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Circ J. Author manuscript; available in PMC 2016 March 18. Published in final edited form as: Circ J. 2015 December 25; 80(1): 4–10. doi:10.1253/circj.CJ-15-0997.
Calpains and CoronaryVascular Disease Brittany A. Potz, MD, Ashraf A. Sabe, MD, M. Ruhul Abid, MD/PhD, and Frank W. Sellke, MD Cardiothoracic Surgery Research Division, Institution: Warren Alpert Medical School Brown University
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Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates for or receive suboptimal revascularization.. Calpains are a class of calcium activated non-lysosomal cysteine proteases which serve as a proteolytic unit for cellular homeostasis. Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac ischemia. Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia reperfusion. Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases including CAD.
Keywords
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Calpain; Coronary Artery Disease; Metabolic Syndrome
Introduction
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Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates for or receive suboptimal revascularization.. Incomplete revascularization in patients with CAD who undergo surgical intervention is a predictor for operative and peri-operative morbidity and mortality. With the increased prevalence of obesity and metabolic syndrome (MS) in the United States, the incidence of severe CAD not amenable to surgical treatment is likely to increase. Inducing a pro-angiogenic state through medical therapies remains a promising therapeutic option for these patients. To achieve this goal, , a deeper understanding of the pro-angiogenic and anti-angiogenic pathways in the setting of hypercholesterolemia and chronic ischemic disease is necessary to treat this complicated and growing population of patients. (1) Calpains are a class of calcium activated non-lysosomal cysteine proteases which serve as a proteolytic unit for cellular homeostasis (2). Cysteine proteases contain the amino acid cysteine in the active site of the molecule. The three major groups of cysteine proteases include cathepsins, caspases and calcium dependent calpains. (3) Under basal conditions,
Corresponding Author: Brittany Potz, Division of Cardiothoracic Surgery, Cardiovascular Research Center, Warren Alpert Medical School Brown University, 2 Dudley Street MOC 360, Providence, RI 02905.
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calpain is predominantly situated in the cytosol and translocates to the membrane on activation. Activation of calpain results in proteolysis of several cellular proteins including cytoskeletal proteins, membrane proteins, enzymes, cytokines, and transcription factors all of which are known to play important roles in transducing signals of cell migration, differentiation and proliferation. (4). Excessive activation of calpains has been implicated in the pathophysiology of several disorders including inflammation, diabetes mellitus (DM), ischemia reperfusion injury, and trauma. (3) (5). Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases including CAD. [Figure 1] Calpain Structure
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15 calpain isoforms have been reported so far. In mammals there are 14 large subunit members (80-kDa catalytic subunit), one small subunit member (30-kDA subunit) and one endogenous inhibitor (calpastatin). (6) Both subunits can bind calcium resulting in the activation of calpain. Calpain activity can also be regulated by auto proteolysis and by calpastatin, suggesting that calpains are part of a regulatory proteolytic system. (7) [Figure 2] Most calpain isoforms contain four structural domains. Calpains are divided into two groups based on the structure of domain IV: Typical and Atypical. Typical calpains (1,2,3,8,9,11,12,14) contain a penta-EF motif in domain IV that can bind calcium, the calpain small subunit (only calpains 1,2 and 9 have been shown to dimerize) or calpastatin. Atypical calpains (5,6,7,10,12 and 15) lack a penta-EF motif in domain IV and are unable to bind the calpain small subunit or calpastatin. (6)
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Some calpains are ubitiquitously expressed (calpains 1,2,4,5,7 and 10) while others are found in specific tissues (calpain 1&2- endothelia cells, calpain 3- skeletal muscle, calpain 6- placenta, calpain 8- smooth muscle, calpain 9- stomach, calpain 11- testes, calpain 12skin after birth and calpain 13- tests and lung). (8). Among the 15 calpain isoforms, 10 isoforms are expressed in the heart. The most well studied of these calpains are Calpain 1 (U-calpain) and calpain 2 (m-calpain) which require micro and milli molar concentrations of calcium for their activation respectively. (3)
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In atypical calpains the function of domain 1 is largely unknown. However, domain 1 for calpain 10 contains a mitochondrial targeting sequence. In calpains 1, 2 and 9, domain I is cleaved after Ca2+ activation resulting in autolysis and autoactivation. Domain II contains the catalytic active site with the ‘catalytic triad’ of cysteine, asparagines and histidine. The catalytic triad is conserved throughout the entire family (except for calpain 6 that lacks proteolytic activity). Domain II can also bind two atoms of Ca2+ and assists in calpain activation. Domain III contains two Ca2+ binding sites and a phospholipid-binding motif which is conserved throughout the family, except for Calpain 10. Domain III is also believed to regulate calpain activity through specific electrostatic interactions and to be involved in substrate recognition. (6) Calpastatin is an endogenous protein that specifically inhibits calpains. The fact that the calpain's active site is conserved throughout the entire family makes it difficult to generate
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isoform specific inhibitors. Interestingly, structural and biochemical data indicate that calpastatin may bind preferentially to calcium activated calpains, suggesting that calpastatin controls activated calpain activity. This suggests that calpastatin limits calpain activation without interfering with basal calpain activity, which is required for normal protein homeostasis. (9) This fact is critical and should be taken into consideration while considering the use of calpain inhibitors for the treatment of cardiovascular disease. Coronary Artery Disease and Metabolic Syndrome It has been well demonstrated that co morbid diseases which constitute MS, such as DM, obesity, and hypercholesterolemia have a deleterious impact on endothelial function and growth. With the increasing prevalence of these co morbid conditions, it is important to understand mechanisms by which these diseases contribute to or exacerbate endothelial dysfunction and CAD.
