Different Ion Channels behind One Pathology

#### **Chapter 1**

## Ion Channels and Neurodegenerative Disease Aging Related

*Marika Cordaro, Salvatore Cuzzocrea and Rosanna Di Paola*

#### **Abstract**

Many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, and age-related disorders are caused due to altered function or mutation in ion channels. Ion channels are important in maintaining cell homeostasis because they affect membrane potential and play a critical role in neurotransmitter secretion. As a result, it appears that a potential antiaging therapy strategy should consider treating multiple diseases at the same time or focusing on identifying a common target among the biological processes implicated in aging. In this chapter, we will go over some of the fundamental ideas of ion channel function in aging, as well as an overview of how ion channels operate in some of the most common aging-related disorders.

**Keywords:** aging, ion channels, neurodegeneration, therapeutic targets

#### **1. Introduction**

Aging is a natural part of life that comprises both physical and mental changes. In distinct organs, aging occurs at molecular, cellular, and histological levels, including in the central nervous system (CNS) and specifically in the brain [1, 2]. The molecular, chemical, and physical properties of neurons change as we become older, resulting in memory loss, altered behaviors, loss of cognition functions, dementia, and reduced immunological responses. In addition, aging is a major risk factor for neurological diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and others. Although the basic reasons of aging are unknown, there is widespread agreement that its etiology is multifaceted [3]. Aging theories can be classified into two categories: those that explain aging as the outcome of damage accumulation and those that explain aging as the result of controlled death processes [4]. It is likely that the interaction of these two basic systems influences the aging process, albeit there is a lot of variation across people. Two of the most accredited molecular alteration involved in brain aging are inflammation and oxidative stress that, when happen lead to cells failure. Different studies reported that reactive oxidative species (ROS), and subsequent oxidation of proteins, involved also ion channels [5].

Ion channels are integral membrane proteins that allow the passive diffusion of ions across membranes [2]. In neurons and other excitable cells, the harmonious coordination between the numerous types of ion channels shapes and propagates electrical signals [6]. Understanding the biology of aging mechanisms is essential to the pursuit of brain health. The ability to stratify senior populations and forecast clinical trajectories in pre-symptomatic adult groups could be critical to the future of aging research [4]. In this chapter, we will discuss about the role of ion channels in the brain during aging with particular attention on neurodegenerative disease age-related. Additionally, we will consider if ion channels could be used as future therapeutic targets to decelerate brain aging and age-related pathologies.

#### **2. Brain aging: from physiological to pathological**

Scientists have been debating the meaning of aging for a long time. Many people regard aging as an illness in and of itself, while others see it as the gradual loss of function that increases the risk of developing age-related diseases. Scientists view aging as an adaptation to lifelong events, and interventions should support the physiological balance during age-related adaptation, response to acute stress, to avoid disease onset. Adapted capacity in most organs has been shown to occur from the third and fourth decades of life [4]. Aging is a complicated and multifaceted condition marked by a steady decline in physiological and behavioral abilities. Aging happens in all organs at all levels, in the brain [2]. The molecular, chemical, and physical properties of neurons change as we become older, resulting in memory loss, altered behaviors, loss of cognition functions, dementia, and reduced immunological responses. Rather than significant rates of neuron loss, brain aging has been linked to subtle changes in the structure and function of neurons in specific neural circuits. The aging brain compensates for the loss of neurons by growing dendritic arbors and synaptic connections. Dendritic arbors and synaptic connections are lost in the brain in agerelated neurodegenerative disorders. As a result, it is unable to compensate for the loss of neurons [7]. Synaptic degeneration, dendritic regression in pyramidal neurons, deposition of fluorescent pigments, cytoskeletal abnormalities, a reduction of striatal dopamine receptors, and astrogliosis and microgliosis are all prevalent features of brain aging in mammals [8]. Despite the discovery of brain aging characteristics in multiple neural networks, the chemical pathways responsible remain unknown [9]. Oxidative stress, inflammation, and ion channel failure are the most widely accepted theories for the development of age-related neurodegenerative diseases [10].

#### **2.1 Oxidative stress in brain**

In the 1950s, Harman's free radical theory of aging suggested that reactive oxygen and nitrogen species (ROS and RNS) cause oxidative damage in cellular macromolecules, including DNA, proteins, and lipids, leading to decreased biochemical and physiological function through aging [11]. The changes in phospholipid composition show that ROS-induced lipid peroxidation occurs in the brains of elderly humans and animals with CNS dysfunction, such as cognitive impairment. Furthermore, increased formation of malondialdehyde (MDA) in the brain has been postulated as a symptom of aging [12]. Superoxide anions produced by the respiratory chain and various oxidases, hydroxyl radical created by the hydrogen peroxide interaction with Cu<sup>+</sup> or Fe2+, and NO produced in response to elevated intracellular Ca2+ levels are

#### *Ion Channels and Neurodegenerative Disease Aging Related DOI: http://dx.doi.org/10.5772/intechopen.103074*

just two of the most common examples of ROS in neurons [13]. During brain aging, enhanced ROS generation and decreased antioxidants result in redox imbalance, causing age-related disorders. NO-dependent oxidative damage promotes apoptosis in motor neurons. It causes vascular cognitive impairment through the aging of the cerebral cortex [14]. The action of several enzymatic and non-enzymatic systems with cellular detoxification functions, collectively referred to as antioxidants, mediates the hemostasis of intracellular ROS and RNS. The nuclear factor erythroid 2-related factor 2 (Nrf- 2) is the main transcription factor and one of the primary regulators of the antioxidant signaling, such as transcription of endogenous antioxidant enzymes including glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), and heme oxygenase-1 (HO-1). This antioxidant system is collectively the primary defense system that neutralizes ROS generation inside the cells [15]. Additionally, another major antioxidant defense complexes are the heat-shock response (HSR), a cellular response that elevates the number of molecular chaperones to diminish the adverse effects on proteins caused via stressors, oxidative stress, increased temperatures, and heavy metals. Increased stress tolerance and cellular protection against neuronal injury can be achieved by activating heat-shock protein (HSP) synthesis [16]. As a result, in metabolic disturbances such as age-related neurodegenerative diseases and aging, the heat-shock response plays a critical role in creating a cytoprotective environment [2].

#### **2.2 Inflammation in brain**

Another key pathway directly involved in brain aging is represented by inflammation. The immune system is one of the most pivotal protective physiological systems of the organism [17]. Immunosenescence is a concept that describes how aging affects the immune system's function [18]. The participation of senescent cells in host immunity is associated with the release of pro-inflammatory cytokines. This phenomenon is defined as senescence-associated secretory phenotype (SASP). Due to SASP's pro-inflammatory tendency, cellular senescence in various organs and tissues significantly increases inflammation in the aged [19]. NF-κB in response to oncogenic stress and DNA damage initiates the transcription of a host of genes including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, IL-1β over stimulating SASP [20]. NF-κB is a transcription factor that is induced by inflammatory mediators and reactive oxygen species (ROS) and contributes to both detrimental and protective responses, depending on the types of induction that lead to the co-activation of distinct pathways. In addition, NF-kB activates genes that control cell survival, specialization, inflammatory processes, proliferation, and apoptosis [20]. It has been shown that the age-induced increase of pro-inflammatory markers (CRP, IL-6, IL-1β, TNF-α) is associated with cognitive decline [21]. Microglia are the brain resident macrophages providing its innate immune defense. Microglia, a kind of glial cell, arise from erythro-myeloid precursors in the yolk sac, which inter the CNS during development [22, 23]. In the neurological system, microglia play two roles. Microglia are ramified cells with extremely motile processes that continually scan the brain parenchyma in reaction to hazardous substances, neuronal cell damage, or infections in a healthy adult brain. Microglia have a dual function in the aging process. Microglia, on the one hand, release trophic factors and control cytokines [24, 25]. On the other hand, microglia enhanced amounts of an intricate set of mediators, such as TNFα, TGFβ, and IL1β, which are enhanced in elderly individuals [22, 23]. There has been

evidence of a link between neuroinflammatory activation of microglia and neuronal loss, as well as impaired neurobehavioral function and cognitive impairment. Redox sensors found in receptors, transcription factors, and enzymes provide complex communication with oxidizing agents during neuroinflammation. These variables have an impact on the link between neurons and glia, as well as neuronal function, which leads to neurodegenerative alterations [26, 27]. Microglial cells also express a stimulable type of NOS following activation and produce large quantities of NO, which causes oxidative damage to neurons. In neurodegenerative illnesses and brain aging, improper immune cell activation causes functional impairment and synaptic degeneration; when properly controlled, these same cascades play critical roles in neuronal stress tolerance and neuroplasticity. For instance, TNF-α plays a pivotal role in learning, memory, and synaptic plasticity in the hippocampus [28]. Also, astrocytes may potentially play a role in adapting to age-related neuronal stress. These cells clear glutamate from synapses, produce neurotrophic factors and boost neuronal bioenergetic activity. Aging may decrease these astrocyte activities, hence, exacerbating pathogenic neuroinflammatory processes [28–30]. TNFα activates NF-κB which protects cells against neurotoxicity β-amyloid (Aβ)-induced and this activation is required for neuronal survival. NF-κB also promotes anti-apoptotic responses and protects neurons from ischemia and excitotoxic brain injury [31–36]. Furthermore, through its response to TNF-mediated inflammatory stimuli, NF-κB activation plays a critical role in the start and persistence of inflammation, resulting in the stimulation of various chemokines and cytokines [37–42].

