**3. Calcium release and Ryanodine receptor 2**

#### **3.1. Regulation of calcium cycle in cardiac cells**

sankar et al. 2003, Jayasankar et al. 2005). Iwasaki and colleagues reported that recombinant human HGF delivered by ultrasound-mediated destruction of microbubbles into the cardio‐ myopathic hearts prevents cardiac dysfunction in an animal model of doxorubicin-induced cardiomyopathy [57]. In this form of anthracycline induced cardiomyopathy in mice, findings of Esaki and colleagues suggest that HGF gene delivery by adenoviral vector exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium and mitigation of cardiac dysfunction. These beneficial effects appear to be related to HGF-induced MAPK/ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins [58]. Okayama et al demonstrated in transegenic mice that HGF reduced cardiac fibrosis by inhibiting endothelial mesenchymal transition and the transformation of fibroblasts into myofibroblasts. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This pattern was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice [59]. In dogs with intracoronary microembolization-induced heart failure, intramyocardial injections of HGF naked DNA plasmid attenuated the expression abnormalities of the SR Ca2+-cycling proteins, improved regional and global left ventricular function and prevented progressive LV remodeling [60]. In a ventricular rapid pacing heart failure canine model, gene transfection of HGF promoted angiogenesis, improved perfusion, decreased fibrosis and apoptosis, promoted recovery from myocyte atrophy, and thereby attenuated cardiac remodeling and improved myocardial function in the failing canine hearts [61]. The gene therapy with hepatocyte growth factor–complementary DNA plasmids reduced coronary artery ligation-induced cardiac impairment in goats (Shirakawa et al. 2005). Taken together, a number of experimental data support the potential therapeutic value of HGF.

Importantly, the concept of gene therapy using HGF has been used in human as well. The intracoronary administration of adenovirus vector encoding the human HGF gene in patients with coronary heart disease resulted in high levels of gene expression of HGF and its receptor c-Met, as well as increased serum concentrations of HGF. Adenovirus vector encoding the human HGF gene effectively induced temporarily high expression of the HGF gene in peripheral blood mononuclear cells and consequently increased serum HGF levels [62]. Nevertheless, the clinical utility of HGF therapy in the myocardium still remains enigmatic. It is still unclear which mechanisms are the most important for the cardioprotective effect of HGF. For a successful translation to clinical application of a protein, a clearly defined primary mode of action and knowledge on pharmacokinetic properties are necessary for the rational

The role of HGF/c-Met signalling in cardiac tissue is predominantly linked to ischemic damage and little is known about its role in diabetic cardiomyopathy. Since HGF contributes to the protection or repair of vascular endothelial cells and decreased serum and tissue HGF levels are related to the progression of endothelial cell damage induced by diabetes [63], the same might be true for cardiac tissue. In general, increased HGF is believed to be a marker of

development of the protein as a therapeutic [52].

**2.5. HGF/c-Met in diabetic cardiomyopathy**

250 Cardiomyopathies

One of the long reported general hypotheses of cardiac impairment is based on the calcium overload. Fleckenstein's calcium theory of myocardial cell necrosis from 1970' is widely quoted in literature as a general mechanism of myocardial cell damage [66]. It must be noted that intracellular calcium dysregulation is present in all types of advanced cardiomyopathy and apparently is a late stage event that represents a final common pathway for myocardial cell damage and death. There is now increasing evidence that depression of contractility in heart failure is linked to a malfunction of calcium regulation in cardiomyocytes, in particular to sarcoplasmic reticulum (SR) Ca2+ uptake and/or release [67; 68].

Sarcoplasmic reticulum (SR) Ca2+ release is maintained by a macromolecular protein complex consisting of the ryanodine receptor (RyR) – a Ca2+ release channel, calsequestrin (CSQ), triadin, and junctin that is activated by L-type Ca2+ current [69; 70]. Aside from cytosolic Ca2+, RyR activity is also regulated by SR luminal Ca2+ [71; 72]. Its storage and release are under the

acids (Mw: 565 kDa). Three isoforms of RyR have been described in mammalian tissues (RyR1, RyR2 and RyR3) of which RyR2 is predominant in cardiac muscle. The RyR is a tetramer consisting of four subunits and forms a complex with other proteins of which the FK506 binding protein (FKBP), calsequestrin, triadin 1 and junctin were identified in cardiac muscle. FKBPs are known for immunosuppressive properties; however, members of this protein family, FKBP12 and FKBP12.6, also bind to the cytoplasmic part of the RyR in skeletal and cardiac muscle and seem to modulate the gating properties of the RyR. Calsequestrin is a 55 kDa high-capacity calcium binding protein located in the lumen of the cardiac or skeletal junctional SR storing the calcium to be released by the RyR. Both triadin and junctin are transmembrane proteins in the junctional SR which bind directly to the RyR and to calseques‐ trin suggesting that these proteins attach calsequestrin to the RyR. Initially, two functional cardiac isoforms of triadin with apparent molecular weights of 35 kDa (triadin 1) and 40 kDa (triadin 2) were cloned of which triadin 1 is predominant and representing more than 95% of cardiac triadin. Junctin was first identified as a 26-kDa calsequestrin-binding protein in cardiac and skeletal muscle. Triadin and junctin are encoded on different genes but exhibit structural and amino acid similarities with single membrane spanning domains (62% identity within this domain), short cytoplasmic N-terminal segments and long highly-charged basic C-terminal