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In particular, DM is recognized as a potent risk factor for cardiovascular disease including atherosclerosis and microvascular complications. (10) (8) Hyperglycemia has been shown to cause endothelial dysfunction characterized by a loss of endothelium derived nitric oxide (NO). Hyperglycemia also results in increased vascular permeability, increased endothelial adhesiveness, thickening of the basement membrane, abnormal inflammatory signals and increased oxidative stress. Endothelial NO is an important regulator of vascular homeostasis. Loss of NO leads to increased vascular tone, and abnormal endothelial adhesiveness, with resultant increased platelet aggregation and leukocyte trafficking in the vessel wall. Thus the diabetic vasculature experiences increased oxidative stress and abnormal inflammatory signals. (11) (10)
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Furthermore, high glucose induced myocyte apoptosis plays an important role in diabetic cardiac complications. Though the molecular mechanisms by which glucose damages cardiac cells remain incompletely understood, it is thought to be partly mediated through cytotoxic oxidative stress and intracellular calcium overload. (12)
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In order to further study the impact of these co morbidities on CAD our group has utilized a clinically relevant large animal model that has been shown to adequately represent metabolic, microcirculatory and molecular abnormalities seen in MS in adult patients with chronic myocardial ischemia. (13). We have also demonstrated that MS in chronic myocardial ischemia significantly impairs notch signaling (an integral component of cardiac angiogenesis) by down regulating notch receptors, ligands and angiogenic proteins. (14) Expression of neuropeptide Y (one of the most abundant neurotransmitters in the myocardium known to influence cardiovascular remodeling) was found to be altered differentially in the serum and myocardium in patients with DM. Findings from our lab and others suggest that altered regulation of the neuropeptide Y in diabetics may be, in part, responsible for the decreased angiogenesis, increased apoptosis and vascular smooth muscle proliferation leading to CAD and heart failure in the MS patient population. (15) It is important to note that co-morbid factors such as DM, obesity, and hypercholesterolemia will influence and exacerbate endothelial dysfunction leading to CAD. Understanding how these pathological conditions affect each other will help devise therapeutics that would
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effectively treat heart disease. In the following section, we will discuss how calpain may be involved in the pathogenesis of DM, obesity and CAD, making it a potential therapeutic target. Uncontrolled Activation of Calpains leads to Heart Disease
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Calpain activity is required for normal myogenesis, whereas calpain over-activation has been implicated in the development of heart disease. (16). (2). (17) In cardiac muscle calcium plays a crucial role in excitation-contraction coupling. Dysregulation of cellular calcium homeostasis, often results in calcium overload which leads to calpain overactivation and results in myocardial cell injury. (2) Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac, cerebral and hepatocellular ischemia. (18) Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia reperfusion. (5). The exact mechanisms by which calpain is up-regulated are not fully understood. Calpain activity is mainly regulated through alterations in the calcium concentration as calcium binding is required for its proteolytic activity. Evidence has been provided that cellular Ca overload leads to over activation of calpains by auto proteolysis of an N-terminal peptide. (18) Studies also suggest that calpain activity can also be regulated by certain phospholipids, ERK1/2 MAPK, PKC and PKA. In addition, NADPH oxidase has been suggested to induce calpain activation in norepeinephrine stimulated cardiomyocytes. For example, one study suggests that the activation of NADPH oxidase results in reactive oxygen species production which activates calpain through regulation of intracellular calcium levels and ERK1/2 pathway. (9)
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In concurrence with the above discussion, our group and multiple others have demonstrated that the over activation of calpain contributes to cardiovascular disease. ( (9) (19) (2) (3) (18) (1) (20). However, a better understanding of the pro-angiogenic and anti-angiogenic pathways in the setting of hypercholesterolemia, hyperglycemia, and chronic ischemic disease is necessary. Our lab has developed a pig model for chronic myocardial ischemia in the setting of MS (weight gain, glucose intolerance, dyslipidemia and hypertension) to elucidate the molecular mechanisms in a pre-clinical setting. (1) Further studies should focus on identifying mechanisms by which this over activation occurs and to identify novel therapies for calpain inhibition as a potential medical therapy for patient with cardiac risk factors and disease. Calpains Improve Myocardial and Endothelial Cystokeletal Protein Remodeling
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In diseased myocardial tissue, remodeling may be associated with ischemia-induced changes to the contractile proteins. Calpain has been found to cleave myofibrillar specific proteins including troponin T, troponin I, titin and desmin in cardiomyocytes. (9) Cardiac troponin I is a subunit of the troponin complex involved in the control of muscle contraction. Interestingly, troponin I has been found to be preferentially affected in ischemic rat hearts. (21) Maekawa et al found that systolic dysfunction of the stunned myocardium is associated with troponin I degradation and over expression of calpastatin will lessen contractile
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dysfunction of rat hearts subjected to ischemia/reperfusion. In addition, the same model found that degradation of troponin following ischemia/reperfusion was prevented by calpain inhibition. (22) Yoshiro et al found that high calcium intracoronary infusion causes calcium overload induced cardiac dysfunction through proteolysis of cytoskeleton protein alpha-fodrin Yashikawa et all found that calpain inhibition protects the rat heart from ischemia/ reperfusion injury by inhibiting proteolysis of alpha fodrin which they speculate prevents conformational changes to the L-type Ca channel at the cell membrane and results in protection of LV function. (19)
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Endothelial cells play an essential role in cardiovascular pathophysiology through their functions in blood vessel formation, cell barrier, coagulation, vascular tone, inflammation and angiogenesis. Many of these processes require cytoskeletal rearrangements and a number of cystoskeletal proteins are known calpain substrates including talin, filamin, spectrin, tau, Paxillin, Vinculin, alpha tubulin, vimentin, and filamin. (9) (6) (7) (10) In vivo studies have demonstrated that calpain inhibitors strongly improved organization of the endothelial actin and tau protein cystokeleton in newly formed blood vessel suggesting that calpain inhibition restores cystoskeletal organization improves capillary morphogenesis and promotes formation of a functionally improved neovasculature that helps to reduce underlying hypoxia. (23) (24)
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Komuro et al demonstrated that calpains impaired cell-cell interactions through degradation of the cadherin-associated protein complex resulting in LV remodeling after myocardial infarction (MI). Interestingly, they found that calpain mediated proteolysis was increased in the chronic phase (7 days or later) but not in the acute phase (before 24 hours) after MI and profound activation of calpains exacerbated LV remodeling. The border zone of MI hearts in these mice showed a decrease in N-cadherin expression concomitant with an increase in calpain activation and exacerbation of LV remodeling. In cultured cardiomyocytes calpain activation caused degradation of N cadherin and disorganization of cadherin based cell adhesions. (25) Similarly, in cultured cardiomyocytes, calpain activation disassembled cadherin-based cell-cell adhesion consisting of intercalated disc proteins such as B-catenin and Connexin 43. (25) This suggests that calpain over activation may lead to myocardial remodeling after MI through the breakdown of cell- cell interactions.
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Additionally, several groups have reported roles for mitochondrial calpain 1 in endothelial cells. In rat heart microvascular endothelial cells, hyper-homocysteinemia induced extracellular matrix remodeling by matrix metalloproteinase 9 has been shown to be caused by calpain 1 over activation. (6). Activation of calpain results in proteolysis of several cellular proteins including cytoskeletal proteins which are known to play a role in myocardial tissue remodeling after an ischemic insult. This may be one mechanism by which calpain inhibition can help prevent ischemic changes in myocardial tissue after an MI.
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Calpains Attenuate Myocardial Apoptosis
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Calpain plays an important role in cell death signaling in the heart. (2) Numerous studies have documented that calpains are important in ischemia/reperfusion injury in the heart, mitochondrial permeability transition and necrotic/apoptotic cell death. (6) Myocardial calpain 1 is active under normal physiological conditions and serves as an essential component of the ubiquitin/proteasome protein degradation pathway that removes proteins whose abnormal accumulation causes cardiomyocyte degeneration and heart failure. (26) Excessive calpain activity has been shown to play a role in damaging mitochondria and oxidative phosphorylation during cardiac ischemia/reperfusion injury. (6).