#### **3. Ion channels in the brain: from function to dysfunction**

Ion channels are key components of neurons that are responsible for nerve impulse and synaptic transmission triggering (neurotransmitter's release). These channels are divided into two big classes: (I) voltage-gated (Na+ , K+ , Ca2+, Cl− ) and (II) ligandgated (nicotinic acetylcholine receptors (nAChRs), γ-amino butyric acid (GABA), N-methyl-D-aspartate receptors (NMDARs), ryanodine receptors (RyRs)) that are involved in impulse transmission across the synapses [43]. However, during the last several decades, research has found a number of genetic faults or aberrations in channel-forming genes that are linked to a variety of neurological illnesses, including memory loss, movement difficulties, and neuromuscular disorders [44].

Ion channel protein establishes a pathway for ions such as Na<sup>+</sup> , K+ , Ca2+, and Cl− to flow across the lipid bilayer's impermeable barrier [45]. They are known to play three main important functions in regulating membrane physiology: first of all, they set up membrane potential in cells, in which ion movement across the membrane creates a potential gradient that determines resting potentials and generates action potentials; secondary they are involved in the transmission of electrical signals; they are also involved in maintaining electrolytic balance across the cell membrane to regulate cell volume, and last but not least they play a crucial role in the generation of regulatory signals in the cell [46, 47]. Thanks to alternative splicing, their enormous structural variety from monomeric to heteromeric levels support their large functional diversity. The amplitude and duration of the action potential are shaped differently by each cell type's assembly of ion channels [48, 49]. At the intracellular level, ion channels are also present on the surface of the mitochondria, endoplasmic reticulum, and nuclear membrane [50, 51]. The correct functionality of ion channels is necessary to keep physiological homeostasis in the brain [52].

As a result, ion channels have been implicated in a number of age-related dysfunctions [53]. Because aging is associated with physiological changes in ion channel function, aberrant changes in ionic gradients seem to be the core of age-related deterioration in physiological functioning. With age, functional changes in ion channels lead to clinical phenotypes called channelopathies [54].

#### **3.1 K+ channels**

K+ channels are the most ubiquitous and heteogeneous family of ion channels expressed in excitable and non-excitable cells (an extensive review on this topic can be found in [55]). K+ channels can be divided into four classes: inwardly rectifying K<sup>+</sup> channels (Kir), voltage-gated K<sup>+</sup> channels (Kv), two-pore K+ channels (K2P), and Ca2+-activated K+ channels (KCa) [56]. K+ channels serve an important physiological function in the signaling mechanisms that lead to neurotransmitter release in neurons. They modulate the resting membrane potential, the repolarization phase of the action potential, and the firing frequency to govern neuronal excitability. Given the importance of K+ channels in so many cellular functions, it's no surprise that changes in their activity have been linked to the development of a variety of neurodegenerative diseases [57, 58]. Furthermore, in recent years, it has been demonstrated that the apoptotic process, which is the key mechanism for cell selection and death in the CNS associated with physiological aging as well as a variety of neuropathological disorders, is critically dependent on changes in ion homeostasis within neuronal cells [59]. K+ efflux, which results in a drop in intracellular K<sup>+</sup> concentration, maybe a key cause of apoptosis. In fact, in various neuronal populations undergoing apoptosis, an increase in outward K+ currents have been seen as well as it has been demonstrated that apoptosis has been shown to be inhibited by voltage-gated K+ channel blockers, whereas heterologous production of inwardly-rectifying K+ channels has been shown to increase apoptosis in cultured hippocampus neurons [60].

#### **3.2 Ca2+ channels**

Ca2+ is the major trigger of neurotransmitter release, a process that has been thoroughly investigated over the past decades [61–63]. Moreover, it has also become clear that Ca2+ is essential for a variety of other neuronal functions, including neuronal excitability, integration of electrical signals, synaptic plasticity, gene expression, metabolism, and programmed cell death [64]. Given its central role in processes that are fundamental to the excitable nature of neurons, Ca2+ homeostasis is tightly regulated in these cells. Plasma membrane Ca2+ channels allow the passive influx of calcium ions down their electrochemical gradient. These channels are divided into two groups based on the mechanism that controls their transition between open and closed conformations: voltage-gated Ca2+ channels (VOCC) and ligand-gated Ca2+ channels. The potential contribution of altered Ca2+ homeostasis at least to some aspects of brain aging and neurodegeneration was first put forward by Khachaturian in the 1980s, with the formulation of the "Ca2+ hypothesis of aging" [65–67]. Early findings in the field that corroborated this hypothesis examined the major transport pathways of Ca2+ during aging and found that at least in some types of neurons, such as the principal cells in the hippocampal CA1 region, there is an increased Ca2+ influx mediated by increased VOCC activity in aged neurons [68]. Similarly, Ca2+ extrusion through the ATP-driven plasma membrane Ca2+ pump (PMCA) was found to be decreased in aged neurons [69]. Following that, the attention switched to the

intracellular mechanisms of Ca2+ homeostasis and how they degrade with age. The increased release of Ca2+ from the endoplasmatic reticulum (ER) stores via both the inositol 3-phosphate (InsP3) and ryanodine receptors (RyR) has been confirmed in several investigations, leading to the suggestion that release from the RyR receptor might be a valuable biomarker of neuronal aging [70]. The high influx of calcium ions into the postsynaptic spine appears to be the crucial event leading to the induction of long-term potentiation (LTP), which is relevant to the function of Ca2+ dysregulation in memory loss. Importantly, LTP is inhibited by intracellular Ca2+ chelators, whereas LTP is promoted when the postsynaptic cell is Ca2+-loaded [71]. Therefore, it is well established that a significant elevation of postsynaptic Ca2+ concentration is both necessary and sufficient for the induction of hippocampal LTP [72]. Ca2+ homeostasis changes may be directly responsible for neuronal death in some circumstances. Increased intracellular Ca2+ levels can cause severe abnormalities in neurons, eventually leading to neuronal death and degeneration [73]. This process is often specifically mediated or even initiated by the diminished capacity of mitochondria to buffer Ca2+. Given the basic relevance of Ca2+ homeostasis in the biology of all cells, it's not unexpected that a growing number of studies demonstrate that unregulated Ca2+ plays a role in normal aging as well as a variety of pathological disorders. Given the nervous system's incredible cellular variety, a general message emerging from this research is that Ca2+ signaling and homeostasis in the nervous system should be investigated. The Ca2+ homeostasis mechanism is equally variable across neurons, according to the demands of each neuronal subtype [62]. The intrinsic variations in morphology, connectivity, proteome, and Ca2+ homeostatic mechanism of neurons, taken together, are extremely likely to contribute to the selective sensitivity of diverse neuronal populations to different causes of senescence collectively and synergistically. The more we learn about how Ca2+ homeostatic processes interact with distinct neurons' inherent properties, the closer we will be to devising cell-specific therapeutics [62].

#### **3.3 Na<sup>+</sup> channels**

Voltage-gated sodium channels (Nav channels) are fundamental for the origination and transmission of signals in electrically excitable tissues. Na+ channels are abundant in neurons and glia throughout the central nervous system and peripheral nervous system (PNS) [74]. The genesis of neurological disorders, including as idiopathic epilepsy, ataxia, and pain sensitivity, is heavily influenced by mutations in genes encoding Na<sup>+</sup> channels [75]. This is most likely due to changes in the synthesis and/or trafficking of Nav channels, which modify their surface expression and impact the neuron's electrical excitability even while the channel's conducting properties remain unchanged. Changes in the function of voltage-gated Na+ channels have been observed during the aging process [76]. These alterations were attributed to an age-related reduction in excitability, which is controlled by voltage-gated Na<sup>+</sup> channels. Furthermore, age-related changes in voltage-gated Na<sup>+</sup> channel activity have been proposed as a possible explanation for the decreased excitability seen in skeletal muscle fibers of old rats [77]. Considering the fundamental role of Nav channels in the modulation of neuronal responses during pathophysiological conditions, and the fact that RNS and ROS may play a role in neurodegenerative events, the study of Nav channel modulation by these free radical species assumes a particular pathophysiological relevance. Recent evidence shows that oxidant-induced alterations in the characteristics of Nav channels may play a role in membrane excitability and conductance modulation. Na+ currents were also elevated when NOS was inhibited or

*Ion Channels and Neurodegenerative Disease Aging Related DOI: http://dx.doi.org/10.5772/intechopen.103074*

NO• was scavenged by hemoglobin and ferrous diethyl thiocarbamate. These findings suggest that RNS may act as autocrine regulators of Na<sup>+</sup> currents in these neurons, inhibiting them. NO•, on the other hand, could potentiate the inactivation resistant Nav channels currents (INaP) seen in hippocampus neurons and posterior pituitary nerve terminals [1, 13, 78]. The current carried by these channels appears to be increased not only by the significant rise in NO• levels evoked by NO• donors but also by the lesser increase triggered by constitutive NOS activation [1, 13]. In hippocampal and pituitary neurons, NO• can cause an increase in Na<sup>+</sup> currents, but it has the reverse effect in peripheral neuronal cells. As a result, it appears that NO• might have either neuroprotective or neurodegenerative effects due to its dual effects on various neuronal sodium channel populations. These effects are probably due to the variety of Nav channel subtypes expressed in the CNS and PNS.

#### **4. Ion channels and neurodegeneration**

Ion channel deficiencies and/or mutations relate to many forms of neurological diseases. Na+ , K+ , Ca2+, and Cl− channel subtypes, for example, have been connected to the pathophysiology of dyskinesia, seizures, epilepsy, and ataxia [79]. Following we briefly discuss the role of ion channel modification in the most common neurodegenerative disorders age-related.

