Mitochondrial complex-1 as a therapeutic target for cardiac diseases

Abstract

Mitochondrial dysfunction is critical for the development and progression of cardiovascular diseases (CVDs). Complex-1 (CI) is an essential component of the mitochondrial electron transport chain that participates in oxidative phosphorylation and energy production. CI is the largest multisubunit complex (~ 1 Mda) and comprises 45 protein subunits encoded by seven mt-DNA genes and 38 nuclear genes. These subunits function as the enzyme nicotinamide adenine dinucleotide hydrogen (NADH): ubiquinone oxidoreductase. CI dysregulation has been implicated in various CVDs, including heart failure, ischemic heart disease, pressure overload, hypertrophy, and cardiomyopathy. Several studies demonstrated that impaired CI function contributes to increased oxidative stress, altered calcium homeostasis, and mitochondrial DNA damage in cardiac cells, leading to cardiomyocyte dysfunction and apoptosis. CI dysfunction has been associated with endothelial dysfunction, inflammation, and vascular remodeling, critical processes in developing atherosclerosis and hypertension. Although CI is crucial in physiological and pathological conditions, no potential therapeutics targeting CI are available to treat CVDs. We believe that a lack of understanding of CI’s precise mechanisms and contributions to CVDs limits the development of therapeutic strategies. In this review, we comprehensively analyze the role of CI in cardiovascular health and disease to shed light on its potential therapeutic target role in CVDs.

Introduction

Mitochondria are the central players in cellular life and death and are crucial for our survival as they rely on oxygen to function effectively. Mitochondria consume approximately 98% of the oxygen we breathe, highlighting their vital role in the physiology of higher species. These remarkable organelles power cellular processes, enabling muscle contractions in the skeletal, cardiac, lung, and gut tissue. They maintain essential ionic gradients across cell membranes, support the secretion of hormones and neurotransmitters, and provide energy for numerous other functions necessary for sustaining life. Mitochondrial dysfunction can lead to various diseases, ranging from minor changes in tissue function to severe disability or death. In particular, mitochondrial dysfunction plays a significant role in the pathophysiology of cardiovascular diseases (CVDs), including cardiomyopathy, heart failure, coronary artery disease, stroke, myocardial infarction, and pressure overload [1].

Mitochondrial oxidative metabolism is responsible for about 90% of ATP production in the heart and is associated with cardiovascular health. Environmental and genetic risk factors contribute to oxidative stress and mitochondrial DNA damage, primarily by generating reactive oxygen species (ROS) in cardiomyocytes and endothelial cells [2]. Uncoupled electron transport and disassembly of mitochondrial complex-1 (CI) can trigger dysfunction and cardiomyopathy in the heart and lead to heart failure [3–5]. Maintaining CI activity can prevent these adverse effects by preventing incomplete electron reduction and maintaining a balanced respiration ratio. Recent studies have explored potential therapeutic interventions, including posttranscriptional regulators like miR-762 and miR-34a. These have shown promise in rescuing cardiomyocytes from oxygen radicals under hypoxic conditions by inhibiting CI and augmenting glycolysis [6, 7]. Mitochondrial dysfunction is also associated with elevated angiotensin II levels, resulting in pressure overload-induced cardiac hypertrophy, decreased mt-DNA content, and increased ROS in cardiomyocytes [8–10]. Antioxidants and CI inhibitors can mitigate these effects in preclinical studies but fail in human clinical trials [9, 11]. This phenomenon could be due to excess suppression of oxidative stress that might lead to reductive stress [12]. Therefore, further research is needed to understand the relationship between CI-related mitochondrial dysfunction and CVDs. Thus, the comprehensive knowledge from this review will help understand the mechanisms of mitochondrial CI-mediated cardiac dysfunctions and identify therapeutic interventions that positively impact mitochondrial function, leading to effective preventive strategies for CVDs.

Mitochondria

The mitochondrion is a highly specialized organelle found in almost all eukaryotic cells. It plays a crucial role in cellular energy production through oxidative phosphorylation (OXPHOS) [13]. Also, mitochondria are involved in various essential cellular functions such as calcium signaling, cellular metabolic regulation, heme synthesis, steroid synthesis, and programmed cell death (apoptosis) [14]. They are architecturally dynamic organelles with a 16.5-kb circular genome known as mitochondrial DNA (mt-DNA), which encodes 13 respiratory complex subunits, 22 tRNAs, and two rRNAs in mammals [15] (Fig. 1).