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Indeed, several disorders of the SR Ca2+ release complex have been identified as causes of heart disease. Hyperphosphorylation of the RyR by PKA and Ca/Calmodulin-dependent protein kinase II (CaMKII) induces a Ca2+ leak during diastole, which can cause heart failure and lead to fatal arrhythmias [78; 79; 80; 81]. The forced expression of triadin or junctin in rat myocytes resulted in an increase of the RyR open probability or a depressed contractility, respectively [77; 82]. Consistently, the ablation of junctin was associated with enhanced cardiac function and increased Ca2+ cycling parameters in mice [83]. Similarly, overexpression of CSQ induces

Predominantly, diabetic cardiomyopathy is related to diastolic abnormalities. In both Type 1 and Type 2 rodent models of diabetes, altered expression, activity and function of all trans‐ porters involved in excitation–contraction coupling, SERCA2a, NCX, and PMCA, leading to dysfunctional intracellular calcium signalling. In particular, abnormalities of SERCA2a, the major splice variant in the heart have been documented in diabetic cardiomyopathy. Protein,

Depressed SERCA activity causes inefficient sequestration of calcium in the SR, resulting in cytosolic calcium overload, impaired relaxation and hence diastolic dysfunction. On the other hand, cardiac overexpression of SERCA improves Ca2+ homeostasis and contraction in diabetic models [12; 85; 86; 87; 88; 89]. Because heart muscle from diabetic animals exhibits a diastolic dysfunction, SERCA2a has been considered a major site for contractile dysfunction. Indeed, perfusion of hearts with glucose can lead to lowered SERCA2a mRNA levels [2; 13]. Several factors may alter proteins regulating cardiomyocytes calcium homeostasis. The process of advanced glycation has been related directly to alterations in myocardial calcium handling

domains situated in the lumen of the SR [67; 68; 75; 77].

rapid development of heart failure in transgenic mice [84].

**3.2. Calcium regulation abnormalities in diabetic cardiomyopathy**

mRNA, and also activity of this protein decreases in response to diabetes [10].

**Figure 2.** A schematic illustration of potential effects of HGF/c-Met in diabetic cardiomyopathy [24; 42].

control of CSQ [71], whereas triadin and junctin may serve as linker proteins between CSQ and the RyR [70; 73]. The tethering of CSQ to the inner surface of the SR allows it to sequester Ca2+ in the vicinity of the RyR during SR Ca2+ cycling [74]. CSQ may act as a Ca2+ sensor that inhibits the RyR at low SR luminal Ca2+ via interaction with triadin/junctin [75]. An increase of SR luminal Ca2+ disrupts the inhibition of the RyR because the CSQ Ca2+ binding sites become more occupied with Ca2+, resulting in a weakened interaction between CSQ and triadin/junctin and an increased open probability of the channel [76]. Sorcin, a 22-kDa Ca2+-binding protein, also binds to cardiac RyR with high affinity, and its interaction with RyR is facilitated by annexin A7 in a Ca2+-dependent manner. Thus the interaction between these proteins appears to be critical for the regulation of SR Ca2+ release. For relaxation to occur, calcium ions must be removed from the cytosol, the majority of which is pumped back into the SR by cardiac specific SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a), while the remainder is ejected out of the cell through the sarcolemmal NCX (Na2+/Ca2+ exchange), PMCA (plasmamembrane Ca2+-ATPase) or mitochondrial calcium uniport.