#### **4.1 Ion channels and Alzheimer's disease**

AD is a kind of dementia marked by cognitive impairment, memory loss, and neuronal death. A buildup of Aβ peptides, tau hyperphosphorylation, and mutations in the catalytic domain of γ secretase are all elements that contribute to the disease's focused characteristic. The ionic imbalance has been linked to AD development, and in particular an aberrant intracellular concentration of Ca2+, Na+ , K+ , and Cl− [80]. Hardy and Higgins were the first to show that Aβ peptides disrupt Ca2+ homeostasis in neurons and increase intracellular Ca2+, which Mattson and his colleagues later validated [81]. Currently, multiple investigations have established the base for a novel concept: Aβ peptide is dangerous to neurons in part because it forms abnormal ion channels in neuronal membranes, disrupting neuronal homeostasis [82–87]. Normally, an influx of Ca2+ ions is strictly controlled, evoking the release of neurotransmitters like glutamate from presynaptic terminals and triggering downstream signals that regulate cellular processes including synaptogenesis, synaptic transmission, synaptic plasticity, neuronal development, and survival. However, in AD, Ca2+ flux is disrupted as a result of increased oxidative stress and disrupted energy metabolism, which affects the glutamate receptor, glucose transporters, and ion-motive ATPases' normal function [88]. In the hippocampus and cortex region in the brain, for instance, accumulated Aβ has been found to elevate the cellular Ca2+ ion level by plasma membrane L-type Ca2+ channels and Na+ /K+ - ATPase activity causing extreme excitatory responses, i.e., glutamate excitotoxicity and neuronal mortality [89]. Presenilin-1, the catalytic subunit of γ secretase, is also identified to be responsible for leaking Ca2+ ions from the endoplasmic reticulum to the cytoplasm via Ca2+ leak channels, increasing the cellular burden of Ca2+ ion in the AD brain [90]. Additionally, new research has revealed that transient receptor potential (TRP) channels impair Ca2+ homeostasis in Alzheimer's disease. Thus, elevated intracellular Ca2+ ion alters amyloid-β precursor protein (AβPP) processing and influences various downstream pathways, including tau metabolism, housekeeping gene suppression, and autophagic function loss, worsening the symptoms of AD [91]. K+ channel abnormalities have also been identified in AD patients. Because the K<sup>+</sup> channel is essential for the formation of action potentials and the maintenance of the resting potential, any blockage causes poor neurotransmission and neuronal injury. Furthermore, accumulating Aβ has been found in hippocampus neurons to suppress voltagedependent fast-inactivating K<sup>+</sup> currents [92]. Moreover, Kv1.3, Kv1.5, KCNN4/KCa3.1 respectively voltage-gated K+ channels and calcium-activated K+ channel, have been reported to induce neurodegeneration in response to neuroinflammation caused by Aβ peptides via microglial activation [93]. Similarly, the Kv3 subfamilies of K+ channel subunits, which can rapidly repolarize the action potential, have been reported to be impaired and downregulated in AD [94]. As a result of the increased K<sup>+</sup> channel activity, intracellular Ca2+ overload occurs, leading to altered neuronal excitability and perhaps neuronal death [95]. On the plasma membrane of activated microglial cells in the hippocampus of mild AD patients, a novel intracellular chloride channel 1 (CLIC1) was recently discovered. CLIC1 channels become strongly expressed after Aβ stimulation of microglia and are responsible for the change in membrane anion permeability of the cell, resulting in neuronal death [96]. In addition to these channels, nAChR also plays a key role in the AD brain because cholinergic depletion may raise the production of Aβ and exacerbate its neurotoxicity through an alteration of the signal transduction events combined with cholinergic neurotransmission [97]. Additionally, the expressions of nAChR subtypes, are described to be highly expressed in AD-affected brain regions, thereby suggesting a role of these receptors in the AD etiopathology [98]. Tan and colleagues reviewed different calcium channel blockers dihydropyridines, benzothiazepines, and phenylalkylamines [99]. Moreover, Wiseman and Jarvik also reviewed different patents on potassium channel blockers or activators as possible therapies against AD such as 2-(phenylamino) benzimidazole, 2-amino benzimidazole derivatives, bis-benzimidazoles & related compounds, and many others [100]. Last but not least, as possible sodium channel blockers with useful property against AD, Shaikh and colleagues propose Aptiom (eslicarbazepine acetate) [101]. Unfortunately, we are still a long way from real AD therapy.

#### **4.2 Ion channels and Parkinson's disease**

After AD, PD is the most prevalent brain disease, affecting 1% of the elderly population (60–65 years). It is characterized by bradykinesia, postural instability stiffness, and resting tremor. PD pathogenesis is caused by a number of variables, including activities linked to cellular Ca2+ excess, mitochondrial malfunction, oxidative or metabolic stress, and, in particular, a small number of neurotoxins that render neuronal cells more susceptible to cell death [102]. For example, the ATP-sensitive potassium channel Kir6.2, which induces excitotoxicity, is abundantly expressed in dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and has been linked to disease development [103]. Similarly, mutations in the Kir3.2 channel render it nonselective, producing an increase in the conduction of Na+ ions as a replacement for highly selective K<sup>+</sup> ions, resulting in the loss of cerebellar cells and DA neurons in the SNc [55]. An important study by Sheih and colleagues also demonstrate that Kir3.1 and Kir3.2 are involved in the direct degeneration of DA neurons in the PD brain. Similarly, also voltage-gated T-type Ca2+ channels (TTCCs), Ca2+−sensitive voltage-gated A-type K+ channels, voltage-gated LTCCs (L-type Ca2+ channels), and ATP-sensitive K<sup>+</sup> (K-ATP) channels contribute toward basal ganglia dysfunction

*Ion Channels and Neurodegenerative Disease Aging Related DOI: http://dx.doi.org/10.5772/intechopen.103074*

in SNc DA neurons thereby leading to progressive loss of neuronal firing, thus causing PD [55]. In addition to a subset of medial SNc DA neurons, K-ATP channel activation aided the transition from tonic firing to NMDAR-mediated bursting in vivo, resulting in phasic DA release. When glutamate binds to the receptor, it causes the NMDAR channel to open, allowing Ca2+ to flow into the cell. As a result, any changes in glutamate transmission generate dyskinesias in people with PD [104]. Recently, it was discovered that a new ion channel, the Hv1 proton channel, is expressed in human brain microglia and immune tissues and that it is required for NADPH oxidase superoxide generation during the respiratory burst in phagocytic leukocytes, which can lead to neurodegeneration such as PD [105]. Also, in this case, different studies proposed ion channel modulators against PD such as 4-amino-7-chloroquinoline, Safinamide, verapamil (phenylalkylamine), and diltiazem (benzothiazepine) [106, 107].

#### **4.3 Ion channels and Huntington's disease**

HD is a hereditary neurodegenerative disorder characterized by cognitive loss, emotional imbalance, and uncoordinated movements. It is caused by an autosomal dominant mutation in the Huntingtin (Htt) gene responsible for the expansion of CAG trinucleotide repeat >36 that leads to the synthesis of polyglutamine tract, thus mutated HTT (mHTT) protein is prone to aggregation and found to form intracellular accumulations in different cell types [108]. Using mouse models, Tong et al. studied the functional implications of ion channels in several cell types to determine the etiopathology of HD. In mHTT-expressing striatal astrocytes, altered Kir4.1 channel activity impaired extracellular K+ homeostasis, resulting in hyperexcitability, i.e., HD motor symptoms in striatal neurons. However, the normal Kir4.1 channel is one of the most important astrocytal K+ channels, since it is required for cell resting membrane potential and extracellular K+ buffering in the brain [109]. Furthermore, mHTT has been shown to affect the function of high-voltage-activated (HVA) Ca2+ channels in HD [110]. Aside from Ca2+ channel malfunction, also Na+ , K+ , Cl− ion channels have demonstrated lower expression in HD animal models in multiple studies [111]. In striatal neurons of HD transgenic mice models, other researchers discovered the reduced expression of K+ channel subunits. Furthermore, in the R6/2 HD mouse model, expression of the muscular ClC-1 chloride channel is significantly reduced; thus, functional alteration of these channels disrupts ion homeostasis in cortical pyramidal neurons, affecting neurotransmitter release, synaptic integration, and genetic expression, all of which contribute to cortical dysfunction in HD [112, 113]. For HD, different calcium channel modulator has been proposed, such as 6-amino-4-(4-phenoxyphenethyl-amino)quinazoline (EVP4593), Inositol 1,4,5-Trisphosphate (Ip3-sponge), Brilliant Blue G, and others, but the way is still long [114, 115].

#### **4.4 Ion channels in multiple sclerosis and amyotrophic lateral sclerosis**

MS is an immune-mediated central nervous system degenerative condition characterized by progressive demyelination in patches throughout the brain and spinal cord. Loss of coordination, muscle weakness, visual, and lingual problems are common signs of this condition, which affects young people in industrialized cultures the most. The presence of macrophages, T lymphocytes, microglia, and dendritic cells has been associated with inflammatory neuronal injury [116]. Infiltrating lymphocytes and macrophages harm neurons largely by direct cell contact or toxicity mediators such as glutamate or nitric oxide, as well as indirectly through the

loss of oligodendrocytes and myelin sheath. Apart from inflammatory mediators, redistribution of voltage/ligand-gated ion channels and transporters has been linked to intracellular calcium excess, mitochondrial dysfunction, changes in electrical activity, and neuronal death [117]. Further, alterations in the expression pattern of Nav1.2, Nav1.5, Nav1.6, and Nav1.8, specific voltage-gated Na+ channel isoforms have been reported in MS, and their overworking is involved in axonal deterioration followed by cerebellar dysfunction [118]. Furthermore, Nav channels cause a Na+ influx into axons, which raises the amount of intra-axonal Ca2+ ions and interferes with axon myelination, resulting in MS pathogenicity. Different calcium and potassium channel isoforms were also shown to be increased, interfering with conduction in demyelinating axons [119]. ALS is a fatal chronic motor neurodegenerative disease marked by significant motor neuron loss in the motor cortex, brain stem, and spinal cord. Patients develop progressive muscle weakening, fasciculation, and atrophy, which leads to a loss of voluntary movement [120]. However, the specific etiology of ALS remains unknown, however, animal models are being used in research to find a feasible reason. Previous research revealed that the contraction of mammalian denervated muscle fibers is caused by spontaneous activation of the voltage-gated Na<sup>+</sup> channel [121]. Furthermore, in human sporadic ALS, a significant drop in potassium channel expression has been found [122]. Axonal hyperexcitability is caused by continuous Na+ ion conduction followed by a rapid reduction in K+ ion conductance, resulting in ALS symptoms [123]. Furthermore, in ALS, motor neurons that innervate tongue muscles are prone to degeneration, which has been related to VGCC expression differences. In ALS patients and animal models, other investigations have found immunoreactivity with several calcium ion channels [124]. Israelson et al. investigated the role of mitochondrial channelopathy during ALS and discovered that mutant superoxide dismutase 1 (SOD1) inhibits the mitochondrial voltage-dependent anion channel-1 and induces mitochondrial-dependent apoptosis, resulting in lethal paralysis in ALS. However, further study is being conducted to determine the specific mechanism responsible for its etiology [43, 125]. There is a lack of data to address the review question on the efficacy of Na+ channel blockers for people with MS [126]. The K+ channel blocker Fampridine-SR is an authorized MS therapy adjunct that has been demonstrated to help with ambulation, tiredness, and endurance [127]. Silva et al. demonstrated the efficacy of Ca2+ channel blocker CTK 01512-2 in mouse models of MS comparing it with Ziconotide. They found a significant improvement in neuroinflammatory event MS-related [128].

