1. INTRODUCTION:

As per the World Health Organization (WHO) Report 2022, approximately 17% of the world population will be aged 60 years or above by the year 2030. Aging leads to adverse changes in cardioprotective mechanisms resulting in the development of cardiovascular diseases (CVD)like coronary heart disease, peripheral vascular disease, stroke, and heart failure^1-3. Studies have estimated that by 2030, nearly half of the elderly in America will be suffering from some form of CVD⁴.

Thus, it is very important to understand the various parameters that lead to cardiac aging and their underlying mechanisms. The chief molecular mechanisms and cellular processes that are considered to be associated with cardiac aging are a decrease in autophagy activity, mitochondrial dysfunction, telomere attrition, altered insulin-like/insulin growth factor-1 signaling, increased fibrosis, elevated oxidative stress, and calcium release units (CRU) dysfunction¹.

Several studies have correlated Mitochondrial DNA (mtDNA) damage with the development of cardiovascular diseases. Researchers have reported an association between cardiac aging and mtdna damage in the form of mutations and decreased copy number of mtDNA. Although the exact underlying mechanism behind cardiac aging is not fully comprehended yet, the deterioration in mitochondrial function has been contemplated as a fundamental principle behind aging⁵. Despite so many investigations leading to the possible role of mitochondrial dysfunction in aging, no drugs are specifically designated for the regulation of mitochondrial functions as a remedial management for cardiovascular diseases. Thus, the development of specific drugs for the restoration of proper functioning of Mitochondria and efficient drug targeting mechanisms are required as specific targeting leads to effective management⁶.

Presently the management strategy focuses on the mitigation of reactive oxygen species (ROS) in the mitochondria by antioxidants or mitochondria-targeted agents to inhibit cellular and cardiac aging. In the last decade, the key focus has been on the covalent modification of compounds to specific carriers targeted to mitochondria owing to their simple synthesis procedures and efficiency. The negative membrane potential within the inner mitochondrial membrane (IMM) causes the positively charged compounds to aggregate in the mitochondrial matrix against the direction of their concentration gradient. The mitochondrial uptake can be enhanced by associating bioactive agents to lipophilic cations like rhodamine, cyanine, alkyl triphenylphosphonium, and cationic peptides. For many years molecules have been modified by linkage to Triphenylphosphonium to facilitate their targeting to mitochondria⁷. These TPP+ cations have been utilized to deliver probes, pharmacophores, and antioxidants to mitochondria. In the present review, we have compiled the research on the molecular mechanism involved in cardiac aging and have explored the potential role of triphenylphosphonium (TPP+) in its amelioration.

2. PROCESSES AND MOLECULAR MECHANISMS INVOLVED IN CARDIAC AGING:

Accumulating evidence from various research suggests that the muscles and cardiac cells undergo intricate structural and molecular changes during aging. These changes may lead to pathological complications including cardiac hypertrophy, heart failure, structural changes in calcium release units, arrhythmia, myocardial remodeling, and microcirculatory dysfunctions⁸. These changes in the heart not only debilitate normal cardiac functioning but also render the heart vulnerable to various stresses which can further contribute to various cardiovascular diseases (CVD). A number of mechanisms for heart aging have been put forward, for instance, reduced autophagy, dysregulated mTOR signaling pathway, mitochondrial damage, and oxidative stress^9,10. Some of these mechanisms have been discussed in detail below.

2.1. Reduced autophagy:

First observed and described by Ashford and Porter as a self-eating event inside the cell¹¹, autophagy functions via lysosome-mediated degradation facilitating in the elimination and recycling process of damaged cell organelles and misfolded proteins. Thus, promoting cellular longevity and renewal of organelles^12,13. However, another study in 2018 demonstrated that over-activation of autophagosomal pathways can also lead to autophagic cell death¹⁴. Thus, autophagy is not only involved in maintaining homeostasis but in certain settings can also lead to various pathological conditions such as cardiac aging¹⁵.

