**1. Introduction**

"Nothing to do to save his life…" says the Beatles song "Good morning, good morning." Ironically, cardiovascular mortality and life-threatening arrhythmias show a circadian increase in the mornings, and chronoprotective agents are still missing [1, 2]. This chapter highlights the importance of melatonin as a potential life-saving agent for the darkest nights (of antiarrhythmics drugs) and a brightest tomorrow.

The cardioprotective properties of melatonin are remarkable. Most of the preclinical and clinical studies support the protective actions and the safety profile of this indolamine [3, 4]. In this chapter, we briefly introduce the multitarget and versatile properties of melatonin and general concepts of electrophysiology to appreciate its potential as a promising antiarrhythmic agent. The second and third sections of the chapters focus on acute and chronic melatonin's antiarrhythmic effects.

### **1.1 Melatonin properties relevant to heart rhythms**

Endogenous and pharmacological increases of melatonin concentrations protect the cardiovascular system [3–11]. However, the relationships between the cardiovascular and circadian systems are highly complex and should not be interpreted in reductionist ways [5, 12–14]. Furthermore, our understanding of the pleiotropy of melatonin, a highly preserved molecule of protection, is continuously expanding [3–7, 10, 15–24]. Therefore, we will focus on melatonin effects on heart rhythms. Additional information regarding melatonin cardiovascular effects can be found elsewhere and include direct actions in the heart, blood vessels, kidney, and other regulatory mechanisms at the nervous, immune, and endocrine systems [11, 25, 26]. Only the electrophysiological information will be extracted from its protective actions against risk factors like hypertension, metabolic syndrome, obesity, inflammation, and pathologies like ischemia/reperfusion injury, infarction, drug-induced cardiotoxicity, diabetic cardiomyopathy, and heart failure [8, 11, 21, 27].

Melatonin is amphipathic and pleiotropic. Melatonin can act on several targets at cell membranes and at intracellular levels in almost any cell [28, 29]. For this electrophysiological analysis, we present the following division of melatonin mechanisms of action:


#### *1.1.1 Melatonin as an antioxidant*

Melatonin protects against oxidants by several mechanisms. In fact, it has been suggested that one of the main functions of melatonin in all living organisms is to protect them from oxidative stress [30, 31]. Melatonin has a well-characterized and extensively documented antioxidant capacity [31–37]. Melatonin is a powerful antioxidant, with a potency of up to 10 times greater than vitamin E [38].

There are oxidants of different chemical nature. They can be free radicals or non-radical reactive species [39, 40]. Free radicals—molecules with an unpaired electron—are unstable, highly reactive, and often trigger chain reactions, which propagate nearby molecular modifications. The most studied oxidants are the reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species. Under physiological conditions, ROS/RNS act as second intracellular messengers modulating signal transduction pathways [40, 41]. A delicate cellular balance between the production and the removal of free radicals maintains low/ moderate concentrations. Oxidative stress occurs when oxidants increase above healthy levels and represent a severe risk to the molecular integrity of lipids, proteins, and DNA [39, 40]. Therefore, neutralization of reactive species by scavenger molecules like melatonin is a chemical way of counteracting oxidative stress.

The main agent involved in oxidative damage is superoxide anion, but hydrogen peroxide, hydroxyl radical, nitric oxide (NO), peroxynitrite, and nitroxyl also participate in oxidative stress. The mitochondria are the main source of oxidizing species during oxidative phosphorylation. Oxidants are also the product of the activation of non-mitochondrial enzyme systems such as NADPH oxidase, xanthine oxidase, and nitric oxide synthase [40–42].

**51**

*1.1.2 Melatonin receptors*

*Melatonin for a Healthy Heart Rhythm*

glutathione peroxidase [40–44].

*DOI: http://dx.doi.org/10.5772/intechopen.91447*

Cells have antioxidants that prevent damage. An antioxidant is any substance that significantly delays or prevents oxidation of lipids, proteins, or DNA [40]. Lipids are often used as target molecules because they are more reactive to oxidants than proteins or DNA. Nonenzymatic antioxidants include reduced glutathione (GSH), vitamins, and melatonin among others. Melatonin is five times more effective than GSH as scavenger of the highly toxic hydroxyl radical [34]. The main antioxidant enzymes are superoxide dismutase (SOD), catalase, thioredoxin, and

Melatonin efficiently prevents oxidative stress. The aromatic indole ring of melatonin reduces and repairs electrophilic radicals acting as generous electron donor. One molecule of melatonin can neutralize up to 10 toxic reagents, including ROS, RNS, and other free radicals [7, 39, 45–47]. In addition, several metabolites formed when melatonin neutralizes harmful reagents are also antioxidants suggesting that a cascade of reactions increases the efficacy of melatonin [28, 35, 47–49]. Being a highly lipophilic and hydrophilic compound, melatonin crosses all morphological barriers and acts not only in each cell but also within each subcellular compartment. Additionally, melatonin increases the efficacy of vitamin E, vitamin C, and GSH [33, 50]. Therefore, the elimination of free radicals can be carried out

Melatonin stimulates antioxidant enzymes by acting on membrane, cytoplasmic, and nuclear receptors [39, 43, 52]. Low melatonin concentrations increase the expression or activity of SOD, catalase, and glutathione peroxidase [43, 53].