Fig. 1.

Mitochondrial DNA architecture and its encoded genes. Left is the mitochondrion, which shows inner (IMM) and outer membranes (OMM) and localization of mtDNA in the matrix. Right is the mtDNA, a circular, double-stranded molecule approximately 16.5 kilobases in size, distinct from nuclear DNA and maternally inherited. It encodes 37 genes essential for mitochondrial function: 13 proteins, 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs). The protein-coding genes include components of the mitochondrial respiratory chain complexes such as ND1-ND6, CYTB, COX1-3, and ATP6-8. The tRNAs are crucial for mitochondrial protein translation, and the rRNAs (12S and 16S) form part of the mitochondrial ribosomes. The non-coding D-loop region contains elements for replication and transcription initiation, including the origins of replication for the heavy (H) strand and light (L) strand, which are rich in guanine and cytosine, respectively. The H-strand origin is within the D-loop, while the L-strand origin lies outside it, facilitating bidirectional replication.

Mitochondria exist as discrete entities within cells but are interconnected in networks, with multiple copies of mt-DNA in each mitochondrion [15]. The regulatory D-loop region of mt-DNA binds nuclear-encoded mitochondrial transcription factor (TFAM) and other regulatory proteins (mtSSB, TWINKLE, PolY), which is crucial for mt-DNA replication and transcription [15]. The mitochondrial proteome consists of approximately 1500 proteins, with the majority (99%) encoded by nuclear DNA and only ~ 1% by mt-DNA [16]. Oxidative phosphorylation, a significant function of mitochondria, is facilitated by four electron transport chain (ETC) complexes: CI, complex-II (CII), complex-III (CIII), and complex-IV (CIV), along with complex-V (CV) that has ATP synthase activity [13]. These complexes assemble into a respirasome supercomplex, which transfers electrons from CI to CIV via CIII [17]. Respiratory complexes and supercomplexes are assembled through step-by-step processes with precise parameters [17]. Mitochondria possess conserved regulatory systems to maintain their integrity and adequate mitochondrial mass and ensure proper bioenergetic and metabolic processes due to their crucial role in cellular homeostasis and cardiac development [15, 18]. Therefore, targeting mitochondria and enhancing metabolism hold potential as therapeutic approaches to reduce the incidence of clinical diseases [19].

Origin and evolution of mitochondria

The origin and evolution of mitochondria have been the subject of scientific investigation. Like the chloroplast, the mitochondrion is believed to have originated from a bacterial progenitor through endosymbiosis. During this evolutionary stage, it is hypothesized that the chloroplast utilized sunlight to produce oxidizable fuels, while the mitochondrion used these fuels to generate energy-rich biological molecules [20]. One of the most compelling pieces of evidence supporting the independent bacterial origin of mitochondria is the presence of mitochondrial DNA (mt-DNA). Mitochondrial DNA is a circular genome with structural properties that resemble primitive bacteria’s circular DNA. Its existence points to the mitochondrion’s evolutionary roots [20]. Another study shed light on the evolutionary process leading to the formation of mitochondria. A recent study proposed that the ε and ζ subunits can be traced back as pre-endosymbiotic genes, which eventually evolved into the mitochondria as we know them today [21]. While mitochondrial DNA encodes a few essential proteins, as mentioned before, most proteins required for mitochondrial function are encoded in the nucleus and transported to the mitochondria. Human mt-DNA, for example, is a 16.5-kilobase double-stranded DNA circle that codes for only 13 proteins, all components of the respiratory chain.

Additionally, mt-DNA contains 24 additional genes encoding two rRNAs and 22 tRNAs involved in synthesizing these 13 proteins. The fact that a sophisticated organelle like the mitochondrion requires hundreds of proteins raises intriguing questions about mitochondrial growth, replication, coordination with nuclear gene products, and protein import into mitochondria. These structural considerations have implications for understanding mitochondrial function and the potential consequences of disruptions in coordination, which may lead to pathological conditions. In the following sections, we will delve into the structure and function of mitochondria and the implications of mitochondrial CI mutations or deficiencies in CVD. It is crucial to grasp the fundamental principles governing mitochondrial function before comprehending the functional impact of mutations and protein expression [20].