In cardiomyocytes the autophagy is regulated by two signaling cascades; Beclin 1- mediated activation pathway and MLC/FAK/AKT/mTOR-mediated inhibitory pathway¹⁰. In the heart, autophagy plays an eminent role in maintaining homeostasis, minimizing cardiac-associated injury as well as maintaining normal cardiac functioning in due course of aging¹⁶. Nevertheless, a decline in autophagic efficiency has been observed in the aging heart¹⁷. This impairment in the autophagic flux and efficiency leads to cardiac dysfunction and aging. Mutations in the autophagy gene or complete inhibition of autophagy have been observed to contribute to neuro degenerative ailments and age-related cardiomyopathy¹⁸. For instance, the knockdown of the autophagy gene Atg5 led to decreased mitochondrial respiration and accelerated hypertrophy and aging in the heart¹⁹.

Some hallmarks of cardiac aging include mitochondrial dysfunction, fibrosis, myocardial hypertrophy, and fibrosis. One reason for these conditions can be the aggregation of misfolded proteins and damaged cell organelles due to decreased autophagy. Thus, enhancing autophagy in such cells can improve cardiac homeostasis and alleviate cardiac aging. For instance, enhancing autophagy by inhibition of Akt/mTORC1 signaling has been shown to inhibit heart aging²⁰. Several forms of Sirtuin (Sirt) also referred to as longevity protein have pro- autophagic effects. Sirt-1 has been reported to regulate autophagy through deacetylation of various transcription factors and its upregulation leads to increased autophagy in the cells^21. Rabbit bone marrow-derived mesenchymal stem cells treated by Metformin showed elevated Sirt-1 expression which was associated with delayed aging and increased autophagy²². Overexpression of another Sirt protein, Sirt-6, has been reported to induce autophagy and reduce the formation of foam cells²³. These studies demonstrate that inhibiting autophagy has an aging effect on the cardiomyocytes whereas, augmenting autophagy has an alleviating effect on cardiac aging. However, several other reports suggests that hyper-activation of autophagy may also lead to cardiac damage²⁴.

2.2 Dysregulated mTOR signaling pathway:

Demonstrated as a key pathway in aging, the mTOR (Mammalian/mechanistic target of rapamycin) pathway is entailed in sensing diverse forms of signals involving but not limited to hormones, nutrients, amino acids, and mitogens. The responses transduced by these signals may involve the governing of transcription and translation, inflammation, apoptosis, autophagy, lipid metabolism, mitochondrial biogenesis, and glycolysis²⁵. Various studies suggest that mTOR signaling plays a principal role in the pathogenesis of CVD²⁰, obesity²⁶, cancer²⁷, pulse arterial hypertension²⁸, and neurodegenerative diseases²⁹.

mTOR signaling pathway is found and operates in ribosomes and endoplasmic reticulum, and is a key switch of cell metabolism and protein synthesis. An age-dependent elevated activity of mTOR has been observed in the mice³⁰. Various studies also have established the role of mTOR in the aging process of the heart. A recent study has shown that the activation of mTOR activity suppresses autophagy in the cardiac cells and thus induces cardiac fibrosis³¹. The Pyrin domain consisting of protein (NLRP3), a member of the NLR family, has been reported to play an indispensable role in cardiac aging. The deletion of NLRP3 in mice results in the obstruction of the mTOR pathway and induction of autophagic activity³². mTOR signaling has been associated with cardiac dysregulations. Additionally, studies have demonstrated its involvement in the pathogenesis of atherosclerosis^33,34. Among the two complexes of mTOR i.e. mTORC1 and mTORC2, mTORC1 functions as an antagonist of autophagy by inhibiting the formation of autophagosome³⁵. Age-related deterioration of heart function, as a consequence of mTORC1 activity, has been observed in drosophila³⁶. Inhibition of mTORC1 has shown increased autophagy and cardiac functions in various models^19,97. Contrary to this, mTORC2 positively regulates autophagic activity. A study has shown that activation of mTORC2 by heart-specific knockdown of a novel upstream mTORC2 regulator tgfβ-inhibin/activin-like protein DAW aids in alleviating cardiac dysfunction and reinstates autophagy³⁸.