#### **5. Conclusion**

Ion channel dysfunction is steadily becoming connected to neurological disorders, making it an intriguing subject of neuroscience research. It has been linked to memory loss, movement issues, and neuromuscular anomalies in a number of neurological diseases. Since they originate in response to genetic defects in channel coding proteins that disturb the ionic equilibrium in the brain, the majority of these illnesses are classified as neurological channelopathies. Aging is a complex and multidimensional biological process that affects all organ systems. In the core section of them, cellular malfunction and senescent cell accumulation are common. Various aspects of brain aging have been discovered at the molecular, cellular, and tissue levels, according to research in the fields of aging and neurobiology. Aging is the leading risk factor for a broad range of neurodegenerative disorders. According to breakthroughs in the

#### *Ion Channels and Neurodegenerative Disease Aging Related DOI: http://dx.doi.org/10.5772/intechopen.103074*

treatment and prevention of some tough diseases such as cardiovascular disease and malignancies, which have enabled more people to survive past the age of 70, aging brain disorders have lately become the leading cause of disability and death. After providing an overview of recent developments in brain aging, the current review describes it as the result of decreased neurogenesis and synaptic plasticity, as well as altered neurochemical and signaling pathways, such as impaired protein processing, glial cell activation, impaired mitochondrial function, increased oxidative stress, and neuroinflammation. Furthermore, the hippocampus and neocortex are the principal susceptible sections, with varying degrees of molecular and cellular abnormalities in their sub-centers as a result of aging. Although each of these age-related alterations is present during normal aging, their combined influence, when combined with genetic background and environmental variables, may trigger the cytotoxic activation cycle. Transcript factors, proteins, and cell-environmental variables including redox potential are all connected to these alterations. However, the crucial component that governs the entire activity is unclear. One of the first priorities would be to figure out how redox capability influences gene transcription and promotes metabolic responses as the brain matures. Our understanding of brain aging is still in its early stages. More research is needed to discover effective therapy approaches and drugs to combat brain aging. Furthermore, non-pharmacological techniques such as lifestyle adjustments, physical exercise, and calorie restriction, which promote the brain's physiological processes while reducing ROS formation and inflammation, may help to promote healthy aging. Understanding the processes that underpin the hallmarks is crucial for developing future therapeutics to slow or even reverse the aging process in the brain. The primary objective of neurobiology and brain aging research should be to discover methods and techniques for supporting healthy brain aging in all people. The functional activities of ion channels connected to the onset of numerous chronic neurological diseases have been determined. NDDs are accompanied with inflammations, neurotoxic protein accumulations, physiological stress, and mitochondrial dysfunctions, according to experimental findings. These abnormal alterations cause disruptions in normal physiological processes and brain homeostasis, which leads to illness development. The pathogenesis of AD, PD, HD, MS, and ALS has been further clarified in terms of faulty ion channels. Until today many natural compounds or synthetics compounds are identified as a modulator of ion channels (for a very extensive review refer to [43, 129]). Furthermore, channel modulators have been discovered to be important in correcting the chronic consequences of abnormal ion channels. Furthermore, understanding their regulation mechanisms in neurodegeneration might lead to the development of newer, more effective treatment techniques.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marika Cordaro1 , Salvatore Cuzzocrea<sup>2</sup> and Rosanna Di Paola<sup>2</sup> \*

1 Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Messina, Italy

2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy

\*Address all correspondence to: dipaolar@unime.it

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 2**

## Mitochondrial Channels and Their Role in Cardioprotection

*Keerti Mishra and Min Luo*

#### **Abstract**

Mitochondria play a pivotal role in cardioprotection. The major cardioprotective mechanism is ischemic preconditioning (IpreC), through which short periods of ischemia protect a subsequent prolonged acute ischemic episode. Mitochondria channels, particularly the potassium channels (mitoK) such as ATP-dependent and calciumactivated potassium channels, have been suggested as trigger or end effectors in IpreC. Activators of mitoK are promising therapeutic agents for the treatment of the myocardial injury due to ischemic episodes. In this chapter, we are summarizing our current knowledge on the physiology function of different mitochondrial channels with a focus on the potassium channels and their mechanism in cardioprotection. Furthermore, the currently under development therapy by targeting the mitochondrial channels for the treatment of heart failure are also discussed.

**Keywords:** cardioprotection, ischemic preconditioning (IpreC), ischemic postconditioning (IpreC), oxidative phosphorylation, reactive oxygen species (ROS), cell death, mitochondrial permeability transition pore, mitochondrial potassium channels, ischemia, reperfusion, heart failure

#### **1. Introduction**

Heart failure is a major public health issue that is still having a poor prognosis despite all the advancements in scientific research and technologies [1]. The approaches for the drug development of heart disease are majorly relying on the pathophysiology of the cellular mechanisms and inter and intracellular channels in the failing heart. Heart being an organ of extensively high energy demand and mitochondria being the powerhouse of the eukaryotic organisms, they are meant to be closely connected. Any change in mitochondrial function inevitably affects the health of the heart irrespective of the etiology. Recent advances in the field indicate that besides having a compromised powerhouse, mitochondrial malfunctioning accompanies certain pathogenic mechanisms leading to heart failure [2, 3]. Current therapies like ischemic pre- and postconditioning provide symptomatic benefit but do not address the abnormalities at a molecular level. Since the mitochondria play an important role in the pathophysiology of a failing heart, understanding its mechanism can potentially improve the approaches for the therapies for direct improvement of cardiac functions. Among the abnormalities shown by the mitochondria, ruptured

electron transport chain, excessive formation of reactive oxygen species (ROS), perturbed ion homeostasis are the basic concerns [4]. An important and potential substrate for therapeutics in heart failure is mitochondrial channels [5]. In this chapter, we intend to discuss the available information about the mitochondrial channel with regards to its pathophysiological effects on heart health and their responses to the ischemic conditioning alongside the available agonist for the mitochondrial channel.

#### **2. Mitochondria and its functions specific to heart cells**

Due to high energy demand, the number of mitochondria in the heart cells is excessively high, with a daily production of approximately 65 kg ATP through oxidative phosphorylation [6]. In the neonatal cardiac myocytes, the mitochondria are highly motile in the cytosol generating energy through glycolysis and glucose metabolism. Whereas, in an adult myocyte the mitochondria have reduced motility, and energy generation occurs from the metabolism of fatty acid [7].

Mitochondria are known to arise billions of years ago through the engulfment of alpha proteobacteria by the precursors of modern eukaryotic cells and it evolved to become an essential multifunctional organelle [8]. Mitochondria are made up of an outer comparatively permeable and inner highly folded relatively impermeable lipid bilayer. The folded inner membrane with a high surface area contains the complexes for the generation and transportation of adenosine triphosphate (ATP) through oxidative phosphorylation. In the myocardial cells, the substrates are oxidized to produce acetyl coenzyme A, which in turn drives the Krebs cycle to produce nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide (FADH2) in the mitochondrial matrix. The oxidation of NADH and FADH2 leads to the establishment of proton motive force later fetched by F1F0 ATP synthase to convert adenosine diphosphate (ADP) and inorganic phosphate to ATP [9].

In the process of energy production, approximately 2% of the electrons flowing in the electron transport cycle are reduced to form a superoxide anion which is reduced to H2O2 followed by H2O generation by antioxidant enzymes. Excessive production of these ROS is toxic to the cell, yet these natural byproducts of oxygen metabolism trigger a variety of oxygen sensing machinery including gene expression, however, the overload of ROS impairs the redox potential of the cell leading to various oxidative damages [10].

The dynamics of Ca2+, an important element to trigger various enzymatic processes and a second messenger for contractile functions, is also organized by mitochondria by either transmembrane Ca2+ transport or ROS-mediated signaling pathways [11]. In case of increased workload rapid mitochondrial Ca2+ uptake is facilitated by Ca2+ uptake channel for elevated ATP production. The elimination of Ca2+ from the mitochondrial matrix however is slower and mediated either directly by Na<sup>+</sup> /Ca2+ exchanger or indirectly by multiple mitochondrial K+ channels with unknown mechanisms [12].

Alongside the role as a life-supporting system, mitochondria can also trigger programmed cell death in the required conditions. The mitochondrial permeability transition pore (MPTP) opens in response to stress and leads to loss of membrane potential, which stops ATP production and release of cytochrome C and other mitochondrial protein causing necrosis and cell apoptosis [13]. In cases of heart failure mitochondria-induced cell death is an important mechanism. Here we intend to discuss important parameters of mitochondrial dysfunction which lead to heart failure, and therapeutic approaches to circumvent the situation.