Structure of mitochondrion

Mitochondria are typically oval-shaped, autonomous organelles, although their appearance can vary depending on dynamic events, ranging from extended reticular networks to rod-shaped structures [22]. The structure of mitochondria is intricately linked to their function, with each feature contributing to highly specialized purposes. Mitochondrial content are surrounded by two membranes: the outer and inner mitochondrial membranes (Fig. 2).

Fig. 2.

Structure of Mitochondrion. The figure provides a detailed overview of the mitochondrion’s structure, emphasizing its critical roles in cellular metabolism and genetic regulation. The mitochondrion is depicted with its outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM), which enclose the mitochondrial matrix. Within the matrix, the processes of glucose oxidation via the citric acid cycle (Krebs cycle) and fatty acid oxidation are highlighted, demonstrating their importance in generating electron carriers NADH and FADH2. These carriers transfer electrons to the electron transport chain (ETC) complexes (I–IV) embedded in the IMM, facilitating oxidative phosphorylation (OXPHOS). The resulting proton gradient drives ATP synthesis by ATP synthase (Complex V), producing the cell’s primary energy currency. The figure also illustrates mitochondrial DNA (mtDNA), emphasizing its circular structure and location within the matrix and the process of mtDNA replication and transcription. Additionally, the Translocase of the Outer Membrane (TOM) complex and the Translocase of the Inner Membrane (TIM) complexes (TIM22, TIM23) are depicted, highlighting their roles in importing cytoplasm-synthesized proteins into the mitochondrion. This figure comprehensively illustrates the mitochondrion’s critical functions in energy production, genetic maintenance, and protein import, underscoring its vital role in cellular metabolism.

These membranes occasionally combine to create junctional complexes or contact sites, similar to gap junctions [23]. The outer membrane separates the organelle from the surrounding cytosol. It has a structure comparable to the cell membrane, allowing lipid-soluble compounds to diffuse into the intermembrane gap. The voltage-dependent anion channel, or porin, is abundantly produced in the outer membrane and facilitates the transport of small proteins (< 5000 Da) and hydrophilic molecules [24]. The inner mitochondrial membrane surrounds the central matrix of the mitochondrion and is impermeable to polar molecules and ions. This membrane is enriched in proteins and cardiolipin compared to the outer membrane, with a higher abundance of proteins required for various metabolic pathways within the organelle [25]. The impermeability of the inner membrane combined with the relatively more porous nature of the outer membrane creates the intermembrane gap, which has a specialized environment similar to the cytoplasm but is specifically adapted for larger proteins with mitochondrial roles [26]. This structural organization significantly influences mitochondrial activity and can pose challenges when targeting mitochondria with drugs.

Although mitochondria have their genome (Fig. 2), which is unusual among animal cell organelles, it only encodes 13 respiratory chain proteins. Consequently, mitochondria depend on the nucleus to synthesize most of their components, which must be imported from the cytoplasm. Larger proteins require specialized mitochondrial targeting sequences to access the mitochondria [16]. The mitochondrial localization signal or mitochondrial targeting sequence, a short peptide with positively charged basic amino acid residues, directs the delivery of a protein to the mitochondria. These typical sequences are found in the N-terminal region of mitochondrial proteins, where they alternately pair hydrophobic and positively charged residues to form an amphipathic helix [25]. The translocator of the outer membrane (TOM) complex and the translocator of the inner membrane (TIM22, TIM23) complex proteins located on the outer and inner mitochondrial membranes, respectively, play vital roles in regulating the import of proteins into different mitochondrial compartments (Fig. 2). The signal sequence of a protein is recognized by a receptor protein in the translocator of the TOM complex, guiding the polypeptide to interact with the translocator of the inner membrane at close contact areas between the inner and outer membranes. This process allows the polypeptide to enter the mitochondrial matrix or travel laterally into the inner membrane [16]. The internal folds of the inner mitochondrial membrane, known as cristae, were traditionally thought to be formed by invagination of the inner membrane. However, recent studies have revealed that cristae structure can vary dramatically among tissues, and the functional implications of these structural variances still need to be fully understood. Cristae biogenesis is influenced by ATP synthase, highlighting the connections between cellular bioenergetics, inner membrane shape, and mitochondrial morphology [27]. Furthermore, discrete subdomains within cristae have been identified, showing enrichment of specific mitochondrial proteins, including OXPHOS proteins [28].