and result in a prolonged action potential duration and arrhythmogenic triggers known as early-after depolarizations (EAD) [64]. Several reviews describe the redox regulation of calcium channel in cardiac myocytes including the ryanodine receptor calcium, the IP3 receptor, and voltage-dependent L-type calcium channel [65–69]. ROS and RNS affect the L-type Ca2+ channel Cav1.2 by regulation of cysteine

residues. However, calcium channel regulation by redox is controversial with reports of increase and decrease of channel functions [66]. Voltage-gated potassium (Kv) channel, mainly responsible for myocardial repolarization, is sensitive to oxidative stress [58, 70–72]. Sulfenic acid modification at a conserved cysteine residue of

Melatonin has receptors in the cellular membranes, in the cytoplasm, and in the nucleus. Melatonin membrane receptors express in several regions of the nervous system and in almost all the organs including the heart, arteries, kidneys, liver, gastrointestinal tract, prostate gland, uterus, skin, and eyes [73]. Melatonin activates two subtypes of G-protein-coupled receptors in the plasma membrane, named MT1 and MT2, according to the official IUPHAR nomenclature (previously called Mel1a and Mel1b) [74]. Both receptors have high affinity to melatonin (*Kd* ~ 0.1 pM). In 2019, Stauch and Johansson reported the crystal structures of the human MT1 and MT2 and set a solid base concerning ligand recognition for both receptors [75, 76].

Kv1.5 under prolonged oxidative stress can induce arrhythmia [58, 72].

Ion channels and many other proteins respond to oxidative stress [54–58]. Amino acid residues are the targets of ROS/RNS. Sulfur atoms like cysteine and methionine, hydroxyl groups from tyrosine, or aromatic rings of histidine, phenylalanine, and tryptophan are vulnerable to reactive species. Those that contain more cysteines are more sensitive to changes because thiol groups (–SH), which exist as thiolates (–S) at physiological pH, tend to react more quickly with ROS/RNS [59]. Many of these proteins are involved in important biological reactions such as oxidative phosphorylation, metabolic regulation, and signal transduction [60, 61]. Oxidative stress can

channel modification [62, 63]

by intracellular interactions independent of any receptor [36, 45, 51].

increase late sodium currents through direct Na+

#### *Melatonin for a Healthy Heart Rhythm DOI: http://dx.doi.org/10.5772/intechopen.91447*

*Melatonin - The Hormone of Darkness and Its Therapeutic Potential and Perspectives*

cardiotoxicity, diabetic cardiomyopathy, and heart failure [8, 11, 21, 27].

Melatonin is amphipathic and pleiotropic. Melatonin can act on several targets at cell membranes and at intracellular levels in almost any cell [28, 29]. For this electrophysiological analysis, we present the following division of melatonin

Melatonin protects against oxidants by several mechanisms. In fact, it has been suggested that one of the main functions of melatonin in all living organisms is to protect them from oxidative stress [30, 31]. Melatonin has a well-characterized and extensively documented antioxidant capacity [31–37]. Melatonin is a powerful

There are oxidants of different chemical nature. They can be free radicals or non-radical reactive species [39, 40]. Free radicals—molecules with an unpaired electron—are unstable, highly reactive, and often trigger chain reactions, which propagate nearby molecular modifications. The most studied oxidants are the reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species. Under physiological conditions, ROS/RNS act as second intracellular messengers modulating signal transduction pathways [40, 41]. A delicate cellular balance between the production and the removal of free radicals maintains low/ moderate concentrations. Oxidative stress occurs when oxidants increase above healthy levels and represent a severe risk to the molecular integrity of lipids, proteins, and DNA [39, 40]. Therefore, neutralization of reactive species by scavenger molecules like melatonin is a chemical way of counteracting oxidative stress.

The main agent involved in oxidative damage is superoxide anion, but hydrogen

peroxide, hydroxyl radical, nitric oxide (NO), peroxynitrite, and nitroxyl also participate in oxidative stress. The mitochondria are the main source of oxidizing species during oxidative phosphorylation. Oxidants are also the product of the activation of non-mitochondrial enzyme systems such as NADPH oxidase, xanthine

antioxidant, with a potency of up to 10 times greater than vitamin E [38].

Endogenous and pharmacological increases of melatonin concentrations protect the cardiovascular system [3–11]. However, the relationships between the cardiovascular and circadian systems are highly complex and should not be interpreted in reductionist ways [5, 12–14]. Furthermore, our understanding of the pleiotropy of melatonin, a highly preserved molecule of protection, is continuously expanding [3–7, 10, 15–24]. Therefore, we will focus on melatonin effects on heart rhythms. Additional information regarding melatonin cardiovascular effects can be found elsewhere and include direct actions in the heart, blood vessels, kidney, and other regulatory mechanisms at the nervous, immune, and endocrine systems [11, 25, 26]. Only the electrophysiological information will be extracted from its protective actions against risk factors like hypertension, metabolic syndrome, obesity, inflammation, and pathologies like ischemia/reperfusion injury, infarction, drug-induced

**1.1 Melatonin properties relevant to heart rhythms**

mechanisms of action:

b.Receptor activation

d.Ion channel modulation

*1.1.1 Melatonin as an antioxidant*

c.Improvement of mitochondrial functions

oxidase, and nitric oxide synthase [40–42].

a.Antioxidant

**50**

Cells have antioxidants that prevent damage. An antioxidant is any substance that significantly delays or prevents oxidation of lipids, proteins, or DNA [40]. Lipids are often used as target molecules because they are more reactive to oxidants than proteins or DNA. Nonenzymatic antioxidants include reduced glutathione (GSH), vitamins, and melatonin among others. Melatonin is five times more effective than GSH as scavenger of the highly toxic hydroxyl radical [34]. The main antioxidant enzymes are superoxide dismutase (SOD), catalase, thioredoxin, and glutathione peroxidase [40–44].

