UNIVERSITÀ DI PISA
DIPARTIMENTO DI FARMACIA
Corso di Laurea Magistrale in “Chimica e Tecnologia Farmaceutiche”
TESI DI LAUREA
“EVALUATION OF THE CARDIOPROTECTIVE PROPERTIES OF NOVEL SYNTHETIC AND NATURAL AGENTS IN AN ACUTE MYOCARDIAL INFARCT
MODEL AND INVESTIGATION OF THE MITOCHONDRIAL TARGETS.”
Relatori:
Prof. Vincenzo Calderone Dott.ssa Lara Testai Corelatore:
Dott.ssa Valentina Citi
Candidato: Lorenzo Zallocco
1 INTRODUCTION ... 1
1.1ISCHEMIA/REPERFUSION INJURY ... 1
1.1.1 Ischemia-induced cell death ... 2
1.1.2 Reperfusion-induced cell death ... 4
1.2MECHANISMS OF CARDIOPROTECTION ... 6
1.2.1 Ischemic preconditioning ... 6
1.2.2 Ischemic postconditioning ... 7
1.2.3 Remote pre and postconditioning ... 7
1.2.4 Mechanism of IPreC ... 8 1.2.4.1 Triggers ... 8 1.2.4.2 Mediators ... 10 1.2.4.3 Transcription factors ... 13 1.2.4.4 Distal mediators ... 14 1.2.4.5 End effectors ... 15 1.2.5 Pharmacological preconditioning ... 17
1.3MITOCHONDRIAL POTASSIUM CHANNELS ... 18
1.3.1 Mito KATP ... 18 1.3.2 Mito KCa ... 21 1.3.2.1 Mito BKCa ... 22 1.3.2.2 Mito IKCa ... 23 1.3.2.3 Mito SKCa ... 24 1.3.4 Mito Kv ... 25 1.3.4.1 Mito Kv 1.3 ... 25 1.3.4.2 Mito Kv 1.5 ... 26 1.3.4.3 Mito Kv 7.4 ... 27
1.3.5 TASK-1 and TASK-3 ... 28
1.3.6 Mito SLO-2 ... 29
1.3.7 Innovative mito-delivered drugs ... 30
1.4GASTRANSMITTER ... 32 1.4.1 H2S ... 32 1.4.1.1 H2S in I/R injury ... 33 1.4.1.2 H2S in preconditioning ... 34 1.4.1.3 H2S in postconditioning ... 35 1.4.1.4 H2S donors ... 36
1.4.2 NO ... 37
1.4.2.1 Endogenous NO in I/R injury and preconditioning ... 38
1.4.2.2 Endogenous NO in postconditioning ... 38
1.4.2.3 NO donors ... 39
1.4.3 CO ... 42
1.4.3.1 Role of endogenous CO in I/R injury and cardioprotection ... 43
1.4.3.2 CO donors ... 44
1.5NATURAL MOLECULES ACTIVE IN CARDIOPROTECTION ... 46
1.5.1 Isosteviol ... 46
1.5.2 Naringenin ... 47
1.5.3 Glucosinolate and isothiocyanates ... 48
2 AIMS OF RESEARCH ... 50
3 MATERIALS AND METHODS ... 51
3.1SOLUTIONS AND COMPOUND ... 51
3.2DEVICES ... 52
3.3ANIMAL EXPERIMENTATION ... 53
3.4INFARCT IN VIVO ... 53
3.4.1 Pharmacological Treatment ... 53
3.4.1.1 Intra-peritoneal injection (I.p.) ... 53
3.4.1.2 Intravenous injection (I.v.) ... 54
3.4.2 Acute myocardial infarct procedure ... 54
3.4.3 Assessment of myocardial infarct damage ... 55
3.5MITOCHONDRIAL ANALYSIS ... 56
3.5.1 Isolation of cardiac mitochondria ... 56
3.5.2 Protein Dosage ... 57
3.5.3 Evaluation of mitochondrial membrane potential ... 58
4 RESULTS AND DISCUSSION ... 59
5 CONCLUSION ... 67
1 Introduction
1.1 Ischemia/reperfusion injury
Ischemic heart disease is the main cause of death in the world (World Health Organization, 2017).
Ischemic cardiomyopathies are related to a reduced blood supply to the organ, due to a partial or total occlusion of the coronary vessels (Lloyd-Jones et al. 2010).
In the ischemic phase the reduced flow of blood does not allow a proper
oxygenation of the tissues and a correct supply of nutrients, leading to cell death.
Currently, the most effective strategy to reduce ischemic damage is an early reperfusion of tissues, but paradoxically reperfusion itself is responsible for an additional damage (Yellon and Hausenloy 2007).
Cell death, in ischemia/reperfusion injury, occurs through two different physiological processes, such as necrosis, during the ischemia and reperfusion phase, and apoptosis, also called "programmed cell death", that may occur especially during the reperfusion phase.
The necrosis cell death is established fairly quickly and can trigger an inflammatory response, leading to multiple ultrastructural changes, such as swelling of the organelles, denaturation and coagulation of the cytoplasmic proteins and the division of the organelles.
These ultrastructural changes cause to cell membrane rupture and are linked to lack of oxygen, ATP depletion and loss of calcium homeostasis (Vanlangenkker et al. 2008, Vanlangenkker et al. 2012).
The mechanism of apoptosis, on the contrary, is a physiological energy-dependent process which activate a cascade of programmed and controlled events, leading to cell death. (Fig 1)
Figure 1- Different cell conformation in necrotic and apoptotic processes.
1.1.1 Ischemia-induced cell death
During the ischemic phase, the cells are in conditions of hypoxia or anoxia and are unable to perform many primary metabolic functions, such as the b-oxidation of fatty acids and the regeneration of ATP through oxidative phosphorylation.
The inhibition of b-oxidation leads to an increase in fatty acid amount, causing arrhythmias and leading to the inhibition of mitochondrial KATP channels (Xu et
al. 2001).
During this phase, the ATP is produced through the glycolysis but in the absence of oxygen, the pyruvic acid is converted by the enzyme lactic dehydrogenase into lactic acid, causing acidification of the intracellular pH which can reach a value of 6.2 in the first ten minutes of ischemia (Garlick et al. 1979).
To counteract the acidity of the cytosol, the excess H+ is extended from the
cytosol by the Na+/H+ antiporter, with a consequent increase in intracellular Na+
The increase of Na+ concentration reverses both the Na+/H+ and the Na+/Ca2+
antiporters, causing a further increase in intracellular acidity and in the concentration of Ca2+ inside the cell.
Furthermore, intracellular Ca2+ level is also increased by the inhibition of
sarcolemmatic and sarcoplasmatic Ca2+ ATPase pumps (Marban et al. 1987).
This ionic imbalance, and in particular the intracellular Ca2+ increase, can lead,
during the subsequent reperfusion phase, to the opening of the mitochondrial permeability transition pore (MPTP), which leads to cell death.
The hypoxic condition during an ischemic episode also causes an increase in Reactive Oxygen Species (ROS).
In particular, ROS have a biphasic behaviour: in the first few minutes of ischemia their concentration is very low but after 20-25 minutes their concentration increases considerably, leading to a gradual and irreversible decline in cellular integrity (Halestrap et al. 1998, Solaini et al. 2005) (Fig 2).
Figure 2 – Schematic representation of events during an ischemia. The ischemia causes the reduction in ATP production (1). The Na+/K+ ATPase pump is inhibited by the lack of ATP (2),
leading to an increase in the concentration of intracellular Na+ (3). The high concentration of Na+
causes the opening of Ca2+ channels voltage-dependent, which is responsible for inhibiting
Na+/Ca2+ antiporter, leading to an overload of Ca2+ in mitochondria (4-5). Furthermore, the high
intracellular concentration of Na+ inhibits the Na+/H+ antiporter, with a consequent decrease in
1.1.2 Reperfusion-induced cell death
The recovery of blood flow during reperfusion allows the nutrients and oxygen to be reintegrated in the tissues affected by the ischemic injury, leading to the generation of ATP and normalization of intracellular pH. These factors are essential for cell survival, but the rapid change of the metabolic conditions and the possible damage caused by the ischemic event contribute in exacerbating the damage in the ischemic area (Sanada et al. 2011).