Alterations in cristae structure have been linked to functional consequences. For instance, the ablation of the fusion protein OPA1, responsible for cristae remodeling, has been shown to affect mitochondrial respiratory chain supercomplex formation and function, ultimately leading to cell death [29]. Similarly, downregulation or inhibition of LonP1, a major mitochondrial protein quality control protease, results in abnormal cristae [30–32]. Studies have also demonstrated that changes in mitochondrial network morphology, including elongation and fragmentation, can occur in response to metabolic shifts in energy transduction and cellular activation, such as in anti-inflammatory and pro-inflammatory macrophages [33]. Despite advancements in our understanding, the critical functions of cristae in mitochondrial maintenance still need to be fully elucidated due to their adaptive capabilities and involvement in complex assembly processes. Additionally, mitochondrial architectures exhibit macroscopic differences between tissues, further emphasizing the diversity of mitochondrial structures [22].

Oxphos

Cellular metabolism involves the synthesis and breakdown of biomolecules to derive energy. This includes catabolic processes such as glycolysis and subsequent pyruvate oxidation for glucose, fatty acid β-oxidation for fatty acids, and oxidative deamination and transamination for amino acids. The molecules derived from these pathways enter the tricarboxylic acid cycle and produce substrates that participate in the ETC for oxidative phosphorylation. Mitochondria serve as the site of adenosine triphosphate (ATP) production, where various catabolic and anabolic pathways occur through OXPHOS. The OXPHOS system consists of five multiprotein enzyme complexes (I–V) and two-electron carriers, cytochrome c and coenzyme Q, embedded in the lipid bilayer of the mitochondrial inner membrane [34, 35]. The primary function of the OXPHOS system is the coordinated transport of electrons and protons, resulting in ATP production. OXPHOS is significantly more efficient than glycolysis, as one glucose molecule can yield up to 36 molecules of ATP in OXPHOS, and fatty acids even more, whereas glycolysis only produces two ATP per glucose molecule; thus, the energy-demanding heart relies on OXPHOS.

During intermediate metabolism (such as the Krebs cycle and fatty acid oxidation), mammalian nutrients such as glucose, amino acids, and fatty acids are converted into reduced forms: NADH, H +, and FADH2. Mitochondria oxidize these reduced equivalents through the ETC to generate energy as ATP. The ETC comprises four multisubunit complexes: CI, -II, -III, and -IV, with mobile electron carriers ubiquinone (coenzyme Q10) and cytochrome c [36] (Fig. 3).

Fig. 3.

Electron transport chain (ETC) complexes in oxidative phosphorylation (OXPHOS). This figure details the electron transport chain (ETC) complexes involved in oxidative phosphorylation, highlighting their composition and function. The ETC, embedded in the inner mitochondrial membrane (IMM), consists of five main complexes (I-V), each composed of subunits encoded by both mitochondrial DNA (mtDNA) and nuclear DNA. Complex I (NADH: Ubiquinone Oxidoreductase) initiates electron transport by oxidizing NADH, transferring electrons to ubiquinone, and pumping protons into the intermembrane space, comprising 45 subunits, 7 of which are encoded by mtDNA (ND1, ND2, ND3, ND4, ND4L, ND5, ND6). Complex II (Succinate: Ubiquinone Oxidoreductase) oxidizes succinate to fumarate, transferring electrons to ubiquinone without proton pumping, and all 4 of its subunits are encoded by nuclear DNA (SDHA, SDHB, SDHC, SDHD). Complex-III (Ubiquinol: Cytochrome c Oxidoreductase) transfers electrons from ubiquinol to cytochrome c, pumping protons into the intermembrane space, comprising 11 subunits, with 1 encoded by mtDNA (CYTB) and 10 by nuclear DNA. Complex IV (Cytochrome c Oxidase) catalyzes the transfer of electrons from cytochrome c to molecular oxygen, forming water and pumping protons into the intermembrane space, with 13 subunits, 3 encoded by mtDNA (COX1, COX2, COX3) and 10 by nuclear DNA. Complex V (ATP Synthase) synthesizes ATP from ADP and inorganic phosphate, driven by the proton gradient created by the previous complexes, consisting of 16 subunits, two encoded by mtDNA (ATP6, ATP8). This comprehensive depiction emphasizes the collaborative roles of mtDNA and nuclear DNA in encoding the subunits essential for mitochondrial oxidative phosphorylation, highlighting the intricate coordination required for efficient cellular energy production.