Melatonin efficiently prevents oxidative stress. The aromatic indole ring of melatonin reduces and repairs electrophilic radicals acting as generous electron donor. One molecule of melatonin can neutralize up to 10 toxic reagents, including ROS, RNS, and other free radicals [7, 39, 45–47]. In addition, several metabolites formed when melatonin neutralizes harmful reagents are also antioxidants suggesting that a cascade of reactions increases the efficacy of melatonin [28, 35, 47–49]. Being a highly lipophilic and hydrophilic compound, melatonin crosses all morphological barriers and acts not only in each cell but also within each subcellular compartment. Additionally, melatonin increases the efficacy of vitamin E, vitamin C, and GSH [33, 50]. Therefore, the elimination of free radicals can be carried out by intracellular interactions independent of any receptor [36, 45, 51].

Melatonin stimulates antioxidant enzymes by acting on membrane, cytoplasmic, and nuclear receptors [39, 43, 52]. Low melatonin concentrations increase the expression or activity of SOD, catalase, and glutathione peroxidase [43, 53].

Ion channels and many other proteins respond to oxidative stress [54–58]. Amino acid residues are the targets of ROS/RNS. Sulfur atoms like cysteine and methionine, hydroxyl groups from tyrosine, or aromatic rings of histidine, phenylalanine, and tryptophan are vulnerable to reactive species. Those that contain more cysteines are more sensitive to changes because thiol groups (–SH), which exist as thiolates (–S) at physiological pH, tend to react more quickly with ROS/RNS [59]. Many of these proteins are involved in important biological reactions such as oxidative phosphorylation, metabolic regulation, and signal transduction [60, 61]. Oxidative stress can increase late sodium currents through direct Na+ channel modification [62, 63] and result in a prolonged action potential duration and arrhythmogenic triggers known as early-after depolarizations (EAD) [64]. Several reviews describe the redox regulation of calcium channel in cardiac myocytes including the ryanodine receptor calcium, the IP3 receptor, and voltage-dependent L-type calcium channel [65–69]. ROS and RNS affect the L-type Ca2+ channel Cav1.2 by regulation of cysteine residues. However, calcium channel regulation by redox is controversial with reports of increase and decrease of channel functions [66]. Voltage-gated potassium (Kv) channel, mainly responsible for myocardial repolarization, is sensitive to oxidative stress [58, 70–72]. Sulfenic acid modification at a conserved cysteine residue of Kv1.5 under prolonged oxidative stress can induce arrhythmia [58, 72].

### *1.1.2 Melatonin receptors*

Melatonin has receptors in the cellular membranes, in the cytoplasm, and in the nucleus. Melatonin membrane receptors express in several regions of the nervous system and in almost all the organs including the heart, arteries, kidneys, liver, gastrointestinal tract, prostate gland, uterus, skin, and eyes [73]. Melatonin activates two subtypes of G-protein-coupled receptors in the plasma membrane, named MT1 and MT2, according to the official IUPHAR nomenclature (previously called Mel1a and Mel1b) [74]. Both receptors have high affinity to melatonin (*Kd* ~ 0.1 pM). In 2019, Stauch and Johansson reported the crystal structures of the human MT1 and MT2 and set a solid base concerning ligand recognition for both receptors [75, 76].

Melatonin membrane receptors can exist as monomers, as well as dimers. The MT1 homodimer forms 3- to 4-fold higher proportion than the MT2 homodimer and the MT1/MT2 heterodimer. Nonmammalian vertebrates present a third lowaffinity receptor termed Mel1c, and a proposed mammalian homologous is the orphan receptor GPR50 [74, 77–79]. This orphan lost its properties to directly interact with melatonin but shows an inhibitory interaction with MT1 receptors by forming heterodimers. More recently, other orphans unable to bind melatonin like GPR61, GPR62, and GPR135 showed a similar indirect inhibitory interaction with MT2 receptors [80]. Other G-protein-coupled receptors like the serotonin receptor 5HT2c can interact with melatonin membrane receptors [79]. These interesting interactions of membrane receptors are not further discussed in this chapter but should be considered in future electrophysiological studies with melatonin.

The MT1 and MT2 inhibit adenylate cyclase-protein kinase A-CREB signaling in target cells by pertussis toxin-sensitive Gαi, β, and γ and toxin-insensitive Gq, β, and γ proteins [74, 79]. The MT1 also increases phosphorylation of mitogenactivated protein kinase 1/2 (MAPK) and extracellular signal-regulated kinase 1/2 (ERK), as well as increasing potassium conductance through inwardly rectifying (Kir3.x) channels. The later effect on potassium channels could be relevant to heart electrophysiology since Kir3.x channels are highly expressed in cardiomyocytes and usually coupled to acetylcholine and adenosine membrane receptors [81]. MT2 melatonin receptor activation inhibits both forskolin-stimulated cAMP production and cGMP formation, activates protein kinase C (PKC) in the nervous system, and decreases calcium-dependent dopamine release in the retina. Native functional MT1/MT2 heterodimers in mouse rod photoreceptors mediate melatonin's enhancement of scotopic light sensitivity through phospholipase C and PKC pathways [82].