#### **3. Mitochondrial dysfunction and heart failure**

In cardiac cells the energy consumption should meet the energy production rate on a beat-by-beat basis, failing which the stored energy cannot last more than a few seconds. In a pathological remodeling the oxidative metabolism switch from fatty acid metabolism to glycolysis, which only contributes less than 5% of the total ATP demand of an adult heart [14]. On the other hand, during pathological remodeling the required energy increases due to disturbed cardiac geometry, and impaired ATP homeostasis. Studies have shown that the mitochondrial mechanisms involved in pathological remodeling in efforts to restore the energy homeostasis eventually led to a vicious cycle that drives pathological remodeling towards heart failure. The most puzzling scenario suggests that in the failing heart the ATP content is largely maintained after an initial glitch, thus whether the heart failure occurs due to energy starvation or in efforts to fight that starvation is a question yet to be addressed. Further in-depth analysis of the mitochondrial mechanisms can clarify if the efforts of maintaining the energy hemostasis are either helpful or potentially worsen the failing heart.

The catalysis of degradative oxidation of the nutrients through anaerobic dehydrogenases is facilitated by the reduction of oxidized pyridine and flavin nucleotide like NAD(P+ ) and FAD. These coenzymes should be again reoxidized since they are non-replenishable and cannot permeate the cell membrane with the degradation rate. During an ischemic episode since the respiratory chain is impaired the oxidation of the above-stated substrates is also hampered, moreover, the NADH(H+ ) oxidation is carried out by lactate dehydrogenase. Therefore, the anaerobic glycolysis takes over as the only pathway for ATP synthesis provided the phosphocreatinine is depleted with the onset of ischemia. Therefore, in a failing heart the oxidative metabolism switch for alternative carbon sources such as glucose which can be beneficial due to increased ATP production and oxygen uptake but when it takes over the usual fatty acid metabolism the energy production is not sufficient for an adult heart [14]. Increased glycolysis causes anaplerosis, increased lactate production, triggers the heart to go into pathological remodeling, and also inhibits branched-chain amino acid (BCAA) catabolism, and causes the accumulation of BCAA. A hyperacetylation of mitochondrial protein has also been seen as a failing heart the cause of which is not clearly understood [15].

The decrease in ATP concentration causes an immense ionic imbalance across the cell cytoplasm leading to the lowering of the pH of the cell. The inhibition of Na<sup>+</sup> / K+ ATPase, Na+ /H+ , Na+ /Ca2+ antiporters leads to an overload of Ca2+ inside the cells causing hypercontracture and triggering the irreversible opening of mitochondrial permeability transition pore (MPTP) [16]. The frequently converting ATP into ADP and phosphate seeps out of the cell which further contributes to reduced performance of the heart. Opening of only one pore causes frequent depolarization and triggers the opening of other pores, following which the rapid influx of small molecular weight solutes enters the mitochondrial matrix to compensate for the depolarization and causes the mitochondrial matrix to swell. The expansion of the inner mitochondrial membrane leads to the rupture of the outer membrane which releases proapoptotic proteins leading eventually to cell death. Therefore, it is believed that altering the MPTP pore opening can be helpful in the prevention of cardiac reperfusion and cell death [17].

During an ischemic episode, the release of ROS is formed under the physiological and pathological conditions within the mitochondria. In a regular respiratory chain

reaction, 2–4% oxygen undergoes an univalent reaction and produces superoxide [4]. The superoxides that are formed at complex I and complex III level are rapidly transformed by metalloenzymes like superoxide dismutase into hydrogen peroxide. In the first minute, it is small but in a later stage, it increases dramatically, leading to the disruption of mitochondrial membrane potential. Therefore, the consequence of ROS formation has been linked to the opening of the MPTP channel leading to apoptosis. These episodes put together in series lead to a gradual and irreversible decline of the cell integrity.

The opening of MPTP can occur through all the factors mentioned here, such as an increase in Ca2+ ion, depolarization, increase in the ROS, and phosphate concentration [18]. Certain factors like a high concentration of H<sup>+</sup> , Mg2+, and ADP can counteract the MPTP opening and work as antagonists [19, 20]. On the contrary, in the condition of reperfusion, the change in the pH is recovered by the burst formation of ROS in the presence of Ca2+, which creates the most favorable condition for MPTP opening even though the antagonizing effect of membrane potential recovery occurred. In isolated mitochondria, the MPTP opens at a very high Ca2+ concentration which is practically not possible in vivo therefore the increased Ca2+ alone is not responsible for MPTP opening rather can be triggered by several processes like ROS generating Ca2+ dependent enzyme.

#### **4. Cardioprotection**

It is believed that to reduce the damage occurring in the heart cells in a prolonged ischemic episode, the heart cells can be trained beforehand through small and regulated episodes of either cardiac ischemia or reperfusion that resulted from ATP deprivation or concentration increase of ROS and Ca2+ (**Figure 1**). This method has been tested in dogs [21] and higher mammals including humans [22, 23]. This process is known as ischemic preconditioning (IpreC). Similarly, ischemic postcondition

#### **Figure 1.**

*The ischemic/reperfused heart mitochondria in comparison to the cardioprotected mitochondria. In cardioprotected mitochondria MPTP and MCU channels are closed and mitoK channel is opened.*

(IpostC) can also be done in a brief intermittent cycle after a severe event [24]. These are performed by natural or artificial biomolecules which will be discussed later in this chapter.

The process of IpreC and IpostC usually activates protein kinase C isozymes [25] and other kinases [26] whose roles in cardioprotection are very dicey, because as ε isozyme protects the mitochondrial function by activating ALDH2 aldehyde dehydrogenase which removes the lipid peroxidation products, Baines et al. showed that the translocation of ε isozyme prevents the opening of MPTP pore [27]. Whereas δ isozyme of protein kinase C increases the tissue injury by flawed perfusion of myocytes and inhibits ATP and pyruvate dehydrogenase regeneration [27]. Several mitochondrial pathways are activated in the conditioning process contributing significantly to the process of cardioprotection and therefore they are considered attractive pharmacological targets.

#### **5. Mitochondrial channels with an integral role in cardioprotection**

The multifaceted relationship of mitochondria with cell death makes it an ideal target for aiming to preserve cardiomyocytes viability. In the lack of oxygen during ischemia although the ATP synthesis cannot be restored yet can protect through decreasing ATP hydrolysis. Several self-defense mechanisms are triggered by ischemic preconditioning like the depolarization of mitochondrial matrix promotes F1F0 ATPase binding to its natural inhibiter Factor (IF) [28]. A similar effect has been shown by overexpressed the BCL-2 gene in mice hearts, to conclude that ATP hydrolysis is modulated by BCL-2 as well since the oligomycin addition did not possess any additional effect. BCL-2 is upregulated in the preconditioned heart and downregulated by ischemia and reperfusion [29]. However, the cardioprotective effect caused by preconditioning can be abolished by antisense nucleotide in a perfused rat heart [30]. Another way to prevent ATP hydrolysis is by MPTP inhibition, which presents a wide range of protective actions like maintaining Ca2+ homeostasis, NAD+ depletion prevention, and preventing the release of pro-apoptotic protein [20, 31, 32]. The preconditioned heart prevents the opening of MPTP pores conferring stress-tolerant condition of the cardiomyocytes [33, 34].

In addition to protective effects posed by MPTP inhibition, numerous studies have vouched for the supporting effect of the mitochondrial potassium channel, specially mitoKATP and calcium-dependent mitoKCa. The influx of K<sup>+</sup> into the inner mitochondrial matrix causes depolarization, with pH increase and matrix swelling [35–37]. It is suggested that matrix swelling due to K+ uptake compensates for the contraction of the matrix caused by increased potential difference due to lack of oxygen. The K+ uptake and matrix swelling are suggested to increase the recovery of ATP concentration, by preventing the loss of substrate channeling which happened due to increased potential difference at the onset of reperfusion [38].

#### **5.1 Mitochondrial permeability transition pore**

A sudden increase in the permeability of the solute in the inner mitochondrial membrane (IMM) is known as the permeability transition [16]. The MPTP was first described by Haworth and Hunter in 1979, who showed that the addition of high levels of calcium to bovine myocardial mitochondria induced a nonspecific increase in permeability of the inner mitochondrial membrane [39]. Although the occurrence of permeability transition and its inhibitor as adenine dinucleotide has been known since 1950 [40]. Our understanding of mitochondrial physiology and the acceptance of the pore theory of permeability transition is greatly attributed to the study of a mitochondrial channel.

The opening of the MPTP channel causes depolarization, blocks ATP synthesis, releases Ca2+, depletes pyridine nucleotide, inhibits respiration, causes matrix swelling, which subsequently leads to cytochrome C mobilization and outer mitochondrial membrane rupture which ultimately releases endonuclease G and apoptosis-inducing factor (AIF) and other proapoptotic protein to kill the cell (**Figure 1**) [41, 42]. It should be noted though, that this detrimental effect of MPTP opening occurs only when the pore opening is long-lasting [43]. Whereas the short-term opening, both in vivo and in vitro [44], is suggested to be involved in the physiological regulation of Ca2+ and the homeostasis of ROS [4], subsequently providing mitochondria a fast mechanism for Ca2+ release. According to a study performed on a mitochondria calcium uniporter (MCU), null mice had an equal I/R injury as the wildtype littermates overruling cyclophilin-D (CyPD) protection (**Figure 1**). It leads to challenging the established concept and awaits the molecular details of the myocardial reperfusion mechanism and the precise roles of the channels for answers to these contradicting observations. The potential role of MPTP opening in heart failure was recognized way before the discovery of the role of mitochondria in apoptosis.

