise training retard I/R-induced disturbances in myocardial Ca2+ homeostasis?

The current study does not provide a definitive answer to this question. Nonetheless, an intriguing possibility is that exercise training elevates myocardial antioxidants and protects against I/R-induced oxidative stress, which in turn, retards oxidative stress-induced Ca2+ overload within cardiac myocytes. Indeed, our results reveal that exercise attenuated the I/R-induced increase in myocardial oxidative stress (Fig. 9), presumably through an increase in myocardial antioxidant enzymes (Table 3). Specifically, the myocardial increase in the endogenous antioxidant Mn-SOD after several days of exercise training has been shown to play a protective role against I/R-induced oxidative injury (15, 33, 34, 38).

Although the relationships among ROS, Ca2+ homeostasis, and calpain activation during I/R are complex and not completely understood, oxidative stress can increase intracellular Ca2+ through interaction with the sarcoplasmic reticulum and/or plasma membrane (16, 32). Specifically, ROS have been shown to affect Ca2+ homeostasis in several possible ways, such as oxidation of sulfhydryl groups located on Ca2+ transport proteins, peroxidation of membrane lipids, and inhibition of membrane-bound regulatory enzymes (16). Accordingly, Ca2+-ATPases are likely targets for free radicals during I/R. Superoxide, hydrogen peroxide, and the hydroxyl radical have also been implicated in Ca2+-ATPase modification and inhibition (16). In addition, modification of Ca2+-ATPases by free radicals may increase the likelihood of their degradation by calpain (28). Regardless of the specific mechanisms, disruption of these ion transport mechanisms would lead to alterations in Ca2+ homeostasis, resulting in increased calpain activation. Hence, it seems plausible that the exercise-induced increases in myocardial antioxidants and the associated prevention of I/R-associated oxidative stress may serve to maintain free cytosolic Ca2+ homeostasis and, therefore, attenuate calpain activation. Our results are consistent with this possibility, as hearts from exercise-trained animals experienced less oxidative stress and calpain activation after I/R compared with hearts from sedentary control animals (Figs. 3–6 and 9).

Exercise retards I/R-induced SERCA2a and PLB degradation. To further investigate the idea of free radical interaction with Ca2+ handling proteins, we measured protein levels of intact SERCA2a, the most abundant Ca2+-ATPase within the myocardium, as well as PLB, a key SERCA regulatory protein. In this regard, two recent papers have described the loss of intact SERCA2a and PLB protein in the heart after I/R (28, 32). Our present work supports these findings, demonstrating a 60% loss in SERCA2a protein and a 52% loss in PLB protein after I/R. Moreover, work by Temsah et al. (32) reveals that addition of the antioxidants SOD and Cat attenuated the I/R-induced loss of SERCA2a function and mRNA in the heart. These findings are also supported by the present study, which demonstrated an increase in myocardial Mn-SOD and Cat along with a preservation of SERCA2a and PLB protein in exercise-trained animals (Table 3, Figs. 10 and 11). Although, Temsah et al. (32) did not detect preservation of PLB protein after I/R with the administration of their antioxidant treatment, we observed that exercise training preserved PLB protein after I/R (Fig. 11). The explanation for this divergent finding is not clear; however, it is possible that exercise training provides a more potent endogenous antioxidant defense than supplementation of exogenous SOD and Cat. It is also possible that exercise training may prevent PLB degradation through another, unknown, mechanism.

Importantly, the present study is among the first to demonstrate that calpain inhibition prevents the I/R-associated degradation of SERCA2a and PLB protein. Our work suggests that calpain may be in part responsible for cleavage of these proteins, raising the interesting possibility that calpain may regulate itself through a feed-forward mechanism by increasing levels of free cytosolic Ca2+ through the cleavage of SERCA2a and/or PLB. Recent work by Sing et al. (28) also reported attenuation of SERCA2a cleavage with a calpain inhibitor, although the inhibitor used in their study, leupeptin, has also been found to have antioxidant properties. This could be confounding based on the possibility of interaction between free radicals and Ca2+-ATPases.

In synopsis, we postulate that the I/R-induced increase in free radicals may lead to increased oxidative modification of Ca2+ handling proteins such as SERCA2a and PLB, making them targets for calpain cleavage. In addition, the cleavage of these proteins would logically lead to an increase in free cytosolic Ca2+ and, therefore, calpain activation. This hypothesis is supported by the fact that increasing antioxidants (either endogenously or exogenously) and/or inhibiting calpain both attenuate the I/R-induced reduction in SERCA2a and PLB protein.

