Mitochondrial redox status as a target for cardiovascular disease

Abstract

Mitochondria are a major player in cellular energetics, oxidative stress and programmed cell death. Mitochondrial dynamics regulates and integrates these functions. Mitochondrial dysfunction is involved in cardiac hypertrophy, hypertension and myocardial ischemia/reperfusion injury. Reactive oxygen species generation is modulated by the fusion-fission pathway as well as key proteins such as sirtuins that act as metabolic sensors of cellular energetics. Mitochondrial redox status has thus become a good target for therapy against cardiovascular diseases. Recently, there is an influx of studies garnered towards assessing the beneficial effects of mitochondrial targeted antioxidants, drugs modulating the fusion-fission proteins, sirtuins, and other mitochondrial processes as potential cardio-protecting agents.

Introduction

Mitochondria, most commonly termed the “powerhouse” of the cell got this name due to their role in energy metabolism and synthesis of adenosine tri-phosphate (ATP), the energy currency of the cell. However, in recent years it is well appreciated that mitochondria are involved in several integral physiological pathways in the cardiovascular system. This includes calcium homeostasis [1], apoptosis [2] and traumatic injury response [3,4], hence, they have gained significance in becoming a drug target for cardioprotection [5]. Mitochondria are one of the key initiators of reactive oxygen species (ROS). Electrons leak along the mitochondrial electron transport chain and oxidative phosphorylation (OXPHOS) which leads to the generation of oxygen derived free radicals, especially superoxide anion (O₂^.−). During OXPHOS, energy from the nutrients are transferred to oxidized nicotinamide adenine dinucleotide (NAD⁺) thus generating reduced form of nicotinamide adenine dinucleotide (NADH). When NADH is oxidized back to NAD⁺, the electrons are transferred back to electron acceptors and molecular oxygen to generate ATP [6]. Significant amounts of O₂^.− are produced by Complex I under two conditions, (i) when the mitochondria are not synthesizing ATP and the proton-motive force is high while the levels of coenzyme Q are low, or (ii) when the NADH/NAD⁺ ratio is high in the matrix [7]. This initial synthesis of superoxide propagates a chain reaction resulting in the synthesis of more stable and reactive free radicals such as hydrogen peroxide (H₂O₂), which in the presence of transition metals (iron-Fe or copper-Cu), can produce hydroxyl radicals (OH^.) through the Fenton or Haber-Weiss reactions.

Mitochondria are also highly motile organelles and their plasticity controls their function. Evidence in the literature suggests a strong interplay between mitochondrial dynamics (fission-fusion pathways) and ROS generation [8]. Mitochondrial proteins such as the sirtuins that use NAD⁺ for their enzymatic activity also modulate the redox status [9]. In this brief review, we will outline the importance of targeting some of these pathways by drug therapies that show promising mitigation of mitochondrial dysfunction in cardiovascular disease [10]. We will highlight some of the mitochondrial targeted antioxidants, drugs modulating the fusion-fission proteins, sirtuins, as well as other mitochondrial processes.

Though mitochondria have built-in check points to prevent the generation of excess free radicals leading to oxidative stress, it is during the cascade of events following cardiac injury and atherosclerosis (i.e. ischemia/reperfusion injury (I/R)) wherein these check points are overwhelmed. Hence, there is a need to therapeutically supplement external or exogenous defense systems such as mitochondria-targeted antioxidants to increase protection or target mitochondrial dynamic pathways that exacerbate damage for cardioprotection.

Mitochondrial Antioxidants

The most well-known endogenous mitochondrial antioxidant enzyme is superoxide dismutase (SOD) which dismutates superoxide to H₂O₂. Three forms of SOD have been identified, copper-zinc SOD, CuZnSOD (SOD1), manganese SOD, MnSOD (SOD2) and extracellular SOD, EC-SOD (SOD3). SOD2 is the form that is highly expressed in the mitochondria. SOD knockouts are embryonically lethal, however, conditional knockout studies using a Cre-lox system have identified its crucial importance in cardiomyopathy and skeletal muscle damage [11]. Though other endogenous antioxidant enzymes exist (i.e. catalase and glutathione peroxidase), the structural architecture of mitochondria prevents these and other small molecular antioxidants to enter the inner mitochondrial membrane (IMM) (see Figure 1). This has led to the discovery of lipophilic, cationic compounds that can penetrate the membrane potential of the mitochondrial inner and outer membrane (IMM and OMM), (some are highlighted in Table 1).