Cardiac specific ryanodine receptor 2 (RyR2), a Ca2+-activated Ca2+ channel situated in the SR membrane, plays the dominant role in Ca2+ release from the SR in cardiac myocytes. In general, the RyR is a huge tetrameric protein with each monomer constituted of around 5000 amino acids (Mw: 565 kDa). Three isoforms of RyR have been described in mammalian tissues (RyR1, RyR2 and RyR3) of which RyR2 is predominant in cardiac muscle. The RyR is a tetramer consisting of four subunits and forms a complex with other proteins of which the FK506 binding protein (FKBP), calsequestrin, triadin 1 and junctin were identified in cardiac muscle. FKBPs are known for immunosuppressive properties; however, members of this protein family, FKBP12 and FKBP12.6, also bind to the cytoplasmic part of the RyR in skeletal and cardiac muscle and seem to modulate the gating properties of the RyR. Calsequestrin is a 55 kDa high-capacity calcium binding protein located in the lumen of the cardiac or skeletal junctional SR storing the calcium to be released by the RyR. Both triadin and junctin are transmembrane proteins in the junctional SR which bind directly to the RyR and to calseques‐ trin suggesting that these proteins attach calsequestrin to the RyR. Initially, two functional cardiac isoforms of triadin with apparent molecular weights of 35 kDa (triadin 1) and 40 kDa (triadin 2) were cloned of which triadin 1 is predominant and representing more than 95% of cardiac triadin. Junctin was first identified as a 26-kDa calsequestrin-binding protein in cardiac and skeletal muscle. Triadin and junctin are encoded on different genes but exhibit structural and amino acid similarities with single membrane spanning domains (62% identity within this domain), short cytoplasmic N-terminal segments and long highly-charged basic C-terminal domains situated in the lumen of the SR [67; 68; 75; 77].

Indeed, several disorders of the SR Ca2+ release complex have been identified as causes of heart disease. Hyperphosphorylation of the RyR by PKA and Ca/Calmodulin-dependent protein kinase II (CaMKII) induces a Ca2+ leak during diastole, which can cause heart failure and lead to fatal arrhythmias [78; 79; 80; 81]. The forced expression of triadin or junctin in rat myocytes resulted in an increase of the RyR open probability or a depressed contractility, respectively [77; 82]. Consistently, the ablation of junctin was associated with enhanced cardiac function and increased Ca2+ cycling parameters in mice [83]. Similarly, overexpression of CSQ induces rapid development of heart failure in transgenic mice [84].

#### **3.2. Calcium regulation abnormalities in diabetic cardiomyopathy**

control of CSQ [71], whereas triadin and junctin may serve as linker proteins between CSQ and the RyR [70; 73]. The tethering of CSQ to the inner surface of the SR allows it to sequester Ca2+ in the vicinity of the RyR during SR Ca2+ cycling [74]. CSQ may act as a Ca2+ sensor that inhibits the RyR at low SR luminal Ca2+ via interaction with triadin/junctin [75]. An increase of SR luminal Ca2+ disrupts the inhibition of the RyR because the CSQ Ca2+ binding sites become more occupied with Ca2+, resulting in a weakened interaction between CSQ and triadin/junctin and an increased open probability of the channel [76]. Sorcin, a 22-kDa Ca2+-binding protein, also binds to cardiac RyR with high affinity, and its interaction with RyR is facilitated by annexin A7 in a Ca2+-dependent manner. Thus the interaction between these proteins appears to be critical for the regulation of SR Ca2+ release. For relaxation to occur, calcium ions must be removed from the cytosol, the majority of which is pumped back into the SR by cardiac specific SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a), while the remainder is ejected out of the cell through the sarcolemmal NCX (Na2+/Ca2+ exchange), PMCA (plasma-

**Figure 2.** A schematic illustration of potential effects of HGF/c-Met in diabetic cardiomyopathy [24; 42].

Cardiac specific ryanodine receptor 2 (RyR2), a Ca2+-activated Ca2+ channel situated in the SR membrane, plays the dominant role in Ca2+ release from the SR in cardiac myocytes. In general, the RyR is a huge tetrameric protein with each monomer constituted of around 5000 amino

membrane Ca2+-ATPase) or mitochondrial calcium uniport.

252 Cardiomyopathies

Predominantly, diabetic cardiomyopathy is related to diastolic abnormalities. In both Type 1 and Type 2 rodent models of diabetes, altered expression, activity and function of all trans‐ porters involved in excitation–contraction coupling, SERCA2a, NCX, and PMCA, leading to dysfunctional intracellular calcium signalling. In particular, abnormalities of SERCA2a, the major splice variant in the heart have been documented in diabetic cardiomyopathy. Protein, mRNA, and also activity of this protein decreases in response to diabetes [10].

Depressed SERCA activity causes inefficient sequestration of calcium in the SR, resulting in cytosolic calcium overload, impaired relaxation and hence diastolic dysfunction. On the other hand, cardiac overexpression of SERCA improves Ca2+ homeostasis and contraction in diabetic models [12; 85; 86; 87; 88; 89]. Because heart muscle from diabetic animals exhibits a diastolic dysfunction, SERCA2a has been considered a major site for contractile dysfunction. Indeed, perfusion of hearts with glucose can lead to lowered SERCA2a mRNA levels [2; 13]. Several factors may alter proteins regulating cardiomyocytes calcium homeostasis. The process of advanced glycation has been related directly to alterations in myocardial calcium handling and hence contractility. The advanced glycation of SERCA2a has been shown to lead to a decrease in its activity and a prolongation of cardiac relaxation [14].