In conclusion, this work confirms the deleterious role of I/R-induced calpain activation. Importantly, this is the first experiment to investigate the effects of endurance exercise on I/R-induced calpain activation in the heart. Our findings clearly support the hypothesis that endurance exercise training attenuates the I/R-associated increase in myocardial oxidative stress and calpain activity. Importantly, our results also reveal that the mechanism to explain the exercise-induced reduction in calpain activity is not due to diminished myocardial calpain levels or increased levels of the endogenous calpain inhibitor calpastatin. Furthermore, our findings confirm that I/R-induced myocardial oxidative stress is attenuated by the improved myocardial antioxidant capacity of the exercise-trained animals. Finally, we show an attenuation of I/R-associated SERCA2a and PLB degradation with both exercise training and calpain inhibition. Collectively, our findings are consistent with the concept that regulation of redox balance and calpain activation play a significant role in cardioprotection against I/R injury in the myocardium. Additional experiments are required to provide further insight into the specific mechanism(s) responsible for protection against I/R-induced calpain activation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funding from the National Heart, Lung, and Blood Institute (HL-072789, S. K. Powers) and the American Heart Association (AHA 0415135B, J. P. French).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 4. Analysis of calpain-cleaved {alpha}II-spectrin. A: representative Western blot illustrating calpain-mediated cleavage of {alpha}II-spectrin after experimental treatments. Intact {alpha}II-spectrin (260 kDa) and BDP (150 kDa) are as indicated. B: semiquantitative analysis of % calpain-cleaved {alpha}II-spectrin protein present. Note that calpain activation was elevated with I/R (control-I/R and vehicle-I/R groups); however, both exercise (trained-I/R) and calpain inhibition (inhibited-I/R) attenuated the I/R-associated increase in calpain activation. Data are integrated from multiple Western blots. *Significantly different from nonperfused control (P < 0.05).

 


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 7. Analysis of calpain II protein. A: representative Western blot illustrating calpain II protein from all experimental groups. B: semiquantitative analysis of calpain II protein present. Note that calpain II protein was not altered in any of the experimental groups. Data are integrated from multiple Western blots.

 