Several compounds interact with MT1 and MT2 receptors, but blocker luzindole is the only with proven myocardial electrophysiological effects [83]. Luzindole and 4P-PDOT competitively block MT1 melatonin receptors at concentrations higher than 300 nM, and both are inverse agonists in systems with constitutively active MT1 receptors [74, 79].

Melatonin interacts with several enzymes and intracellular proteins. The MT3 receptors is a quinone reductase 2 with an affinity in the nanomolar ranges [84]. This enzyme is possibly involved in the regulation of cellular oxidative status, although the exact regulatory action of melatonin remains unclear [84–87]. Furthermore, the electrophysiological effects of MT3 have not been reported yet.

Melatonin interacts with intracellular proteins such as calmodulin, calreticulin, or tubulin [88]. The low-affinity interaction between melatonin and calmodulin antagonizes the binding of Ca2+ and may be involved in its antioxidant action as well as other electrophysiological signaling processes [89–96].

Melatonin increases the cytoplasmic levels of the heat shock protein 70 in several tissues including the heart [97–102]. Further interaction with this chaperon will be described in Section 3 of the chapter.

Melatonin is a ligand for the retinoid-related orphan nuclear hormone receptor family (RZR/ROR) [74, 79]. RZR/RORα is expressed in a variety of organs, whereas RZRβ is specific for the brain and retina [33]. ROR/RZR has been proposed to work in coordination with the plasma membrane receptors MT1/MT2 to regulate gene expression. We suggest a potential interaction with Vitamin D receptor (VDR), which was elegantly confirmed in recent experiments [97, 103].

#### *1.1.3 Melatonin improves mitochondrial functions*

Mitochondria are critical for cellular metabolism and energy production. They maintain life but also are gatekeepers of cell death [31, 104]. Mitochondria

**53**

*Melatonin for a Healthy Heart Rhythm*

*DOI: http://dx.doi.org/10.5772/intechopen.91447*

produce up to 95% of the cellular energy in the form of ATP in aerobic cells [105]. Mitochondrial oxidative phosphorylation uses a system of oxidoreductase protein complexes (complexes I, II, III, and IV) to transfer electrons during ATP production. Deficiencies in the electron transport chain can result in the leakage of electrons and generate ROS/RNS [40, 41, 106, 107]. Oxidative stress decreases respiratory complex activity, impairs electron transport system, and opens the mitochondrial permeability transition pores leading to cell death [104, 106, 108]. Mitochondria are essentials for the protective actions of melatonin [51, 97, 106, 107, 109–111]. The mechanisms involved include its antioxidant properties and the preservation of complex I and III functions, inhibition of the opening of the permeability transition pores, and the release of cytochrome c. Petrosillo et al. demonstrate that melatonin prevents the opening of the mitochondrial permeability transition pores and its deleterious consequences [51, 110, 112, 113]. We recently reported that melatonin prevents mitochondrial edema, dilation of the ridges, high activity of NADPH oxidase, and apoptosis [97]. Melatonin improves mitofusin-2, which preserves the mitochondrial functional network and prevents apoptosis [114]. The reduction of mitochondrial damage in the heart could be related to the negative regulation of angiotensin II type 1 receptor (AT1) by melatonin [97]. The induction of Hsp70 through melatonin is compatible with an additional mechanism related to Tom 70, a translocase of the outer mitochondrial membrane [97, 115, 116]. The interaction of Hsp70 with Tom 70 initiates mitochondrial import processes [116]. Tom 70 regulates melatonin-induced cardioprotection by preventing

mitochondrial deterioration and oxidative stress [97, 115].

against mitochondrial-mediated lesions.

*1.1.4 Melatonin modulates ion channels*

properties as an antiarrhythmic.

Melatonin's cardioprotection associates with an increase in the number of mitochondria and positive regulation of survival genes such as nicotinamide phosphoribosyl transferase and nicotinamide adenine dinucleotide-dependent deacetylases, called sirtuins [117]. Particularly sirtuin-1 and sirtuin-3 are downstream mediators of the cardioprotective actions of melatonin. Sirtuin-1can modulate fatty acid oxidation, apoptosis, oxidative stress, and autophagy through deacetylation of transduction factors like NF-κB, forkhead box class O, p53, peroxisome proliferatoractivated receptor alpha, thioredoxin-1, and Bcl-xL [117–121]. Sirtuin-3 is a family member that is primarily located in the mitochondria and protects against inflammation and diseases related to oxidative stress. Melatonin elevates sirtuin-3, stimulates superoxide dismutase activity, and suppresses mitochondrial oxidative stress [31, 117, 122, 123]. Additionally, melatonin protects nuclear and mitochondrial DNA [122, 124, 125]. The multiple actions of melatonin provide potent protection

Melatonin exerts its electrophysiological effects by multiple mechanisms. One of the ways for melatonin to interact is through the modulation of ion channels. Whether we consider its role as a drug or as a biological molecule, it should be taken into account how melatonin has been considered an electrophysiological modulator for many physiological and clinical conditions such as control of circadian rhythms, regulation of arterial blood pressure and heart rate in mammals, sleep processes, and antiaging, among others. Its role in the modulation of several ion channels is crucial to understand the molecular mechanism underlying the electrophysiological