NADH supplies electrons to CI, while FADH2 supplies electrons to CII. These electrons are then transferred to ubiquinone and routed to complexes III and IV via CoQ and cytochrome c. The final acceptor of electrons is oxygen (1/2 O₂), which receives electrons from Complex-IV. During this process, proton transfer occurs from the mitochondrial matrix to the intermembrane space at complexes I, III, and IV levels. This process generates an electrochemical proton gradient, known as the proton motive force, across the inner membrane, which can be separated into a transmembrane electric potential difference (ΔΨm) and a pH gradient. Complex-V, or F1/Fo-ATP synthase, utilizes this proton gradient to synthesize ATP from ADP and Pi. During this process, uncouplers can disrupt oxidative phosphorylation by allowing electron transport but preventing the phosphorylation of ADP into ATP. They increase the permeability of the inner membrane to H⁺, which inhibits the formation of proton gradients across the membrane. Although there are reports that these sites are impaired in myocardial ischemia, the related processes are not fully understood. However, targeting the mitochondrial energy conversion cascade to maintain energy transduction and limit the loss of electron propensity from the ETC has been proposed to improve myocardial function [37].

ETC complexes

The mitochondrial ETC consists of transmembrane protein complexes (I-IV) containing ubiquinone and cytochrome c. These complexes are found in folded inner membranes called cristae and are responsible for transferring free mobile electrons. To perform the proper activity, ETC complexes are assembled with F1F0-ATP synthase or Complex-V, known for generating ATP during oxidative phosphorylation [38, 39]. Each complex’s structural and functional concept is based on the whole process of electron transportation and ATP synthesis through the ETC.

CI, also known as NADH: ubiquinone oxidoreductase is the largest ETC complex. It contains a multisubunit enzyme that transfers electrons from matrix NADH to ubiquinone (Fig. 3). Atomic resolution X-ray crystallography studies have been instrumental in determining the structure of CI[40, 41]. The L-shaped eukaryotic structure of Complex-I, observed in Bos taurus heart mitochondria, is considered the best model for human Complex-I. It consists of two domains: the membrane arm submerged in the inner membranes and the matrix arm stretched into the matrix. These domains comprise 14 core subunits conserved in bacterial CI and carry out enzymatic reactions [40]. The matrix arm contains seven core subunits (NDUFS1, NDUFS2, NDUFS3, NDUFS7, NDUFS8, NDUFV1, and NDUFV2) with various cofactors, including flavin mononucleotide (FMN) molecule, FeS clusters, and the electron-accepting iron-sulfur cluster (N2 cluster) [40, 41]. The mitochondrial genome encodes the membrane arm and consists of seven hydrophobic subunits (ND1-6 and ND4L) and additional accessory subunits [42], possibly because the synthesis and trafficking of these proteins cannot be that efficient from the outside.

CII, also known as succinate dehydrogenase, is a Krebs cycle and ETC component. It facilitates the connection between metabolism and oxidative phosphorylation [43]. CII accelerates the oxidation of succinate to fumarate in the Krebs cycle and serves as an entry site for electrons from succinate to ubiquinone via FeS clusters (Fig. 3). CII consists of four subunits, including the hydrophobic membrane anchor proteins CybL and CybS, which have a CoQ binding site and are connected with the inner membrane [42]. The other two subunits are covalently linked to succinate, and the complex contains three FeS clusters and a flavoprotein binding site [44]. The electron transfer process involves the reduction of FAD to FADH2, followed by transferring electrons from succinate to FeS clusters. Finally, electrons from the FeS cluster convert ubiquinone to ubiquinol [45].

CIII, also known as cytochrome bc1 complex or CoQ cytochrome c reductase, transfers electrons from ubiquinol (QH2) to cytochrome c (Fig. 3). It is a symmetric dimer with 11 subunits in each monomer [46]. The complex contains iron-sulfur proteins such as cytochrome b (bL and bH), cytochrome c1, and a high potential (2Fe2S) cluster participating in the catalysis [47]. Complex-III possesses two CoQ binding sites, Qo and Qi, located at each terminal of cytochrome b. The Q cycle enables electron transfer by oxidizing QH2 to Ubisemiquinone (QH) and transferring an electron to the iron–sulfur cluster (2Fe2S), along with the movement of protons from the matrix to the intermembrane space [48].