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Many of the proteins involved in apoptotic signaling are substrates for activated calpain. Partial cleavage of pro or anti-apoptotic proteins by calpain have been shown to activate or inactivate proteins such as caspase 3, 7, 8,9,12, BLC-2, BCl-xl, Bid, Bax and NF-kB. (27) (20) Over expression of calpastatin has been shown to abrogate caspase-3 activation and prevent apoptosis. (27)
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In the past few years, there has been growing evidence that supports a role for mitochondrial calpains in mitochondrial dysfunction. The mitochondrial permeability transition pore (mPTP) is a non specific pore located in the inner mitochondrial membrane that is permeable to small molecules. Opening of the mPTP during ischemia-reperfusion induces apoptosis through the mitochondrial release of proapoptotic proteins and ATP depletion. Inhibitors of mPTP opening limit the size of myocardial infarction in ischemia/reperfusion. mPTP opening is induced physiologically by calcium and is enhanced by mitochondrial calcium overload in ischemia/reperfusion. Shintani-Ishida and Yoshida demonstrated that mitochondrial matrix m-calpain contributes to complex I activation and mPTP opening after post ischemic reperfusion in the rat heart. (28) Similarly, mitochondrial calpains have been reported to be pro-apoptotic proteases by mediating the cleavage of Apoptosis Inducing Factor (AIF) after Ca2+ overload. AIF is bound to the inner mitochondrial membrane and when there is mitochondrial damage, AIF is cleaved allowing it to translocate to the nucleus and induce DNA degradation. Chen et al showed that in the presence of exogenous Ca2+ in isolated heart cells, mitochondrial calpain causes activation of AIF. (6) They also showed that pre-treatment with MDL28170, a calpain inhibitor, prevents release of AIF in isolated heart mitochondria, suggesting that inhibition of mitochondrial calpain 1 prevents the release of AIF resulting in reduced cardiac cell death. (6)
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Another mechanism through which calpain induces cardiac cell injury is through proteolysis of PKC resulting in a constitutively active protein kinase M which leads to stunned myocardium in ischemia/reperfusion injury. (22). Specifically, it is thought that during oxidized-LDL-stimulated apoptosis, intracellular Ca2+ increases and induces calpain activation which leads to Bid cleavage, cytochrome c release, apoptosome formation and caspase 3 activation. The increase in oxidized-LDL activation of calpain and induction of apoptosis in atherosclerotic areas suggests that this type of apoptosis is important in atherothrombotic events. . (22) (28)
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Additional calpain substrates include pro apoptotic proteins p53 and transcription factors YY1, c-mos, c-jun and c-fos. (21) (7) (16) Vousden et al found that calpain inhibits p53 to generate an N terminally truncated protein and treatment of cells with a calpain inhibitor induced rapid accumulation of p53 protein, plausibly as a result of enhanced protein stability. (7) Lee et al suggest that myogenic calpain activity functions through cleavage of the multifunctional transcription factor YYI which is capable of repressing myogenic transcription via its DNA-binding activity. (16) Interestingly, they found that calpain inhibition served to stabilize YY1 in myoblasts. Of particular importance is that calpain inhibition only stabilized YY1 in settings of elevated calcium influx. (16) This suggests that calpain over activation could affect the transcription of genes important in cell death.
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Oxidative stress in myocytes induces calpain activation which in turn further increases oxidative stress. Studies indicate that hypoxia induces calpain activity in retinal, lung and umbilical vein endothelial cells. (23). However, it is difficult to determine whether oxidative stress or calpain activation occurs first. Published results do suggest that calpain activation may promote oxidative stress which in turn potentially leads to cellular apoptosis (2) (20) Future work is needed to elucidate the relationship between calpain activation and oxidative stress. Inhibition of calpain over activity has been reported to protect the myocardium against myocardial ischemia-reperfusion injury and cardiac hypertrophy by reducing infarct size, preventing LV remodeling and protecting LV function (2) (6) (5) This suggests that calpain inhibition may be a good medical therapy for patients with CAD. Calpains Attenuates Angiogenesis in Ischemic Tissue
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Moderate calpain inhibition in the setting of hypercholesterolemia and chronic myocardial ischemia has been found to improve proangiogenic protein expression, microvascular relaxation and myocardial perfusion. (1) (20) Vascular Endothelial Grown Factor-A is essential for angiogenesis in a variety of important pathologies including ischemia and wound repair, proliferative retinopathies, psoriasis, rheumatoid arthritis and cancers. Interestingly VEGF induces a highly abnormal vasculature in pathological settings and has been shown to induce calpain activity in endothelial cells . Khalil et al showed that through VEGF calpain inhibition reduced infarct size and improved hemodynamics and contractile function in a large animal model of cardiac ischemia/ reperfusion. (18)
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VEGF potently works through induction of NO production through PI3K/AKT and/or AMPK-dependent phosphorylation of endothelial nitric oxide synthase (eNOS) . (4) (29) (30) Exrin is a member of the exrin/radixin/moesin (ERM) protein complex that is classically involved in cytoskeletal remodeling. Youn et al found that exrin-dependent membrane-specific translocation and activation of calpain by VEGF precedes AMPK and AKT dependent phosphorylation of eNOS and production of NO, suggesting a critical role for calpain interaction in modulating endothelial cell function. (24)
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Calpain activity has also been shown to induce hypoxic retina and calpain hyper-activation has been implicated in this retinal pathology. (23) Hoang et al studied the relationship between VEGF and calpain both in vivo and in vitro. To determine the consequences of calpain inhibition on hypoxia induced neovascularization, Hoang et al used their established mouse model of ischemic retinopathy to illustrate that neovessels do not relieve hypoxia and that moderation of calpain activity offers a novel strategy for normalizing pathological retinal neovascularization and restoring normal oxygenation. They found that moderate calpain inhibition improved neovascular architecture and function as measured by reduction in abnormal vascular tufts and vascular leakage. Administration of calpain inhibitors also significantly improved vascular re-growth as measured by improved vascular coverage of retina. Most importantly, the improvement in neovascularization provided by calpain inhibitors resulted in marked reduction of underlying retinal hypoxia. In vivo studies found that calpain inhibitors strongly improved organization of the endothelial actin and tau protein cystokeleton in newly formed blood vessel. Their research suggests that calpain inhibition restores cystoskeletal organization, improves capillary morphogenesis and promotes formation of a functionally improved neo-vasculature that reduces underlying hypoxia. (23). Notably, while appropriately moderate doses of calpain inhibitors improved vascular coverage in all cases higher doses inhibited retinal neovascularization. In vitro capillary morphogenesis experiments indicated that optimal improvements were observed at 30-35% calpain inhibition (indicated by reduced formation of abnormal vascular tufts and focal leakage within 5 days of treatment). (23) Complete suppression of calpain resulted in non functional cells (indicated by cell rounding). (24) . (6)
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Scalia et al found that calpain activity is increased in diabetic vasculature and that inhibition of calpain activity attenuates the vascular dysfunction associated with chronic DM (10) More specifically they found that increased levels of endothelial NO and attenuated expression of ICAM-1 and VCAM-1 are key mechanisms of the anti-inflammatory action of calpain inhibition in the diabetic vasculature in vivo. (Timothy J. Stalker, 2005) They also found reduced availability of endothelial NO and decreased hsp90/eNOS association in the microcirculation of diabetic rats. And that inhibition of calpain activity: 1) restored the association of eNOS with hsp90 2) increased NO release and 3) attenuated leukocyte trafficking in the microcirculation. This supports a theory that hsp90 plays a key role in activating eNOS by serving as s docking site for AKT-dependent phosphorylation of eNOS and that calpain over activation regulates hsp90 function. (11) Calpain Over Activation Promotes Inflammatory Pathways
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Recent studies have reported that calpain inhibition exerts anti-inflammatory effects. (10) Leukocyte-endothelium interaction is an important aspect of the inflammatory process. The vascular endothelium directs leukocytes to sites of vascular injury in response to inflammatory stimuli in order to prevent infection. Abnormal initiation of the inflammatory response during pathologic conditions may cause damage to blood vessels and surrounding tissue due to the production of toxic mediators by the dysfunctional endothelium or by activated leukocytes. It is thought that pathologic leukocyte trafficking may have a role in the development of macro vascular disease. (10) The inappropriate activation of calpain has been found to change platelet function and partially degrade proteins resulting in
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hyperaggregability. Some experimental results indicate an increase in calpain activity and leukocyte trafficking in the microcirculation of NIDDM (noninsulin dependent DM) rats and that inhibition of calpain activity significantly attenuates leukocyte endothelium interactions. (8) One of the cytokines released after ischemia/reperfusion injury is TNF-alpha. Calpain has been implicated to play a role in TNF-alpha mediated apoptosis in cardiomyocytes. (27) It is thought that calpain activation causes over expression of TNF-alpha and NO which are known to depress myocardial contractile function. (3).
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Another possible mechanism is that calpain activation leads to degradation of IkB which is an essential step in the translocation of transcription factor NF-kB from the cytosol to the nucleus. (10) NF-KB then binds to the promoter region of genes encoding various inflammatory mediators or proteins such as TNF-alpha, IL-1, IL-6, vascular molecule-1, inducible nitric oxide synthase, iNOS and cyclooxygenase-2 (Cox-2). (3) (6). Li et al used cultured adult cardiomyocytes and an in vivo model of endotoxaemia to investigate the role of calpain inhibitor calpastatin in LPS-stimulated calpain activation and myocardial function. Their results suggest that over expression of calpastatin inhibits caspase-3 activation and TNF-alpha expression in LPS-stimulated cardiomyoctyes and improves cardiac function in endoteoxaemia. (9)
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Inhibition of calpain activity may improve myocardial function by the means of the attenuation of tissue leukocyte infiltration and endothelial cell activation in the inflamed coronary vasculature. (3) Tissier et all found that calpain inhibitors attenuated leukocyte endothelial in the mesentery venule in a rat model of endoteoxemia which is consistent with previous studies showing that organ injury is attenuated by anti-leukocyte therapies. They also found that calpain inhibition improved myocardial contractile dysfunction in the same rat model. (3) Calpain inhibition via its anti inflammatory effects may help to reduce atherosclerosis in diseased myocardium. . Calpain Over Activation Is Implicated in Diabetes and Obesity
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Interestingly, calpain over activation has been implicated in the pathogenesis of many of the co morbidities associated with CAD including diabetes and obesity. Calpains have been found to play a role in cardiac hypertrophy of diabetic cardiomyopathy. (10) (8) The mechanism through which this occurs has been the subject of much research. S. Kumar et al investigated the role of high glucose in intracellular calcium mobilization and calpain-1 activation in cardiomyocyte cells. They found that high glucose induces calpain-1 activation through up-regulation of calcium. Activated calpain-1 promoted cellular apoptosis, scavenging of ROS, and chelation of excessive calcium. Inhibition of calpain-1 significantly reduced calpain activation and prevented cellular apoptosis during elevated glucose conditions. The pro-apoptotic role of high glucose induced proteolytically cleaved calpain-1 was facilitated through activation of capase-12 and caspase-9. (12).
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Endothelial calpain is known to be activated acutely by hyperglycemia. IDDM has been found to activate endothelial calpain through the PKC signaling cascade to induce endothelial dysfunction. Interestingly, down regulation of protein kinase C activity seems to be an important factor to maintain the proper phosphorylation level of insulin receptors. (8). PKC is also a known target of calpain activity. (26). Inhibition of calpain activity improves vascular dysfunction linked with chronic DM, suggesting that calpain plays a critical role in the pathophysiology of diabetic vascular disease. (8). Calpain-10 is the first DM gene to be identified through the genome scan. Calpain 10 has been suggested to play a role in facilitating the actin reorganization required for glucose stimulated insulin release. Calpain inhibition has been found to block insulin secretion. (31)
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Interestingly, expression of calpain in obese rats was found to be greater than in lean rats in a rat model of non insulin dependent diabetes. Exercise and low body weight were found to play a significant role in reducing calpain expression. Moreover, calpain has also been shown to reduce glucose transport turnover in skeletal muscle suggesting it may play role muscle glucose equilibrium and muscle mass. (8) Exercise reduced calpain mRNA expression in a rat model of NIDDM suggesting that exercise may be related to inhibition of calpain over activity. (8). Calpain has also been implicated in obesity, polycystic ovary syndrome, diabetic nephropathy and cataract formation. (31)
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Using a large animal model that has been shown to adequately represent metabolic, microcirculatory and molecular abnormalities seen in adult patients with chronic myocardial ischemia and MS our group has studied the effect of calpain inhibition on myocardial angiogenesis (1). We have observed and reported that calpain activity was significantly increased in the ischemic myocardium of pig with MS and that calpain inhibition improved collateral dependent perfusion, and increased expression of proteins involved in vasodilatation. Furthermore, calpain inhibition promoted expression of the survival proteins, decreased oxidative stress and inhibited apoptotic pathway in animals with MS and chronic myocardial ischemia. (20) (1)
Conclusion
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Excessive activation of calpains has been implicated in the pathophysiology of several disorders including coronary arterty disease, myocardial ischemia/reperfusion injury, DM and obesity. Calpain over activation is involved in number of cellular processes including: cystoskeletal remodeling, cellular apoptosis, tissue angiogenesis, and endothelial inflammation. Calpain Inhibition, therefore, may offer a novel potential medical therapy for patients suffering from a number of diseases including CAD. Calpain inhibitors are experimental drugs and have not yet been developed as therapeutic agents in patients. Though the results of these studies are encouraging further studies are merited to help elucidate the mechanism and extent by which calpain inhibitors exert their beneficial effects. [Figure 3]
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Acknowledgments This work was supported in part by the National Heart, Lung, and Blood Institute (R01HL46716, R01HL69024) to Dr. Sellke; NIH Training grant (5T32-HL094300), to Dr. Sabe; NIH/NIGMS Training Grant 2T32 GM065085 to Dr. Potz; NIGMS Centers of Biomedical Research Excellence grant (GM1P20GM103652 (Project-3) and American Heart Association Grant-in-Aid (14GRNT20460291) to Dr. Abid,
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Figure 1.
Calpain Overactivation Leads to Heart Disease
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Schematic of Calpain Structure
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Figure 3.
Calpain Inhibition Attenuates Heart Disease
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Circ J. Author manuscript; available in PMC 2016 March 18. Published in final edited form as: Circ J. 2015 December 25; 80(1): 4–10. doi:10.1253/circj.CJ-15-0997.
Calpains and CoronaryVascular Disease Brittany A. Potz, MD, Ashraf A. Sabe, MD, M. Ruhul Abid, MD/PhD, and Frank W. Sellke, MD Cardiothoracic Surgery Research Division, Institution: Warren Alpert Medical School Brown University
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Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates for or receive suboptimal revascularization.. Calpains are a class of calcium activated non-lysosomal cysteine proteases which serve as a proteolytic unit for cellular homeostasis. Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac ischemia. Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia reperfusion. Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases including CAD.
Keywords
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Calpain; Coronary Artery Disease; Metabolic Syndrome
Introduction
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Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates for or receive suboptimal revascularization.. Incomplete revascularization in patients with CAD who undergo surgical intervention is a predictor for operative and peri-operative morbidity and mortality. With the increased prevalence of obesity and metabolic syndrome (MS) in the United States, the incidence of severe CAD not amenable to surgical treatment is likely to increase. Inducing a pro-angiogenic state through medical therapies remains a promising therapeutic option for these patients. To achieve this goal, , a deeper understanding of the pro-angiogenic and anti-angiogenic pathways in the setting of hypercholesterolemia and chronic ischemic disease is necessary to treat this complicated and growing population of patients. (1) Calpains are a class of calcium activated non-lysosomal cysteine proteases which serve as a proteolytic unit for cellular homeostasis (2). Cysteine proteases contain the amino acid cysteine in the active site of the molecule. The three major groups of cysteine proteases include cathepsins, caspases and calcium dependent calpains. (3) Under basal conditions,
Corresponding Author: Brittany Potz, Division of Cardiothoracic Surgery, Cardiovascular Research Center, Warren Alpert Medical School Brown University, 2 Dudley Street MOC 360, Providence, RI 02905.
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calpain is predominantly situated in the cytosol and translocates to the membrane on activation. Activation of calpain results in proteolysis of several cellular proteins including cytoskeletal proteins, membrane proteins, enzymes, cytokines, and transcription factors all of which are known to play important roles in transducing signals of cell migration, differentiation and proliferation. (4). Excessive activation of calpains has been implicated in the pathophysiology of several disorders including inflammation, diabetes mellitus (DM), ischemia reperfusion injury, and trauma. (3) (5). Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases including CAD. [Figure 1] Calpain Structure
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15 calpain isoforms have been reported so far. In mammals there are 14 large subunit members (80-kDa catalytic subunit), one small subunit member (30-kDA subunit) and one endogenous inhibitor (calpastatin). (6) Both subunits can bind calcium resulting in the activation of calpain. Calpain activity can also be regulated by auto proteolysis and by calpastatin, suggesting that calpains are part of a regulatory proteolytic system. (7) [Figure 2] Most calpain isoforms contain four structural domains. Calpains are divided into two groups based on the structure of domain IV: Typical and Atypical. Typical calpains (1,2,3,8,9,11,12,14) contain a penta-EF motif in domain IV that can bind calcium, the calpain small subunit (only calpains 1,2 and 9 have been shown to dimerize) or calpastatin. Atypical calpains (5,6,7,10,12 and 15) lack a penta-EF motif in domain IV and are unable to bind the calpain small subunit or calpastatin. (6)
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Some calpains are ubitiquitously expressed (calpains 1,2,4,5,7 and 10) while others are found in specific tissues (calpain 1&2- endothelia cells, calpain 3- skeletal muscle, calpain 6- placenta, calpain 8- smooth muscle, calpain 9- stomach, calpain 11- testes, calpain 12skin after birth and calpain 13- tests and lung). (8). Among the 15 calpain isoforms, 10 isoforms are expressed in the heart. The most well studied of these calpains are Calpain 1 (U-calpain) and calpain 2 (m-calpain) which require micro and milli molar concentrations of calcium for their activation respectively. (3)
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In atypical calpains the function of domain 1 is largely unknown. However, domain 1 for calpain 10 contains a mitochondrial targeting sequence. In calpains 1, 2 and 9, domain I is cleaved after Ca2+ activation resulting in autolysis and autoactivation. Domain II contains the catalytic active site with the ‘catalytic triad’ of cysteine, asparagines and histidine. The catalytic triad is conserved throughout the entire family (except for calpain 6 that lacks proteolytic activity). Domain II can also bind two atoms of Ca2+ and assists in calpain activation. Domain III contains two Ca2+ binding sites and a phospholipid-binding motif which is conserved throughout the family, except for Calpain 10. Domain III is also believed to regulate calpain activity through specific electrostatic interactions and to be involved in substrate recognition. (6) Calpastatin is an endogenous protein that specifically inhibits calpains. The fact that the calpain's active site is conserved throughout the entire family makes it difficult to generate
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isoform specific inhibitors. Interestingly, structural and biochemical data indicate that calpastatin may bind preferentially to calcium activated calpains, suggesting that calpastatin controls activated calpain activity. This suggests that calpastatin limits calpain activation without interfering with basal calpain activity, which is required for normal protein homeostasis. (9) This fact is critical and should be taken into consideration while considering the use of calpain inhibitors for the treatment of cardiovascular disease. Coronary Artery Disease and Metabolic Syndrome It has been well demonstrated that co morbid diseases which constitute MS, such as DM, obesity, and hypercholesterolemia have a deleterious impact on endothelial function and growth. With the increasing prevalence of these co morbid conditions, it is important to understand mechanisms by which these diseases contribute to or exacerbate endothelial dysfunction and CAD.
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In particular, DM is recognized as a potent risk factor for cardiovascular disease including atherosclerosis and microvascular complications. (10) (8) Hyperglycemia has been shown to cause endothelial dysfunction characterized by a loss of endothelium derived nitric oxide (NO). Hyperglycemia also results in increased vascular permeability, increased endothelial adhesiveness, thickening of the basement membrane, abnormal inflammatory signals and increased oxidative stress. Endothelial NO is an important regulator of vascular homeostasis. Loss of NO leads to increased vascular tone, and abnormal endothelial adhesiveness, with resultant increased platelet aggregation and leukocyte trafficking in the vessel wall. Thus the diabetic vasculature experiences increased oxidative stress and abnormal inflammatory signals. (11) (10)
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Furthermore, high glucose induced myocyte apoptosis plays an important role in diabetic cardiac complications. Though the molecular mechanisms by which glucose damages cardiac cells remain incompletely understood, it is thought to be partly mediated through cytotoxic oxidative stress and intracellular calcium overload. (12)
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In order to further study the impact of these co morbidities on CAD our group has utilized a clinically relevant large animal model that has been shown to adequately represent metabolic, microcirculatory and molecular abnormalities seen in MS in adult patients with chronic myocardial ischemia. (13). We have also demonstrated that MS in chronic myocardial ischemia significantly impairs notch signaling (an integral component of cardiac angiogenesis) by down regulating notch receptors, ligands and angiogenic proteins. (14) Expression of neuropeptide Y (one of the most abundant neurotransmitters in the myocardium known to influence cardiovascular remodeling) was found to be altered differentially in the serum and myocardium in patients with DM. Findings from our lab and others suggest that altered regulation of the neuropeptide Y in diabetics may be, in part, responsible for the decreased angiogenesis, increased apoptosis and vascular smooth muscle proliferation leading to CAD and heart failure in the MS patient population. (15) It is important to note that co-morbid factors such as DM, obesity, and hypercholesterolemia will influence and exacerbate endothelial dysfunction leading to CAD. Understanding how these pathological conditions affect each other will help devise therapeutics that would
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effectively treat heart disease. In the following section, we will discuss how calpain may be involved in the pathogenesis of DM, obesity and CAD, making it a potential therapeutic target. Uncontrolled Activation of Calpains leads to Heart Disease
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Calpain activity is required for normal myogenesis, whereas calpain over-activation has been implicated in the development of heart disease. (16). (2). (17) In cardiac muscle calcium plays a crucial role in excitation-contraction coupling. Dysregulation of cellular calcium homeostasis, often results in calcium overload which leads to calpain overactivation and results in myocardial cell injury. (2) Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac, cerebral and hepatocellular ischemia. (18) Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia reperfusion. (5). The exact mechanisms by which calpain is up-regulated are not fully understood. Calpain activity is mainly regulated through alterations in the calcium concentration as calcium binding is required for its proteolytic activity. Evidence has been provided that cellular Ca overload leads to over activation of calpains by auto proteolysis of an N-terminal peptide. (18) Studies also suggest that calpain activity can also be regulated by certain phospholipids, ERK1/2 MAPK, PKC and PKA. In addition, NADPH oxidase has been suggested to induce calpain activation in norepeinephrine stimulated cardiomyocytes. For example, one study suggests that the activation of NADPH oxidase results in reactive oxygen species production which activates calpain through regulation of intracellular calcium levels and ERK1/2 pathway. (9)
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In concurrence with the above discussion, our group and multiple others have demonstrated that the over activation of calpain contributes to cardiovascular disease. ( (9) (19) (2) (3) (18) (1) (20). However, a better understanding of the pro-angiogenic and anti-angiogenic pathways in the setting of hypercholesterolemia, hyperglycemia, and chronic ischemic disease is necessary. Our lab has developed a pig model for chronic myocardial ischemia in the setting of MS (weight gain, glucose intolerance, dyslipidemia and hypertension) to elucidate the molecular mechanisms in a pre-clinical setting. (1) Further studies should focus on identifying mechanisms by which this over activation occurs and to identify novel therapies for calpain inhibition as a potential medical therapy for patient with cardiac risk factors and disease. Calpains Improve Myocardial and Endothelial Cystokeletal Protein Remodeling
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In diseased myocardial tissue, remodeling may be associated with ischemia-induced changes to the contractile proteins. Calpain has been found to cleave myofibrillar specific proteins including troponin T, troponin I, titin and desmin in cardiomyocytes. (9) Cardiac troponin I is a subunit of the troponin complex involved in the control of muscle contraction. Interestingly, troponin I has been found to be preferentially affected in ischemic rat hearts. (21) Maekawa et al found that systolic dysfunction of the stunned myocardium is associated with troponin I degradation and over expression of calpastatin will lessen contractile
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dysfunction of rat hearts subjected to ischemia/reperfusion. In addition, the same model found that degradation of troponin following ischemia/reperfusion was prevented by calpain inhibition. (22) Yoshiro et al found that high calcium intracoronary infusion causes calcium overload induced cardiac dysfunction through proteolysis of cytoskeleton protein alpha-fodrin Yashikawa et all found that calpain inhibition protects the rat heart from ischemia/ reperfusion injury by inhibiting proteolysis of alpha fodrin which they speculate prevents conformational changes to the L-type Ca channel at the cell membrane and results in protection of LV function. (19)
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Endothelial cells play an essential role in cardiovascular pathophysiology through their functions in blood vessel formation, cell barrier, coagulation, vascular tone, inflammation and angiogenesis. Many of these processes require cytoskeletal rearrangements and a number of cystoskeletal proteins are known calpain substrates including talin, filamin, spectrin, tau, Paxillin, Vinculin, alpha tubulin, vimentin, and filamin. (9) (6) (7) (10) In vivo studies have demonstrated that calpain inhibitors strongly improved organization of the endothelial actin and tau protein cystokeleton in newly formed blood vessel suggesting that calpain inhibition restores cystoskeletal organization improves capillary morphogenesis and promotes formation of a functionally improved neovasculature that helps to reduce underlying hypoxia. (23) (24)
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Komuro et al demonstrated that calpains impaired cell-cell interactions through degradation of the cadherin-associated protein complex resulting in LV remodeling after myocardial infarction (MI). Interestingly, they found that calpain mediated proteolysis was increased in the chronic phase (7 days or later) but not in the acute phase (before 24 hours) after MI and profound activation of calpains exacerbated LV remodeling. The border zone of MI hearts in these mice showed a decrease in N-cadherin expression concomitant with an increase in calpain activation and exacerbation of LV remodeling. In cultured cardiomyocytes calpain activation caused degradation of N cadherin and disorganization of cadherin based cell adhesions. (25) Similarly, in cultured cardiomyocytes, calpain activation disassembled cadherin-based cell-cell adhesion consisting of intercalated disc proteins such as B-catenin and Connexin 43. (25) This suggests that calpain over activation may lead to myocardial remodeling after MI through the breakdown of cell- cell interactions.
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Additionally, several groups have reported roles for mitochondrial calpain 1 in endothelial cells. In rat heart microvascular endothelial cells, hyper-homocysteinemia induced extracellular matrix remodeling by matrix metalloproteinase 9 has been shown to be caused by calpain 1 over activation. (6). Activation of calpain results in proteolysis of several cellular proteins including cytoskeletal proteins which are known to play a role in myocardial tissue remodeling after an ischemic insult. This may be one mechanism by which calpain inhibition can help prevent ischemic changes in myocardial tissue after an MI.
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Calpains Attenuate Myocardial Apoptosis
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Calpain plays an important role in cell death signaling in the heart. (2) Numerous studies have documented that calpains are important in ischemia/reperfusion injury in the heart, mitochondrial permeability transition and necrotic/apoptotic cell death. (6) Myocardial calpain 1 is active under normal physiological conditions and serves as an essential component of the ubiquitin/proteasome protein degradation pathway that removes proteins whose abnormal accumulation causes cardiomyocyte degeneration and heart failure. (26) Excessive calpain activity has been shown to play a role in damaging mitochondria and oxidative phosphorylation during cardiac ischemia/reperfusion injury. (6).