Melatonin regulates anionic and cationic selective channels by multiple pathways, at different doses and time-dependent responses. It is important to remember the wide spectrum of action this molecule has. For example, results regarding the pathophysiology of lung fibrosis show that volume-regulated anion

#### *Melatonin for a Healthy Heart Rhythm DOI: http://dx.doi.org/10.5772/intechopen.91447*

*Melatonin - The Hormone of Darkness and Its Therapeutic Potential and Perspectives*

Melatonin membrane receptors can exist as monomers, as well as dimers. The MT1 homodimer forms 3- to 4-fold higher proportion than the MT2 homodimer and the MT1/MT2 heterodimer. Nonmammalian vertebrates present a third lowaffinity receptor termed Mel1c, and a proposed mammalian homologous is the orphan receptor GPR50 [74, 77–79]. This orphan lost its properties to directly interact with melatonin but shows an inhibitory interaction with MT1 receptors by forming heterodimers. More recently, other orphans unable to bind melatonin like GPR61, GPR62, and GPR135 showed a similar indirect inhibitory interaction with MT2 receptors [80]. Other G-protein-coupled receptors like the serotonin receptor 5HT2c can interact with melatonin membrane receptors [79]. These interesting interactions of membrane receptors are not further discussed in this chapter but should be considered in future electrophysiological studies with melatonin.

The MT1 and MT2 inhibit adenylate cyclase-protein kinase A-CREB signaling in target cells by pertussis toxin-sensitive Gαi, β, and γ and toxin-insensitive Gq, β, and γ proteins [74, 79]. The MT1 also increases phosphorylation of mitogenactivated protein kinase 1/2 (MAPK) and extracellular signal-regulated kinase 1/2 (ERK), as well as increasing potassium conductance through inwardly rectifying (Kir3.x) channels. The later effect on potassium channels could be relevant to heart electrophysiology since Kir3.x channels are highly expressed in cardiomyocytes and usually coupled to acetylcholine and adenosine membrane receptors [81]. MT2 melatonin receptor activation inhibits both forskolin-stimulated cAMP production and cGMP formation, activates protein kinase C (PKC) in the nervous system, and decreases calcium-dependent dopamine release in the retina. Native functional MT1/MT2 heterodimers in mouse rod photoreceptors mediate melatonin's enhancement of scotopic light sensitivity through phospholipase C and PKC pathways [82]. Several compounds interact with MT1 and MT2 receptors, but blocker luzindole is the only with proven myocardial electrophysiological effects [83]. Luzindole and 4P-PDOT competitively block MT1 melatonin receptors at concentrations higher than 300 nM, and both are inverse agonists in systems with constitutively active

Melatonin interacts with several enzymes and intracellular proteins. The MT3 receptors is a quinone reductase 2 with an affinity in the nanomolar ranges [84]. This enzyme is possibly involved in the regulation of cellular oxidative status, although the exact regulatory action of melatonin remains unclear [84–87]. Furthermore, the electrophysiological effects of MT3 have not been reported yet. Melatonin interacts with intracellular proteins such as calmodulin, calreticulin, or tubulin [88]. The low-affinity interaction between melatonin and calmodulin antagonizes the binding of Ca2+ and may be involved in its antioxidant action as

Melatonin increases the cytoplasmic levels of the heat shock protein 70 in several tissues including the heart [97–102]. Further interaction with this chaperon will be

Melatonin is a ligand for the retinoid-related orphan nuclear hormone receptor family (RZR/ROR) [74, 79]. RZR/RORα is expressed in a variety of organs, whereas RZRβ is specific for the brain and retina [33]. ROR/RZR has been proposed to work in coordination with the plasma membrane receptors MT1/MT2 to regulate gene expression. We suggest a potential interaction with Vitamin D receptor (VDR),

Mitochondria are critical for cellular metabolism and energy production. They maintain life but also are gatekeepers of cell death [31, 104]. Mitochondria

well as other electrophysiological signaling processes [89–96].

which was elegantly confirmed in recent experiments [97, 103].

*1.1.3 Melatonin improves mitochondrial functions*

**52**

MT1 receptors [74, 79].

described in Section 3 of the chapter.

produce up to 95% of the cellular energy in the form of ATP in aerobic cells [105]. Mitochondrial oxidative phosphorylation uses a system of oxidoreductase protein complexes (complexes I, II, III, and IV) to transfer electrons during ATP production. Deficiencies in the electron transport chain can result in the leakage of electrons and generate ROS/RNS [40, 41, 106, 107]. Oxidative stress decreases respiratory complex activity, impairs electron transport system, and opens the mitochondrial permeability transition pores leading to cell death [104, 106, 108].

Mitochondria are essentials for the protective actions of melatonin [51, 97, 106, 107, 109–111]. The mechanisms involved include its antioxidant properties and the preservation of complex I and III functions, inhibition of the opening of the permeability transition pores, and the release of cytochrome c. Petrosillo et al. demonstrate that melatonin prevents the opening of the mitochondrial permeability transition pores and its deleterious consequences [51, 110, 112, 113]. We recently reported that melatonin prevents mitochondrial edema, dilation of the ridges, high activity of NADPH oxidase, and apoptosis [97]. Melatonin improves mitofusin-2, which preserves the mitochondrial functional network and prevents apoptosis [114]. The reduction of mitochondrial damage in the heart could be related to the negative regulation of angiotensin II type 1 receptor (AT1) by melatonin [97]. The induction of Hsp70 through melatonin is compatible with an additional mechanism related to Tom 70, a translocase of the outer mitochondrial membrane [97, 115, 116]. The interaction of Hsp70 with Tom 70 initiates mitochondrial import processes [116]. Tom 70 regulates melatonin-induced cardioprotection by preventing mitochondrial deterioration and oxidative stress [97, 115].