CIV, known as cytochrome c oxidase, transfers electrons from cytochrome c to the terminal electron acceptor O₂, producing water (Fig. 3). It consists of four redox-active metal centers: CuA, heme a (Fea), heme a3 (Fea3), and CuB [49, 50]. The mitochondrial DNA encodes core subunits I, II, and III, while the nuclear genome encodes ten auxiliary subunits. Subunit I contains heme a and the binuclear center responsible for electron transfer to O₂, while subunits II and III play essential roles in stabilization and proton pumping [51]. The allosteric ATP-mediated inhibition of complex-IV, regulated by the ATP/ADP-ratio, influences physiological activity through nuclear gene-encoded subunits [52, 53].

CV, also known as F1F0-ATP synthase, consists of two functional domains: F0 and F1 (Fig. 3). The F0 domain is embedded in the inner mitochondrial membrane. It contains subunits that form the proton channel, including a subunit c ring, subunits a, b, d, F6, oligomycin sensitivity conferring protein, and accessory subunits [54]. The F1 domain is located in the mitochondrial matrix. It is composed of soluble subunits, including three α subunits, three β subunits, a γ subunit, a δ subunit, and an ε subunit [54, 55]. Proton transfer from the intermembrane space to the matrix through F0 drives the conformational changes in F1, leading to ATP synthesis [56].

CI structure and function

The first and largest enzyme of the mitochondrial respiratory chain and OXPHOS, CI (nicotinamide adenine dinucleotide [NADH]: ubiquinone oxidoreductase), is responsible for transferring electrons from reduced NADH to coenzyme Q10 (CoQ10, ubiquinone), as well as pumping protons to maintain the electrochemical gradient across the inner mitochondrial membrane [57]. The complexes of the mitochondrial ETC assemble as a supercomplex called respirasomes, which have been explored and studied using techniques such as non-denaturing blue native polyacrylamide gel electrophoresis and cryo-electron microscopy. These studies have revealed the association of CI with CIII and CIV, with monomeric-dimeric structures observed for CI, single subunit for CIII, and monomeric forms for CIV [58, 59].

CI alone has an approximate molecular mass of 1 MDa [60] and comprises 45 protein subunits (Fig. 4). Mitochondrial DNA (mt-DNA) genes encode seven subunits, while nuclear genes encode the remaining ones. CI is the largest multisubunit complex of the mitochondrial ETC and is commonly referred to as NADH: ubiquinone oxidoreductase. It plays a crucial role in the assembly, regulation, and stabilization of the OXPHOS system [61]. In addition to the core subunits, eukaryotic CI contains 32 supernumerary or accessory subunits, which are believed to be involved in structural stability and assembly rather than direct enzymatic activity [62]. The subunits of CI are organized into three main constituents: the N module responsible for NADH oxidation, the Q module where ubiquinone reduction occurs, and the P module involved in proton translocation across the membrane [63].

Fig. 4.

Mitochondrial Complex-I structure with their subunits. The figure details the structure and function of mitochondrial Complex I, NADH: Ubiquinone Oxidoreductase, a crucial electron transport chain (ETC) enzyme. Complex I is the largest of the ETC complexes, playing a vital role in cellular respiration and ATP production through oxidative phosphorylation. It has an L-shaped structure consisting of peripheral and membrane arms. The peripheral arm extends into the mitochondrial matrix and contains sites for NADH oxidation and electron transfer, while the membrane arm is embedded in the inner mitochondrial membrane (IMM) and is involved in proton translocation to inter-membrane space (IMS). Complex I comprises 45 subunits, with 14 core subunits forming the minimal functional unit conserved across species. These core subunits are divided into seven hydrophilic subunits in the peripheral arm and seven hydrophobic subunits in the membrane arm. Seven subunits of CI are encoded by mitochondrial DNA (mtDNA)—ND1, ND2, ND3, ND4, ND4L, ND5, ND6—and are integral to the membrane arm, primarily involved in proton translocation across the IMM. The remaining subunits are encoded by nuclear DNA and include iron-sulfur (Fe-S) cluster proteins and other accessory proteins that play roles in electron transfer and structural stability, such as NDUFS1, NDUFS2, NDUFS3, NDUFS7, and NDUFS8, which contain Fe–S clusters (green letter subunits) and facilitate electron transfer from NADH to ubiquinone, and NDUFV1 and NDUFV2 (red letter subunits), which are involved in binding NADH and flavin mononucleotide (FMN) for the initial electron transfer. Complex I catalyzes the oxidation of NADH, transferring electrons to ubiquinone and reducing it to ubiquinol, with this electron transfer coupled to the translocation of four protons from the mitochondrial matrix to the intermembrane space, contributing to the proton gradient used by ATP synthase (Complex V) for ATP production. Electrons are transferred from NADH to FMN, forming FMNH2, then passed through Fe-S clusters to ubiquinone, with the energy released driving conformational changes in the membrane arm subunits to facilitate proton translocation. This comprehensive depiction of Complex I highlights its intricate structure, comprising both mtDNA- and nuclear DNA-encoded subunits, and underscores its critical role in mitochondrial energy metabolism and ATP production.

