Targeting Mitochondrial Dynamics and Redox Regulation in Cardiovascular Diseases

Abstract

Accumulating evidence highlights the pivotal role of mitochondria in cardiovascular diseases (CVDs). Understanding the molecular mechanisms underlying mitochondrial dysfunction is crucial for developing targeted therapeutics. Recent years have seen substantial advancements in unraveling mitochondrial regulatory pathways in both normal and pathological states. This review presents recent findings on regulators of mitochondrial dynamics and reactive oxygen species, critical factors influencing mitochondrial function in CVDs. Despite the development of potent drugs, specific delivery of drugs into the mitochondria is still a challenge. So, we also discussed advancements in drug delivery strategies aimed at overcoming the technical barrier in targeting mitochondria for CVD treatment.

Targeting dysfunctional mitochondria in cardiovascular diseases

Altered vascular cell functions normally precede cardiovascular diseases (CVDs). The mitochondrial network plays a critical role in determining cellular functions by coordinating with other major cell organelles (Box 1). Dysfunctional mitochondria are among the major contributors to CVDs [1]. To rescue mitochondrial function, we face two major challenges. First, mitochondria are highly dynamic and regulated by numerous exogenous and endogenous molecules in different vascular cells. Generally, the mitochondria are mainly regulated on two interrelated aspects, fission/fusion dynamics and the redox status (the balance between mitochondrial reactive oxygen species (mtROS) and the antioxidant system). Studies indicate that fission/fusion imbalance and mtROS overproduction contribute to atherosclerosis [12-14], a common pathological process that leads to various CVDs (Box 2). This review summarizes novel findings on regulators of mitochondrial dynamics and mtROS that present opportunities for drug targeting. Significant emphasis will be placed on targeting mitochondrial dysfunction during atherosclerosis. Another challenge is how to specifically deliver drugs into the mitochondria. The clinical transition of mitochondrial therapies remains slow and suboptimal [2]. However, novel delivery systems targeting mitochondria have shown promising results in animals. We will review the new progress in recent years and briefly discuss their therapeutic potential in humans.

Box 1. Multifaceted roles of the mitochondria contribute to CVDs.

Mitochondria, essential sub-organelles in mammalian cells, are traditionally known as the cell's "powerhouses," which generate ATP through oxidative phosphorylation. However, recent research has unveiled their multifaceted roles in cell differentiation [1], autophagy [2], innate immunity [3], and programmed cell death [4]. They interact dynamically with various cellular organelles, such as the plasma membrane [5], nucleus [6], endoplasmic reticulum [7], and endosome systems [8], to coordinate cellular functions. Dysfunctional mitochondria are implicated in various diseases, making them prime targets for therapeutic interventions. While recent reviews have highlighted mitochondrial therapy in cancers [9], autoimmune diseases [10], and neurodegenerative disorders [11], there's growing interest in their role in cardiovascular diseases (CVDs).

Box 2. Atherosclerosis is a chronic inflammatory pathological process leading to various CVDs.

Atherosclerosis is characterized by the deposition of lipid-enriched cellular materials and cell debris, forming atherosclerotic plaques in the walls of arteries. These plaques generally develop for decades asymptomatically until the plaque ruptures, causing abnormal blood clots that disrupt blood flow. In the most severe cases, a complete blockage of blood flow can be inflicted, leading to heart attack (myocardial infarction), stroke (cerebral infarction), and even death [83]. In addition, atherosclerosis is also the dominant cause of other CVDs such as heart failure and peripheral arterial disease [84]. Atherosclerosis is initiated when apolipoprotein B-containing lipoproteins accumulate underneath the endothelial layer, the reason of which is still a mystery. These lipoproteins induce the surrounding endothelial cells to secrete pro-inflammatory cytokines such as MCP-1 to attract circulating monocytes to the local lesion sites, where monocytes differentiate into macrophages and secrete more cytokines for the vicious cycle of chronic inflammation [85]. Over decades, a variety of immune cells (mostly macrophages), smooth muscle cells, and dead cells (necrotic core) build up into the plaques, causing the blood vessels to narrow and harden in advanced stages [86]. Importantly, mitochondrial dysfunctions appear to contribute to the progression of atherosclerosis, which is elaborated in the main text.

Regulation of mitochondrial fission/fusion dynamics

Mitochondria exhibit dynamic responses to stimuli, undergoing fission and fusion events mediated by various proteins to maintain metabolic and signaling integrity [16]. The fission and fusion events reach a delicate balance to maintain the proper distribution of metabolites, proteins, and other small molecules for metabolism and signaling [3]. Dysregulated mitochondrial dynamics exacerbate CVD severity, including atherosclerosis, heart failure, myocardial infarction, and arrhythmias [18].

Mitochondrial fission regulators

Fission occurs through a series of events on the outer mitochondrial membrane (OMM). Briefly, a receptor called mitochondrial fission factor (MFF) accumulates on OMM, thereby creating docking sites for dynamin-related protein 1 (Drp1). After docking, Drp1 oligomerizes into a ring-like structure around OMM, which fastens mitochondria until it is split [4]. Although there are other receptors of Drp1, namely FIS1, MID49, and MID51, Drp1 primarily binds to MFF. Recent findings suggest that while Drp1 deletion led to decreased fission, MFF deletion did not significantly reduce fission due to the presence of other OMM receptors of Drp1 [5]. Therefore, Drp1 appears to be a more effective target for fission regulation compared to its receptors, owing to compensatory effects by other receptors. This section therefore focuses primarily on newly uncovered mechanisms of Drp1 regulation that alter the fission/fusion balance and subsequently CVDs. We start with the description of an exogenously developed Drp1 inhibitor, followed by endogenous extracellular molecules/bioparticles, and then intracellular signaling pathways.

Mdivi-1

Mdivi-1 was identified as a small chemical compound that inhibited mitochondrial fission in yeast and mammalian cells through a screening assay [6]. It selectively attenuates Drp1 activity and facilitates mitochondrial elongation due to reduced fission events. Earlier in vitro studies showed that Mdivi-1 protected cardiomyocytes against ischemia/reperfusion (I/R) injury [7, 8]. Interestingly, a recent work in mice demonstrated that Mdivi-1 reduced atherosclerosis plaque formation and dampened pro-inflammatory phenotypes in macrophages [9]. Mechanistically, it inhibited Drp1-mediated mitochondrial fission, which subsequently suppressed mtROS and NLRP3 inflammasome signaling. However, caution is advised when considering Mdivi-1 as a drug, as another study showed that it can lead to defective efferocytosis, potentially resulting in the accumulation of dead cells in our body [10]. Although Mdivi-1 effectively inhibits Drp1 and decreases plaque size in atherosclerosis, it induces side-effects like muscle twitching, tetanus, and contractile stress in skeletal muscles [11]. This highlights the need to explore additional and more specific regulators of Drp1.

Oxidized LDL

Oxidized LDL (oxLDL) is endogenously produced when both proteins and lipids in native LDL react with and are modified by ROS. OxLDL is well-known to promote atherosclerosis by facilitating foam cell formation and chronic inflammation [12-14]. Mechanistically, oxLDL binds to macrophage surface scavenger receptor CD36 and stimulates downstream signaling and metabolic effects. Among these effects, we have recently shown that oxLDL upregulates Drp1, accompanied by a highly fragmented mitochondrial network and continuous induction of mtROS in macrophages [15] which is a pre-requisite for atherosclerotic plaque development. Another study showed similar effects from oxLDL on the induction of Drp1-mediated fission and mtROS, which led to myocardial injury [16]. These studies indicate that an imbalance of the mitochondrial dynamics induced by Drp1 upregulation contributes to CVD progression.

Apoptotic cells

The concept of “one size fits all” does not apply to cell biology, as what is considered harmful in one context can be beneficial in another. The case of Drp1 is a good example. During the early stages of atherosclerosis, dyslipidemia triggers Drp1 to initiate mitochondrial fission, ROS, and death in macrophages. However, in the later stages of atherosclerosis, Drp1-mediated mitochondrial fission plays a helpful role in the removal of apoptotic cells by macrophages, which reduces the necrotic core [10]. The clearance of apoptotic cells (ACs) by phagocytes is a process known as efferocytosis. Defective efferocytosis contributes to many autoimmune and chronic inflammatory diseases, including atherosclerosis [17]. To maintain efficient efferocytosis, phagocytes must continuously internalize multiple ACs. An interesting study demonstrated that the uptake of multiple ACs by macrophages necessitates Drp1-mediated mitochondrial fission, a process triggered by the uptake of ACs [10]. Specifically, when the first AC encounters macrophages, they upregulate Drp1 expression to increase mitochondrial fission, which reduces the mitochondrial calcium sequestration capacity, leading to elevated cytosolic calcium levels. Consequently, calcium promotes intracellular vesicular trafficking and new phagosome formation for the second AC uptake. However, when mitochondrial fission is disabled by the removal of Drp1 in macrophages, AC-induced cytosolic calcium signaling is blunted and efferocytosis of second AC is inhibited. Mitochondrial calcium sequestration is mediated by the mitochondrial calcium uniporter (MCU) [18]. Interestingly, defective efferocytosis was rectified through the silencing of MCU, which makes it a potentially better drug target than Drp1 for advanced stages of atherosclerosis which involves defective efferocytosis.

Micro-RNAs

The role of micro-RNAs (miRs) in cellular homeostasis is an emerging branch of science that inspires research to screen the role of different miRs as potential targets for the regulation of cellular processes [19]. Recent studies have highlighted the role of miRs in mitochondrial dynamics. In a study by Cui et al., it was found that in oxLDL-treated human umbilical vascular endothelial cells (HUVECs) downregulation of miR-199b-5p was associated with increased ROS, mitochondrial fission and apoptosis, whereas these changes were markedly reversed by enhanced miR-199b-5p expression or the application of Mdivi-1. A downstream target of miR-199b-5p was confirmed as A-kinase anchoring protein 1 (AKAP1). The anti-apoptotic effects of miR-199b-5p were reversed by AKAP1 overexpression, facilitated by the increased interaction between AKAP1 and DRP1. Furthermore, human coronary atherosclerotic endothelial tissues exhibited downregulation of miR-199b-5p, upregulation of AKAP1, and excessive mitochondrial fission [20]. This study inspires the idea of screening additional miRs to fine tune Drp1 functions to achieve specificity and efficacy. This could make a great impact in a less explored avenue of combating CVDs.

Inositol-AMPK signaling

AMPK is an energy-sensing intracellular kinase, and its activation in various vascular cells appears to be cardioprotective by regulating mitochondrial homeostasis [21]. In an interesting study, a decrease in inositol level triggered the activation of AMPK and mitochondrial fission. Conversely, the accumulation of inositol prevented AMPK-dependent mitochondrial fission. When cells and mice experienced metabolic stress or mitochondrial damage, inositol levels decreased, resulting in AMPK-dependent mitochondrial fission. Inositol was found to bind directly to AMPKγ and competed with AMP for binding to AMPKγ. This competition limited AMPK activation and subsequently mitochondrial fission. Thus, AMPK functions as a sensor for inositol, and its inactivation by inositol serves as a mechanism to restrict mitochondrial fission [22]. Drugs targeting the inositol-AMPK pathway hold promise for alleviating CVDs associated with dysfunctional mitochondrial dynamics.

CD137 signaling

CD137, a transmembrane protein, is a member of TNFR family for pro-inflammatory and cell-proliferative responses in T cells. These responses have been studied for their potential in the therapeutics of cancer research, but their role in atherosclerosis is less explored. Xu et al., showed that continued activation of CD137L by agonist (anti-CD137L antibody) led to increased necrotic core formation and macrophage apoptosis within the plaques of atherosclerotic mice model. Notably, CD137 signaling promoted mitochondrial fission mediated by Drp1. Inhibition of Drp1 using Mdivi-1 resulted in reduced expression of pro-apoptotic proteins and a decrease in the number of apoptotic macrophages induced by CD137. Moreover, the CD137-induced mitochondrial dysfunction (i.e. mitochondrial membrane potential loss, cytochrome c release, and the generation of mtROS) was alleviated by p38 MAPK inhibitor SB203580 [23]. Therefore, regulating the CD137-MAPK-Drp1 axis could prove to be beneficial in the regulation of mitochondrial fission and could thus be helpful in reducing CVD events.

Epigenetic enzymes

Expression of Drp1 is not only dependent on transcription factors and other proteins but it is also regulated by epigenetic modulation machinery. S-adenosylhomocysteine (SAH) is a potent inhibitor of methyltransferase activity. SAH breaks down into adenosine and homocysteine catalyzed by an enzyme called S-adenosylhomocysteine hydrolase (SAHH). Lower expression of SAHH led to higher SAH levels, which was positively correlated with increased vascular senescence, an early symptom of atherosclerosis. Mechanistically, upregulated SAH repressed DNA methylation in endothelial cells causing overexpression of Drp1, which eventually led to higher mtROS and atherosclerosis [24]. These studies offer a novel therapeutic strategy targeting upstream regulators of Drp1.

Melatonin

Melatonin is an endogenous hormone secreted by the pineal gland. It is also widely used as a drug and dietary supplement in treating sleep disorders due to its role in regulating circadian rhythms [25]. Interestingly, Melatonin regulates both fission and fusion events (its role in fusion is summarized in the next section). Ding et al. reported that Melatonin reduced diabetes-induced cardiac dysfunction by reducing Drp1-mediated mitochondrial fission and decreasing cardiomyocyte apoptosis [26]. Mechanistically, Melatonin downregulates Drp1 in a SIRT1/PGC-1α-dependent manner, as PGC-1α directly controls Drp1 expression by binding to its promoter. The role of melatonin in Drp1 regulation opens an unexplored avenue of circadian rhythm biology in the regulation of mitochondrial dynamics.

Mitochondrial fusion regulators

Mitochondrial fusion involves two steps in both membranes of mitochondria. The first step entails the fusion of OMMs, facilitated by mitofusins (MFNs) 1 and 2 which are large GTPases located on the OMM. MFNs undergo conformational changes, leading to the formation of homotypic (MFN1–MFN1 or MFN2–MFN2) or heterotypic (MFN1–MFN2) complexes, which then facilitate the tethering of adjacent OMMs. The second step is the fusion of inner mitochondrial membranes (IMMs), which is orchestrated by GTPase called optic atrophy 1 (OPA1). OPA1 forms homotypic (OPA1–OPA1) and heterotypic (OPA1–cardiolipin) complexes. It has two isoforms of different lengths: the short isoform S-OPA1 and the long isoform L-OPA1 both of which are required for effective IMM fusion. Newly identified fusion regulators are summarized below.

Melatonin

Vascular calcification is a prominent feature of advanced atherosclerotic plaques. As reported in a recent study, Melatonin upregulated AMPK and OPA1 expression in vascular smooth muscle cells (VSMCs). This upregulation activated mitochondrial fusion and mitophagy, which reduced apoptosis, oxidative stress, and calcium deposition, all of which collectively inhibited VSMC calcification [27]. Melatonin thus demonstrates a dual action, as previously explained, by not only inhibiting Drp1-mediated mitochondrial fission but also enhancing OPA-1-mediated mitochondrial fusion. Also, since Melatonin has already been widely used, it is a potential therapeutic strategy to repurpose it against CVDs by rescuing mitochondrial dysfunction.

Homocysteine

Homocysteine is a homologue of the amino acid cysteine and can be synthesized endogenously. A high circulating homocysteine level is an independent risk factor for atherosclerosis and contributes to CVDs [28, 29]. A recent report showed that exposure to homocysteine enhanced the binding of c-Myc to the DNMT1 promoter. This, in turn, led to hypermethylation of the MFN2 promoter, resulting in the suppression of MFN2’s transcription. Consequently, this contributed to the abnormal proliferation of VSMCs during the formation of atherosclerotic plaques [30]. Although no direct evidence was presented on mitochondrial fusion in that study, it emphasizes that breaking the balance of the fission/fusion dynamics in either direction will bring adverse effects. For drug development targeting the mitochondria, it is better to re-establish the balance than to push the system in one direction.

S89, a small molecule agonist

S89 is a newly developed small molecule agonist of mitochondrial fusion. It directly engages with a loop region situated in the helix bundle 2 domain of MFN1, thereby promoting GTP hydrolysis and vesicle fusion. The presence of S89 rectified mitochondrial and cellular dysfunctions arising from mitochondrial DNA mutations, oxidative stress inducers, and ferroptosis inducers, primarily by enhancing the expression of endogenous MFN1. Remarkably, S89 effectively mitigates mitochondrial damage caused by ischemia/reperfusion (I/R) and safeguards the mouse heart from I/R injury. These findings shed light on the priming mechanism for MFNs and offer a therapeutic approach in situations where additional mitochondrial fusion proves advantageous [31].

Opioid receptors

K-opioid receptor is a GPCR which is mainly associated with mood, anxiety, stress, and depression, but its role is now being explored beyond the coordinative biology. A recent study found that activation of the κ-opioid receptor with its specific agonist, U50,488H, induced mitochondrial fusion and increased the heart’s resistance to MI/R injury. These protective effects were counteracted by nor-BNI, a selective κ-opioid receptor antagonist. Furthermore, κ-opioid receptor activation led to elevated STAT3 phosphorylation and OPA1 expression, both of which were blocked by nor-BNI [32]. Targeting STAT3-OPA1 pathway regulated by κ-opioid receptor activation may offer a potential therapeutic strategy for ischemia/reperfusion injury.

Long noncoding RNAs

Long noncoding RNAs (LncRNAs) regulate gene expression in various ways and are known to contribute to CVDs [33]. Recently, a novel lncRNA called Punisher was discovered to promote beneficial effects by regulating mitochondrial fusion in VSMCs [34]. As reported, Punisher was downregulated in human atherosclerotic plaques. Mechanistically, Punisher functioned as a competing endogenous RNA by directly binding to miR-664a-5p. This interaction subsequently regulated the expression of its target, OPA1, mitochondrial fusion, and ultimately impacted the biological function of VSMCs. Notably, Punisher overexpression significantly suppressed neointima formation and VSMC apoptosis [34]. Thus, both lncRNA Punisher and miR-664a-5p have the potential to serve as novel targets for the diagnosis and treatment of cardiovascular diseases.

Regulation of mitochondrial redox status

In chemistry, oxidation and reduction (or redox reactions) always occur simultaneously, which essentially is the transfer of electrons from one molecule (reductants) to another one (oxidants). In mammalian cells, the mitochondrion is the only organelle that harbors an electron transport chain (ETC), consisting of five ETC complexes located at the IMMs. As a byproduct, electrons can leak from the ETC complex I and III, leading to the generation of ROS, typical types of cellular oxidants. Mitochondrial ROS (mtROS) have physiological roles [35], but excessive ROS are cytotoxic as they modify DNA, proteins, and lipids to alter their normal functions. Therefore, cells develop an antioxidant system to control ROS levels. Redox status refers to the balance between antioxidants and ROS. Since mitochondria are the major sites of ROS production, their redox status is critical for the proper functions of cells and tissues. Excess mtROS are well known to contribute to atherosclerosis and CVDs [36]. Here, we summarize the newly defined regulatory mechanisms (Figure 2) and discuss their therapeutic potential.

Figure 2. Overview of the recently defined cellular pathways that regulate mtROS and, subsequently, atherosclerosis.

Coenzyme Q10 and its downstream signaling up-regulate OPA1, which induces the fusion of mitochondria into large network-like structures. Sirtuin-3 (Sirt3) inhibits mtROS by up-regulating antioxidant systems. Activation of Sirt3 using small chemical agonists becomes a promising future strategy to alleviate CVD progression. Oxidized Low-Density Lipoprotein (ox-LDL) binds to and up-regulates scavenger receptor CD36, which facilitates the transport of the long-chain fatty acids (LCFA) into the mitochondria. LCFA accumulation causes mtROS probably due to electron leakage. Aquaporin 8, present on the inner mitochondrial membrane, causes transmembrane transport of H₂O₂ from mitochondria to cytoplasm, which increases cellular oxidative stress. The figure was created using BioRender.com.

CoQ10-AMPK-OPA1 pathway

Coenzyme Q10 (CoQ10) is an endogenously synthesized coenzyme, which is a component of the ETC. It acts as an electron carrier during the electron transfer and, therefore, can serve as a ROS-scavenging antioxidant. CoQ10 has already been widely used as a dietary supplement. Earlier clinical trials using CoQ10 showed that its long-term treatment reduced the mortality among patients with heart failure [37] and lowered inflammation in coronary artery disease [38]. So far, except for some minor issues, CoQ10 appears to be well-tolerated among the general human population with no serious side effects observed. A recent publication has revealed the molecular mechanism of CoQ10 in the atherosclerosis-prone Apoe^−/− mice [39]. When mice were fed a high-fat diet to induce atherosclerosis, oral gavage of CoQ10 significantly suppressed oxidative stress and alleviated inflammation. Moreover, CoQ10 improved the mitochondrial function in ATP production, and reduced serum total cholesterol, LDL-cholesterol, and triglyceride levels. Mechanistically, CoQ10 activated AMPK, which upregulated OPA1 for the maintenance of mitochondrial integrity [39]. Thus, CoQ10 is a promising anti-atherosclerosis drug. However, more human trials with long-term treatment and follow-up studies are required to understand its efficacy and underlying mechanisms in human bodies.

OP2113, a new class of mtROS blocker

A potential caveat for nonselective ROS scavengers is that they may interfere with the physiological functions of ROS. That may explain the largely disappointing results from human trials on antioxidants [40]. So recent studies focus on the development of more specific ROS inhibitors. OP2113 is a small chemical compound newly discovered to specifically block mtROS production from ETC complex I without affecting oxidative phosphorylation [41]. In two recent studies in rats, OP2113 reduced myocardial infarct size during ischemia/reperfusion injury [42] and had protective effects on chronic hypoxia-induced pulmonary hypertension [43]. Nevertheless, there has been no study to test its effect during atherosclerosis yet. No human trial has been conducted on OP2113.

SIRT3/5 agonists

Sirtuins (SIRT) are a family of protein deacetylases regulating cell metabolism and inflammation. SIRT3 and SIRT5 are both mitochondrial proteins, which regulate mtROS levels by controlling the activities of the antioxidant enzymes [44, 45]. Depletion of SIRT3 in a hypertension mouse model promoted endothelial dysfunction, vascular inflammation, and organ damage [46]. In a recent study on human diabetic patients, SIRT3 levels were inversely associated with the progression of atherosclerosis [47]. Thus, activating mitochondrial sirtuin proteins may be a promising strategy against CVDs. Accordingly, selective SIRT3 and SIRT5 agonists (1,4-DHP derivatives) were developed, which bind to the catalytic cores and stimulate substrate turnover [48]. However, the effects of these compounds on mtROS production await future investigations.

OxLDL-CD36 pathway

As mentioned earlier, oxLDL promotes a pro-atherogenic phenotype in macrophages by binding to a surface scavenger receptor CD36, which is a dual functional receptor for both signaling and lipid metabolism (we reviewed it in [49]). This unique feature of CD36 makes it a potential drug target for metabolic syndromes including atherosclerosis. In the past several years, our lab has been working on a pro-atherogenic signaling pathway, which links extracellular signals to mtROS overproduction in macrophages [15]. Briefly, we have discovered that oxLDL/CD36 signaling leads to macrophage metabolic reprogramming from oxidative phosphorylation to glycolysis for ATP production. However, while the fatty acid oxidation pathway (metabolism upstream of oxidative phosphorylation) is also downregulated, oxLDL upregulates CD36 and its downstream machinery to import long-chain fatty acids (LCFAs) to the mitochondria. These effects result in the accumulation of LCFAs in the mitochondria, which disrupts the IMMs and induces electron leakage for mtROS production. In addition, we showed continuous mtROS induction in both circulating leukocytes and aortic macrophages, which contribute to atherosclerosis in mice [15]. Since CD36 null humans are commonly found in many subpopulations with no serious health issues [49], the CD36 functions don’t appear to be essential for life. Thus, using genetic or epigenetic methods to shut down the macrophage oxLDL/CD36 signaling may be a feasible strategy.

Aquaporin 8 and mitochondrial H₂O₂ transfer

Hydrogen peroxide (H₂O₂) is one of the most well-studied ROS endogenously produced by various organelles including mitochondria. By modifying enzymes, H₂O₂ controls their activity and sometimes stability to fine-tune metabolic processes. It is important not only for normal cellular functions but also for cell adaptation to the changing stressful environments [50]. However, its overproduction causes oxidative stress and heart diseases. Besides using antioxidants to suppress its level as discussed earlier, a novel strategy emerges, which is to limit its transportation across the mitochondria membrane and lower its damaging effects on the nuclei DNA, enzymes, and lipids outside of mitochondria. Originally speculated to diffuse across cell membranes freely, H₂O₂ actually relies on membrane protein channels to cross cell membranes efficiently [51]. Aquaporin 8 belongs to the family of membrane channel proteins that transport water (H₂O) across the cell membrane. New studies show that aquaporin 8 is expressed in IMMs and is important for H₂O₂ transport across the mitochondria membranes [52, 53]. More interestingly, human GWAS have linked SNPs of its encoding gene, AQP8, to atherosclerotic plaque formation, which implicates AQP8 expression in the development of atherosclerosis [54].

Delivery strategies specifically targeting the mitochondria

Numerous strategies have been devised to efficiently deliver bioactive reagents to mitochondria. A classical approach involves incorporating bioactive agents with a mitochondria-targeting backbone, enhancing its pharmacokinetic profile and mitochondrial accumulation. Recent advancements in nano-drug delivery systems enable specific targeting of cells within atherosclerotic lesions, facilitating precise treatment directed at dysfunctional mitochondria in pathological tissues [55]. These novel systems also permit the delivery of mitochondrial mtDNA, mtDNA editors, mitochondrial constituents, and even intact mitochondria. In this section, we will introduce novel strategies and future potentials that are aimed at effectively delivering pharmaceutical agents with high specificity for targeting mitochondria in CVDs (Figure 3 and Table 1).

Figure 3. Graphical representation of different drug delivery systems to deliver cargo into mitochondria for the regulation of ROS and mitigating CVDs.

LIPOPHILIC CATIONS: Antioxidant drugs in conjugation with lipophilic cations, such as triphenylphosphonium (TPP), inhibit CVDs by reducing ROS generation in mitochondria. MITOCHONDRIA TARGETED PEPTIDES: Synthetically produced peptide SS-31 inhibits ROS by inhibiting electron leakage from mitochondria to cytoplasm thereby reducing the CVDs. LIPOSOMES: Bilayered liposomal vesicles called MitoPorter facilitates delivery of respiration regulatory drugs inside mitochondria to mitigate symptoms of atherosclerosis and CVDs. LIPID NANOPARTICLES: Lipid Polymer Hybrid Nanoparticle consists of a single-layered phospholipid and a polymer core. The polymer core helps in the delayed and prolonged release of drugs inside mitochondria for long-lasting results. Polymer core, when infused with antioxidant drugs, inhibits ROS generation, thus reducing CVDs. MitoScript has a more complex structure with multiple proteins containing various domains on its membrane. One of the domains, mitochondrial DNA binding domain (MBDB), binds to and inhibits the ND6 gene, which is located within the mitochondrial genome. This binding leads to the silencing of ND6 and mtROS induction. Although MitoScript is Designed for increased ROS generation, it can also be redirected to maintaining mitochondrial functions by regulating the mitochondrial genome. The figure was created using BioRender.com.

Table 1.

Examples of delivery strategies for percise CVD treatment

Delivery plateform	Bioactive ingredients	Treatment outcomes	Reference#
SS Peptide	ROS scavenger	decreasing mtROS production and protective effect on renal IR injury in vivo	59
MITO-Porter: a liposome-based system fuse with mitochondrial memberane	Resveratrol	enhanced mitochondrial respiratory capacity in myocardial cells	63
	mtDNA	decreased mutation rate and resulted in patient derived myocardial cells replenishment of cellular respiration levels	64
Nanoparticles composed of polypeptide-peptide	Lonidamine	facilitated drug delivery to mitochondria without altering mitochondria structure in vitro	65
Mitochondria-targeted lipid-polymer hybrid nanoparticles	Herbal antioxidant	high drug accumulation in heart and improved anti-myocardial infarction activity in vitro and in vivo	66
MitoScript: a nanoparticle-based synthetic mitochondrial transcription regulator	NADH dehydrogenase ND6 subunit gene silencing suppressor	transporting suppressors of ROS-lowering genes of mitochondria	67
Reconstituted HDL nanoparticles	Inhibitor of CD40–TRAF6 signalling	decreased inflammation, reduced plaque sizes in atherosclerotic plaques in vivo	68
Cyclodextrin-based phospholipid nanoparticles	Simvastatin	decreased plaque size in aorta and brachiocephalic arteries	69
Platelet-derived extracellular vesicles	NLRP3-inflammasome inhibitor	better targeting damaged aorta and better efficacy than free drug,reduced atherosclerotic plaques and lesion site inflammation in vivo	74
Liposome-based multivesicular vesicles	Pro-efferocytotic exosomes derived from MSCs	release exosomes into plaques microenvironment, accelerate apoptotic cell clearance, tinhibit thrombosis in vivo	76
Mitochondria-rich extracellular vesicles	Intact mitochondria derived from cardiomyocyte	restored the intracellular adenosine triphosphate production and improved contractile profiles in hypoxia-injured cardiomyocytes	77

Lipophilic cations and mitochondria-targeted peptides, the conventional strategies

A classic strategy for targeting mitochondria is to conjugate the bioactive molecules with lipophilic cations and mitochondria-targeted peptides [2]. These delivery strategies lead to heightened drug accumulation within the mitochondria and are commonly used for therapies targeting mtROS. As one example, triphenylphosphonium (TPP) is a widely used lipophilic cation compound, which drives a conjugated cargo into the mitochondria [56]. Recent studies have demonstrated the efficacy of TPP-based mitochondria-targeted strategies in dampening mtROS and rescuing mitochondrial functions [57, 58]. Another example is the use of Szeto-Schiller (SS) peptides. These peptides can condense conjugated compounds within mitochondria, with their specificity achieved through selective binding to the inner membrane. As a result, the compounds primarily accumulate in the inner membrane rather than the matrix [59]. Among the SS peptides, SS-31 has exhibited protective effects in reducing atherosclerosis [60], and rescuing cardiac mitochondrial dysfunctions in mice [61].

Although promising, these lipophilic cation-based conventional approaches have several limitations. Firstly, they generally rely on the normal mitochondrial membrane potential for the mitochondrial-specific uptake [56]. However, some cardiac injury conditions may compromise the mitochondrial membrane integrity with loss of the membrane potential [62], which renders these methods ineffective. Secondly, they lack tissue specificity, as the compounds prefer to accumulate in tissues with high mitochondrial content, which may not always be their ideal targets. Thirdly, once delivered into the mitochondria, lipophilic cations tend to accumulate and disrupt the IMMs, which may interfere with the mitochondrial functions. Fourthly, these exogenous peptides, once delivered in vivo, may be quickly cleared by immune cells before reaching their targets. These pitfalls limit the application of novel treatments targeting mitochondria dynamics, as the targets in many cases are not within the IMMs and the bioactive reagents may not be suitable for the conjugation. Thus, alternative strategies are needed to overcome these limitations.

Liposome, lipid nanoparticles, and nano-drug delivery system

Nanocarriers, as highly promising therapeutic vehicles, have garnered considerable attention in the pharmaceutical field in recent years. Over the past two decades, substantial progress has been achieved in the development of precise nano-drug delivery systems for treating atherosclerosis and cardiovascular diseases [55]. Specifically, nanoparticles coated with polymers are engineered to evade clearance by the mononuclear phagocyte system, thereby extending their circulation in the bloodstream and facilitating a continuous release of therapeutic agents at target sites. When combined with mitochondria-directing peptides, the nano-drug delivery system enables the effective delivery of drugs to mitochondria, offering a novel strategy for mitochondrial treatments targeting mitochondria dynamic proteins and mtROS.

MITO-Porter is a liposome-based carrier system. It releases its cargo into the mitochondria by specifically fusing with the mitochondrial membrane. In myocardial cells, MITO-Porter successfully delivers reagents to improve mitochondrial respiratory capacity [63]. In another recent study, MITO-Porter effectively transports wild-type mtDNA into cells derived from patients with mtDNA mutations. This results in a decreased mutation rate and increase in mitochondrial respiration [64].

In contrast to bilayer membrane liposomes, lipid nanoparticles consist of a single phospholipid outer layer. Recently developed nanoparticles, comprising polypeptide-peptide complexes, have demonstrated the ability to facilitate drug delivery to mitochondria without altering mitochondrial structure [65]. Another noteworthy development is a mitochondria-targeted lipid-polymer hybrid nanoparticle delivery system that co-loaded herbal antioxidants, exhibiting high drug accumulation in the heart and improved anti-myocardial infarction activity in vivo [66]. Another recent innovation, MitoScript, is a nanoparticle-based synthetic mitochondrial transcription regulator. By modulating mtDNA transcription, MitoScript silences NADH dehydrogenase ND6 subunit gene, regulating mtROS generation and mitochondrial function [67].

Nanocarriers also can be specifically designed to recognize and deliver drug cargo to atherosclerotic lesion sites, overcoming challenges faced by conventional therapies, including low bioavailability, poor target specificity, and high toxicity [55]. As an example, reconstituted HDL nanoparticles have demonstrated the ability to specifically deliver bioactive agents to macrophages, reducing inflammation in atherosclerotic plaques in Apoe^−/− mice [68]. As another example, cyclodextrin-based phospholipid nanoparticles exhibit a high affinity for the cholesterol-rich microenvironment of plaques. In Apoe^−/− mice fed a high-fat diet, the nanoparticle delivery system effectively reduces inflammation burden in atherosclerotic plaques and decreases plaque areas [69].

Extracellular vesicles

Extracellular vesicles (EVs) are nano-sized membranous vesicles released by cells into the extracellular matrix. EVs carry many bioactive molecules and play a significant role in cell-to-cell communication and regulate mitochondrial functions [70]. They are implicated in various pathophysiological processes [71]. EVs exhibit a natural tendency to accumulate at atherosclerotic plaque sites. Consequently, EVs can serve as effective vehicles for delivering drugs to plaque sites, and they may even accommodate large cargo such as intact mitochondria [72]. Due to the recent progress in technologies for EV isolation, characterization, and manipulation [73], the EVs-based delivery system opens up new possibilities for targeting mitochondrial dynamics and mtROS, thereby expanding the range of therapeutic agents available.

In a recent report, engineered platelet-derived EVs loaded with the NLRP3-inflammasome inhibitor significantly reduce the formation of atherosclerotic plaques and inflammation at lesion sites in vivo [74]. In another study, mesenchymal stem cells (MSC) derived EVs were fabricated with platelet membranes to create platelet-mimetic MSC-EVs. These EVs delivered MSC-secreted miRs into macrophages through lysosomal escape. As a result, lesional macrophages transitioned into an anti-inflammatory phenotype, leading to reduced ROS and lipid deposition, ultimately attenuating the progression of atherosclerosis [75]. MSC-derived EVs can be further engineered into liposome-based multivesicular vesicles. These vesicles release exosomes with pro-efferocytotic agents in the plaque microenvironment, accelerating efferocytosis to clear apoptotic cells. This process also inhibits thrombosis and prevents in-stent restenosis [76]. Finally, EVs are capable of improving mitochondrial functions by delivering exogenous intact mitochondria. Human-induced pluripotent stem cell-derived cardiomyocytes produce mitochondria-rich EVs in a conditioned medium. These EVs, rich in mitochondria, successfully transfer functional mitochondria into hypoxia-injured cardiomyocytes. The transferred mitochondria fuse with the endogenous mitochondrial networks, significantly restoring intracellular adenosine triphosphate production and improving contractile profiles [77]. In a reverse way, the cardiomyocytes also eject EVs containing dysfunctional mitochondria, which are then taken up and cleared by surrounding macrophages [78]. Another study on obesity reported that adipocytes underwent mitochondrial stress and responded by releasing small EVs containing damaged mitochondria, which entered circulation [79]. More interestingly, these small EVs were taken up by cardiomyocytes, and induced ROS production. This subsequently resulted in compensatory antioxidant signaling in the heart and protected cardiomyocytes from further oxidative stress. A single injection of small EVs from stressed adipocytes limited cardiac I/R injury in mice [80]. Taken together, EVs represent endogenous mechanisms that maintain mitochondrial functions and protect the hearts. There are recently completed and ongoing clinical trials testing the safety and potential usefulness of EVs in various diseases including vascular diseases (NCT04313647, NCT03384433, NCT01668849). Although no connection between EVs and mitochondrial functions has been tested in human body yet, we expect this field to be more intensively explored with exciting outcomes in the years to come.

Concluding remarks and future perspectives

Decades of studies have accumulated strong evidence that mitochondrial dysfunction is a critical contributing factor for CVDs. Mitochondrial dysfunction is often displayed as an imbalance between fission and fusion dynamics as well as between the mtROS and antioxidants. Therefore, it has been and will remain, a central topic how to design mitochondrial-targeted drugs to reinstall those balances to reduce atherosclerosis and CVDs. Human trials using newly developed nanosized drug delivery systems have demonstrated their promising future. In combination with physical and chemical engineering methods to achieve tissue and cell type-specific mitochondrial delivery, mitochondrial therapy against CVDs is becoming a more and more feasible and efficient strategy.

Although numerous efforts have been made to uncover the regulatory mechanisms of mitochondrial functions in various cell types under distinct conditions, there are many knowledge gaps in this field (see Outstanding questions) that limit our pace of mitochondrial-targeted drug development. One major reason therapeutically targeting mitochondria is so challenging is that mitochondria are highly dynamic. Most of their parameters are constantly changing even within the same cell. As a brief example, macrophages generally display a highly connected mitochondrial network phenotype with efficient oxidative phosphorylation when they are not activated or in an anti-inflammatory status. However, they show a fragmented mitochondrial phenotype with disrupted oxidative phosphorylation and elevated mtROS when they encounter pathogen antigens (e.g. LPS) and get pro-inflammatory activated [81]. Since this phenotypic change supports their physiological functions in our body, it becomes difficult to define what kind of mitochondrial phenotype marks its dysfunction. To further complicate the scenario, single-cell RNA sequencing data from both animal models and humans have clearly shown that atherosclerosis plaques contain a variety of immune cells, among which macrophage population can even be divided into subpopulations with high heterogeneity in the mitochondrial gene expression [82]. Thus, we emphasize here that mitochondrial dysfunction is an imbalance beyond one cell and even beyond one cell type. Future drug designs should take intercellular communication into consideration and reestablish a balance at least at the organ levels.

Outstanding questions.

• How do different types of cells sense, regulate, and balance mitochondrial dynamics and redox status under physiological and pathological conditions?

• Is there a quantifiable parameter as a reliable indicator of mitochondrial dysfunction?

• Is mitochondrial dysfunction the fundamental driving force of atherosclerosis? If so, what type(s) of cells play the key role and should be the major target(s) for mitochondrial therapy?