The blood flow allows a very rapid recovery of the extracellular pH, generating however an imbalance between the extra and intracellular pH.
The strong ionic gradient generated leads to the activation of the Na+/H+
antiporter, which involves a restoration of the correct intracellular pH and a considerable increase in Na+ ions. The increase in intracellular sodium
concentration leads to an osmotic swelling of the cell and to the activation in "reverse mode" of the Na+/Ca2+ antiporter, with the consequent accumulation
of intracellular calcium (Schäfer et al. 2001).
During the first 30/60 minutes of reperfusion the cell can’t to restore the homeostasis of the intracellular calcium.
The accumulation of calcium during the first minutes of reperfusion is responsible for a series of phenomena that increase cellular damage, such as the activation of lipases, proteases and nucleases, hypercontraction of myofibrils of the contractile apparatus (causes band necrosis) and finally the opening of the MPTP (Garcia-Dorado et al. 2012).
The MPTP is a high conductance pore, anchored between the outer and inner membrane of the mitochondrion. Its activation allows the passage of molecules up to 1500 Da and the causes a rapid depolarization of the membrane potential, inducing the inhibition of the enzymes involved in oxidative phosphorylation (Lemasters et al. 2009).
Furthermore, the opening of the MPTP causes a swelling of the mitochondria, with consequent rupture of the outer mitochondrial membrane, causing the release in the cytosol of the molecules contained in the inter-membrane space, including Cytochrome C which activates the apoptotic processes (Murphy et al. 2008).
However, its opening mainly occurs in the reperfusion phase, because the presence of acidic pH in the ischemic phase prevents its assembly (below pH 7 a total inhibition occurs) (Halestrap 1991) (Fig 3).
The reperfusion allows a recovery of normal oxygen level which are responsible for the high production of ROS, although this event is necessary for the physiological process of the cell.
ROS production is mainly due to some enzymes involved in the electron transport chain (NADH dehydrogenase, Succinate Dehydrogenase, Cytochrome C reductase) but also to other mitochondrial oxidative enzymes (xanthine oxidase, monoamine oxidase and aconitase) (Kevin et al. 2003, Kalogeris et al. 2012, Chen et al. 2014), which are responsible for the accumulation of the superoxide anion (O2-).
Under normal physiological conditions, O2- anions are converted into hydrogen
peroxide (H2O2) by the superoxide dismutase, and finally transformed in H2O
and O2 by catalase.
The ischemic event compromising the endogenous antioxidant mechanisms producing an insufficient response to counteract the ROS accumulation which induces severe damage to cell structures, enzymes and membrane channels (Sanada et al. 2011).
Figure 3 – Schematic representation of events during the reperfusion. The oxygenation of tissues during reperfusion promotes recovery of ATP levels (1). The Na+/K+ ATPase pump
activity is restored (2), with the consequent normalization of the concentration of intracellular Na+ and K+ (3). The physiological pH combined with the high Ca2+ concentration causes the
1.2 Mechanisms of cardioprotection
Cardiologists observed that patients with acute myocardial infarction who had experienced episodes of prodromal angina, often exhibited less chest pain, less variation in the ST segment of ECG, less cardiac dysfunction and smaller myocardial infarct size.
More detailed studies of this phenomenon highlighted that previous cycles of short ischemia and reperfusion made the myocardium more resistant to subsequent episodes of acute infarction.
The application of cycles of ischemia/reperfusion before a severe ischemic event is called preconditioning, whereas the application of cycles is applied during the early stages of reperfusion, is defined postconditioning.
Figure 4 – Schematic representation of the differences among preconditioning, postconditioning and pharmacological preconditioning (Testai et al. 2015).
1.2.1 Ischemic preconditioning
In 1986, the ischemic preconditioning (IPreC) has been described for the first time in a canine model. Through the exposition of the heart to short cycles of non-lethal ischemia (five minutes of ischemia followed by five minutes of reperfusion), the myocardium increased its resistance to a subsequent more severe and prolonged episode of ischemia (Murry et al. 1986).
This phenomenon is considered one of the most powerful endogenous mechanisms of cardioprotection.
Its effectiveness has been found in several other species, including: rabbits, pigs, rats and subsequently its activity has been ascertained also in humans (Edwards et al. 2000, Yellon et al. 2003, Kloner et al. 2006, Heusch et al. 2013). Ischemic preconditioning has thus been highlighted as a new possible target for pharmacological and physio pathological research.
Subsequent studies distinguished this phenomenon in two phases.
The first stage is called "classic IPreC", it lasts about three hours after the beginning of the ischemic phase; in this stage, some trigger, mediator and end effector, activate pro-survival mechanisms and inhibit the action of pro-death signals.
The second stage is called "second window of IPreC", it begins approximately twenty-four hours after the beginning of the ischemic event and it lasts about three days; in this phase, the transcription of stress-responsive genes is activated, leading to the synthesis of proteins with cardioprotective properties (Edwards et al. 2000, Millar et al. 1996, Bianes et al. 2003).
1.2.2 Ischemic postconditioning
The clinical application of ischemic preconditioning is limited, due to the unpredictability of myocardial infarct and for this reason it has been necessary to develop a new reliable method to mimic the cardioprotection process.
Interestingly, Zao et al. thought to act at the beginning of the reperfusion; indeed, they applied short and intermittent cycles of ischemia (thirty seconds) and reperfusion (thirty seconds), immediately after reperfusion.
This protocols significantly reduce the extension of the ischemic area, tissue edema and post-ischemic endothelial dysfunction (Zhao et al. 2003).
As for the ischemic preconditioning this phenomenon has been ascertained in different animal species and in humans (Skyschally et al. 2009, Staat et al. 2005).
1.2.3 Remote pre and postconditioning
Schmidt et al. have shown that short and alternating ischemic cycles in the limbs offered significant protection during an episode of myocardial infarct, thus
preserving cardiac function, also reducing the typical arrhythmias of the reperfusion phase (Schmidt et al. 2007).
Subsequent studies demonstrated that this type of protection occurs through an inhibition of oxidative stress and has also been shown that ischemia can occur in any districts of the body, producing the same protective effect on cardiac tissues (Li et al. 2006).
1.2.4 Mechanism of IPreC
After observing that the cardioprotective effects of the pre- and post-ischemic conditioning did not act synergistically, in animals treated simultaneously with both protocols, it was possible to hypothesize that these effects are due to the same transduction pathways (Restaldo et al. 2006).
The transduction pathways involved in the cardioprotection process, include triggers, mediators and end effector.
The main signalling pathways involved in a IPreC stimulus are schematized in Figure 5.
1.2.4.1 Triggers
Following the pre or post ischemic conditioning stimulus, adenosine (Liu et al. 1991), bradykinin (Wall et al. 1994) and opioids (Schultz et al. 1995), activate their receptors on the cell membrane and give rise to a series of complex intracellular signals.
Although the three receptors act through different transduction pathways, it has been proposed that the action of these receptors have additive effects (Goto et al. 1995).
• Adenosine
Increasing adenosine levels have been observed during short periods of ischemia.
Adenosine induces the reduction of cardiac inotropism and vasodilation, through the interaction with the endothelial receptors (Liu et al. 1991). Adenosine binding to its receptor A1, a G protein coupled receptor, activates
the Phospholipase C (PLC), which catalyses the hydrolysis of phosphatidylinositol-4,5-diphosphate (PIP2) into Inositol-1,4,5-triphosphate
This event stimulates two different isoforms of PKC (𝛼 and 𝛿) that phosphorylates the MitoKATP channels (Yang et al. 2010, Cohen et al. 2001).