The core subunits of CI are primarily embedded in the inner mitochondrial membrane and contain the catalytic sites for electron transfer. These core subunits, encoded by the mitochondrial genes ND1, ND2, ND3, ND4, ND5, ND6, and ND4L, are surrounded by peripheral arm subunits in the matrix [64]. The peripheral arm, also known as the matrix arm, is hydrophilic and consists of core subunits such as NDUFS1, NDUFV1, NDUFS2, NDUFV2, NDUFS3, NDUFS7, and NDUFS8 [64]. It also contains the primary electron acceptor FMN (flavin mononucleotide) and eight iron-sulfur (Fe–S) clusters, which form the main electron transfer pathway connecting the N and Q sites [64]. The membrane arm of CI comprises subunits ND1, ND3, ND4L, and ND6, creating a recognized proton translocation pathway known as the E-channel near the core subunits. The structural arrangement of CI involves the association of core subunits with supernumerary subunits, which contribute to the stability and assembly of the complex [58]. The assembly of CI involves numerous assembly factors (Table 1), with 20 factors known for their roles in biogenesis [60]. These factors assist in the correct folding, insertion of cofactors, and assembly of CI subunits, ensuring the proper formation and function of the complex [65, 66].

Table 1.

Mitochondrial complex I entities and subunits involved in forming modules and their corresponding assembly factors

S.N	Module	Subunits	Entity of subunits	Module assembly factors
1	N	NDUFV1, NDUFV2, NDUFS1, NDUFA2	Peripheral NADH-oxidization at matrix arm	NUBPL
2	Q	NDUFA5, NDUFS2, NDUFS3, NDUFS7, NDUFS8	Q site formation at the interface of matrix and membrane arm	NUBPL, NDUFAF3, NDUFAF4, NDUFAF5, NDUFAF6, NDUFAF7
3	PP-a	ND1, NDUFA3, NDUFA8, NDUFA13	Q site formation at the membrane-embedded portion	TIMMDC1
4	PP-b	ND2, NDUFC1, NDUFC2, ND3, ND4L, ND6	Form E-channel and first antiporter-like subunit	ACAD9, ECSIT, NDUFAF1, COA1, TME, EM126B, TMEM186
5	PD-a	NDUFB5, NDUFB10, NDUFB11, NDUFB6, ND4	Found around the second antiporter-like subunit	FOXRED1, ATP5SL, TMEM126A52, TMEM70
6	PD-b	NDUFAB1, NDUFB7, NDUFB3, NDUFB8, ND5, NDUFB9, NDUFB2	Found around the third antiporter-like subunit	DMAC1

Mitochondrial encoded genes of CI

CI comprises seven membrane-bound core subunits encoded by mitochondrial DNA (mt-DNA), namely ND1, ND2, ND3, ND4, ND4L, ND5, and ND6. These genes encode the ‘mitochondrially encoded NADH: ubiquinone oxidoreductase core subunits’ or NADH-ubiquinone oxidoreductase chain. CI plays a crucial role in catalyzing the transfer of electrons from NADH to ubiquinone, an electron acceptor, through the equation: (NADH⁺ H⁺) + CoQ + 4 H⁺(matrix)—> NAD⁺ + CoQH2 + 4 H⁺(intermembrane) [67]. However, mutations and deficiencies in CI have been associated with various cardiac pathologies, including mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS), Leigh syndrome, mitochondrial CI deficiency, mitochondrial type 1 (MC1DM1), hypertrophic cardiomyopathy, and Leber’s hereditary optic neuropathy (LHON) [68]. For instance, studies have shown that a triple mutation (D199G/L200K/A201V) in the human MT-ND1 gene (m.3902–3908inv7) impairs electron transfer and proton translocation, resulting in an inability to maintain a balanced NADH/NAD⁺ ratio and produce ATP, primarily through the loss of its enzymatic activity [69].