   FOOTNOTES
 

Address for reprint requests and other correspondence: S. K. Powers, Dept. of Applied Physiology and Kinesiology, Center for Exercise Science, Univ. of Florida, Gainesville, FL 32611 (e-mail: spowers{at}hhp.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bolli R. Causative role of oxyradicals in myocardial stunning: a proven hypothesis. A brief review of the evidence demonstrating a major role of reactive oxygen species in several forms of postischemic dysfunction. Basic Res Cardiol 93: 156–162, 1998.[CrossRef][ISI][Medline]
  2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  3. Buss H, Chan TP, Sluis KB, Domigan NM, and Winterbourn CC. Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 23: 361–366, 1997.[CrossRef][ISI][Medline]
  4. Chen M, He H, Zhan S, Krajewski S, Reed JC, and Gottlieb RA. Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. J Biol Chem 276: 30724–30728, 2001.[Abstract/Free Full Text]
  5. Chen M, Won DJ, Krajewski S, and Gottlieb RA. Calpain and mitochondria in ischemia/reperfusion injury. J Biol Chem 277: 29181–29186, 2002.[Abstract/Free Full Text]
  6. Chen Z, Siu B, Ho YS, Vincent R, Chua CC, Hamdy RC, and Chua BH. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol 30: 2281–2289, 1998.[CrossRef][ISI][Medline]
  7. Coombes JS, Powers SK, Demirel HA, Jessup J, Vincent HK, Hamilton KL, Naito H, Shanely RA, Sen CK, Packer L, and Ji LL. Effect of combined supplementation with vitamin E and alpha-lipoic acid on myocardial performance during in vivo ischaemia-reperfusion. Acta Physiol Scand 169: 261–269, 2000.[CrossRef][ISI][Medline]
  8. Coombes JS, Powers SK, Hamilton KL, Demirel HA, Shanely RA, Zergeroglu MA, Sen CK, Packer L, and Ji LL. Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and {alpha}-lipoic acid. Am J Physiol Regul Integr Comp Physiol 279: R2149–R2155, 2000.[Abstract/Free Full Text]
  9. Criswell D, Powers S, Dodd S, Lawler J, Edwards W, Renshler K, and Grinton S. High intensity training-induced changes in skeletal muscle antioxidant enzyme activity. Med Sci Sports Exerc 25: 1135–1140, 1993.[ISI][Medline]
  10. Frangogiannis NG, Smith CW, and Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res 53: 31–47, 2002.[Abstract/Free Full Text]
  11. Goll DE, Thompson VF, Li H, Wei W, and Cong J. The calpain system. Physiol Rev 83: 731–801, 2003.[Abstract/Free Full Text]
  12. Hamilton KL, Staib JL, Phillips T, Hess A, Lennon SL, and Powers SK. Exercise, antioxidants, and HSP72: protection against myocardial ischemia/reperfusion. Free Radic Biol Med 34: 800–809, 2003.[CrossRef][ISI][Medline]
  13. Hamilton KL, Quindry JC, French JP, Staib J, Hughes J, Mehta JL, and Powers SK. MnSOD antisense treatment and exercise-induced protection against arrhythmias. Free Radic Biol Med 37: 1360–1368, 2004.[CrossRef][ISI][Medline]
  14. Hearse DJ and Sutherland FJ. Experimental models for the study of cardiovascular function and disease. Pharmacol Res 41: 597–603, 2000.[CrossRef][ISI][Medline]
  15. Iwamoto H, Miura T, Okamura T, Shirakawa K, Iwatate M, Kawamura S, Tatsuno H, Ikeda Y, and Matsuzaki M. Calpain inhibitor-1 reduces infarct size and DNA fragmentation of myocardium in ischemic/reperfused rat heart. J Cardiovasc Pharmacol 33: 580–586, 1999.[CrossRef][ISI][Medline]
  16. Kourie JI. Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol Cell Physiol 275: C1–C24, 1998.[Abstract/Free Full Text]
  17. Lennon SL, Quindry J, Hamilton KL, French J, Staib J, Mehta JL, and Powers SK. Loss of exercise-induced cardioprotection after cessation of exercise. J Appl Physiol 96: 1299–1305, 2004.[Abstract/Free Full Text]
  18. Lennon SL, Quindry JC, French JP, Kim S, Mehta JL, and Powers SK. Exercise and myocardial tolerance to ischaemia-reperfusion. Acta Physiol Scand 182: 161–169, 2004.[CrossRef][ISI][Medline]
  19. Marban E, Kitakaze M, Koretsune Y, Yue DT, Chacko VP, and Pike MM. Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion. Circ Res 66: 1255–1267, 1990.[Abstract/Free Full Text]
  20. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159–163, 1985.[Abstract]
  21. Molinari M and Carafoli E. Calpain: a cytosolic proteinase active at the membranes. J Membr Biol 156: 1–8, 1997.[CrossRef][ISI][Medline]
  22. Park JL and Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thorac Surg 68: 1905–1912, 1999.[Abstract/Free Full Text]
  23. Pontremoli S and Melloni E. The role of calpain and protein kinase C in activation of human neutrophils. Prog Clin Biol Res 282: 195–208, 1988.[Medline]
  24. Powers SK, Demirel HA, Vincent HK, Coombes JS, Naito H, Hamilton KL, Shanely RA, and Jessup J. Exercise training improves myocardial tolerance to in vivo ischemia-reperfusion in the rat. Am J Physiol Regul Integr Comp Physiol 275: R1468–R1477, 1998.[Abstract/Free Full Text]
  25. Powers SK, Quindry J, and Hamilton K. Aging, exercise, and cardioprotection. Ann NY Acad Sci 1019: 462–470, 2004.[Abstract/Free Full Text]