Although the molecular nature and precise composition of MPTP remain unknown it is believed that some proteins regulate the function of MPTP like CyPD (**Figure 1**). After the observation that cyclosporin (CsA) is a potent inhibitor of MPTP opening [45, 46], Halestrap et al. demonstrated that it occurred due to an inhibition of a peptidyl-prolyl cis-trans isomerase PPIase in the matrix [47]. They further purified and demonstrated the protein to be CyPD, which is an 18 kDa matrix protein. A range of other CsA analogs and sanglifehrin A (SfA) that showed their potency in preventing MPTP opening also acted as inhibitors of PPIase of CyPD. On the other hand, the MPTP opening is also inhibited by ATP and ADP but their complexes with Mg2+ and other nucleotides like AMP, GTP, or GDP fail to show a similar effect, it is worth noting that none of them are transported by the adenine nucleotide translocase (ANT) [48]. Furthermore, the increased sensitivity of MPTP opening towards Ca2+ is attributed to the inhibition in the binding of the ATP and ADP with ANT either by depleting the matrix of adenine nucleotides or by modifying ANT by thiol [49]. Helstrap group developed a model for MPTP, where CyPD binds to ANT and they undergo conformational changes to induce pore formation under Ca2+ trigger, and they showed that matrix Ca2+ favored 'C' conformation for ANT. Several matrices facing glutamate and aspartate residues on ANT are present whose carboxyl groups might play the role of Ca2+ binding as there is no Ca2+ binding motif established on ANT [49]. Another data consistent with the model showed the coprecipitation of CyPD specifically with ANT and the bonding increases with rising oxidative stress and decreases with the introduction of CsA but not with inactive CsH analog [50, 51]. The crystal structure of bovine ANT1 [52] showed a constriction provided by 3 helices, block the channel and if these are rearranged by the change facilitated by CyPD, then an extensive conformational change might account for MPTP formation. Phosphate ion has been known as an MPTP activator and carboxyatractyloside (CAT) prevents ANT from binding the phenyl arsine oxide (PAO) column but still does not prevent MPTP activation, which suggests that PAO can have an additional MPTP activation site apart from the ANT. When CAT treated beef heart mitochondria was passed through the PAO column phosphate carrier protein (PiC) was bound to

the column [53]. Pretreatment of the column with MPTP inhibitors like ubiquinone (UQo) prevents the PiC binding to the column which suggests a key role in MPTP formation. Other proteins have also been suggested to have the structural and regulatory role in MPTP formation like peripheral benzodiazepine receptor and voltagedependent anion channel, hexokinase, creatinine kinase, BCL2 proteins, and Bcl 2 associated X (BAX) proteins may also be associated with MPTP, but which proteins eventually constitute the formation of the pore is still unknown [54, 55].

Recently, another theory of multiple pores in MPTP has been proposed. Studies have been supporting the potential roles of ANT, PiP, F1F0 ATP synthase, and CyPD to be inner membrane component but all of them has shown CsA sensitive permeability despite the genetic deletion of the responsible gene, which raises a question on the hypothesis and further investigation led to propose the multiple pore-forming mechanisms. Deletion of the C subunit of F1F0 ATP synthase showed that CsA induced MPTP synthesis showed much lower conductance as compared to wild-type MPTP [56]. This C-subunit lacking channel could be inhibited by an ANT inhibiter bongkrekic, therefore it was suggested that a classic MPTP was not formed in the knockout mitochondria. It was concluded that the MPTP formation could be enhanced through other proteins e.g. ANT in the lack of c-subunit. Another study proposed that dimer of F1F0 ATP synthase, ANT and PiC can assemble into synthasome complex, and it requires CyPD for disassembly into its components. They further suggest that ATP synthasome assembles and disassembles in high work conditions and MPTP formations respectively. Low ADP, high calcium enhancement leading to increase the membrane potential, and ROS formation trigger the disassembly of ATP synthasome leading to MPTP formation. Additional studies will be required to completely understand all the components of synthasome in generating MPTP [57].

As a result of its central role in myocardial infarction, MPTP poses itself as an obvious target for cardioprotection. A wide variety of cardioprotective protocols have been demonstrated to prevent MPTP opening during reperfusion. Certain drugs directly inhibit MPTP like CsA and SfA and their non-immunosuppressant derivatives like 4-methyl-val-CsA and D-3-MeAla-4-EtVal-CsA etc. and certain protocols that decrease oxidative stress and pH for inhibiting MPTP pore opening such as ischemic preconditioning [34] and ischemic postcondition [58], temperature preconditioning [59], Na+ /H+ exchanger inhibiter like cariporide [60], mitochondrial ubiquinone antioxidants [61], the anesthetic propofol [62], urocortin [63], antioxidants including pyruvate [64].

The drugs that directly inhibit MPTP pose great value in protecting the heart during cardiac surgery, it has been shown that CsA improved cardiac performance following angioplasty treatment [65]. However, CsA and Sfa administration pose unwanted side effects because they interact with other cyclophilins like CypA moreover, their MPTP opening inhibition is overruled by the intensity of the pore opening stimulus [66]. This situation requires the development of new MPTP inhibitor drugs which can overcome these constraints. The development of new drugs requires structural insight into the MPTP pores.

#### **5.2 Inner mitochondrial anion channel (IMAC)**

The inner mitochondrial anion channel (IMAC) was the first mitochondrial channel to be identified using the patch-clamp method [67]. The pharmacological drug testing on the cardiomyocytes, for analysis of the mitochondrial matrix swelling, led to the discovery of its role in membrane potential perturbation. Its activity

is promoted under stressed oxidizing conditions [68]. O'Rourke and co-workers proposed that the arrhythmias and electrophysiological alteration in cardiomyocytes are the results of disturbed membrane potential due to failed cellular mitochondrial network under oxidative stress [69]. The inhibition of IMAC mediated mitochondrial membrane potential oscillation with 4-chlorodiazepam showed a significant reduction and stabilization of the sarcolemmal action potential [70]. High-resolution optical action potential mapping showed that the introduction of 4-chlorodiazepam facilitates the restoration of action potential duration and prevents ventricular fibrillation. The thiol oxidants trigger the oscillation of membrane potential, glutathione, and NADH, which in turn increases the ROS concentration [71]. The inhibition of IMAC activity is triggered by the binding of 4-chlorodiazepam with benzodiazepine receptors. The inhibited IMAC preserves the membrane potential; however, the prohibited efflux of superoxide from IMAC further increases the ROS concentration [72]. The increasing ROS and decreasing glutathione concentration in the mitochondrial matrix trigger the opening of the MPTP pore, and therefore, IMAC can be considered as an instigator of MPTP opening [71].

#### **5.3 Mitochondrial Ca2+ uniporter**

The macromolecular structural assembly responsible for mitochondrial Ca2+ uptake machinery is known as the mitochondrial calcium uniporter (MCU) complex. It was initially assumed that an active uptake and passive release are required for the transport of Ca2+ across the inner mitochondrial membrane [73], but multiple groups showed that the uptake is energetically favored whereas efflux requires electrogenic ion-exchange [74].

Ca2+ uptake in mitochondria results from a single transport mechanism by a Ca2+ sensitive channel of mitochondria known as MCU (**Figure 1**). The molecular identification of the MCU protein complex which was closely connected to a comprehensive protein compendium MitoCarta was done in 2008 [75]. Following the establishment of a compendium Ca2+ sensing regulator, mitochondrial Ca2+ uptake 1(MICU1) was discovered in 2010 [76]. MICU1 was predicted to contain no transmembrane domain and was therefore not considered forming a pore. Later 40 kDa two transmembrane domains were identified termed MCU in 2011 [77, 78] followed by the identification of other regulatory subunits.

The concentration Ca2+ increases in the mitochondrial matrix during ischemia and reperfusion and this increase is proposed to activate MPTP opening [16]. Therefore, the inhibition of mitochondrial calcium uniporter is studied to reduce cell damage in I/R. Studies from MCU knockout mice in the germline [79] and MCU mutated gene [80], in both the cases the Ca2+ uptake was hindered leading to no MPTP opening, but neither of the situations reduced the size of cardiac infarct at the onset of I/R. In contrast, where the MCU was deleted after birth in adult hearts showed cardioprotection in an in vivo model [81]. The reason for this kind of difference is not very clear but apparently, the MCU knockout before birth could generate a more robust MPTP pore not regulated by CsA as well. Alongside MCU, the other two core structural components are mitochondrial calcium uniporter b MCUb and an ion transport component termed "essential MCU regulator" or EMRE. MCUb is closely related to MCU with 50% amino acid homology, containing two similar transmembrane domains linked with coiled-coil domain. On the other hand, EMRE is a 10 kDa protein span in the inner mitochondrial membrane that contains an aspartate-rich, highly conserved, C-terminal region, whose topology however is still unclear [82]. It was proposed by

Mootha et al. that EMRE is required for Ca2+ channeling activity and also helps in keeping the MICU1/MICU2 intact to the MCU complex [83].

Altering the levels of regulators of MCU complex the calcium uptake can also be regulated in mitochondria, subsequently altering the susceptibility to MPTP-induced cell death. A mitochondria Ca2+ uptake protein1 (MICU1) mutation causing a loss of function in a human patient is associated with ataxia, attributed to mitochondrial Ca2+ overload [84, 85]. In a failing heart, an increase of MICU1 and Na+ /Li+ /Ca2+ exchanger (NCXL) has been observed to compensate for the Ca2+ overload [86]. MICU2 on the other hand has been observed to increase, with cardiovascular disease in both humans and mice, at the transcriptional level [87]. Mice with deletion of MICU2 showed a certain degree of diastolic dysfunction. The low ratio of MICU/MCU maintains a low threshold of calcium entry in mitochondria and the overexpression of MICU1 causes contractile dysfunction to the heart. Therefore, the rise in MICU1 and MICU2 with age and disease alters the susceptibility of calcium overload and MPTP inhibition.