Melatonin's cardioprotection associates with an increase in the number of mitochondria and positive regulation of survival genes such as nicotinamide phosphoribosyl transferase and nicotinamide adenine dinucleotide-dependent deacetylases, called sirtuins [117]. Particularly sirtuin-1 and sirtuin-3 are downstream mediators of the cardioprotective actions of melatonin. Sirtuin-1can modulate fatty acid oxidation, apoptosis, oxidative stress, and autophagy through deacetylation of transduction factors like NF-κB, forkhead box class O, p53, peroxisome proliferatoractivated receptor alpha, thioredoxin-1, and Bcl-xL [117–121]. Sirtuin-3 is a family member that is primarily located in the mitochondria and protects against inflammation and diseases related to oxidative stress. Melatonin elevates sirtuin-3, stimulates superoxide dismutase activity, and suppresses mitochondrial oxidative stress [31, 117, 122, 123]. Additionally, melatonin protects nuclear and mitochondrial DNA [122, 124, 125]. The multiple actions of melatonin provide potent protection against mitochondrial-mediated lesions.

## *1.1.4 Melatonin modulates ion channels*

Melatonin exerts its electrophysiological effects by multiple mechanisms. One of the ways for melatonin to interact is through the modulation of ion channels. Whether we consider its role as a drug or as a biological molecule, it should be taken into account how melatonin has been considered an electrophysiological modulator for many physiological and clinical conditions such as control of circadian rhythms, regulation of arterial blood pressure and heart rate in mammals, sleep processes, and antiaging, among others. Its role in the modulation of several ion channels is crucial to understand the molecular mechanism underlying the electrophysiological properties as an antiarrhythmic.

Melatonin regulates anionic and cationic selective channels by multiple pathways, at different doses and time-dependent responses. It is important to remember the wide spectrum of action this molecule has. For example, results regarding the pathophysiology of lung fibrosis show that volume-regulated anion currents do not respond to acute exposure of cells to melatonin in hypotonic solutions [126]. However, when cells are pre-incubated with melatonin concentrations from 1 to 100 μM for 30–60 min, the anionic currents in response to hypotonicity are blunted in a dose-dependent manner. These time- and dose-dependent responses could support the electrophysiological effect during regional ischemia after 20–30 minutes of melatonin exposure in isolated rat hearts, because during ischemia cardiomyocyte swelling activates anionic currents, and melatonin downregulation of these currents is a potential explanation [127, 128]. Additionally, these MT receptor interactions described in fibroblast deserve further evaluation in myocardial tissue.

From the perspective of the interaction between melatonin and its target, it will be crucial to increase the knowledge about the allosteric contact between melatonin and an ion channel. For example, melatonin blocks the potassium channels (Kv1.3) in a reversible manner through the interaction with different binding sites on the human peripheral blood T lymphocytes [129]. However, the inhibitory effects require high extracellular melatonin in the mM range [129]. Cardiomyocytes do not express this specific potassium channel, but a homologous mechanism can exist for other channels waiting to be reported.

Most of the information regarding the role and effect of melatonin in the organisms has been described in the nervous system. One of the most popular is melatonin-related circadian rhythm. In particular, how melatonin influences circadian phase and electrical activity thanks to the interaction with Kir3.x channels presents them as a therapeutic target for diseases related to circadian disruption and melatonin signaling features [130]. In addition, the effects of melatonin in this pacemaker of circadian rhythm could be due also to its modulation of inwardly rectifying potassium channels (Kir3.1/Kir3.2) via MT1 receptors [131]. Moreover, melatonin is also necessary for circadian regulation of sleep. This effect was described to be driven by the suppression of GABAergic neurons by melatonin in the lateral hypothalamus (crucial function for wakefulness), via interaction with MT1 receptor in order to inactivate hyperpolarization-activated cyclic nucleotidegated channels [132].

Melatonin is a potential neuroprotective molecule thanks to its interaction in a mitochondrial pathway involving the closing of permeability transition pore and opening of ATP sensitive potassium channels (KATP) [133]. The opening of KATP contributes to melatonin antiseizure effect [134]. The preventive actions on the permeability transition pore have been reported in myocardial tissue as well [51, 112, 113]. However, opening of KATP channels with high concentrations of melatonin could be proarrhythmic [135, 136].

Melatonin modulates most of the voltage-activated calcium channel subtypes (L, P, Q, N, and R) with different effects [137–141]. Melatonin inhibits voltagedependent calcium entry in cultured rat dorsal root ganglia neurons, regulates calcium entry into pineal cells, and has dose-dependent inhibitory effects on free [Ca2+]i in mouse brain cells [137]. Melatonin has no effect on voltage-activated calcium channels in cultured human aortic smooth muscle cells [141]. Melatonin accutelly increase L type calcium currents in chick cardiac membranes [140, 141]. An early study shows that melatonin downregulates voltage-sensitive calcium channels in the heart [142]. These results indicate that melatonin may have differnt acute and chronic implications for normal cardiac physiology and for the pharmacological manipulation of the heart [142].