• Bradykinin
Bradykinin is an endogenous inflammation mediator that acts on the circulatory system, increasing the permeability of blood vessels, causing vasodilation.
Goto et al. observed that this molecule is released in the myocardium during short periods of ischemia (Goto et al. 1995).
This peptide binds the B2 receptor and activates the PI3K/AKT/iNOS
transduction pathway.
PI3K determines the phosphorylation of AKT, which activates the endothelial isoform of nitric oxide synthase (eNOS), which in turn synthesizes nitric oxide (NO). NO activates the Guanylate Cyclase, with consequent increase of cytosolic cGMP, which in turn activates the Protein Kinase G (PKG) (Cohen et al. 2001, Cohen et al. 2007, Oldenburg et al. 2004).
• Opioids
The presence of opioid receptors, µ, k and d, G protein-coupled receptors, in the myocardium has been demonstrated and an ischemic event leads to the increase of intracellular opioid peptides, such as encephalin (Maslov et al 2014).
The opioid peptides are believed to carry out their action through metal-mediated transactivation of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor that, when activated, dimerizes and auto phosphorylates the tyrosine residues in its intracellular domain, thus triggering a cascade of signals that leads to the activation of PI3K (Cohen et al. 2007). Then, as for Bradykinin, PI3K leads to PKC activation.
These triggers act simultaneously, but to achieve a cardioprotective effect they have to reach a threshold value (Goto et al. 1995).
ROS are also considered triggers for cardioprotective cascade.
In fact, the exposure to oxygen free radicals mimics the protective effects of ischemic preconditioning (Tritto et al. 1997) and the exposure to antioxidants can abolish this effect (Baines et al. 1997).
1.2.4.2 Mediators
• Protein kinase C (PKC)
PKC is a serine/threonine kinase, activated by lipid cofactors and derives from the degradation of membrane phospholipids by the Phospholipase C (PLC).
There are different isoforms of PKC in the heart:
o a, b and g that depend on the DAG and the concentration of Ca2+, o e, d and h which are Ca2+ independent and are activated only by the
DAG,
o V is not dependent on either the DAG or the Ca2+.
Activation of PKC requires translocation of the enzyme from the cytosol to the binding site in the sarcolemma membrane (Liu et al. 1994).
The cardioprotective activity of this enzyme has been demonstrated through the use of selective inhibitors such as staurosporin and polymyxin B, which abolishes the protective effect of the myocardial preconditioning.
Similarly, PKC activators, like the esters of phorbole, mimic protective effects (Ytrehus et al 1994).
At the end of this cascade of reactions, the PCK transmits the signal received by phosphorylating the target proteins.
PKC is present within the mitochondrion in two isoforms: o e isoform when activated has a cardioprotective effect,
o d isoform if activated mediates many of the processes that damage the tissues (Chen et al. 2001).
In particular, PKCe, once activated, phosphorylates and activates mitochondrial aldehyde dehydrogenase 2 (ALDH2), which removes the products of lipid peroxidation, thus protecting the mitochondrial functions (Chen et al. 2008). Another mechanism of cardioprotection of this protein is given by the direct phosphorylation of the MPTP thus inhibiting the pore opening (Ping et al. 1997, Baines et al. 2002, Bianes et al. 2003).
PKCd, if activated, phosphorylates the mitochondrial pyruvate dehydrogenase kinase, thus inhibiting pyruvate dehydrogenase and consequently the regeneration of ATP (Churchill et al. 2005).
significant effects during the subsequent clinical trial (Mochly-Rosen et al. 2011).
• Protein kinase G (PKG)
PKG is a cGMP dependent serine/threonine protein kinase; its activation through the PI3K pathway promoting the opening of MitoKATP channels, which
are responsible for cardioprotective effect (Costa et al. 2005).
It has also been shown that PKG is able to inhibit the opening of MPTP (Takuma et al 2001) (Hausenloy et al. 2006).
Other studies suggest that PKG activation may reduce Gap-junction conduction (Kwak et al. 1995).
• Protein kinase A (PKA)
Shanda et al. reported that the activation of PKA during ischemic preconditioning phase leads to a protective effect (Sanada et. al 2004), while its activation during an ischemia produces harmful effects (Makaula et al. 2005).
The mechanism of these effects has not been fully clarified, however it has been shown that PKA inhibits Rho GTPase and Rho kinase.
Its activation seems to be related to the generation of cAMP (Inserte et al. 2004) (Sanada et al. 2004).
• P38 MAPK
p38 mitogen activated protein kinases exists in four main isoforms: a, b, g and d (Widmann et al. 1999).
The isoforms a and b are expressed in the heart and have opposite roles in the mechanisms of cardioprotection.
Noteworthy, the p38a mediates processes of cell death and is inhibited following an IPreC.
On the contrary, p38b contributes to the survival of the cell and is activated by the IPreC (Nemoto et al. 1998) (Saurin et al. 2000).
The p38 appears to be activated by the PKC and ROS production (Hausenloy et al. 2006).
The targets of this protein are MAPK-activating protein kinase 2 (MAPKAPK2), a kinase able to phosphorylate small proteins such as HSP27, which confers stability to the cytoskeleton and the crystalline 𝛼B, whose phosphorylation and activation leads to a cardioprotective effect (Armstrong et al. 1999) (Eaton et al. 2001).
Further studies showed the activation of Connexin-43 by the p38, leading to a reduction in the conductance of the Gap-junctions (Schulz et al. 2003). • BMK-1
The Big MAP protein kinases 1 (BMK-1 or Erk5) plays a crucial role in the proliferation of normal and cancerous cells.
It is activated by oxidative stress, hyperosmotic and growth factors (Kato et al. 2000) and regulates the entry into the S phase of the cell cycle (Kato et al. 1998).
Therefore, BMK-1 inhibition prevents the pass into S phase of the cycle by stabilizing the cyclin dependent protein kinase (CDK). (Perez-Madrigal et al. 1999).
Further studies have shown an increase in activity of this protein in preconditioned pig hearts (Takeishi et al. 1999).
This potential cardioprotective effect may be linked to a reduction in Gap-Junction conductance, with the probable involvement of Connexin 43 (Cameron et al., 2004) and an inhibitory effect of the proapoptotic factor BAD, via phosphorylation. (Pi et al. 2004)
• PI3K/AKT
Phosphatidylinositol-3-kinase (PI3K) is an enzyme able to phosphorylate the hydroxyl in position 3 of phosphatidylinositol bisphosphate (PIP2) phosphorylated in PIP3.
PIP3 can activate the protein serine/threonine kinase AKT (also called PKB), involved in the NO production and activation of PKCe (Tong et al. 2000). Other targets of AKT are: phosphorylation and inhibition of the proapoptotic factors BAX, BAD and GSK-3b and the phosphorylation and activation of the antiapoptotic factor Bcl-2.
• GSK-3b
Glycogen Synthase Kinase-3b is a protein that is phosphorylated and inhibited by different kinases such as AKT, PKC and ERK 1/2 (Tong et al. 2002).
The inhibition of this protein seems to delay and inhibit the opening of MPTP (Murphy et al 2005) and a reduction in the activation of the BAX proapoptotic factor (Murphy et al 2008).
1.2.4.3 Transcription factors
In the late phase of the preconditioning process, 12-24 hours after the ischemic event, the activation of transcription factors mediates the ex-novo synthesis of proteins, also called distal mediators.
• Nuclear Factor-kB
In IPreC models, on rabbit hearts it was shown that, the cardioprotective effect was abolished by inhibiting NFkB and this transcription factor is activated by NO, ROS, PKC and TK (Xuan et al. 1999).