In a cardiac muscle, constant rhythmic cycles of contraction are dependent on permanent uptake and release of Ca2+ in the cytoplasm and buffering organelles [88]. After a myocardial contraction, the removal of the Ca2+ from the cytoplasm is provided by Na<sup>+</sup> /Ca2+ exchanger (NCX) in the endoplasmic reticulum, on the other hand in non-muscle cells the cytosolic Ca2+ signals and Ca2+ buffering depends on mitochondrial Ca2+ uptake [89]. Although this mitochondrial Ca2+ uptake in cardiomyocytes possesses a very low MCU current and constitutes less than 1% of total Ca2+ uptake [88, 90], it plays a key role in coordinating between excitation and metabolism coupling [91]. In a healthy heart, two models of mitochondrial Ca2+ dynamics have been suggested by Cao et al., the first model suggests that Ca2+ concentration oscillates in a beat-to-beat manner in cardiomyocytes whereas, the second model emphasizes gradual Ca2+ uptake by cardiac mitochondria. On the contrary, in the damaged heart the Ca2+ mishandling within the mitochondria is well documented [92]. The MICU1 protein content is significantly low following I/R due to inhibition of translocase expression of the outer membrane. Furthermore, treatment attempts using siRNA on myocardial MICU1 aggravated the ischemic episode increasing tissue damage and depressing cardiac function due to apparent Ca2+ overload [86].

As it is quite evident that the uncontrolled influx of Ca2+ is disastrous for cardiomyocytes, and MCU is the major route for Ca2+ entry. Therefore, alteration in the expression of MCU can be a promising target for cardioprotection. For the inhibition of MCU ruthenium red and its derivatives are generally used, however, ruthenium red has nonspecific activity towards other ion channels [93] as well which does not make it a suitable inhibitor and prevents it from usage as a therapeutic agent. Recently, two new highly selective MCU inhibitors were developed one is DS16570511 prevents Ca2+ overload and raises cardiac contractility without affecting heart rate [94]. The second one is Ru265 is negligibly toxic and prevents hypoxia in the cell model [95]. Mitoxanthrone, an anticancer drug that showed its efficiency in inhibiting MCU [96], similarly kaempferol known as an anticancer [97] and cardioprotective drug [98] could prevent Ca2+ created arrhythmias [99]. These can be promising drugs in preventing Ca2+ related risks to cardiomyocytes but they require more animal study, and careful modeling and validation before adapting as therapeutics.

#### **5.4 Mitochondrial potassium channel**

On one side where the opening of mitochondrial mega channels like MPTP and Ca2+ uniporter represents a hallmark of cell death, on the other hand, the transport of K+ through ion channels is known to play a central role in neural and cardioprotection [100–102]. The membrane potential and permeability of the inner mitochondrial membrane are strictly controlled for efficient ATP production. The presence of an electrophoretic pathway for entry and antiporter mechanism for the exit of K+ has been well established and they critically regulate the mitochondrial volume and function. The transport of K<sup>+</sup> ions from the cytosol to the mitochondrial matrix is carefully conducted through ion channels by utilizing electrogenic transport, where the proton ejection by the electron transport system generates enough membrane potential for the influx of K+ . There are four kinds of mitochondrial K+ channels (**Figure 2**) present in the inner membrane the ATP regulated [103], Ca2+ regulated [104], Twin pore TASK channel [105], and voltage-gated Kv1.3 potassium channel [106]. These channels resemble the plasma membrane potassium channels in their basic biophysical properties and are regulated to avoid the membrane potential collapse.

#### *5.4.1 ATP sensitive potassium channel*

Several mitochondrial K<sup>+</sup> channels (**Figure 2**) have been discovered so far but ATP sensitizing uptake of K<sup>+</sup> has gazed maximum attention. The cardiac ischemic conditioning was first believed to be working on KATP of the plasma membrane counterpart but based on pharmacological analysis with channel openers and inhibitors shown to affect mitochondrial KATP channel. The mitoKATP channel was first identified in rat liver mitochondria using the patch-clamp method [90] and was later found in the inner mitochondrial membrane [107, 108]. They are situated at the crossroad of metabolism and membrane sensitivity. The molecular identities of a mitoKATP were recently determined by Angela Paggio et al. [109], which is similar to its plasma membrane counterparts that consisting of pore-forming potassium channel CCDC51 (MITOK) and ATP-binding cassette (ABC) transporter ABCB8 (MITOSUR); however, the detailed assembly and function mechanism is still unknown due to the missing of structural information. The plasma membrane KATP is heterooctameric, containing four inward rectifying potassium channel subunits of Kir6.1 and four

#### **Figure 2.**

*Mitochondrial potassium channels (a) voltage-gated potassium channels (Kv 1.3, Kv 1.1, Kv 1.5), (b) small and large conductance mitoKATP channel (IKCA, BKCa), (c) ATP sensitive K+ channel, (d) twin pore K<sup>+</sup> channel. mitoKATP and mitoBKCa having extensively studied for cardioprotection.*

#### *Mitochondrial Channels and Their Role in Cardioprotection DOI: http://dx.doi.org/10.5772/intechopen.101127*

sulfonylurea receptor subunits, which belong to the ABC transporter family [110]. Whether this newly identified mitoKATP is occupying a similar octameric assembly as the plasma membrane KATP is still unknown and moreover, it may not present the only version of mitoKATP as channels such as Kir6.1 has also been suggested in the formation of mitoKATP [111]. Nevertheless, they are believed to play a central role in cardioprotection, since the bizarre method of cardioprotection called ischemic conditioning was introduced. Ischemic preconditioning was first observed with plasma membrane using KATP channels as effectors, but later mitochondrial potassium channel became an interesting target for the same. The pathway involves activating the protein kinase and generating ROS, but the precise role of mitoKATP is not very well established. Therefore, evidence of molecular structure for the mitoKATP will subside the pharmacology-based arguments of its existence and role in the process of preconditioning. According to a study by Peng Duan et al., mitoKATP channel opening is helpful in the optimal expression of protein kinase B (p-AKT) and forkhead box protein O1 (pFoxo1) in an insulin-resistant cell. The increased p-Foxo1 is phosphorylated by p-AKT, reducing its transcriptional efficiency, and transferred out of the nucleus, and prevents the expression of pro-apoptotic protein, thereby preventing apoptosis [112].

A study done by Garlid et al. in 2006, showed that the K<sup>+</sup> ion uptake in the mitochondria leads to increased ROS production as they explain that the opening of the mitoKATP channel will lead to a small amount of K+ uptake but this lowered potential will increase the matrix volume and pH by a persistent steady state. Valinomycin was used to induce the mitoKATP opener and an increased pH caused an increased ROS production, when the acetic acid influx increased for compensation of the alkaline matrix the ROS production also reduced proving that the alkalinity is a cause for ROS production. It was also proved by them that the ROS was generated from the complex I of the electron transport system [113].

#### *5.4.2 Ca2+ activated potassium channel*

These channels were first discovered in the glioma cell line LN-229 and have been extensively studied in the brain and cardiac cells since then [104]. The calciumactivated potassium channel is of two types small and intermediate conductance K+ channel and large or big conductance K<sup>+</sup> channel. The small and intermediate channels are only calcium-dependent and not voltage-dependent they possess calmodulin for the Ca2+ binding at the C-terminal region. Their major function is promoting proliferation and migration of dendritic cells and smooth muscle [114].

The first evidence of its presence in the inner mitochondrial membrane was found in the late 90s by Siemen and coworkers although showing that it possesses a conductance of 300 pS [104]. Its role in protecting the heart from ischemic insult was first discovered by Xu et al. and their structural characterization in the plasma membrane indicates that it originates from potassium calcium-activated channel subfamily M alpha 1 (Kcnma1) gene containing extracellular N terminus and intracellular C-terminus [115]. It has been proven in 2013 that the BKCa pore-forming alpha subunits are encoded by the same genes (Kcnma1) as the basis of why they possess the same physiological properties [116].

The big conductance Ca2+ sensitive K+ channel also known as MitoBKCa on the other hand is intuitive to voltage and mechanical stress alongside Ca2+ sensitivity. The knockout experiments have proven for the MitoBKCa channels to have a cardioprotective effect by reversing the ROS production and opening MPTP. It has also

been shown that these channels form a multiprotein complex with several proteins involved in apoptotic machinery [117].

mitoBKCa channel [118] represent themselves as a key pathophysiological target due to their sensitivity towards calcium, voltage, and a range of cellular components. Several small molecular openers for BKCa and pharmacological agents have provided very insightful information to decipher the role of BKCa channel. Pharmacological agents like NS1619 and NS11021 have been used to activate BKCa can potentially play a vital role in cardioprotection. However, they fail to reach the clinical applications due to their non-specificity [119–122]. Although it is posing a great deal of difficulty in developing the BKCa activators, it becomes essential considering that expression of BKCa is vital for cardioprotection [116, 123].

The first representation BKCa playing an essential role in cardioprotection from I/R injury was performed by using NS1619, whose effect was blocked by praxilline [115]. A 3 mM NS1619 preconditioning showed an improved reduction of infarct size, possibly by modulated Ca2+ and ROS concentration [117]. BKCa mediated cardioprotection involves ROS, Ca2+, and MPTP and their interplay. It is anticipated that reduction of deleterious ROS through BKCa activation prevents the excess release of Ca2+ from the endoplasmic reticulum subsequently reducing the influx and overload in mitochondria preventing the cell from injury.