Figure 1. Mitochondrial dynamics within a cardiomyocyte.

A. Diagram of cardiomyocyte mitochondrial architecture within the myofibrils. a. capillary and red blood cell, b. lysosome, c. closely packed mitochondria between myofibril layers, d. Z disks, A band, I band, and M Line of myofibrils, c. mitochondria undergoing autophagy (mitophagy). B. Fusion and fission targets and their respective therapeutic drugs. C. Diagram of reactive oxygen production and scavenging in relation to mitochondrial fusion and fission proteins (DRP1, Fis1, Mfn1, Mfn2 OPA1) and Sirt regulators of apoptosis, ROS regulation and fusion and fission.

TABLE 1.

Mitochondrial targeted antioxidants:

Class	Compounds	Mechanism of action and Relevance to CVD	Ref
Triphenylphosphonium (TPP) Derivatives:	MitoQ	It consists of a TPP attached to the ubiquinone moiety of the CoQ10. Scavenges superoxide, peroxyl and peroxynitrite. I/R, spontaneous hypertension, fatty liver and other kidney diseases	[30]
	MitoTempol, MitoVitE, MitoPBN	Either directly act as SOD mimetic (MitoTempol) by scavenging O2− or by detoxifying Fenton reactions, others (Mito VitE and MitoPBN) act more on preventing lipid peroxidation either as a chain-breaking antioxidant or scavenging carbon-centered radicals. I/R, hypertension	[30]
Skulachev-ion derivatives	SkQ1	TPP conjugated to plastoquinone acts by either recycling the fatty acids thus creating a mild uncoupling reaction or by preventing lipid oxidation of cardiolipin. I/R	[31]
Szeto-Schiller peptides	SS-01, SS-20, SS-31	Small aromatic-cationic peptides scavenge H2O2, peroxynitrite and inhibit lipid peroxidation (probably attributed to the tyrosine residues). MI, hypertension	[32]
SOD/catalase mimetics	Salen Mn complexes, EUK-134, EUK-189, EUK-207	I/R, hypertension	[33]

Mitochondrial Dynamics

Mitochondria undergo constant fusion and fission to maintain a balance between mitochondrial biogenesis and mitochondrial autophagy (mitophagy) or apoptosis [12,13]. Several of the proteins involved in mitochondrial dynamics have been well characterized. Mitofusin 1 (Mfn1), mitofusin 2 (Mfn2) and autosomal dominant optic atrophy-1 (Opa1) are the major proteins involved in the mitochondrial fusion process. Dynamin related protein-1 (Drp1) and fission protein 1 (Fis1) are involved in the fission process. The fusion process is activated during conditions of increased mitochondrial bioenergetics. The fission process is activated during mitochondrial degradation through the autophagosome (autophagy) [14]. The nuclear protein, peroxisomal proliferative activated receptor-γ coactivator 1α (PGC-1α), is a key mediator of mitochondrial biogenesis and an inducer of Mfn2.

Perturbations of the IMM and OMM during ROS leakage are exacerbated by disrupted fusion and fission regulatory pathways. For instance, the dynamin related IMM protein Opa1 has been shown to maintain cristae and Opa1 +/− mice displayed enlarged mitochondria and disrupted cristae leading to cardiomyopathy [15]. Similarly, a tamoxifen-inducible cardiac-specific Drp1 knockout mouse line had mitochondrial dysfunction and impaired mitophagy leading to cardiomyopathy [16]. Interestingly, protection of cardiac tissue by lowering mitochondrial metabolism can occur with DRP1 inhibition [17]. While ablation of MFN1 in the adult heart did not cause a cardiac phenotype [18], ablation of both MFN1 and MFN2 in mice resulted in a lethal cardiomyopathy and mitochondrial fragmentation and cristae disorder [19].