2.3 Oxidative stress:

The free radical theory of aging hypothesized by Beckman and Ames in 1998 asserts that the elevation in oxidative damage to the delicate cellular structure is related an-associated decline in cellular and molecular functions³⁹. These damages are accumulated as a function of imbalance in the production and mitigation of free radicals such as nitrogen (RNS) and reactive oxygen species (ROS)⁴⁰. Various catabolic and metabolic pathways like the tricarboxylic acid (TCA) cycle and endogenous processes such as the electron transport chain (ETC) are involved in the generation of ROS. Although, implied as an important phenomenon for maintaining homeostasis and survival of the cell⁴¹, hyper-production or accumulation of ROS can give rise to various cardiovascular diseases including cardiac aging⁴².

The mitochondria being the main endogenous producer of ROS due to ETC has been intensively studied in cardiac aging⁴³. Numerous studies have highlighted the eminent role of mitochondrial ROS and oxidative stress in the aging of cardiomyocytes⁴² and death in myocardial ischemia/reperfusion (IR)⁴⁵. Leakage of electrons from ETC accounts for approximately 90% of ROS produced within the mitochondria. Further, the ROS generated leads to the reduction in the functioning ability of ETC thus, setting up a vicious cycle that increases the oxidative stress and aggravates cardiac aging⁴⁶. The mouse model study consisting of the resistant and accelerated aging groups reported increased age-dependent oxidative stress in the accelerated aging group. Moreover, cardiac cells from mice in the accelerated aging group exhibited an exhausted ROS scavengers’ pool and reduced activity of ETC complexes with a significant increase in the oxidative stress markers⁴⁷. The oxidative stress can also lead to defects in mechano-energetic coupling and age-associated cardiac dysfunction by protein oxidative modifications⁴⁸. Activation of the TGF-β pathway in vivo by oxidative stress causes physiological senescence of the cardiac cells and blocking the pathway has shown therapeutic benefits⁴⁹. Further studies done using transgenic mice for overexpression of mitochondria-targeted catalase supported that attenuating prolonged exposure to ROS could alleviate cardiac aging⁵⁰.

3. THE INTERPLAY OF ORGANELLAR ROS IN MITOCHONDRIAL DYSFUNCTION:

In a cell, there are various subcellular compartments known as organelles, and all of these organelles work in synchronization to maintain cellular homeostasis. During various metabolic processes going on inside these organelles, various intermediates and byproducts are formed which, if not taken care of can cause damage to the delicate cellular structure. Production of superoxide and peroxide is one such phenomenon, as their increased concentration inside the cell can cause oxidative stress leading to cell death. Although being the primary source of ROS production, mitochondria can also be impacted by the ROS produced by other organelles.

3.1 Endoplasmic reticulum stress:

Figure 1(A). Interplay of organaller ROS leading to the mitochondrial dysfunction. Disruption of connection between SR and mitochondria in the young heart cardiomyocytes led to an age-dependent decline in Ca²⁺ handling and transfer, signifying that any change or defect in this delicate physiological connection leads to dysregulated Ca²⁺ exchange, thus contributing towards mitochondrial dysfunction and decreased contractile activity in aged heart. The H₂O₂ produced in peroxisome leaked to the surrounding medium, demonstrate that the peroxisomal membrane is permeable to the H₂O₂ and it can readily diffuse through it. This high leakage of ROS from the peroxisomes is very likely to be responsible in pathological conditions. Experiments on S.cerevisiae showed that deletion of peroxisomal Cta1p reduces lifespan of organism.

(B). Redox crosstalk between the endoplasmic reticulum (ER) and mitochondria (MT) takes place at mitochondria-associated ER membranes (MERCs), where various mechanisms encourages the generation of reactive oxygen species (ROS). These mechanisms include calcium flux from the ER to mitochondria through the IP3R/VADC calcium channels, the oxidative folding activity facilitated by the ER chaperone Ero- 1a, and the electron transport facilitated by p66Shc at the mitochondrial electron transport chain (ETC). The substantial ROS generation at MERCs creates redox nanodomains at the interface between the ER and mitochondria, thereby influencing the apposition of ER and mitochondria.

Endoplasmic reticulum (ER)contributes to numerous cellular processes for instance, calcium ion (Ca²⁺) homeostasis, protein synthesis and folding, and their participation towards peroxisome and autophagosome generation⁵¹. Apart from being involved in protein synthesis and folding the ER also functions as an intracellular Ca²⁺ reservoir which is crucial for the contraction and relaxation of the muscles⁵². For instance, the sarcoplasmic reticulum (SR), a type of muscle-specific ER, governs the Ca²⁺ fluxes in cardiomyocytes and regulates the excitation-contraction coupling of the heart⁵³. Several key proteins play important roles in the utilization and release of Ca²⁺ in the ER/SR. For instance, IP3R and RyR regulate the cytosolic release of Ca²⁺from lumen and SERCA⁵⁴.

Almost all the organelles in the cell produce some amount of ROS as the product of metabolic pathways. However, some organelles produce more ROS than others. Recent investigations have demonstrated that the ER is also a potent producer of ROS, although it has a low impact on intracellular oxidative stress in comparison to mitochondria⁵⁵. ROS in the ER is predominantly produced by the oxidative protein folding and by the Nox4 and cytochrome P450 members⁵⁶. Aggregation of unfolded and misfolded proteins in the endoplasmic reticulum due to various stresses and/or aberration in the Ca²⁺ regulation results in the stimulation of ER-associated unfolded protein response (UPR^ER), an important stress response pathway in the ER. (UPR^ER) initially functions as an adaptive process to reinstate ER homeostasis, however, its chronic activation leads to impaired molecular cell machinery and CHOP-initiated apoptosis⁵⁷. In other organs, this apoptotic clearance of dysfunctional cells may have a beneficial effect on the overall organ function, but in the heart, it can have detrimental effects as cardiomyocytes don’t regenerate.

The decline in normal functions of the heart due to aging is a major contributor to cardiac diseases ⁵⁸. In cardiomyocytes, aging has been linked with dysfunction in mitochondria and several cellular processes such as metabolic imbalance, increased oxidative stress, and autophagy⁵⁸. During aging, metabolic modifications such as reduced fatty acid oxidation and enhanced glucose metabolism sensitize the heart toward stress⁵⁹. Also, a reduction in the mitochondrial calcium ion uptake has been observed in the aged hearts. This phenomenon has been associated with elevated mitochondrial oxidative stress and declined NAD(P)H production⁶⁰. Furthermore, faulty communication, in cardiomyocytes, between voltage-dependent anion channel (VDAC) of mitochondria and RyR of SR has been linked with dysregulated handling of Ca²⁺ in aged hearts. Disruption of connection between SR and mitochondria in the young heart cardiomyocytes led to an age-dependent decline in Ca²⁺ handling and transfer, signifying that any change or defect in this delicate physiological connection leads to dysregulated Ca²⁺ exchange, thus contributing towards mitochondrial dysfunction and decreased contractile activity in the aged heart. Moreover, a decline in the UPR^ER’s protective adaptive response and an increase in pro-apoptotic signaling in due course of aging has been observed in various organs including the heart⁶¹. Also, various key regulator proteins, enzymes, and chaperones in the ER become damaged in due course of aging⁶². Particularly, chaperones undergo increased oxidation with aging, resulting in their functional decline, which in turn is associated with aberrant cellular response to ER stress in aging heart⁶³. Increased ER-stress-mediated apoptosis and decreased contractile activity were seen in cardiac-specific Sirtuin 1 (Sirt1) knockdown mice, signifying the role of Sirt1 as a probable ER stress response modulator to protect the heart from ER stress-mediated apoptosis ⁴⁶.

3.1. Peroxisomes generated ROS:

Although several studies have highlighted the role of peroxisomes in H₂O₂⁶⁴, their involvement in various metabolic pathways has also been described⁶⁵. Peroxisomes are involved in the oxidation of fatty acids, anabolism of lipids, and metabolism of glyoxylate and amino acids⁶⁶. The enzymes catalyzing these reactions in the peroxisomes also give rise to ROS⁶⁷.

Previous studies have reported that about 35 percent of rat liver peroxide is of peroxisomal origin⁶⁸. In addition to this, the fact that 20-60 percent of the H₂O₂ produced in peroxisome leaked to the surrounding medium, demonstrates that the peroxisomal membrane is penetrable to H₂O₂ and it can readily diffuse through it. On the other hand, the Pxmp2 channel facilitates the diffusion of peroxide⁶⁹. This high leakage of ROS from the peroxisomes is very likely to be responsible for pathological conditions⁶⁷. Petriv and Rachubinski in 2004 ⁷¹, through their experiment on S. cerevisiae, showed that deletion of peroxisomal Cta1p reduces the lifespan of the organism. The same authors also showed that deletion of Ctl-2, a peroxisomal catalase, in C.elegans, led to a decreased lifespan of long-lived Δclk-1 mutants. Additionally, it was observed that the decrease in the lifespan was also associated with alteration in the peroxisomes’ morphology and dysfunction⁷¹. In humans various health conditions have been linked with the mutation in the catalase genes⁷² and targeting peroxisomes with functional catalase in the catalase-deficient cell lines led to enhanced hydrogen peroxide detoxification and reinstatement of fatty acid levels and cellular plasmalogen⁷³.

Regardless of their morphological, functional, and evolutionary differences⁷⁴ mitochondria and peroxisomes seemingly crosstalk to maintain cellular homeostasis and carry out their linked functions coordinately. Most notable is their coordination in the oxidation of fatty acids. Both, mitochondria and peroxisomes in the animal cell have distinct β-oxidation systems that work in a synchronized manner for fatty acid degradation⁷⁵. The functions of both organelles are closely linked to each other. Generation of ROS and dysfunction of peroxisomes and mitochondria have been linked with various pathological conditions such as carcinogenesis, aging, and neurodegradation⁷⁶. In late-passage human cells elevating the catalase level in the peroxisomes has been linked with restoration of mitochondrial integrity⁷⁷, Nevertheless, decreased catalase activity has been associated with mitochondrial dysfunction⁷⁸. In addition to this, an increase in the lifespan of the transgenic mouse was reported by targeting catalase in the mitochondrial matrix⁷⁹. All these observations put together indicate that there exists an intricate relationship between mitochondria and peroxisomes and any disruption to it leads to pathophysiological conditions.

3.2. Cytosolic ROS:

Several soluble components of the cell for instance, hydroquinones, flavins, thiols, and catecholamines can undergo oxidation and reduction reactions which lead to the generation of ROS⁸⁰. In addition to that, various cytosolic enzymes also contribute towards ROS production in due course of their catalytic activity. Perhaps, xanthine oxidase is an extravagantly investigated intracellular ROS-producing cytosolic enzymes. In normal heart tissues, xanthine dehydrogenase (XHD) catalyzes the oxidation of hypoxanthine to xanthine as an intermediate and further to uric acid as an end product. However, in damaged cells,the XHD gets converted to oxidase form either due to irreversible proteolysis triggered by Ca²⁺-stimulation or due to reversible oxidation of cysteine residues. This oxidase form during hypoxanthine oxidation transfers the electrons to the molecular oxygen which leads to the production of superoxide radical⁸¹. ROS generated in the cytosol functions as a secondary messenger and regulates several signaling pathways. However, its hyper-accumulation can lead to detrimental effects and apoptosis⁸¹. A plethora of evidence suggests the crosstalk between mitochondria and cytosol to maintain proteostasis is present⁸³. Also, it has been reported that the mitochondrial membrane permits the extra- mitochondrial release of the superoxide⁸⁴ and this can be attributed to the presence of mitochondrial permeability transition pore and inner membrane anion channel⁸⁵. The presence of these channels enhances the permeability of the mitochondrial membrane for superoxide and peroxide and given the intense relation between the cytosol and mitochondria in maintaining homeostasis it will not be extravagant to assume that the cytosolic production and accumulation of ROS causes mitochondrial damage and dysfunction. Nevertheless, further detailed studies are needed to develop a complete comprehension of this ROS crosstalk between cytosol and mitochondria and its pathophysiological consequences.

4. THERAPEUTIC POTENTIAL OF TARGETING MITOCHONDRIA:

Healthy cardiomyocytes require a high amount of energy and this elevated energy demand is fulfilled by the breakdown of branched amino acids, fatty acids, and ketone bodies to function as a fuel in the TCA cycle and production of ATP via ETC. In contrast, the glycolysis pyruvate plays a very little role in the production of ATP in the heart⁸⁶. However, in due course of disease initiation and progression, this delicate metabolic structure gets disrupted. Cardiomyocyte metabolic reprogramming has been reported in various cardiomyopathies⁸⁷. The whole molecular mechanism behind this reprogramming remains to be elucidated however, the role of various transcription factors has been proposed in accordance with animal studies⁸⁸. Altogether, these observations indicate that targeting mitochondrial metabolism could be of therapeutic potential.

L-carnitine is well known to promote mitochondrial fatty acid uptake from cytosol. Supplementation of l-carnitine in myocardial infarction patients has been shown to reduce arrhythmias, heart failure severity and incidence, and cardiac death⁸⁹. Nevertheless, successive studies were unable to establish any conclusive effect of l-carnitine^90. In patients with conditions such as congestive HF and angina pectoris β-oxidation inhibitors have also been investigated. Etomoxir, a potent and irreversible inhibitor of β-oxidation showed inconclusive results⁹¹. Contrariwise, two different β-oxidation inhibitors trimetazidine and perhexiline, with similar action as etomoxir, are approved for clinical use as antianginal in many countries⁹². The therapeutic effects of trimetazidine and perhexiline have been postulated to be due to the rebalancing of the fluxes which are linked to activation of autophagy⁹³. However, further clinical trials on both of the drugs have been stopped in the USA due to very narrow therapeutic potential⁹⁴. Acadesine, a compound formed as an intermediate during inosine monophosphate synthesis and reported as a strong inducer of AMP-activated protein kinase (AMPK) which regulates mitochondrial metabolism and biogenesis⁹⁵, has shown encouraging results in targeting mitochondrial metabolism⁹⁶. However, clinical trials evaluating the long-term therapeutic potential of acadesine were stopped due to a lack of promising results⁹⁷.

Dysfunction in mitochondrial dynamics and proteins that regulate mitochondrial fission and fusion have been correlated to pathological conditions⁹⁷. This mitochondrial remodeling is essential for the homeostasis of mitochondria, at least to some extent as the fission results in the mitophagy of dysfunctional mitochondria⁹⁸. Various genetic predispositions and defects have led to mitochondrial dynamics impairment causing various cardiovascular disorders. DNM1L, a mitochondrial fission-fusion regulator, is necessary for mitochondrial fragmentation to adapt to elevated energy needs in cardiomyocytes⁹⁹ and conditional deletion of its one copy has led to mitophagy impairment in the mice¹⁰⁰. Also, transgenically overexpressing DNM1LC452F, a variant of DNM1L, leads to significant defects in mitophagy along with dilated cardiomyopathy¹⁰¹.

Mdivi1, a chemical inhibitor of DNM1L, has exhibited cardioprotective in cardiomyopathy and myocardial IR injury in rodent models¹⁰². Although some studies have questioned the mdivi1 specificity¹⁰³, other DNM1L inhibitors for instance Dynasore and P110 have shown similar mitochondrial protective effects¹⁰⁴. Although these compounds have shown promising results in the preliminary studies, there remains an urgent requirement for clinical trials to evaluate their efficacy in the clinical setting.

5. TARGETING MITOCHONDRIA BY TRIPHENYLPHOSPHONIUM (TPP) CONJUGATES:

Conjugating the molecules with TPP to expedite mitochondrial targeting has long been known. Initially, TPP⁺- conjugates were used to study mitochondrial coupling and kinetics but later on Murphy and his coworkers reinvented and redefined the TPP⁺ for the mitochondrial delivery of pharmacophores, antioxidants, and probes¹⁰⁵. The benefits of using TPP⁺ over other mitochondria-targeting molecules include but are not limited to, its relatively easy synthesis, stability in the biological systems, low cytotoxicity, and presence of both hydrophilic and lipophilic properties. TPP⁺ takes advantage of inner mitochondrial membrane potential for facilitating and targeting the moiety inside the mitochondria. According to the Nernest equation, for every 60mV membrane potential, the intake of lipophilic cations such as TPP⁺enhances by 10 folds thus leading to a significant accumulation of these molecules within mitochondria¹⁰⁶. The electrochemical gradient formed between inner mitochondrial membrane potential and cell membrane potential acts as a potent driving force for mitochondrial uptake of these molecules and since no other organelle possesses the high negative membrane potential as mitochondria, selective delivery to mitochondria is achieved. Various antioxidants and pharmacophores have been tagged with TPP⁺ to increase their mitochondrial bioavailability. Multiple in-vitro and in-vivo investigations have demonstrated favorable therapeutic results.

Among many, Coenzyme Q10 (CoQ10), a liposoluble antioxidant, is well known for its lipid peroxidation inhibition. Various in-vivo CVD models have established that treatment with CoQ10 results in cardioprotection and mitigation of structural damage to mitochondria. However, due to its hydrophobic characteristics and substantial molecular mass, Coenzyme Q10 (CoQ10) displays limited bioavailability ¹⁰⁷. To enhance mitochondria targeting and optimize antioxidant therapy, CoQ10 has been conjugated with the triphenylphosphonium (TPP) carrier, resulting in the formulation of mitoquinone (MitoQ). Positioned at the inner mitochondrial membrane (IMM), MitoQ functions as a potent antioxidant, countering lipid peroxidation and peroxynitrite scavenging. Experimental models, both in vitro and in vivo studies, have shown the cardioprotective outcome of MitoQ treatment. In vitro studies reveal that MitoQ averts oxidative damage and cellular demise induced by hydrogen peroxide(H₂O₂) and chemically triggered ischemia-reperfusion (IR)¹⁰⁸. The ROS- scavenging activity of MitoQ is implicated in the restoration of the function of mitochondria and cardiac performance in acute IR rodent models¹⁰⁹. Furthermore, in a murine heterotopic heart transplantation model, administering MitoQ in the storage solution to the donor's heart shields against IR injury by inhibiting oxidative damage to the graft and suppressing the recipient's early pro-inflammatory response¹¹⁰. Notably, prolonged MitoQ treatment (8 weeks) in spontaneously hypertensive rats results in diminished systolic blood pressure and mitigated cardiac hypertrophy¹¹¹. In a murine model subjected to significant pressure overload, MitoQ mitigated impaired contractile function and enhanced the integrity of the mitochondrial network, along with the alignment of mitochondria and the sarcoplasmic reticulum (SR). At the molecular level, MitoQ disrupted the detrimental interaction among the pro-fibrotic factors and redox signaling linked with mitochondria, concurrently normalizing mitochondrial dynamics. Notably, MitoQ rectified the imbalance in several redox-sensitive noncoding RNAs implicated in cardiac remodeling¹¹².

SkQ1, another lipophilic cation utilizing triphenylphosphonium (TPP) and featuring the chloroplast-derived analogue plastoquinone in place of ubiquinone, interacts with cardiolipin in mitochondria. Cardiolipin, a phospholipid specialized to the IMM, plays a pivotal role in regulating the architecture of cristae, maintaining the integrity of respiratory chain complexes, and organizing supercomplexes¹⁰⁷. Research reveals that the interaction between cardiolipin and cytochrome C transforms the respiratory chain electron carrier into a peroxidase. The association of SkQ1to cardiolipin, disrupts this detrimental interaction, thereby averting cytochrome c-mediated oxidative damage within the mitochondria¹¹³. It has been shown that the SkQ1 is potent enough to ameliorate both, chronic and acute aging¹¹⁴. In various in vivo and ex vivo rat models, SkQ1 has demonstrated protective effects against hydrogen peroxide-induced injury, myocardial infarction, and cardiac arrhythmia¹¹⁵. Another study investigating SkQ1 showed a protective effect on myocardial mitochondrial post-hemorrhagic shock by mitigating excess ROS and showing anti-inflammatory properties¹¹⁶. Owing to these properties of SkQ1 it is the first mitochondrial-targeted drug being utilized clinically¹¹⁷. Although great benefits have been observed in targeting mitochondria using antioxidant SkQ1, it has also been observed to act as a pro-oxidant ¹¹⁸and thus more work needs to be done for its future human cardiac clinical applications.

Mito-TEMPO, a targeted antioxidant for mitochondria has been intensively investigated for its protective effects in numerous heart ailments. It plays a principal role in protecting against oxidative harm to these cellular organelles. Possessing scavenging properties for both superoxide and alkyl radicals, Mito-TEMPO has been demonstrated to aid in the liberation of proapoptotic proteins from the mitochondria. This action mitigates necrosis and apoptosis mediated by ATP-depletion recovery. Additionally, Mito-TEMPO has proven effective in counteracting adverse impacts on the cardiac Na+ channel within cardiomyocytes. The use of Mito-TEMPO holds promise as a potentially effective therapy for burn-induced cardiac dysfunction, as it can significantly reduce the inflammatory cell infiltration caused due to burn and fibrogenesis by mitigating cardiac mitochondrial dysfunction¹¹⁹. In another experiment on murine models of Cardiac dysfunction induced by abdominal Ischemia- Reperfusion Mito-TEMPO alleviated contractile dysfunction caused by impaired calcium homeostasis¹²⁰. In addition to all these effects, the Mito-TEMPO has also been reported to exhibit cardioprotective activity against 5-fluorouracil-induced toxicity while decreasing the levels of cardiac injury markers¹²¹. The protective effect exhibited by Mito-TEMPO is mainly due to its ROS scavenging activity. It is further confirmed by its antiarrhythmic in aged rats by protecting against mitochondrial ultrastructural remodeling, increased ROS, and mitochondrial fission.

6. CURRENT CHALLENGES AND FUTURE PROSPECTS:

Addressing the current challenges and exploring future prospects of utilizing triphenylphosphonium (TPP)- conjugated antioxidants for targeting cardiac mitochondria is crucial for advancing therapeutic strategies. One significant challenge lies in achieving efficient and targeted delivery, considering issues related to bioavailability, particularly in systemic administration. Ensuring the safety profile of TPP-conjugated antioxidants is paramount, given the intricate nature of mitochondrial dynamics and their impact on cellular functions. Additionally, the heterogeneity of mitochondrial populations within cardiac cells poses a challenge in achieving uniform and effective targeting. A comprehensive understanding of the interactions between TPP-conjugated antioxidants and specific mitochondrial components is essential for optimizing therapeutic efficacy and minimizing unintended consequences. These understandings of intricate functions are also required as the TPP+ accumulation inside the mitochondria can lead to the alteration in the membrane potential which may disrupt normal mitochondrial function. Some studies have also shown that the antioxidants especially the ones tagged with TPP+ can also act as pro-oxidants and thus may show unnecessary effects. More studies related to the efficacy of these drugs and compounds in the human population need to be done.

Looking ahead, advancements in nanotechnology offer promise in enhancing targeted drug delivery to cardiac mitochondria, addressing bioavailability concerns. Precision medicine approaches, tailored to individual variations in mitochondrial function and genetics, may enhance treatment outcomes and minimize adverse effects. Exploring synergistic effects with other interventions and identifying reliable biomarkers associated with mitochondrial health will further contribute to the field. Additionally, expanding the preclinical and clinical studies to validate the safety and efficiency of TPP-conjugated antioxidants in diverse cardiac conditions will be crucial for their prospering translation into clinical practice. Navigating these challenges and capitalizing on future prospects will be instrumental in realizing the full therapeutic potential of TPP-conjugated antioxidants for addressing age-related cardiac impairments and associated diseases.

7. CONFLICT OF INTERESTS:

The authors declare that they don’t have any known competing interests. It is also declared that all the authors have studied the final manuscript and have agreed to it

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Received on 25.07.2024 Revised on 21.01.2025

Accepted on 24.03.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3408-3418.

DOI: 10.52711/0974-360X.2025.00492

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Sadhana Jaiswal1, Deepali Rajwade1*, Preeti Mehta2, Hemant Kumar3, Sumit Rajput4