• What is (are) the molecular mechanism(s) of intercellular communication to synchronize or maintain mitochondrial functions within a microenvironment?

• Can we engineer naturally occurring EVs into a better mitochondrial-targeted drug delivery system?

Figure 1. Overview of the recently defined fission and fusion regulators that control atherosclerosis progression and CVDs.

Left panels: fission regulators. MicroRNA, miR-199b-5p, up-regulates AKAP (A-kinase anchor protein) signaling, which increases Dynamin-related Protein (Drp1) levels leading to fission of mitochondria and subsequent increase in arterial thickening. Enzyme S-adenosylhomocysteine hydrolase (SAHH) breaks down S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferase (DNMT1) activity which eventually increases Drp1 levels. Melatonin indirectly inhibits Drp1 levels by up-regulating PGC1α through Sirt1 signaling, which is an inhibitor of Drp1. CD137 agonists increase Drp1 protein levels by up-regulating p38MAPK signaling. Mitochondrial division inhibitor (Mdivi-1) inhibits Angiotensin2-GPCR signaling, which causes an increase in Drp1 phosphorylation at Ser616, a Drp1 activation site. Inositol binds to the γ-domain of the AMPK enzyme, rendering it inactive. Consequently, the AMPK substrate mitochondrial fission factor (MFF) is in a dephosphorylated status, which results in abnormally low mitochondrial fission and CVDs. Right panels: fusion regulators. Melatonin increases AMPK signaling, resulting in unregulated OPA1 levels and increased mitochondrial fusion. This will lead to thread-like long-networked mitochondria, eventually reducing atherosclerosis and other CVDs. Long non-coding RNA called Punisher inhibits microRNA miR-66a-5p, which in turn inhibits OPA1 and fusion events. K-opioid receptor, when stimulated by its agonist, causes phosphorylation of STAT3 (Signal transducer and activator of transcription 3). Activated STAT3 increases OPA1 protein levels and a reduction in CVD symptoms. Small molecule agonist S89 brings a conformational change in Mitofusin1 (MFN1) by binding to the HB1 domain, thereby activating the GTPase domain responsible for MFN-MFN attachment and mitochondria fusion. Homocysteine enhances the binding of c-myc to DNMT1 promoter, leading to suppression of MFN2 expression. The figure was created using BioRender.com.

Glossary

AMPK

AMP-activated protein kinase is a serine/threonine kinase that senses intracellular energy levels and regulates energy molecule production.

Dynamin-related protein 1 (Drp1)

Drp1 is a member of the dynamin GTPase superfamily proteins. It uses energy from GTP to mediate mitochondria fission.

Epigenetic modulation

a process of modulating gene expression without changing DNA sequences. It is normally achieved by enzymes modifying either DNA directly or histone proteins. Additional mechanisms include microRNAs targeting mRNA stability or any other processes affecting the chromosome structures.

Electron transport chain (ETC)

a series of mitochondrial inner membrane protein complexes (complex I-V) that transfer electrons, which is coupled with transfer of protons across the inner membrane. The net result is to generate a proton gradient for ATP synthesis within the mitochondrial matrix with the consumption of oxygen.

GWAS/SNPs

genome-wide association studies are to identify genes that are associated with a particular disease. This method normally searches for small DNA sequence variations, e.g. single nucleotide polymorphisms (SNPs), in a certain gene that change its expression.

Inositol

endogenously produced sugar molecules that can serve as second messengers by direct interaction with cellular enzymes including AMPK.

Ischemia/reperfusion (I/R) injury

a tissue injury induced by initial restriction of blood supply to a tissue (ischemia), followed by restoration of the blood supply (reperfusion), which triggers inflammation and oxidative stress damage.

Liposome

small artificial vesicles with spherical shape surrounded by one or more lipid bilayers. They are widely used to deliver exogenous enzymes, DNAs, or siRNAs to the cells.

Lipid nanoparticles

structurally similar to liposomes, but they have a single phospholipid outer layer that encapsulates the interior, which may be non-aqueous. Compared to liposomes, lipid nanoparticles have the advantage of lower toxicity, lower immunogenicity, and higher stability for better drug delivery.

Long noncoding RNAs (LncRNAs)

a large class of RNA molecules that are longer than 200 nucleotides. Although they do not code any genes, some of them are found to regulate gene expression by controlling transcription and mRNA processing.

Micro-RNAs (miRs)

small, single-stranded, non-coding RNAs normally containing 20-23 nucleotides. They are endogenously produced and silence gene expression by binding to mRNAs, leading to mRNA degradation.

Mitochondrial membrane potential

the electrical potential across the inner membrane of the mitochondria. It is mainly contributed by a proton gradient across the inner membrane, which is generated through electron transport chain reactions.

Mitochondria-targeted peptides

cell-penetrating peptides that can deliver conjugated proteins or other cargos into the mitochondria.

Oxidative phosphorylation

a metabolic pathway in which cells use energy released during nutrient oxidation to produce ATP. In eukaryotes, the process takes place only in the mitochondria.

Reactive oxygen species (ROS)

highly reactive chemicals formed from diatomic oxygen, water, and hydrogen peroxide. Examples of endogenous ROS include hydrogen peroxide, superoxide, and hydroxyl radicals. ROS often have physiological signaling functions, but excessive ROS lead to oxidative stress.

Single-cell RNA sequencing

a recently developed technology that examines cell transcriptomics at the single-cell level. Generally, this method can measure mRNA concentrations of thousands of genes (transcriptomics) in each cell and simultaneously determine transcriptomics from thousands of cells from a given population. Compared to conventional methods such as bulk RNA sequencing, it is a more powerful tool to reveal heterogeneity in signaling or metabolic pathways from cell populations.