Additionally, both fusion and fission proteins have been implicated in vascular smooth muscle (VSMC) proliferation and pulmonary arterial hypertension. MFN2 was found to suppress proliferation of VSMC and the concomitant increase in fatty acid oxidation and decrease in glucose oxidation [20]. Down regulation of MFN2 and PGC-1α may lead to pulmonary arterial hypertension as MFN2 activity is needed to keep the activity of DRP1 at bay. Thus, fusion/fission proteins are potential targets for therapy. Treatment with the small molecule inhibitor of Drp1, mdivi-1, was shown to prevent the progression of pulmonary arterial hypertension [21]. It should be noted, however, that the benefits of intervention in these cases may only be short-lived as tipping the scales too far in the direction of fusion could lead to downstream complications if worn-out mitochondria cannot be cleared [13].

Thus, a deleterious feedback loop between ROS and dynamics leads to mitochondrial ROS dysfunction and subsequent apoptosis. While detrimental in any cell type, cardiomyocytes are especially vulnerable since mitochondria account for 30% of the volume of cardiomyocytes and the large myofilaments and rigid cytoskeleton likely impede their movements. This is complicated by the fact that mitochondria are damaged during I/R injury and need to be removed and replaced. Some of the drugs targeting the fusion/fission pathway are also being tested in cardiovascular diseases (see Table 2 and Figure 1).

Table 2.

Mitochondrial targeted drugs in consideration for cardiovascular diseases (CVD)

Number	Mito Target	Drug Name	Relevance in CVD	References
1	Mitochondrial fission/fusion drugs	DRP1, Opa1 targets- mdivi-1, Dynasore, P110,	I/R injury, inhibits proliferation	[13,21]
		Mfn targets- S3 (15-oxospiramilactone)		[34]
2	Sirtuins	SRT2104 and SRT2379, SRT1720, SRT2379	Metabolic diseases, atherosclerosis	[35]
3	Respiratory chain inhibitors	Complex I-Amobarbital, Metformin, Rotenone	I/R, TIA, ischemic stroke	[36]
		Complex III- Antimycin A, Myxothiazole	I/R	[37]
		Complex IV-H2S (Complex IV)	MI, arrhythmia, hypertrophy, fibrosis, IR and Heart failure	[38]
4	F1F0-ATPase inhibitors/Proton channel blockers	Aurovertin, Resveratrol	Ischemia, I/R, cardiomyopathy	[39]
		PK1119, Bz-423, Diindolyl methane, Apoptolidin, Oligomycin	Cardiac fibrosis	[40]
5	PT pore inhibitors	4′-chlorodiazepam (ro5-4684)	Arrhythmia, I/R	[41]
		Cyclosporin (CsA), Sanglifehrin A, Debio 025, NIM811	Reperfusion injury, Post-MI injury	[42]
		TRO40303	I/R	[43]
6	NO analogs	MitoSNO1, Nitrolipids, Nitrite, SNO-MPG	I/R, CHF	[44]
7	Potassium channel openers	Atpenin A5, BMS-191095, Cromakalim, Diazoxide, Malonate, Minoxidil, NS11021, NS1619, Pinacidil	I/R, MI, infarct size-limiting effect of ischemic preconditioning, myocardial reperfusion hyperoxygenation, improved cardiac contractile activity	[45]
8	RISK pathway modulators	Lithium, SB216763	Ischemic stroke, I/R	[46]
		Statins	prevent acute life-threatening coronary events	[47]
9	Aldehyde dehydrogenase 2	alpha-lipoic acid	Hypertension, I/R,	[48]
		Alda1	ventricular functioning/HF	[49]
10	Metabolic modulators	A-769662, Acadesine (AICAR), DCA, Etomoxir, GIK, Idebenone, L-arginine, Oxfenicine, perhexiline, ranolazine, trimetazidine	Hypertension, Ischemia, heart remodeling,	[50]

Mitochondrial Sirtuins

Sirtuins are modulators of energy metabolism, DNA repair, oxidative stress and play important roles in cardiovascular disease [22]. Sirt-1 is both a nuclear and cytoplasmic protein and has been observed in mitochondria, while Sirt-3, 4, and 5 are mitochondrial proteins. Sirt-1 has been shown to regulate both biogenesis and degradation of mitochondria. Its expression decreases upon trauma and hemorrhage in rats leading to decreased PGC-1α expression. Since PGC-1α activates both peroxisome proliferator-activated receptor alpha (PPARα) and nuclear respiratory factor-1 (Nrf-1) to promote mitochondrial biogenesis, Sirt-1 modulates the creation of new mitochondria during tissue repair in cases of cardiac injury [23]. Indeed, Sirt-1 has the widest known regulatory activity among the Sirt family and can deacetylate forkhead box O (FoxO), nuclear factor- kappa B (NF-κ B), protein kinase B (Akt), and p53, thereby modulating fatty acid oxidation, cardiac hypertrophy, apoptosis, oxidative stress, and autophagy. In cardiomyocytes, activated FoxO upregulates MnSOD, catalase, and thioredoxin1, and anti-apoptotic factors, like B-cell lymphoma-extra large (Bcl-xL) [24]. Several therapeutic pharmacological activators of Sirt-1 (see Table 2), as well as some natural activators such as caloric restriction, stachydrine (found in citrus), omega 3 fatty acids and vitamin E are currently under clinical investigation for treatment of CVD.

NAD⁺ levels increase during starvation as well as during altered redox status, and Sirt-1 and Sirt-3 activity are part of a response to that oxidative condition. In relation to the redox status, Sirt-3 increases the transcription factor, Forkhead box O3 (FOXO3), binding to the mitochondrial superoxide scavenger genes SOD2/MnSOD, cytochrome C oxidase assembly protein (SCO2), and catalase [25], thus modulating oxidative stress. Sirt-3 can also directly activate SOD2 activity by deacetylating it [26]. To regulate ATP production, Sirt-3 also plays important roles in the electron transport chain. It interacts with and deacetylates members of Complex I. Additionally, Sirt-3 is responsible for maintaining ATP levels in the heart and kidney [27]. A component of Complex II and tricarboxylic cycle (TCA) enzyme, succinate dehydrogenase complex subunit A (SdhA), is a substrate for Sirt-3. Here, Sirt-3 stimulates succinate dehydrogenase activity lowering levels of NAD⁺ [28]. A decrease in Sirt-3 increases I/R injury, mostly in aging hearts and in pulmonary artery hypertension. Sirt-4 represses malonyl CoA decarboxylase and its knockdown results in increased β-oxidation. Due to its availability to modulate hypoxia-induced cardiomyocyte viability and apoptosis, Sirt-4 activation can be beneficial in ischemic heart disease. Sirt-5 is known to be downregulated by oxidative stress in cardiomyocytes and to act as a safeguard against ROS by reducing ROS-induced cell death [29].

Other mitochondrial targets

In addition to the targets mentioned above, there are several drugs that are being targeted to other mitochondrial proteins such as, the F1F0 ATPase inhibitors, potassium channel openers, nitric oxide analogs, drugs that target aldehyde dehydrogenase etc. (Table 2). Many of these compounds listed in Table 2, are being tested in cardiovascular diseases.

Conclusion

Mitochondria are a major player in bioenergetics but also participate in other metabolic pathways. Cardiac and vascular tissues are highly dependent on mitochondrial homeostasis. Several drugs over the years have targeted mitochondrial proteins in the hopes of providing cardioprotection. The majority of these drugs did not reach clinical significance due to limited bioavailability or toxicity. Future discovery of mitochondrial-targeted drugs will be focused on improvement in cytotoxicity and efficacy.

Highlights.

• Mitochondrial ROS and dynamics play a role in cardiovascular diseases

• Mitochondrial fusion-fission pathways regulate cardiomyocyte function

• Mitochondrial targets are useful in cardioprotection