The role of this transcription factor in IPreC was subsequently confirmed through the pharmacological activation of the MitoKATP channels or the
stimulation of Adenosine A3AR receptors (Zhao et al., 2002).
• Activator protein-1
It is a stress-dependent transcription factor, often activated in conjunction with NFkB (Li et al. 2000).
Its activation triggered by TNF-a and regulated by PKCe, ERK 1/2 and JNK and leads to a nuclear translocation of the factors c-Jun and c-Fos (Dawn et al. 2004).
• JAK-STAT
The Janus Kinase-Signal Transducer an Activator of Transcription is a stress-responsive transduction pathway that connects the transcription signal from the cell membrane to the nucleus (Bolli et al. 2003).
After being activated by PKCe, RAF1 or ERK 1/2, JAK acts by phosphorylating the tyrosine residue of STAT 1/3, which leads to the synthesis of COX-2 and iNOS distal mediators (Xuan et al. 2003) (Xuan et al. 2005).
• HIF-1a
Hypoxia inducible factor 1a is a subunit of the HIF1 factor; under normal oxygenation conditions it is immediately degraded by the prolylhydroxylase domain (PHD) and the Von Hippel-Lindau (VHL) protein.
Under hypoxic conditions, however, PHD is inhibited and HIF-1a binds to HIF-1b by forming the complete transcription factor (Semenza et al. 2011). HIF1 promotes the transcription of many proteins for cell survival in hypoxic conditions, such as glycolytic enzymes, for the synthesis of ATP in the absence of oxygen, and the vascular endothelial growth factor (VEGF), which promotes angiogenesis.
The cardioprotective effect of HIF-1a is attributed to the activation of transcription factors such as heme oxygenase (Ockaili et al. 2005) and iNOS (Natarajan et al., 2006).
1.2.4.4 Distal mediators
They represent transcribed proteins that mediate cardioprotection about 24 hours after the preconditioning stimulus.
• iNOS
The inducible Nitric Oxide Synthase is an enzyme that catalyse the formation of NO from arginine.
It was shown that by inhibiting these enzyme before the preconditioning stimulus, the cardioprotective effect of late IPreC was abolished (Takano et al. 1998).
Subsequently, a biphasic behaviour for the activation of cardiac NOS has been hypothesized.
In fact, following a preconditioning stimulus the endothelial isoform of the enzyme (eNOS) is immediately overexpressed, while the iNOS is very active about 24 hours after the stimulus (Xuan et al. 2000).
The interaction between iNOS and COX-2 was also studied, observing that COX-2 is probably activated before iNOS (Shinmura et al. 2002).
Interestingly, 72 hours after the preconditioning stimulus, the iNOS is no longer active, while COX-2 remains overexpressed (Wang et al. 2004). • COX-2
The Cyclooxygenase-2 is an enzyme that participates in the cascade of arachidonic acid for the synthesis of prostaglandins.
Using rabbit model, about 24 hours after a preconditioning stimulus, COX-2 was overexpressed, leading to an increase of PGE2 and 6-keto-PGF1a
production (Shinmura et al. 2000).
Subsequent studies have shown that COX-2 mediates late IPreC induced by d1 agonists (Kodani et al. 2002), H2S (Hu et al. 2008) and diazoxide, but it
doesn’t mediate the late IPreC induced by the stimulation of A1AR and A3AR
• RISK
The Reperfusion Injury Salvage Kinase is a transduction pathway composed by PI3K, AKT and ERK ½, activated after an ischemic event (Hausenloy et al. 2004).
1.2.4.5 End effectors • MPTP
The Mitochondrial Permeability Transition Pore is formed by three main subunits:
o the Voltage-Dependent Anionic Channel (VDAC), located on the outer membrane of the mitochondrion (Shoshan-Barmatz et al. 2006), which exists in three isoforms, VDAC1, VDAC2 and VDAC3.
VDAC1 is the most expressed isoform and is very important because induces apoptosis (Zaid et al. 2005).
On the contrary, VDAC2 inhibits the apoptotic process (Cheng et al. 2003), while the role of VDAC3 in the mechanisms of cell death has not been clarified yet.
The VDAC channels also play an important role in regulating the metabolic and energy flow through the outer mitochondrial membrane; in fact, they are involved in the transport of ATP, ADP, some metabolites and in Ca2+
homeostasis. (Shoshan-Barmats et al. 2003)
o Adenine Nucleotide Transferase (ANT) is one of the most common mitochondrial proteins and is the central component of MPTP (Halestrape-Brenner et al. 2003).
It transports ATP from the mitochondrion to the cytosol, the ADP from the cytosol to the mitochondrial matrix and interacts with VDAC and CyP-D to form the MPTP.
Also, this protein exists in three isoforms: ANT1 is expressed in the heart muscle tissue, ANT2 is expressed only in tumour cells, while ANT3 is expressed in all other tissues.
This protein can have two conformations, the "c" conformation which binds the CyP-D, increasing the sensitivity to Ca2+ and the "m" conformation
which binds ATP and ADP by desensitizing MPTP to Ca2+ (Halestrap et al.
o Cyclophyline D (CyP-D) is located in the inner membrane of the mitochondrion and directly controls the MPTP opening.
When the levels of Ca2+ in the mitochondrial matrix are high, CyP-D
promotes the "c" conformation of ANT, promoting the opening of the MPTP. A reduction in the expression of this protein increases the ability of the mitochondria to retain Ca2+, preventing the swelling and the pore
formation (Schinzel et al. 2005), it also preserves the cell death and the excessive accumulation of Ca2+ in the mitochondrion (Baines et al. 2005).
The over expression of CyP-D leads instead to the swelling of the matrix and the opening of the MPTP with consequent cell death (Li et al. 2004) (Baines et al. 2005).
• Gap-Junction
The Gap-Junctions are intercellular connections made up of proteins called Connexins.
Their inhibition, mediated by proteins such as MAPK and PKC, through the phosphorylation of Connexin 43, seems to mediate a cardioprotective effect (Garcia-Dorado D et al., 2002).
However, recent studies demonstrated that Connexin 43 may act independently from the Gap-junctions and interestingly its translocation was observed at the mitochondrial level during preconditioning.
Moreover, the cardioprotection doesn’t occur in isolated cardiomyocytes, it can’t develop gap-junction (Heinzel et al 2005).
• Mito KATP
The activation of mitochondrial potassium channel is also involved in the cardioprotective process.
Figure 5 – Signalling pathways involved in ischemic conditioning
1.2.5 Pharmacological preconditioning
The discovery of these phenomena led to the development of novel pharmacological strategies, in order to promote protection effects by using through substances able to interact with those targets, involved in the cardioprotective process.
Mitochondria play a pivotal role in the mechanism of cell death during the acute myocardial infarction and in the protective mechanism during the ischemic conditioning.
For this reason, those organelles have been studied in greater detail, also developing some drugs that allow the activation of cardioprotective processes. Another example of pharmacological preconditioning is represented to endogenous gastransmitter and some natural molecules.
These molecules may modulate the signalling pathways involved in pre or post ischemic conditioning leading to the cardioprotective effects.
1.3 Mitochondrial Potassium Channels
The mitochondrial potassium channels play a fundamental role in mitochondrial activity.
The mitochondrial channels involved in the cardioprotective mechanisms are: KATP, KCa, Kv, TASK and SLO-2 (Fig. 6).
Figure 6 – Mitochondrial potassium channels characterized in inner membrane (Citi et al. 2017)
1.3.1 Mito K
ATPATP-sensitive potassium channels are the first potassium channels recognized as a target for pharmacological preconditioning (Yao et al. 1994, Grover et al. 1995a, Grover et al. 1995b, Grover et al. 1996).
The presence of these channels was initially identified in the sarcolemma (Sarc KATP)of skeletal muscles, pancreatic tissues and cardiomyocytes (Noma et al.
1983).