  26. Quindry J, French J, Hamilton K, Lee Y, Mehta JL, and Powers S. Exercise training provides cardioprotection against ischemia-reperfusion induced apoptosis in young and old animals. Exp Gerontol 40: 416–425, 2005.[CrossRef][ISI][Medline]
  27. Rappaport L. Ischemia-reperfusion associated myocardial contractile dysfunction may depend on Ca2+-activated cytoskeleton protein degradation. Cardiovasc Res 45: 810–812, 2000.[Free Full Text]
  28. Singh RB, Chohan PK, Dhalla NS, and Netticadan T. The sarcoplasmic reticulum proteins are targets for calpain action in the ischemic-reperfused heart. J Mol Cell Cardiol 37: 101–110, 2004.[CrossRef][ISI][Medline]
  29. Squier MK, Sehnert AJ, Sellins KS, Malkinson AM, Takano E, and Cohen JJ. Calpain and calpastatin regulate neutrophil apoptosis. J Cell Physiol 178: 311–319, 1999.[CrossRef][ISI][Medline]
  30. Steenbergen C, Murphy E, Levy L, and London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 60: 700–707, 1987.[Abstract/Free Full Text]
  31. Taylor RP, Ciccolo JT, and Starnes JW. Effect of exercise training on the ability of the rat heart to tolerate hydrogen peroxide. Cardiovasc Res 58: 575–581, 2003.[Abstract/Free Full Text]
  32. Temsah RM, Netticadan T, Chapman D, Takeda S, Mochizuki S, and Dhalla NS. Alterations in sarcoplasmic reticulum function and gene expression in ischemic-reperfused rat heart. Am J Physiol Heart Circ Physiol 277: H584–H594, 1999.[Abstract/Free Full Text]
  33. Trumbeckaite S, Neuhof C, Zierz S, and Gellerich FN. Calpain inhibitor (BSF 409425) diminishes ischemia/reperfusion-induced damage of rabbit heart mitochondria. Biochem Pharmacol 65: 911–916, 2003.[CrossRef][ISI][Medline]
  34. Urthaler F, Wolkowicz PE, Digerness SB, Harris KD, and Walker AA. MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKC{epsilon} proteolysis in reperfused ferret hearts. Cardiovasc Res 35: 60–67, 1997.[Abstract/Free Full Text]
  35. Wang KK, Roufogalis BD, and Villalobo A. Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca2+-ATPase. Arch Biochem Biophys 267: 317–327, 1988.[CrossRef][ISI][Medline]
  36. Wang KK and Yuen PW. Development and therapeutic potential of calpain inhibitors. Adv Pharmacol 37: 117–152, 1997.
  37. Yamashita N, Hoshida S, Otsu K, Asahi M, Kuzuya T, and Hori M. Exercise provides direct biphasic cardioprotection via manganese superoxide dismutase activation. J Exp Med 189: 1699–1706, 1999.[Abstract/Free Full Text]
  38. Yoshikawa Y, Hagihara H, Ohga Y, Nakajima-Takenaka C, Murata KY, Taniguchi S, and Takaki M. Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics. Am J Physiol Heart Circ Physiol 288: H1690–H1698, 2005.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 FASEB J.Home page
J. P. French, K. L. Hamilton, J. C. Quindry, Y. Lee, P. A. Upchurch, and S. K. Powers
Exercise-induced protection against myocardial apoptosis and necrosis: MnSOD, calcium-handling proteins, and calpain
FASEB J, August 1, 2008; 22(8): 2862 - 2871.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
C. Pitsavos, S. A. Kavouras, D. B. Panagiotakos, S. Arapi, C. A. Anastasiou, S. Zombolos, P. Stravopodis, Y. Mantas, Y. Kogias, A. Antonoulas, et al.
Physical Activity Status and Acute Coronary Syndromes Survival: The GREECS (Greek Study of Acute Coronary Syndromes) Study
J. Am. Coll. Cardiol., May 27, 2008; 51(21): 2034 - 2039.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. N. Kavazis, J. M. McClung, D. A. Hood, and S. K. Powers
Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli
Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H928 - H935.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
A. Medeiros, N. P. L. Rolim, R. S. F. Oliveira, K. T. Rosa, K. C. Mattos, D. E. Casarini, M. C. Irigoyen, E. M. Krieger, J. E. Krieger, C. E. Negrao, et al.
Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice
J Appl Physiol, January 1, 2008; 104(1): 103 - 109.
[Abstract] [Full Text] [PDF]

Home page
 Anesth. Analg.Home page
J. An, Z. J. Bosnjak, and M. T. Jiang
Myocardial Protection by Isoflurane Preconditioning Preserves Ca2+ Cycling Proteins Independent of Sarcolemmal and Mitochondrial KATP Channels
Anesth. Analg., November 1, 2007; 105(5): 1207 - 1213.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Bartoli, N. Bourg, D. Stockholm, F. Raynaud, A. Delevacque, Y. Han, P. Borel, K. Seddik, N. Armande, and I. Richard
A Mouse Model for Monitoring Calpain Activity under Physiological and Pathological Conditions
J. Biol. Chem., December 22, 2006; 281(51): 39672 - 39680.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/H128    most recent
00739.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by French, J. P.
Right arrow Articles by Powers, S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by French, J. P.
Right arrow Articles by Powers, S. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH