Calpains are intracellular Ca2+- activated proteases and important mediators of the actions of calcium. The regulated cleavage by calpain is critical in a variety of calcium-regulated cellular processes such as muscle contraction, neuronal excitibility, secretion, signal transduction, cell proliferation, differentiation, cell cycle progression, and apoptosis. Deregulation of calpain caused by a disruption of calcium homeostasis during cardiac pathologies such as atrial fibrillation, heart failure, hypertrophy, or ischemia reperfusion, critically contributes to myocardial damage.^1-5

The term “calpain” was originally used for classical µ- and m-calpains that are heterodimers composed of a large 80 kDa catalytic subunit encoded by either CAPN1 for µ -calpains (calpain 1), or by CAPN2 for m-calpains (calpain 2), and a common 30 kDa regulatory subunit encoded by CAPNS1 (calpain 4). Whereas the small subunit is identical for both enzymes, the large subunits share 55-65% sequence homology.^5-6 The µ- and m-calpains differ in their requirements of intracellular Ca2+, which need to be micromolar and millimolar in concentration, respectively. Based on sequence homology, 14 human genes have been identified as members of the calpain large catalytic 80 kDa family, and 2 human genes for the small regulatory 30 kDa family.^7-8

The typical calpains are composed of four domains originally found in μ- and m-calpains (Figure 1 ).^7-8 The N-terminal region of domain I of the large subunit comprises a single -helix responsible for stabilizing of the domain arrangement. Domain II contains the catalytic site (Cys, His, and Asn residues) and is composed of the subdomains IIa and IIb. Domain III binds phospholipids in a Ca2+-dependent manner and is supposed to mediate Ca2+-dependent membrane translocation of calpains. Domain IV, at the C-terminal end of the large subunit, is a Ca2+-binding domain containing five EF-hand motifs. The large catalytic subunit interacts with the small regulatory subunit through the fifth EF-hand motifs in domain IV and VI to form a heterodimeric calpain. Atypical calpains lack EF-hand motifs and, therefore, cannot form a dimer with the small regulatory subunit.

Figure 1. Domain structure of calpains.

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The large subunit of the typical calpains comprises four domains, while the small subunit has two domains. The atypical calpains varies from the domain organisation originally found in μ- and m-calpains. All atypical calpains lack EF-hand motifs and therefore cannot form a dimer with the small subunit. Alternatives for domain I: Zn, or does not exist (dne); for domain III: SOH, PBH, or does not exist; Alternatives for domain IV: III`, T, or does not exist.

Most calpains are ubiquitously expressed, but the expression of some calapins, including calpains 3, 6, 8, 9, and 12, is rather tissue-specific.^6-8 Although calpains are cytoplasmic enzymes, several studies have shown that µ-calpain, m-calpain, and calpain 10 are present also in mitochondria.^9-10 Calpains exists in the cytosol as inactive enzymes, which translocate to membranes in response to increased intracellular calcium levels. As mentioned before, at the membrane, calpains become activated in the presence of calcium and phospholipids.^11-12

Based on data from both in vitro and in vivo studies, calpain can play a regulatory role in important processes including remodeling of cytoskeletal proteins, modulation of signal transduction pathways, degradation of cell cycle-regulating enzymes, regulation of gene expression, and initiation of apoptotic pathways. The proteolytic activity of calpains is not specific for certain amino acid residues or motifs, but recognizes the overall three-dimensional structure of its substrates.^7-8 Many proteins have been identified as potential substrates of calpain. These include a large number of cytoskeletal and myofibrillar proteins (myosin, troponin, tropomyosin, titin),^13-15 membrane-associated proteins (receptors, ion channels),^16-24 metabolic enzymes,^25-26 signaling-modulated kinases and phosphatases (PKC, calcineurin),^27-31 transcription factors (NF-κB),^32-33 and proteins involved in apoptotic signaling.^10,34-³⁹

Under physiological conditions, the activity of calpain is thightly regulated. Regulatory mechanisms include the existance of a specific endogenous inhibitor, calpastatin. The binding of calpastatin to calpain is calcium-dependent and reversible.⁷ Calpastatin contains four equivalent inhibitory domains, each bearing three conserved regions (A, B, and C). The regions A and C bind to domain IV of the large subunit and to domain VI of the small subunit of calpain, respectively.⁴ The region B shows inhibitory activity itself.^7,40 Both µ- and m-calpains exhibit similar sensitivity to calpastatin. The presence of the two calmodulin-like domains IV and VI are necessary for effective inhibition of calpastatin. Thus, calpastatin can inhibit only dimeric calpain like µ-, m-calpain, and calpain.⁹ The atypical calpains are resistant to the inhibitory actions of calpastatin.⁴⁰ The cloning and molecular characterization of calpastatin revealed the existence of potential phosphorylation sites for protein kinase C, cAMP- and cGMP-dependent protein kinase, and casein kinase II.⁴¹ Thus, these motifs are supposed to play an important role in molecular regulation of the calpain/calpastatin system.

An additional mechanism for regulation of calpain activity is its phosphorylation by protein kinase A (PKA)⁴² or by extracellular-signal regulated kinase (ERK).^43-44 Glading et al.⁴⁴ provided evidence that m-calpain can be activated after direct phosphorylation at Ser-50 by ERK in the absence of cytosolic calcium influx. However, more recent studies indicated that the phosphorylation of calpain by ERK and PKA regulates the enzyme indirectly by controlling its cellular redistribution. The phosphorylation by ERK promotes the translocation of m-calpain to the membrane and the binding to phospolipids which in turn facilitates the activation of calpain. In contrast, PKA–dependent phosphorylation of calpain at Ser-369 prevents domain movement and the formation of the calpain active site.^41-43

Most recently, it was shown that intracellular acidosis attenuates the activation of calpain.⁴⁵ The study by Inserte demonstrated that the prolongation of intracellular acidosis during reperfusion by either post-conditioning or by acidic perfusion of isolated rat hearts has cardioprotective effects through inhibition of calpain activation. Further investigations are necessary to demonstrate a relation between delayed acidosis and the putative involvement of kinases in the cardioprotective mechanisms.

Finally, results of many in vitro and in vivo studies support the potential value of cardioprotective strategies based on interfering with calpain activation. The first generations of synthetic calpain inhibitors inactivate calpains by forming a covalent bond within the active site of the calpain catalytic domain (E-64, leupeptin, ALLN, ALLM),^46-49 or by interacting with the Ca2+-binding domain of the calpain large subunit (PD150606).⁵⁰ Persisting problems resulting from low membrane permeability and target selectivity led to the continuing development of calpain inhibitors. Several studies have used screening approches to identify new calpain inhibitor templates, and these have produced a wide range of peptidic, peptidmimetic, and non-peptide inhibitors.⁵¹ They target the active site of calpain and exhibit an improved pharmacokinetic profile

Amongst the synthetic calpain inhibitors developed so far, calpastatin peptide analogues and their truncated versions stand out due to their unique selectivity and affinity profile.^59-60 Calpastatin is the only known inhibitor with absolute specificity for both µ- and m-calpains, but its large molecular mass (110 kDa), making it membrane-impermeable, limits its therapeutic use.

Pathophysiologically, atrial fibrillation (AF) goes along with profound changes of the electrophysiologic and structural appearance of atrial tissue.^4,53-⁵⁹ Previous studies have shown that AF is characterized by an initial intracellular calcium overload, which causes atrial electrical remodeling.⁴,⁵⁷ In addition, abnormal calcium handling during AF contributes to mechanical atrial dysfunction after successful cardioversion.^1,60-⁶² The histopathology of fibrillating atria is similar to that of chronically ischemic ventricular myocardium.^53,63 Ischemia/reperfusion injury is mediated by increased levels of cytosolic calcium, which causes activation of the calcium-dependent proteases µ- and m-calpain.^4,64-⁶⁵

Interaction With Other Calcium-Dependent SignalPathways During AF

Altered intracellular calcium homeostasis and activation of atrial renin-angiotensin-system have been identified as important factors contributing to the oxidative stress in the endocardium of fibrillating atrial tissue. Elevated levels of intracellular Ca2+ and reactive oxygen species (ROS) are major activators of the immediate early response transcription factor NF-κB, which is a redox sensor in atrial tissue. Typical target genes of NF-κB are pro-inflammatory cytokines such as interleukin-8 and tumor necrosis factor α (TNF-α), but also the endothelial adhesion molecules.^82-84 Interestingly, regulation of NF-kappaB signaling is linked to antioxidant levels, oxidative stress, and enhanced cell death. Concomitantly, calpain becomes activated, and treatment with calpain inhibitors, therefore, is able to restore NF-kappaB-p65 levels and to increase cell viability. Thus, calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death.⁹⁰

Calcium-dependent phosphatase, calcineurin (CnA), is also activated in atrial tissue during AF.^73,91-⁹² Rapid pacing causes an upregulation of CnA activity that is associated with increased dephosphorylation of the transcription factor NFATc3, allowing dephosphorylated NFATc3 to enter the cell nucleus. Thereby, NFATc3 increased the transcription of genes responsible for the atrial hypertrophic cellular response, as demonstrated by an increased expression of ANP and -myosin heavy chain.^4-91 In accordance with this finding, a study by Wang et al. indicated that calpain 1 is responsible for the formation of constitutively active calcineurin in the ventricular myocardium from patients with heart failure.⁹²

Finnaly, there is apparent crossinteraction between activated calpain and calcium-dependent singalling pathways during atrial fibrillation (Figure 2 ). The calpain-dependent apoptosis of atrial cardiomycytes and degradation of contractile proteins contribute to the occurrence of contractile dysfunction. Moreover, activation of calcium-dependent singalling pathways (NF-κB, Calcineurin) influence the extracellular matrix and support the progress of structural remodeling in atrial tissue. These all processes collectively change the architectur of atrial tissue and may further contribute to sustained alterations in the atrial contractile performance

Figure 2. Schematic representation of accompanying pathways involved in µ-calpain activation during atrial fibrillation (AF).

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Intracellular calcium overload and subsequent sequestration into mitochondria results in alteration in mitochondrial morphology and function. Concomitantly, AF causes an increased expression and activity of µ-calpain. These changes are accompanied by degradation of myofilaments and reduced amounts of troponin T (TnT). Furthermore, persistent calcium overload leads to the reduction of L-type Ca2+ channel proteins. The activation of atrial renin-angiotensin-system and altered intracellular calcium homeostasis are important factors contributing to the oxidative stress in AF. Elevated in [Ca2+]i levels and reactive oxygen species (ROS) are major activators of the immediate early response transcription factor NF-κB, which is a redox sensor in atrial tissue. Rapid pacing causes an upregulation of calcineurin (CaN), a phosphatase responsible for the translocation of transcription factor NFAT to the nucleus where it induces the expression of pro-hypertrophic genes.

Calpains are important modulators of the normal signal transduction, gene expression and muscle contraction. Deregulation of their activity is associated with cardiovascular pathologies such as heart failure, myocardial infarction, and atrial fibrillation. The increased expression and activity of calpains during atrial fibrillation may result from the deregulation in calcium homeostasis and contributes to the occurrence of contractile dysfunction due to degradation of contractile proteins. Moreover, activation of calpains initiates apoptosis processes and mediates damage of atrial cardiomyocytes. Results of in vitro and in vivo studies have shown the beneficial effects of calpain inhibitors in different models of atrial fibrillation. Therefore, this protease has attracted a special attention in the medical science as a potential drug target. Nevertheless, calpain inhibitors have not been tested in clinical trials, yet, to validate the role of calpain inhibition in the clinical setting. Persisting problems resulting from the lower target selectivity, membrane permeability, and pharmacokinetic properties hinder the introduction of calpain inhibitors into clinical trails. Therefore, efforts should be dedicated to development of compounds resolving these problems.

The work was supported by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement HEALTH-F2-2010-261057.

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