Subsequently, their presence was also demonstrated in the inner mitochondrial membrane (Mito KATP) (Garlid et al. 2003, Murata et al. 2001).
KATP channels are structurally formed by a hetero-octameric complex, containing
K+ sensitive subunit (K
IR 6.X) and sulphonyl urea receptor (SUR1 and SUR2)
(Szabo et al. 2014, Szewezyk et al. 2006, Miki et al. 2002, Wojtovich et al. 2013).
MitoKATP and SarcKATP isoforms show some structural differences; indeed, the
mitochondrial isoform has been shown to be avoided of the classic SUR1 or 2 subunits, probably they possess a mitochondrial specific variant generated by an intra-exonic splicing of classical SUR2 subunit (Ye et al. 2009).
Other authors have demonstrated that by knocking down KIR proteins, MitoKATP
Recent studies have shown the presence of renal outer medullary potassium (ROMK) channels in the inner membrane of mitochondria of myocardiocytes. ROMK are ATP-Sensitive channels located in the cortical portion of the nephron, with the function of transporting the K+ outside the cell and probably ROMK may
be a structural subunit of the MitoKATP channels (Foster et al. 2012).
MitoKATP channel modulators
The first experiments performed to confirm the cardioprotective properties of KATP activators were performed with molecules having a benzopyran nucleus, in
particular cromakalim has been the first KATP activator to be tested (Fig. 7).
However, this compound has shown no selectivity for the mitochondrial potassium channel isoform: its cardioprotective effect was firstly attributed to the activation of SarcKATP channels, located in the smooth muscle of vessels and
cardiomyocytes (Mannhold 2004).
To highlight a possible selectivity of a molecule for MitoKATP channels, selective
blockers, were used.
Glibenclamide a well-known KATP blocker does not show selectivity for
mitochondrial or sarcoplasmic isoform (Mannhold 2004), while 5-hydroxydecanoate (5-HD) is able to selectively inhibit the mitochondrial channel isoform (Fig. 7).
Indeed, in isolated rat cardiac mitochondria, 5-HD inhibited diazoxide-induced flux of K+ ions (Jaburek et al. 1998).
Diazoxide, a prototype of the benzothiadiazine class, is an activator of the MitoKATP channels at low concentration, while at higher concentration can induce
also the activation of SarcKATP channels (Garlid et al. 1996, Correia et al. 2010)
(Fig. 7).
Beside its effect, diazoxide is able to inhibit the mitochondrial succinate dehydrogenase (Wojtovich et al. 2009, Busija et al. 2008) and shows side effects, such as vasodilation and hyperglycaemia (Calderone et al. 2010, Antoine et al. 1992, Pirotte et al. 1993).
The Bristol-Myers Squibb Company in order to develop molecules with high selectivity for MitoKATP channels, has produced two hybrid molecules,
BMS180448 and BMS19095, combining the benzopyranic portion of cromakalim with a cyanoguinidine nucleus derived from pinacidil, which is another important activator of KATP channels (Mannhold et al. 2004, Garlid et al. 1997) (Fig. 7).
Both these molecules showed a good cardioprotective profile, but BMS19095 was thirty times more selective versus MitoKATP channels.
However, this molecule can’t be used in clinical practice because of its neuronal toxicity (Grover et al. 2002).
Subsequently, new molecules were developed, adding a 2-carboxyalkylindole or 2-carboxyalchylindoline nucleus in position 4 of the benzopyranic nucleus. The activity of these two molecules is completely abolished in the presence of 5-HD, confirming their selectivity towards the MitoKATP channels, but
BMS191095 showed a better cardioprotective activity both in vitro and in vivo and a weak vasorelaxation activity (Lee et al. 2003).
Moreover, the analogue of the benzopyranilindole, KR-31466, showed a good cardioprotective activity, reducing the damage of hypoxia in H9c2 cardiomyoblast cells (Fig. 7).
Its mechanism of action seems to be linked to the opening of the MitoKATP
channels, but also to the activation of the PKC (Jung et al. 2003).
Then, many molecules were developed by replacing the portion 4 of the benzopyranic nucleus with spiromorpholonic or spiromopholenic groups, which give the molecule greater rigidity, increasing its selectivity for the MitoKATP
channels (Calderone et al. 2010, Breschi et al. 2008, Rapposelli et al. 2011). Among these molecules, the N-acetylspiromorpholone derivate (F163) showed a significant cardioprotective effect in ex vivo models of ischemia/reperfusion (Fig. 7).
More detailed studies on cardiomyoblasts and on in vivo models of acute myocardial infarction have shown that the cardioprotective effect of F163 is related to a selective opening of the MitoKATP channels (Calderone et al. 2010).
Moreover, the activity of this molecule is also enantioselective because only the levorotant enantiomer is active (Rapposelli et al. 2011).
Figure 7 Chemical structures of non-selective KATP openers (cromakalim, diazoxide and
pinacidil), selective Mito KATP openers (BMS-180448, BMS-191095, F163 and KR-31466) and
selective Mito KATP blockers (5-HD and Gliburide).
1.3.2 Mito K
CaThese channels are activated and opened by an increase of cytosolic calcium and three different potassium channels, have been described in the inner membrane of the mitochondrion:
• Large conductance calcium regulated potassium channel (BKCa) (Siemen
et al. 1999),
• Intermediate conductance calcium regulated potassium channel (IKCa)
(De Marchi et al. 2009),
• Small conductance calcium regulated potassium channel (SKCa) (Stowe
et al. 2013).
The difference among these channels lies in their primary amino acid sequence which confers them a different pharmacological and physiological profile.
1.3.2.1 Mito BKCa
Large conductance calcium regulated potassium channels have been identified in mitochondria using different techniques (Siemen et al. 1999):
• western blotting, • electron microscopy,
• immunofluorescence microscopy.
Structurally these channels are constituted by four a subunits, with seven transmembrane domains, four b and four g subunits.
b and g subunits regulate the activity of the a subunit, modulating the opening of the channel thought a conductance of 250-300 pS (Contreras et al. 2013). Mito BKCa channels have double regulation: both the membrane depolarization
and Ca2+ and Mg2+ intracellular concentration activates the channel in a separate
but also synergistically manner (Brelidze et al. 2003).
Mito BKCa channel modulators
The first molecule used to characterize BKCa channels was NS1619, a
benzimidazole derivative (Fig. 8).
NS1619 showed cardioprotective properties, reducing the ischemic area in a model of ischemia/reperfusion.
These cardioprotective effects are antagonized by selective blockers of BKCa
channels, such as paxillin (Xu et al. 2002) (Ardeali & O'rourke 2005) (Fig. 8). The cardioprotective activity of NS1619 is mediated by an anti-inflammatory effect, that prevents adhesion between leukocytes and endothelial cells, preserving mitochondrial functionality (Liu et al. 2012, Wang et al. 2010). However, very low concentrations of this molecule can provoke abnormal effects at the mitochondrial level, including a decrease in respiratory chain control, insensitivity to the selective blockers of the MitoBKCa channel and a significant
decrease in membrane potential, even in the absence of K+ ions (Bednarczyk et
al. 2008, Bentzen et al. 2009).
High concentrations of NS1619 instead cause the inhibition of L-type calcium channels, voltage dependent calcium channels, K+ and Na+ channels.
These nonspecific effects complicate the study of the role of MitoBKCa channels
It exerts protective effects through the activation of the MitoBKCa channels and
these effects are completely antagonized by paxillin.
Noteworthy, at nanomolar concentrations, NS11021 increases the bioenergetics performance of cardiac mitochondria, through an increase of K+ uptake and a
slight swelling of the membrane, without significant changes in the membrane potential (Debska et al. 2002, Aon et al. 2010).
Another interesting compound is CGS7184, which exerts its cytoprotective action by reducing ROS production in isolated rat mitochondria (Kulawiak et al. 2008) and membrane potential depolarization in EAhy926 endothelial cells (Fig. 8) (Wrzosek et al. 2009). In particular, these effects may be related to an interaction with the sarcoplasmic reticulum that modulates calcium homeostasis (MacPherson et al. 1997, Wrzosek et al. 2012).
Stumpner et al. reported the involvement of BKCa channels in desflurane induced
postconditioning, in a mice model of acute myocardial infarction (Fig. 8) (Stumpner et al. 2012).
Desflurane is a volatile anaesthetic used to induce or maintain anaesthesia during surgery.
Other molecules, initially not described as activators of the BKCa channels, are
for example cilostazol and sildenafil, two phosphodiesterase inhibitors, which can mimic the pharmacological preconditioning (Wang et al. 2008, Fukasawa et al. 2008).
1.3.2.2 Mito IKCa
Intermediate conductance calcium regulated potassium channels are expressed in different types of cells, such as endothelial cells, blood cells and epithelial cells (Szabo et al. 2014).
Unlike BKCa channels, their opening is not regulated by voltage, but their activity
depends only to the bond with the ions Ca2+ through the interposition of the
calmodulin.
Calmodulin is a small protein found in the cytosol, with high affinity for Ca2+
ions; by binding 2 or 3 ions of Ca2+, it changes its conformation increasing its
affinity with the membrane transport proteins.
Structurally, these channels are made by four subunits with six transmembrane domains; no differences were found between the mitochondrial and sarcoplasmic isoforms (Dale et al. 1996).
To date, they aren’t described in cardiac mitochondria and their conductance is significantly lower than the BKCa and was recorded around 20-85 pS.
Mito IKCa channel modulators
The pharmacological role of this channel has not been investigated yet, but it has been shown that the use of an IKCa inhibitor such as TRAM-34, causes
hyperpolarization of the mitochondrial membrane, according to the typical profile of mitochondrial potassium channel activators (Fig. 8) (Sassi et al. 2010).
1.3.2.3 Mito SKCa
Small conductance calcium-regulated potassium channels are very similar to IKCa channels: their opening is not regulated by the voltage but only by the
interaction with the Ca2+ ions through calmodulin.
Structurally they are formed by homo-tetramers with six transmembrane domains that form the pore (Stowe et al. 2013).
Their presence has been demonstrated in the inner membrane of cardiac mitochondria and in guinea pig neuronal cells, through immune-electron microscopy techniques and purified with isoelectric focusing (Dolga et al. 2013). Their conductance is very low, approximately 10 pS.
Mito SKCa channel modulators
Among the activators of the SKCa channel, the DCEBIO molecule induced
cardioprotective effects, which were abolished by NS8593 (Sørensen et al. 2008) and TBAP, two blockers of the SKCa channels (Fig. 8).
Moreover, TBAP is a scavenger of peroxynitrite (superoxide dismutase mimetic) and its use has allowed to hypothesize that the opening of this channel is mediated by the activation of superoxide dismutase (Stowe et al.2013).
Another blocker of the SKCa and IKCa channels, is the NS309, which has been
successfully used to demonstrate the involvement of MitoSKCa channels in the
protection of dopaminergic neurons in a rotenone toxicity model (Dolga et al. 2014).
Figure 8 Chemical structures of BKCa openers (NS-1619, NS-11021, CGS-7184 and
desflurane), BKCa blocker (paxillin), SKCa opener (DCEBIO), SKCa blockers (NS-8593 and TBAP)
and IKCa blocker (TRAM-34).
1.3.4 Mito Kv
Voltage-gated potassium channels are the potassium channels more expressed in many districts of the human organism.
Their presence has been highlighted both in excitable cells, where they control the membrane potential at rest, and in non-excitable cells, where they play a fundamental role in the feedback regulation of membrane potential (Gutman et al. 2005).
These channels are encoded by 40 of the 90 human genes and are divided into 12 families (Kv1-12) (Gutman et al. 2005).
1.3.4.1 Mito Kv 1.3
The expression of these channels was reported in lymphocytic T (Szabo et al. 2005) and cerebral mitochondria (Bednarczyk et al. 2010); they are made up of four subunits with six transmembrane domains and have an activation threshold between –50 and -60 mV.
These channels are therefore very present in the cells of the immune system and are involved in the regulation of the apoptotic process, but they haven’t been found at the cardiac level.
Moreover, some studies have highlighted the presence of Kv 1.3 and 1.5 channels in mitochondria of J774 macrophages, and their inhibition may lead to apoptosis of the macrophage (Leanza et al. 2012, Szab`o et al. 2010).
Mito Kv 1.3 channel modulators
The Mito Kv 1.3 selective drugs described so far are very few.
Initially the inhibitors Psora-4 and clofazimine have been described, and they can induce cell death in different human and murine cell lines (Fig. 9) (Cahalan et al. 2009).
More recently, two variants of Psora-4, PAPTP and PCARBTP, have been synthesized.
They are two inhibitors of the Mito Kv 1.3 channels, which have a positively charged portion of triphenylphosphonium (TPP+) (Fig. 9).
The TPP+ confers lipophilicity that allows the drug to penetrate much more easily
into the mitochondrial matrix (Leanza et al. 2017).
1.3.4.2 Mito Kv 1.5
Mito Kv 1.5 as Kv 1.3 are implicated in tissue differentiation ad cell growth (Felipe et al. 2006).
This channel is expressed in the immune system, kidney, brain and skeletral and smooth muscle (Coma et al. 2003, Vicente et al. 2003, 2006, Villalonga et al. 2008, Bielanska et al. 2012).
Kv1.5 current contributes to the ultra-rapid activating K+ current in the heart,
known as Ikur and plays a role in the repolarization of the action potential (Lesage et al. 1992).
The activation of this channel in the heart seems to be linked to the stimulation of the heart rhythm and, in pathological cases, of atrial fibrillation.
Mito Kv 1.5 channel modulators
In order to limit and control atrial fibrillation, two blockers, MK0048 and XEN-D0103, were investigated.
The use of these blockers led to a reduction in sinus rhythm but prolonged the duration of fibrillation (Ford et al. 2016, Loose et al. 2014).
Furthermore, it has been observed that Kv 1.3 blockers such as Psora-4, tetraethylammonium and 4-aminopyridine also block the Kv 1.5 channel (Grissemer et al. 1994) (Fig. 9).
1.3.4.3 Mito Kv 7.4
Their presence has been described in cardiac (Testai et al. 2016), muscle (Haick et al. 2016) and neuronal mitochondria (Gribkoff 2003), where they play an important role in the regulation of neuronal excitability.
Moreover, their role in cardioprotection against ischemic damages has been demonstrated.
Mito Kv 7.4 channel modulators
Testai et al. described for the first time the presence of this channel in cardiac mitochondria, using retigabine and flupirtine as selective activators of Kv 7.4 channels (Fig. 9) (Testai et al. 2016).
Retigabine is a very studied drug, actually used in the clinic practice for the treatment of epilepsy (Gunthorpe et al. 2012).
Recent studies demonstrated that this drug reduces neuronal hyperexcitability in motoneurons and could be used in neurodegenerative processes (Noto et al. 2016).
Flupirtine is currently used as an analgesic drug and acts at the neuronal level, inducing opening of the potassium channels.
Figure 9 Chemical structure of Mito Kv 1.3 blockers (PAPTP, clofazimine and PCARBTP), Mito Kv 1.3 and 1.5 blockers (Tetraethilammonium, Psora-4 and 4-Aminopyridine), Mito Kv 7 blocker (XE-991) and Mito Kv 7.4 openers (Flupirtin and Retigabin).
1.3.5 TASK-1 and TASK-3
Mitochondrial tandem pore domain acid-sensitive potassium channels are divided into: TASK-1, TASK-2, TASK-3, TASK-4 and TASK-5, based on the pH value which they are activated at.
TASK1 and TASK-3 are the more expressed channels in the heart.