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Many of the proteins involved in apoptotic signaling are substrates for activated calpain. Partial cleavage of pro or anti-apoptotic proteins by calpain have been shown to activate or inactivate proteins such as caspase 3, 7, 8,9,12, BLC-2, BCl-xl, Bid, Bax and NF-kB. (27) (20) Over expression of calpastatin has been shown to abrogate caspase-3 activation and prevent apoptosis. (27)
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In the past few years, there has been growing evidence that supports a role for mitochondrial calpains in mitochondrial dysfunction. The mitochondrial permeability transition pore (mPTP) is a non specific pore located in the inner mitochondrial membrane that is permeable to small molecules. Opening of the mPTP during ischemia-reperfusion induces apoptosis through the mitochondrial release of proapoptotic proteins and ATP depletion. Inhibitors of mPTP opening limit the size of myocardial infarction in ischemia/reperfusion. mPTP opening is induced physiologically by calcium and is enhanced by mitochondrial calcium overload in ischemia/reperfusion. Shintani-Ishida and Yoshida demonstrated that mitochondrial matrix m-calpain contributes to complex I activation and mPTP opening after post ischemic reperfusion in the rat heart. (28) Similarly, mitochondrial calpains have been reported to be pro-apoptotic proteases by mediating the cleavage of Apoptosis Inducing Factor (AIF) after Ca2+ overload. AIF is bound to the inner mitochondrial membrane and when there is mitochondrial damage, AIF is cleaved allowing it to translocate to the nucleus and induce DNA degradation. Chen et al showed that in the presence of exogenous Ca2+ in isolated heart cells, mitochondrial calpain causes activation of AIF. (6) They also showed that pre-treatment with MDL28170, a calpain inhibitor, prevents release of AIF in isolated heart mitochondria, suggesting that inhibition of mitochondrial calpain 1 prevents the release of AIF resulting in reduced cardiac cell death. (6)
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Another mechanism through which calpain induces cardiac cell injury is through proteolysis of PKC resulting in a constitutively active protein kinase M which leads to stunned myocardium in ischemia/reperfusion injury. (22). Specifically, it is thought that during oxidized-LDL-stimulated apoptosis, intracellular Ca2+ increases and induces calpain activation which leads to Bid cleavage, cytochrome c release, apoptosome formation and caspase 3 activation. The increase in oxidized-LDL activation of calpain and induction of apoptosis in atherosclerotic areas suggests that this type of apoptosis is important in atherothrombotic events. . (22) (28)
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Additional calpain substrates include pro apoptotic proteins p53 and transcription factors YY1, c-mos, c-jun and c-fos. (21) (7) (16) Vousden et al found that calpain inhibits p53 to generate an N terminally truncated protein and treatment of cells with a calpain inhibitor induced rapid accumulation of p53 protein, plausibly as a result of enhanced protein stability. (7) Lee et al suggest that myogenic calpain activity functions through cleavage of the multifunctional transcription factor YYI which is capable of repressing myogenic transcription via its DNA-binding activity. (16) Interestingly, they found that calpain inhibition served to stabilize YY1 in myoblasts. Of particular importance is that calpain inhibition only stabilized YY1 in settings of elevated calcium influx. (16) This suggests that calpain over activation could affect the transcription of genes important in cell death.
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Oxidative stress in myocytes induces calpain activation which in turn further increases oxidative stress. Studies indicate that hypoxia induces calpain activity in retinal, lung and umbilical vein endothelial cells. (23). However, it is difficult to determine whether oxidative stress or calpain activation occurs first. Published results do suggest that calpain activation may promote oxidative stress which in turn potentially leads to cellular apoptosis (2) (20) Future work is needed to elucidate the relationship between calpain activation and oxidative stress. Inhibition of calpain over activity has been reported to protect the myocardium against myocardial ischemia-reperfusion injury and cardiac hypertrophy by reducing infarct size, preventing LV remodeling and protecting LV function (2) (6) (5) This suggests that calpain inhibition may be a good medical therapy for patients with CAD. Calpains Attenuates Angiogenesis in Ischemic Tissue
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Moderate calpain inhibition in the setting of hypercholesterolemia and chronic myocardial ischemia has been found to improve proangiogenic protein expression, microvascular relaxation and myocardial perfusion. (1) (20) Vascular Endothelial Grown Factor-A is essential for angiogenesis in a variety of important pathologies including ischemia and wound repair, proliferative retinopathies, psoriasis, rheumatoid arthritis and cancers. Interestingly VEGF induces a highly abnormal vasculature in pathological settings and has been shown to induce calpain activity in endothelial cells . Khalil et al showed that through VEGF calpain inhibition reduced infarct size and improved hemodynamics and contractile function in a large animal model of cardiac ischemia/ reperfusion. (18)
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VEGF potently works through induction of NO production through PI3K/AKT and/or AMPK-dependent phosphorylation of endothelial nitric oxide synthase (eNOS) . (4) (29) (30) Exrin is a member of the exrin/radixin/moesin (ERM) protein complex that is classically involved in cytoskeletal remodeling. Youn et al found that exrin-dependent membrane-specific translocation and activation of calpain by VEGF precedes AMPK and AKT dependent phosphorylation of eNOS and production of NO, suggesting a critical role for calpain interaction in modulating endothelial cell function. (24)
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Calpain activity has also been shown to induce hypoxic retina and calpain hyper-activation has been implicated in this retinal pathology. (23) Hoang et al studied the relationship between VEGF and calpain both in vivo and in vitro. To determine the consequences of calpain inhibition on hypoxia induced neovascularization, Hoang et al used their established mouse model of ischemic retinopathy to illustrate that neovessels do not relieve hypoxia and that moderation of calpain activity offers a novel strategy for normalizing pathological retinal neovascularization and restoring normal oxygenation. They found that moderate calpain inhibition improved neovascular architecture and function as measured by reduction in abnormal vascular tufts and vascular leakage. Administration of calpain inhibitors also significantly improved vascular re-growth as measured by improved vascular coverage of retina. Most importantly, the improvement in neovascularization provided by calpain inhibitors resulted in marked reduction of underlying retinal hypoxia. In vivo studies found that calpain inhibitors strongly improved organization of the endothelial actin and tau protein cystokeleton in newly formed blood vessel. Their research suggests that calpain inhibition restores cystoskeletal organization, improves capillary morphogenesis and promotes formation of a functionally improved neo-vasculature that reduces underlying hypoxia. (23). Notably, while appropriately moderate doses of calpain inhibitors improved vascular coverage in all cases higher doses inhibited retinal neovascularization. In vitro capillary morphogenesis experiments indicated that optimal improvements were observed at 30-35% calpain inhibition (indicated by reduced formation of abnormal vascular tufts and focal leakage within 5 days of treatment). (23) Complete suppression of calpain resulted in non functional cells (indicated by cell rounding). (24) . (6)
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Scalia et al found that calpain activity is increased in diabetic vasculature and that inhibition of calpain activity attenuates the vascular dysfunction associated with chronic DM (10) More specifically they found that increased levels of endothelial NO and attenuated expression of ICAM-1 and VCAM-1 are key mechanisms of the anti-inflammatory action of calpain inhibition in the diabetic vasculature in vivo. (Timothy J. Stalker, 2005) They also found reduced availability of endothelial NO and decreased hsp90/eNOS association in the microcirculation of diabetic rats. And that inhibition of calpain activity: 1) restored the association of eNOS with hsp90 2) increased NO release and 3) attenuated leukocyte trafficking in the microcirculation. This supports a theory that hsp90 plays a key role in activating eNOS by serving as s docking site for AKT-dependent phosphorylation of eNOS and that calpain over activation regulates hsp90 function. (11) Calpain Over Activation Promotes Inflammatory Pathways
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Recent studies have reported that calpain inhibition exerts anti-inflammatory effects. (10) Leukocyte-endothelium interaction is an important aspect of the inflammatory process. The vascular endothelium directs leukocytes to sites of vascular injury in response to inflammatory stimuli in order to prevent infection. Abnormal initiation of the inflammatory response during pathologic conditions may cause damage to blood vessels and surrounding tissue due to the production of toxic mediators by the dysfunctional endothelium or by activated leukocytes. It is thought that pathologic leukocyte trafficking may have a role in the development of macro vascular disease. (10) The inappropriate activation of calpain has been found to change platelet function and partially degrade proteins resulting in
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hyperaggregability. Some experimental results indicate an increase in calpain activity and leukocyte trafficking in the microcirculation of NIDDM (noninsulin dependent DM) rats and that inhibition of calpain activity significantly attenuates leukocyte endothelium interactions. (8) One of the cytokines released after ischemia/reperfusion injury is TNF-alpha. Calpain has been implicated to play a role in TNF-alpha mediated apoptosis in cardiomyocytes. (27) It is thought that calpain activation causes over expression of TNF-alpha and NO which are known to depress myocardial contractile function. (3).