#### *5.4.3 Voltage-gated potassium channel*

These are the most diverse family of K<sup>+</sup> channels. They are grouped into 12 families comprising 40 of the 90 genes present in human cells [124]. These channels mostly consist of six transmembrane helices (S1-S6) where two of them (S5-S6) form the loop and four of them are proceedings to the loop (S1-S4). The fourth positively charged loop senses the change in the membrane potential. These channels have a wide variety and therefore, they represent a fine regulation of K<sup>+</sup> flux in the homeostasis and pathological processes. The mitochondrial counterpart mitoKv1.3 is found in lymphocytes [125] and many carcinogenic cells [126, 127], they present similar physiological functions as the plasma membrane counterpart and, they are translated from the same gene. mitoKv1.3 is a target for pro-apoptotic protein Bax [128]. Their complex prevents the opening of mitoKv1.3 channel for K+ influx and therefore causes the disturbance in membrane potential and eventually leads to apoptotic cell death. Therefore, mitoKv1.3 has represented itself as a new tool for targeted cell death for many tumor cells by triggering mitochondriainduced apoptosis. Their presence in cardiac cells has not been reported. Similar to Kv1.3 other potassium channels like Kv1.1 and Kv1.5 have also shown dual origin in both mitochondrial and plasma membrane causing cell apoptosis by targeting macrophages [129, 130].

#### *5.4.4 Twin pore potassium channel*

The mitochondrial TASK-3 was discovered in human keratinocyte HeCaT cells using the patch-clamp method [131]. It shows similarity with its plasma membrane counterpart and its activity is inhibited at acidic pH [132]. Lidocaine and low pH completely block the task channel activity in mitochondria. TASK-3 is essential for the survival of WM35 melanoma cells [133] but its activity in the mitochondrial dysfunction in cardiac reperfusion is not known.

#### **6. Mitochondria channels as a therapeutic target of heart failure**

The above discussions have made it clear that since the inner mitochondrial channels regulate the onset of apoptosis and cell death, they present an important target for cardioprotective therapeutics. Not only mitochondrial potassium channel but also MPTP, MCU, connexin-43, and protein uncoupling have shown their potential roles in reducing myocardial infarct size and preventing heart failure.

A sudden opening of MPTP can be triggered through a high concentration of Ca2+, high amount of ROS production, and decrease in mitochondrial membrane potential, which results in the loss of proton gradient appearing as an uncoupling effect, which prevents ATP formation and promotes its hydrolysis [40]. Subsequently, the proton gradient utilizes the Ca2+ uptake and causes the matrix swelling as an approach of the MPTP to prevent the detrimental rise in Ca2+ in the mitochondrial matrix [134]. It is known that opening of MPTP for a short duration can proceed without affecting cell viability and can also contribute to cardioprotection through participating in pre ischemic conditioning and it later prevents the opening of MPTP pore during ischemic reperfusion preventing cell damage and the onset of heart failure [17]. Although the evidence to support its cardioprotective functions is majorly based on the pharmacology and genetic observation that avoided MPTP opening. It has been shown that the administration of cyclosporin A shows a cardioprotective effect by preventing MPTP opening in the mice model; however, the results are mixed for the large mammalian model [135].

As mentioned earlier an increase in the Ca2+ concentration contributes to the opening of the MPTP channel, it is also necessary to mention that the Ca2+ is essential for the key enzyme activation in the oxidation of the substrates that fuel the respiratory chain, followed by ATP formation. It is very unfortunate that despite the advancement in technologies we are still unable to determine the physiological and pathological concentration of Ca2+ in the mitochondrial matrix [136]. Nevertheless, the Ca2+ homeostasis is maintained within the mitochondrial matrix by the uptake of Ca2+ through uniporters and the release is catalyzed by Na+ /Ca2+ exchanger. The understanding of the molecular nature of Ca2+ uniporter has advanced our knowledge about Ca2+ homeostasis. The deletion of mitochondrial calcium uniporter gene from the embryonic and adult mice has shown completely contradicting results and the reasons of which have not yet been fully understood. However, the results that appeared in adult mice fully support the role of calcium overload leading to MPTP opening and eventually cell death. This is further supported by the Na<sup>+</sup> /Ca2+/Li+ exchanger knockout mice which showed the overload of Ca2+ leading to MPTP opening on the onset of ischemic reperfusion leading to cell death. Therefore, the drugs that intend to target Ca2+ uniporter for therapeutics need to validate the contrast effects before large animal and clinical testing [137].

Connexin43(Cx43) is a well-known channel for the intercellular connections by forming the gap junctions, but apart from the plasma membrane occurrence, they are also known to be present in cellular organelle like subsarcolemmal mitochondria [138], nucleus [139], and exosomes [140]. Cx43 plays an important role in ischemic-reperfusion injury and its prevention. According to pharmacological evidence the concentration of Cx43 increases with the introduction of diazoxide DZX or fibroblast growth factor 2 to prevent myocardial injury, but this increase is not observed after the ischemic preconditioning protocol [141]. It also interacts with the mitochondrial potassium channel [141] and regulates nitric oxide

formation. The role of Cx43 is certain in cardioprotection but the exact mechanism and function remain to be elucidated.

At last, the presence of several mitochondrial K<sup>+</sup> channels and their activity in the failing heart presents them as a crucial target in the therapeutics of myocardial dysfunction. The K<sup>+</sup> uptake and release play a central role in the maintenance of mitochondrial matrix volume. The electrophoretic influx of K<sup>+</sup> is balanced by the K+ /H<sup>+</sup> antiporter [142]. Valinomycin triggers the uncontrolled K<sup>+</sup> influx disturbing the mitochondrial polarization and causing the swelling of the matrix. The function of potassium channels is basically to maintain the matrix volume. Initially, surface K+ channel was suggested to play an essential role in ischemic pre and postconditioning but later when diazoxide (DXZ), which was involved in cardioprotection of non-contractable heart, did not show any effect on surface K<sup>+</sup> channel, whereas drugs that were only targeting surface K<sup>+</sup> did not show cardioprotective effect. On the contrary, the isolated mitochondria showed restored activity of ATP inhibited flux and showed inhibition caused by 5-hydroxydecanoate (5HD) [143]. This shifts the attention to the mitochondrial K channel for cardioprotection but ever since the DXZ and 5HD also affect mitochondrial physiology in general it requires the molecular structure information and in vivo attempts for concrete statements. The structural information will provide tools for determining its exact function in myocardial ischemia/reperfusion in a failing heart, Ca2+ transport and MPTP opening, and protein involved in ROS formation, followed by improving therapeutic approaches [144]. Similar to the ATP activated K<sup>+</sup> channel, Ca2+ and voltage-activated channels are also pharmacologically proven to play a similar role as mitoKATP, the ischemic conditioning protocol triggers the formation of protein kinase C which is helpful in an increased opening mitoKCa channel.

#### **7. Current progress in the field**

The strategies used to protect the heart from opening MPTP and mitochondrial calcium uniporter pores and in the case of ischemia conditioning, the opening of mitoKATP and BKCa channel plays a vital role in the cardioprotection. CsA is a well-known desensitizer for MPTP, but it did not prove to be the best option in clinical trials [65]. CsA exerts its activity by binding to CyPD, but in cases of intense stimuli, the pore opening becomes independent of CyPD. Therefore, there is a need of developing more pharmacological agents that can directly inhibit the MPTP openings, but they require further information about the structural insight of the pore. Although CyPD activates pore opening the complete mechanism is still unclear. Similarly, although it is evident that ROS and Ca2+ influence the MPTP opening but without knowing the structural details of MPTP we fail to conclude how they do so.

Mitochondrial calcium regulates a range of myocyte functions alongside energy production like cell division and trophism. With the development of MCU structures over the years, they have emerged as a very important target for cardioprotection, but the development of a reliable drug is still in process. Potassium channels are also widely accepted as an important target and are closely linked to modulating the apoptotic process. This information is present due to the pharmacology of the channel openers and inhibitors. Very limited knowledge is present to show concrete evidence. The molecular structure can be helpful in understanding and curing several mitochondria-associated diseases.

The inhibition of MPTP channel opening and the mitoK channel both elicit the cardioprotection and are likely to be related. Uptake of K+ through mitoK decreased the mitochondrial membrane potential which reduces the mitochondrial Ca2+, which in turn decreases the possibility of MPTP opening. Along with ATP production, and ROS regulation, mitochondrial channels like MPTP, Ca2+ channel, and mitoK channels are established to play a crucial role in cardioprotection. The mechanism, however, for their connection and coordination with each other in the process of cardioprotection is far from conclusive.

Recent attempts to translate cardioprotective strategies that target some of these mitochondrial ion channels have been hugely disappointing, and the translational of these strategies in clinical settings have not been successful. Several drugs have been tested on various animal models that have shown certain cardioprotective mechanisms. However, the lack of knowledge about the underlying mechanism of protective actions needs a lot of following studies to design modulators specific for mitochondrial channels with regards to cardioprotection in human trials. In the light of studies available, we still have a long way to go in the depth of the cardioprotective mechanism.

#### **8. Conclusions**

Conclusively, heart failure is an outcome of cardiac injury that originated due to a variety of etiologies and denotes a complex clinical syndrome. Several mitochondrial channels associated mechanisms have been recognized that drive the depletion of cardiomyocytes before cell death. These observations not only provide a link of overall heart health with mitochondrial channel opening and closing but also inspires therapeutic approaches. The core molecular identity of some mitochondrial channels like MCU and mitoKATP are discovered recently, whereas most mitochondrial potassium channels are in their intermediate state. These channels act as switches to control the development of ischemic injury either towards recovery or the loss of viability. The progress towards understanding the molecular identity and mechanism of channel opening and inhibition will help to translate the experimental approaches into promising therapeutic development to combat a deadly health concern.

#### **Acknowledgements**

We thank Dr. Nileshkumar Dubey for his help with the figures. **Figure 1** was created with Biorender.com.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Keerti Mishra and Min Luo\* Department of Biological Sciences, National University of Singapore, Singapore

\*Address all correspondence to: dbslmin@nus.edu.sg

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 2