In particular TASK-1 is expressed in all the cardiac tissues (Duprat et al. 1997); however, TASK-3 is more active at a slightly acidic pH (6.7) and therefore is more involved during an acute myocardial infarction event.
TASK-3 has been shown in mitochondria of keratocytes of melanoma cells (HaCaT cell line) (Szewczyk et al. 2009, Quast et al. 2012, Szewczyk et al. 2006), in the rat hippocampus (Kajma et al. 2012), in the epithelium of intestinal cells (Kovacs et al. 2005) and in rat heart (Decher et al. 2015, Karschin et al. 2001).
In the human heart the expression of this channel appears to be low, the higher expression is found in the sinoatrial node and in atrioventricular node.
The activity of TASK-1 and TASK-3 channels seems to be linked to the cardiac conduction system, in fact blockers of these channels, such as amiodarone (Gierten et al. 2010), dronedarone (Schmidt et al. 2012) or carvedilol (Staudacher et al. 2011) induce an antiarrhythmic activity (Fig. 10).
At the moment, there are very few selective blockers of these channels and one of the most studied is the A293, a selective blocker of the TASK-1 channel (Fig. 10) (Putzke et al. 2007).
1.3.6 Mito SLO-2
In mammals two isoforms of this channel have been identified, SLO-2.1 and SLO-2.2 (Wojtovich et al. 2011).
The activation of these channels is due to the increase in the intracellular concentration of Na+.
Their structure is similar to the other potassium channels, the subunit forming the pore is constituted by six transmembrane domains, with the C- and N-terminal portions located in the mitochondrial matrix.
Using mice that only expressed the Slo2.2 channel, it was observed that these channels are involved in cardioprotective mechanisms and a preconditioning mechanism was activated using volatile anaesthetics such as isoflurane (Fig. 10).
Contrarily using mice expressing only the Slo2.1 channel, it has been shown that this isoform is not involved in the cardioprotection mechanisms (Wojtovich et al. 2016).
Mito SLO-2 channel modulators
SLO-2 channels appear to be insensitive to potassium channel inhibitors such as 5-HD, iberiotoxin, charybdotoxin and apamin; on the contrary, they are activated by the bithionol (Fig. 10) (Wojtovich et al. 2011).
Bithionol is an anthelmintic drug that is widely used in veterinary medicine but is also suitable for human use.
Figure 10 Chemical structure of TASK blockers (amiodaron, dronedaron and carvedilol), selective TASK-1 blocker (A293), SLO 2.2 opener (isoflurane) and SLO 2 opener (bithionol)
1.3.7 Innovative mito-delivered drugs
Ideal MitoK activators should easily penetrate the cell, reach the mitochondria and selectively activate MitoK channels.
For this purpose, during the design of new molecules, some "mitochondria-addressed" cations, such as triphenyl phosphonium (TPP+), are often added to
the pharmacophore moieties, linked to the molecule through an appropriate aliphatic chain (Fig. 11).
The lipophilicity of the molecule depends on the length of the aliphatic chain and it influences the ability of the drug to cross the membranes (Kelso et al. 2001, Smith et al. 1999).
TPP+ is a lipophilic cation and it rapidly accumulates within the mitochondria,
depending on the membrane potential (about -180 mV) (Porteous et al. 2010, Rodriguez-Cuenca et al. 2010).
The lipophilic cations are very useful because of low reactivity and poor interaction with the other cellular components.
In addition to lipophilic cations, some peptides have been demonstrated to effectively accumulate into the mitochondria.
In particular, Szeto-Schiller (SS) peptides are able to bind selectively the inner membrane of the mitochondrion, independently of the membrane potential
Contrarily to lipophilic cations, these peptides exert their effect in the mitochondria without being influenced by the molecule which they are linked to. In particular, SS-02 and SS-31 (Fig. 11) have the ability to reduce in vitro concentrations of hydrogen peroxide, hydroxyl radical and peroxynitrite (Zhao et al. 2004).
Their cardioprotective activity has therefore been attributed to the ability of promoting mitochondrial respiration (consequently increasing the synthesis of ATP) and reducing ROS generation (Szeto 2008).
1.4 Gastransmitter
Endogenous gastransmitter are a family of molecules involved in the regulation of many physiological and pathological functions in mammalian tissues.
It is now widely recognized that gastransmitter such as nitric oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO), are involved in a plethora of
physiological functions (Caliendo et al. 2010; Szabo et al. 2010; Peers and Steele et al. 2012).
In the cardiovascular system, the role of NO, H2S and CO includes vasodilation,
stimulation of angiogenesis, cardioprotection (Szabo et al. 2010, Coletta et al. 2012), and vasodilation (Muchova et al. 2007).
1.4.1 H
2S
Du Vigneud et al. first described the presence of H2S in the body in 1942,
observing the release of this gas by incubating sulphur-containing amino acids in liver homogenates (Binkley and DuBigneud 1942).
After this discovery for many years the research focused on the toxicity of this molecule. Only in the recent years the role of H2S has been studied in normal
physiological processes, highlighting its pharmacological potential at very low concentrations.
Within the organism, H2S can be synthesized from organic and non-organic
sources.
L-Cysteine is the main organic source of H2S in the body, this amino acid can
be degraded by three enzymes: cystathionine 𝛽-synthase, cystathionine 𝛾-lyase and 3-mercaptosulfotransferase assisted by cysteine aminotransferase (Abe et al. 1996, Hosoki et al. 1997, Shibuya et al. 2009).
• Cystathionine 𝛽-synthase (CBS) is an enzyme mainly found in the brain, kidneys, liver and pancreas; this enzyme catalyses two reactions that lead to the release of H2S: the transulfurization of homocysteine into
L-cystathionine and L-cysteine into L-serine.
CBS uses heme as a cofactor, through the oxidation of Fe2+ in Fe3+ (Fig. 12).
• Cystathionine 𝛾-lyase (CSE) is mainly expressed in the cardiovascular system, where it catalyses the degradation of L-cystathionine in cysteine,
∝-subsequently into L-thiocysteine. In turn, L-Thiocysteine can be converted into L-cysteine with the formation of H2S (Fig. 12).
• Cysteine aminotransferase (CAT) catalyses the reaction between L-cysteine and ∝-ketoglutarate, leading to the formation of L-glutamate and 3-mercaptopyruvate.
3-mercaptopyruvate can be desulfurized by 3-mercaptosulfurtransferase (3-MST), obtaining H2S and pyruvate (Fig. 12).
There are also many inorganic sources of H2S within the body. For example, in
erythrocytes, sulphur can be reduced by reducing molecules obtained by glucose oxidation (Searcy et al. 1998).
Enterobacterial flora can also be a source of H2S, through the degradation of
chyme (Fiorucci et al. 2006, Furne et al. 2001).
1.4.1.1 H2S in I/R injury
The H2S-generating enzymes are all expressed at cardiac level, however the
CSE is much more expressed than CBS and 3-MST; for this reason, to obtain an inhibitory effect on the synthesis of H2S in order to study the role of endogenous
H2S, propargylglycine (PRG) or cyano-L-alanine (BCA) are used to selectively
inhibit the CSE (Asimakopoulou et al. 2013).
The role of H2S in the protection of an acute myocardial infarction was shown
by inhibiting the CSE with PRG, observing an increase of the ischemic area (Bliksoen et al. 2008).
The administration of L-cysteine, the endogenous substrate of CSE, led to a reduction in ischemia/reperfusion injury and the co-admistration of PRG attenuated the cardioprotective effect. (Elsey et al. 2010).
Furthermore, the overexpression of CSE led to a reduction of the damage, while the lack of this enzyme increased the area of the lesion (Elrod et al. 2007) (King et al. 2014).
Figure 12 Endogenous synthesis of H2S (Szabo and Papapetropoulos 2017)
1.4.1.2 H2S in preconditioning
During an ischemic event the production of H2S is reduced, it compared to
normal physiological conditions.