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Another possible mechanism is that calpain activation leads to degradation of IkB which is an essential step in the translocation of transcription factor NF-kB from the cytosol to the nucleus. (10) NF-KB then binds to the promoter region of genes encoding various inflammatory mediators or proteins such as TNF-alpha, IL-1, IL-6, vascular molecule-1, inducible nitric oxide synthase, iNOS and cyclooxygenase-2 (Cox-2). (3) (6). Li et al used cultured adult cardiomyocytes and an in vivo model of endotoxaemia to investigate the role of calpain inhibitor calpastatin in LPS-stimulated calpain activation and myocardial function. Their results suggest that over expression of calpastatin inhibits caspase-3 activation and TNF-alpha expression in LPS-stimulated cardiomyoctyes and improves cardiac function in endoteoxaemia. (9)
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Inhibition of calpain activity may improve myocardial function by the means of the attenuation of tissue leukocyte infiltration and endothelial cell activation in the inflamed coronary vasculature. (3) Tissier et all found that calpain inhibitors attenuated leukocyte endothelial in the mesentery venule in a rat model of endoteoxemia which is consistent with previous studies showing that organ injury is attenuated by anti-leukocyte therapies. They also found that calpain inhibition improved myocardial contractile dysfunction in the same rat model. (3) Calpain inhibition via its anti inflammatory effects may help to reduce atherosclerosis in diseased myocardium. . Calpain Over Activation Is Implicated in Diabetes and Obesity
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Interestingly, calpain over activation has been implicated in the pathogenesis of many of the co morbidities associated with CAD including diabetes and obesity. Calpains have been found to play a role in cardiac hypertrophy of diabetic cardiomyopathy. (10) (8) The mechanism through which this occurs has been the subject of much research. S. Kumar et al investigated the role of high glucose in intracellular calcium mobilization and calpain-1 activation in cardiomyocyte cells. They found that high glucose induces calpain-1 activation through up-regulation of calcium. Activated calpain-1 promoted cellular apoptosis, scavenging of ROS, and chelation of excessive calcium. Inhibition of calpain-1 significantly reduced calpain activation and prevented cellular apoptosis during elevated glucose conditions. The pro-apoptotic role of high glucose induced proteolytically cleaved calpain-1 was facilitated through activation of capase-12 and caspase-9. (12).
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Endothelial calpain is known to be activated acutely by hyperglycemia. IDDM has been found to activate endothelial calpain through the PKC signaling cascade to induce endothelial dysfunction. Interestingly, down regulation of protein kinase C activity seems to be an important factor to maintain the proper phosphorylation level of insulin receptors. (8). PKC is also a known target of calpain activity. (26). Inhibition of calpain activity improves vascular dysfunction linked with chronic DM, suggesting that calpain plays a critical role in the pathophysiology of diabetic vascular disease. (8). Calpain-10 is the first DM gene to be identified through the genome scan. Calpain 10 has been suggested to play a role in facilitating the actin reorganization required for glucose stimulated insulin release. Calpain inhibition has been found to block insulin secretion. (31)
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Interestingly, expression of calpain in obese rats was found to be greater than in lean rats in a rat model of non insulin dependent diabetes. Exercise and low body weight were found to play a significant role in reducing calpain expression. Moreover, calpain has also been shown to reduce glucose transport turnover in skeletal muscle suggesting it may play role muscle glucose equilibrium and muscle mass. (8) Exercise reduced calpain mRNA expression in a rat model of NIDDM suggesting that exercise may be related to inhibition of calpain over activity. (8). Calpain has also been implicated in obesity, polycystic ovary syndrome, diabetic nephropathy and cataract formation. (31)
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Using a large animal model that has been shown to adequately represent metabolic, microcirculatory and molecular abnormalities seen in adult patients with chronic myocardial ischemia and MS our group has studied the effect of calpain inhibition on myocardial angiogenesis (1). We have observed and reported that calpain activity was significantly increased in the ischemic myocardium of pig with MS and that calpain inhibition improved collateral dependent perfusion, and increased expression of proteins involved in vasodilatation. Furthermore, calpain inhibition promoted expression of the survival proteins, decreased oxidative stress and inhibited apoptotic pathway in animals with MS and chronic myocardial ischemia. (20) (1)
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Excessive activation of calpains has been implicated in the pathophysiology of several disorders including coronary arterty disease, myocardial ischemia/reperfusion injury, DM and obesity. Calpain over activation is involved in number of cellular processes including: cystoskeletal remodeling, cellular apoptosis, tissue angiogenesis, and endothelial inflammation. Calpain Inhibition, therefore, may offer a novel potential medical therapy for patients suffering from a number of diseases including CAD. Calpain inhibitors are experimental drugs and have not yet been developed as therapeutic agents in patients. Though the results of these studies are encouraging further studies are merited to help elucidate the mechanism and extent by which calpain inhibitors exert their beneficial effects. [Figure 3]
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Acknowledgments This work was supported in part by the National Heart, Lung, and Blood Institute (R01HL46716, R01HL69024) to Dr. Sellke; NIH Training grant (5T32-HL094300), to Dr. Sabe; NIH/NIGMS Training Grant 2T32 GM065085 to Dr. Potz; NIGMS Centers of Biomedical Research Excellence grant (GM1P20GM103652 (Project-3) and American Heart Association Grant-in-Aid (14GRNT20460291) to Dr. Abid,
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Figure 1.
Calpain Overactivation Leads to Heart Disease
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Schematic of Calpain Structure
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Figure 3.
Calpain Inhibition Attenuates Heart Disease
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