Modulation of cardiomyocyte Ca2+ handling by RyR2 is long known to occur by caffeine and tetracaine, which increase RyR2 open probability. More recently, flecainide was reported to prevent catecholamine polymorphic ventricular tachycardia as a result of decreasing RyR2 conductance and RyR2 open time RyR2s from these hearts were S-nitrosylated and depleted of FKBP12.6, resulting in leaky RyR2 channels and a diastolic SR-Ca2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca2+ channel stabilizer S107, a novel RyR2-specific benzothiazepine derivative compound, inhibited the SR-Ca2+ leak and prevent‐ ed arrhythmias in vivo. Similarly in skeletal muscle, S107 which binds to RyR1 and recovers the binding of FKBP12.6 to the nitrosylated channel inhibits SR Ca2+ leak, improves muscle function, and increases exercise performance in muscular dystrophic-deficient mouse model [90; 91]. Taken together, these data opens new era of new drugs – stabilizers of RyR complex (rycals), in regulation of calcium in various cells what could have an impact also in treatment

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This article was supported by the grant EFSD New Horizons 2012 *The role of HGF/c-Met signalling in diabetic end-organ damage* from the European Foundation for the Study of Diabetes - New Horizons, Collaborative Research Initiative and the grant APVV-0887-11 *Molecular aspects of drug induced heart failure and ventricular arrhythmias* from the Slovak Research and

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in

[1] Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, Branwood AW, Grishman A, New type of cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol 30

[2] Boudina S, Abel ED, Diabetic cardiomyopathy revisited. Circulation 115 (2007)

[3] Bell DS, Diabetic cardiomyopathy. Diabetes Care 26 (2003) 2949-2951.

of diabetic cardiomyopathy in the future.

**Acknowledgements**

Development Agency.

**Author details**

Bratislava, Slovak Republic

(1972) 595-602.

3213-3223.

Jan Klimas

**References**

Recently, attention has been focused on abnormalities of calcium release in diabetic conditions. In diabetic subjects, oxidative stress arises from an imbalance between production of reactive oxygen and nitrogen species and capability of the system to readily detoxify reactive inter‐ mediates. Importantly, it is now well established that RyR channels are highly susceptible to modification by various endogenous redox agents. Furthermore, RyR channels serve a role as intracellular redox sensors, via redox induced Ca2+ release and they are likely to connect cellular redox state with Ca2+ signaling cascades. Indeed, endogenous redox active molecules enhance RyR2 channel activity and RyR2 is one of the well-characterized redox-sensitive ion channels in heart. In general, oxidizing conditions increase RyR2 activity and so stimulate SR Ca2+ and causing Ca2+ leak (*see figure 3)*. In addition, RyR2 is activated also by reactive nitrogen species and S-nitrosylation increases RyR open probability in cardiac muscle and leads to increased Ca2+ leak [14]. Redox reactions by biological oxidants and antioxidants have been shown to alter the kinetics of Ca2+-induced Ca2+ release in the heart tissue. Besides several potential phosphorylation sites, the tetrameric RyR2 channel contains ~84 free thiols and is Snitrosylated in vivo. S-Nitrosylation of up to 12 sites (3 per subunit) led to progressive channel activation that was reversed by denitrosylation. RyR2 is activated also by reactive nitrogen species. For example, nNOS is expressed in SR and can supply NO to RyR2 in the immediate vicinity for S-nitrosylation, which increases RyR2 open probability in cardiac muscle and leads to increased Ca2+ release. Thus, sulfydryl-oxidizing agents, hydrogen peroxide and diamide, diminished RyR2-FKBP12.6 binding [90].

**Figure 3.** Intracellular calcium regulation and influence of oxidizing molecules (ROS, reactive oxygen species; RNS, re‐ active nitrogen species) on RyR2 function.

Modulation of cardiomyocyte Ca2+ handling by RyR2 is long known to occur by caffeine and tetracaine, which increase RyR2 open probability. More recently, flecainide was reported to prevent catecholamine polymorphic ventricular tachycardia as a result of decreasing RyR2 conductance and RyR2 open time RyR2s from these hearts were S-nitrosylated and depleted of FKBP12.6, resulting in leaky RyR2 channels and a diastolic SR-Ca2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca2+ channel stabilizer S107, a novel RyR2-specific benzothiazepine derivative compound, inhibited the SR-Ca2+ leak and prevent‐ ed arrhythmias in vivo. Similarly in skeletal muscle, S107 which binds to RyR1 and recovers the binding of FKBP12.6 to the nitrosylated channel inhibits SR Ca2+ leak, improves muscle function, and increases exercise performance in muscular dystrophic-deficient mouse model [90; 91]. Taken together, these data opens new era of new drugs – stabilizers of RyR complex (rycals), in regulation of calcium in various cells what could have an impact also in treatment of diabetic cardiomyopathy in the future.