The inhibition of H2S synthesis is significantly attenuated submitting the heart
to cycle of ischemic preconditioning, thus demonstrating the involvement of this gastransmitter in the signalling pathways involved in preconditioning (Bian et al. 2006).
Even in isolated cardiomyocytes, the inhibition of CSE, through the use of PRG, led to a decrease in the effects of ischemic preconditioning, also decreasing the production of endogenous H2S (Pan et al. 2006) (Bian et al. 2006).
The cardioprotective activity of H2S in IPreC occurs through modulation of
numerous signalling pathways:
• H2S has anti-inflammatory properties, preventing leukocyte adherence
(Zanardo et al. 2006) and inhibiting the translocation of the transcription factor NF-kB (Oh et al. 2006).
o It inhibits the opening of MPTP (Yao et al. 2010),
o It leads to the activation of signalling pathway PI3K/Akt/Erk1/2/eNOS (Yong et al. 2008),
o It promotes activation of PKC𝜀. (Yong et al. 2008)
• Calvert et al. demonstrated the antioxidant activity of this gastransmitter which acts mainly through the stimulation of the Nrf2 transcription factor. In turn, Nrf2 regulates the expression of numerous enzymes with antioxidant activity, such as superoxide dismutase (SOD) (Calvert et al., 2009).
• Finally, the H2S-mediated cardioprotection also occurs through the
regulation of ion channels, in particular it interacts with the Ca2+ L-type
channels (Murata et al. 2001, Wang et al. 2001), the voltage-gated sodium channels, the sarcolematic and mitochondrial KATP channels (Zhao et al.
2001, Sivarajah et al. 2009).
1.4.1.3 H2S in postconditioning
H2S plays also an important role in ischemic postconditioning, in fact the
synthesis of H2S is increased during the first phase of reperfusion in
postconditioned hearts (Yong et al 2008).
The administration of PRG both before and after the ischemic phase led to an attenuation of the cardioprotective effect of the PostIC (Huang et al., 2012) (Yong et al 2005).
The overexpression of CSE, as in preconditioning, led to a significant reduction of tissue lesion (Elrod et al 2007).
Other studies confirmed this hypothesis by administering NaHS through a continuous infusion for 2 minutes or through six cycles of 10 seconds each, at the beginning of the reperfusion phase in isolated rat hearts, observing a marked improvement in cardiac function, a reduction of arrhythmias and a reduction in ischemic damage (Zhang et al. 2007, Yong et al. 2008, Ji et al. 2008).
In studies of IpostC, hydrogen sulfide was able to activate the signalling pathways JAK2/STAT3 and Akt/PKC/eNOS (Yong et al. 2008, Luan et al. 2012).
1.4.1.4 H2S donors
In isolated hearts, H2S perfusion led to a reduction of damage following
ischemia/reperfusion.
However, the administration of gaseous H2S is very limited and dangerous, due
to the difficulty of ensuring a correct dosage and due to the toxicity of this gas at high doses.
For this reason, compounds able to release H2S in a controlled manner have
been developed and characterized (Martelli et al. 2012).
Sodium hydrosulphide (NaHS) is considered a prototype of H2S-donor which
releases H2S very quickly and often used as a reference drug for experimental
procedures.
Some natural derivatives have shown a slow release of H2S, for example: the
alliin, a sulfurized amino acid contained in Allium sativum L. is a molecule that is rapidly converted into allicin (diallyltytiosulpinate) (Fig. 13).
In turn, allicin is transformed into more stable organosulfuric derivatives, such as dialilpolysulfides and ajoene (Fig. 13).
Dialilpolysulphides are slow-releasing H2S donors which require the presence of
endogenous thiols, such as reduced glutathione (Benavides et al. 2007).
In addition to these natural polysulphides some synthetic molecules, such as GYY-4137 have also been developed (Fig. 13) (LI et al., 2008), which was used in industrial processes for rubber vulcanization.
The administration of GYY-4137 in rats showed an increase in the plasma concentration of H2S, reaching a peak after 30 minutes and an effect lasting for
many hours (Li et al., 2008).
Another group of H2S-donor molecules is the hybrid drugs, obtained by
combining molecules already described in the literature with a H2S-releasing
moiety.
Examples of this strategy are the conjugation of FANS, such as: Aspirin, Diclofenac, Sulindac, and Naproxen with a portion of dithiolithione (HPDTT), which is currently the most used moiety to synthesise H2S-Donors hybrids (Fig.
Figure 13 Chemical structure of natural H2S-donors (alliin, allicin, ajoene) and synthetical H2
S-donors (GYY-4137, H2S-aspirin, H2S-diclofenac and H2S-sulindac).
1.4.2 NO
Nitric Oxide (NO) within the cell is synthesized by enzymatic and non-enzymatic pathways.
Under normal physiological conditions the non-enzymatic pathway has a fairly marginal relevance while the enzymatic represents the main pathway.
During an ischemic event, however, this path becomes the main source of NO, because the acidic pH and the absence of oxygen limits the enzymatic pathway (Tennyson-Lippard 2011).
The non-enzymatic mechanisms occur in fact by reduction of nitrites and nitrates assimilated with the diet, therefore they do not require the presence of oxygen.
The enzymatic pathway is represented by the family of enzymes, Nitric Oxide Synthase (NOS).
Structurally, the NOS are formed by three subunits:
• an oxidizing subunit which, using the heme and the oxygen as a cofactor, oxidizes the L-arginine’s nitrogen, forming NO and Citrulline,
• a reducing subunit that, using NADPH, FAD and FMN as cofactors, gives electrons to the oxidizing subunit, thus allowing its activity,
• a subunit interposed between the previous two which, using Calmodulin as cofactor, regulates the flow of electrons.
There are several NOS isoforms, two of which are constitutive (eNOS and nNOS) and one inducible by immunological stimuli (iNOS) (Knowles and Moncada 1994,
Morris and Billiar 1994, Nathan and Xie 1994, Sessa 1994, Stuehr and Griffit 1992).
The eNOS was first discovered in the vascular endothelium, but its presence was also detected in the platelets (Radomsky et al. 1990) and in some neuronal populations inside the brain (Dinerman et al. 1994).
The nNOS, on the other hand, was initially identified in neuronal cells but was subsequently also described in skeletal muscles (Kobzik et al. 1994), in the bronchial epithelium and in the tracheal epithelium (Kobzik et al. 1993).
1.4.2.1 Endogenous NO in I/R injury and preconditioning
Endogenous nitric oxide plays a fundamental role in many transduction pathways.
In particular, NO plays a key role in cardioprotection, interacting with PKG and with the RISK and SAFE transduction pathways (Schulz and Ferdinandy 2013, Murphy et al. 2012), however the accumulation during an ischemic event and the reaction with superoxide (O2.-), generated during reperfusion, leads to the
formation of peroxynitrite anion (ONOO-); that it’s a strong oxidizing and nitrosylating agent and then can damage many cellular components (Pacher et al. 2007).
Moreover, it has been observed that the administration of NOS-inhibitors before an ischemic event decreases the ischemia/reperfusion lesion, by decreasing the formation of peroxynitrite, but also the cardioprotective effects of a possible ischemic preconditioning are lost, thus highlighting the importance of NO in the modulation of ischemic damage (Ferdinandy et al 2007).
The role of NO has not yet been fully clarified, although the increase in iNOS expression seems to be fundamental (Ferdinandy and Shulz 2003, Jones and Bolli 2006), it has also been shown that iNOS expression induced in preconditioning does not lead to an accumulation of NO during ischemia (Bencsik et al. 2010).
1.4.2.2 Endogenous NO in postconditioning
Several studies have demonstrated the fundamental role of NO in ischemic postconditioning, also highlighting very different physiological mechanisms