Melatonin mediates vasodilation of cerebral arteries through the activation of large-conductance Ca2+-activated K+ (BKCa) channels via both melatonin receptordependent and melatonin receptor-independent modes, increasing BKCa channel current density but not the KV channel current density [143]. Small-conductance

**55**

*Melatonin for a Healthy Heart Rhythm*

channels in their hippocampi [144].

change KCNQ and KCNH2 gene expression profiles [145].

in the case of tachyarrhythmia and slower in bradyarrhythmia.

Arrhythmias also increase with age and heart diseases [146–148].

ent risk factors would be one of the explanations [158].

and chronic antiarrhythmic effects of melatonin.

**1.2 Electrophysiology and arrhythmias**

*DOI: http://dx.doi.org/10.5772/intechopen.91447*

Ca2+-activated K (SK) channels are also modulated by the action of melatonin [144]. Upregulation of SK channels plays a role in memory loss and indicates that melatonin reverses memory deficits in rats by downregulation of SK1, SK2, and SK3

Additional information was brought about KCNQ from the aorta and related with vascular tone, and KCNH2 in the left ventricle was associated with QT duration in rats where melatonin was able to prevent the increase in blood pressure and

Melatonin effects on connexin proteins will be extensively analyzed in the second and third sections of the chapters for its proven relationship with both acute

The heart pumps blood under a synchronized electrical control. Arrhythmias are the electrical problems in the rhythm of the heart. The heartbeats may be faster

Fatal arrhythmic events follow a circadian pattern [2]. Arrhythmogenesis decreases during nighttime when the melatonin levels increase 30 to 70 folds. Lifethreatening cardiac arrhythmias (ventricular tachycardia, ventricular fibrillation, and sudden cardiac death) are more likely to occur in the morning after waking.

Disturbances in membrane excitability or conduction cause arrhythmias. Excitability manifests as action potentials and involves coordinated ion movements across the cell membrane through ion channels, exchangers, and ATPases [149]. Conduction is the propagation of bioelectrical signal throughout the heart. Action potentials automatically originate at the sinoatrial node, spread to the atria, and, after a small delay in the atrioventricular node, rapidly and synchronously activate the ventricles via the His-Purkinje system. Action potentials propagate from cell to cell using low-resistance pathways known as gap junctions. Connexin proteins assemble into intercellular channels at gap junctions. Connexin 43 (Cx43) is the most abundant connexin in the heart [150]. Gap junctions couple the cells and allow the flow of electrical current and small molecules. The largest accumulation of connexins occurs in a specialized structure at the ends of cardiomyocytes called intercalated discs. Cardiac propagation is anisotropic, particularly more rapid in the longitudinal direction of the cell than in the transverse direction. The lateral borders of the myocytes usually show variable amount gap junctions depending on age or disease. Cardiovascular diseases are the leading cause of death in the world [151]. Most deaths occur suddenly [152]. Catastrophic sudden death events motivate us to search for causes and possible solutions [153]. This is a great scientific and social health challenge. The approaches of recent years have reduced the burden of cardiovascular disease, but there is still much to improve [154]. A case occurs with arrhythmias. The rhythm disorders motivated emergency interventions, especially during the first hour of the manifestation of coronary heart disease. Cardiopulmonary resuscitation, ambulances, and cardiodefibrillation were response strategies to unexpected events. Unfortunately, they are still unexpected due to the limited understanding of the causes at a level that would allow us to predict, avoid, or control the occurrence of an event [155]. In that sense, the strategies that attempt to determine risks grew in order to establish a more efficient direction of interventions [156, 157]. Today they allow us to expect more lethal events in severely ill people. However, risk factors are still far from being effective and much less efficient. The changes that occur in physiology as a result of exposure to differ-

#### *Melatonin for a Healthy Heart Rhythm DOI: http://dx.doi.org/10.5772/intechopen.91447*

*Melatonin - The Hormone of Darkness and Its Therapeutic Potential and Perspectives*

in myocardial tissue.

gated channels [132].

other channels waiting to be reported.

nin could be proarrhythmic [135, 136].

manipulation of the heart [142].

large-conductance Ca2+-activated K+

currents do not respond to acute exposure of cells to melatonin in hypotonic solutions [126]. However, when cells are pre-incubated with melatonin concentrations from 1 to 100 μM for 30–60 min, the anionic currents in response to hypotonicity are blunted in a dose-dependent manner. These time- and dose-dependent responses could support the electrophysiological effect during regional ischemia after 20–30 minutes of melatonin exposure in isolated rat hearts, because during ischemia cardiomyocyte swelling activates anionic currents, and melatonin downregulation of these currents is a potential explanation [127, 128]. Additionally, these MT receptor interactions described in fibroblast deserve further evaluation

From the perspective of the interaction between melatonin and its target, it will be crucial to increase the knowledge about the allosteric contact between melatonin and an ion channel. For example, melatonin blocks the potassium channels (Kv1.3) in a reversible manner through the interaction with different binding sites on the human peripheral blood T lymphocytes [129]. However, the inhibitory effects require high extracellular melatonin in the mM range [129]. Cardiomyocytes do not express this specific potassium channel, but a homologous mechanism can exist for

Most of the information regarding the role and effect of melatonin in the organisms has been described in the nervous system. One of the most popular is melatonin-related circadian rhythm. In particular, how melatonin influences circadian phase and electrical activity thanks to the interaction with Kir3.x channels presents them as a therapeutic target for diseases related to circadian disruption and melatonin signaling features [130]. In addition, the effects of melatonin in this pacemaker of circadian rhythm could be due also to its modulation of inwardly rectifying potassium channels (Kir3.1/Kir3.2) via MT1 receptors [131]. Moreover, melatonin is also necessary for circadian regulation of sleep. This effect was described to be driven by the suppression of GABAergic neurons by melatonin in the lateral hypothalamus (crucial function for wakefulness), via interaction with MT1 receptor in order to inactivate hyperpolarization-activated cyclic nucleotide-

Melatonin is a potential neuroprotective molecule thanks to its interaction in a mitochondrial pathway involving the closing of permeability transition pore and opening of ATP sensitive potassium channels (KATP) [133]. The opening of KATP contributes to melatonin antiseizure effect [134]. The preventive actions on the permeability transition pore have been reported in myocardial tissue as well [51, 112, 113]. However, opening of KATP channels with high concentrations of melato-

Melatonin modulates most of the voltage-activated calcium channel subtypes (L, P, Q, N, and R) with different effects [137–141]. Melatonin inhibits voltagedependent calcium entry in cultured rat dorsal root ganglia neurons, regulates calcium entry into pineal cells, and has dose-dependent inhibitory effects on free [Ca2+]i in mouse brain cells [137]. Melatonin has no effect on voltage-activated calcium channels in cultured human aortic smooth muscle cells [141]. Melatonin accutelly increase L type calcium currents in chick cardiac membranes [140, 141]. An early study shows that melatonin downregulates voltage-sensitive calcium channels in the heart [142]. These results indicate that melatonin may have differnt acute and chronic implications for normal cardiac physiology and for the pharmacological

Melatonin mediates vasodilation of cerebral arteries through the activation of

dependent and melatonin receptor-independent modes, increasing BKCa channel current density but not the KV channel current density [143]. Small-conductance

(BKCa) channels via both melatonin receptor-

**54**

Ca2+-activated K (SK) channels are also modulated by the action of melatonin [144]. Upregulation of SK channels plays a role in memory loss and indicates that melatonin reverses memory deficits in rats by downregulation of SK1, SK2, and SK3 channels in their hippocampi [144].

Additional information was brought about KCNQ from the aorta and related with vascular tone, and KCNH2 in the left ventricle was associated with QT duration in rats where melatonin was able to prevent the increase in blood pressure and change KCNQ and KCNH2 gene expression profiles [145].

Melatonin effects on connexin proteins will be extensively analyzed in the second and third sections of the chapters for its proven relationship with both acute and chronic antiarrhythmic effects of melatonin.

### **1.2 Electrophysiology and arrhythmias**

The heart pumps blood under a synchronized electrical control. Arrhythmias are the electrical problems in the rhythm of the heart. The heartbeats may be faster in the case of tachyarrhythmia and slower in bradyarrhythmia.

Fatal arrhythmic events follow a circadian pattern [2]. Arrhythmogenesis decreases during nighttime when the melatonin levels increase 30 to 70 folds. Lifethreatening cardiac arrhythmias (ventricular tachycardia, ventricular fibrillation, and sudden cardiac death) are more likely to occur in the morning after waking. Arrhythmias also increase with age and heart diseases [146–148].

Disturbances in membrane excitability or conduction cause arrhythmias. Excitability manifests as action potentials and involves coordinated ion movements across the cell membrane through ion channels, exchangers, and ATPases [149]. Conduction is the propagation of bioelectrical signal throughout the heart. Action potentials automatically originate at the sinoatrial node, spread to the atria, and, after a small delay in the atrioventricular node, rapidly and synchronously activate the ventricles via the His-Purkinje system. Action potentials propagate from cell to cell using low-resistance pathways known as gap junctions. Connexin proteins assemble into intercellular channels at gap junctions. Connexin 43 (Cx43) is the most abundant connexin in the heart [150]. Gap junctions couple the cells and allow the flow of electrical current and small molecules. The largest accumulation of connexins occurs in a specialized structure at the ends of cardiomyocytes called intercalated discs. Cardiac propagation is anisotropic, particularly more rapid in the longitudinal direction of the cell than in the transverse direction. The lateral borders of the myocytes usually show variable amount gap junctions depending on age or disease.

Cardiovascular diseases are the leading cause of death in the world [151]. Most deaths occur suddenly [152]. Catastrophic sudden death events motivate us to search for causes and possible solutions [153]. This is a great scientific and social health challenge. The approaches of recent years have reduced the burden of cardiovascular disease, but there is still much to improve [154]. A case occurs with arrhythmias. The rhythm disorders motivated emergency interventions, especially during the first hour of the manifestation of coronary heart disease. Cardiopulmonary resuscitation, ambulances, and cardiodefibrillation were response strategies to unexpected events. Unfortunately, they are still unexpected due to the limited understanding of the causes at a level that would allow us to predict, avoid, or control the occurrence of an event [155]. In that sense, the strategies that attempt to determine risks grew in order to establish a more efficient direction of interventions [156, 157]. Today they allow us to expect more lethal events in severely ill people. However, risk factors are still far from being effective and much less efficient. The changes that occur in physiology as a result of exposure to different risk factors would be one of the explanations [158].