The assembly of mitochondrial CI is assisted by various subunits, including NDUFA12, which acts as a molecular chaperone [70]. These subunits are involved in electron transfer from NADH to ubiquinone in the respiratory chain. Notably, NDUFAB1 has been identified as a required subunit for the viability of HEK293T cells, demonstrating its unique survival property. Knockout of NDUFAB1 leads to cell death in galactose media due to the lack of CI assembly [60]. Further research will continue to provide valuable insights into the mechanisms by which mutations interfere with the function of CI. In this regard, age-dependent impairment of CI function has also been reported due to the accumulation of mt-DNA mutations [71]. To address this, wild-type mitochondrial genes have been expressed from the nucleus through allotopic expression, with partial or sufficient recovery of the disease phenotype explicitly observed for the ND4 gene [60, 72, 73]. However, achieving full mt-DNA complementation and stable expression requires nuclear codon optimization, making gene therapy a potential avenue for treatment in CI deficiency diseases [74].

CI deficiency in humans

Research on mitochondrial CI deficiency in human disease has revealed significant heterogeneity, with impaired oxidative phosphorylation affecting approximately five out of every 10,000 live births. The severity of the condition varies from neonatal to adult stages, resulting in diverse clinical phenotypes. These include non-specific encephalomyopathy, macrocephaly with progressive leukodystrophy, cardiomyopathy, liver disease, Leigh syndrome, Leber’s hereditary optic neuropathy, and neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease. Regarding inheritance, nuclear type mitochondrial CI deficiency consistently follows autosomal recessive transmission. Several specific sub-types of mitochondrial CI deficiency have been identified, each with distinct clinical manifestations. For instance, mitochondrial CI deficiency, nuclear type 5, is characterized by muscular hypotonia, assessed with pathological significance and a loss of catalytic activity [75]. In the case of mitochondrial CI deficiency, nuclear type 6 hypertrophic cardiomyopathy is observed in three out of five patients, while Leigh syndrome occurs in all affected individuals. Biochemical analysis has shown that these patients exhibit an unaltered Km value for ubiquinone-1 but experience a loss of catalytic activity due to mutations [76].

In a case series involving patients diagnosed with mitochondrial CI deficiency, nuclear type 8, unrelated individuals presented with symptoms such as hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. The condition was associated with CI disassembly, limited enzyme activity, and pathogenesis [77]. Another study on the same nuclear type deficiency revealed reduced enzymatic action due to highly unstable and aggregated protein [78–80]. Mitochondrial CI deficiency, nuclear type 2, has been linked to encephalomyopathy as a clinical phenotype in children and the late onset of Leigh syndrome in adults. These individuals exhibit significant pathological impressions, reduced enzymatic activity, and impaired CI assembly [80, 81].

Mitochondrial CI deficiency, nuclear type 37, manifests as developmental delay, microcephaly, abnormalities related to development, and epilepsy. It is characterized by decreased CI enzymatic activity and reduced NDUFA8 protein levels. The limited enzymatic activity may compromise CI assembly, affecting the integration of CIII or CII and CIII dimer formation. Consequently, alterations in the mitochondrial network and morphology are observed in fibroblasts [82]. The disassembly of CI assembly factors in fibroblasts and transformed mitochondria morphology is evident in patients with mitochondrial CI deficiency, nuclear type 26 [83]. Mitochondrial CI deficiency, nuclear type 10, affects CI assembly and activity, leading to progressive encephalopathy [70].

Mitochondrial CI deficiency, nuclear type 28, presents with early-onset hypotonia, dyskinesia, sensory deficiencies, and mitochondrial CI instability, accompanied by limited NDUFA13 protein expression [84]. Mitochondrial CI deficiency, nuclear type 32, is associated with a clinical phenotype similar to Leigh-like encephalomyopathy and is caused by nucleotide substitutions or missense mutations [85]. Mitochondrial CI deficiency, nuclear type 36, significantly reduces CI assembly and protein levels [67]. Although respiratory chain impairment induces stress conditions for survival and proliferation of CI-deficient cells, cytosolic malic enzyme one has been found to restore defective NADPH levels instead of consuming ATP. A study suggests that non-CI mutant cells exhibit enhanced CI metabolism under galactose conditioning to restore reduced NADPH through the pentose phosphate pathway [86]. Recent studies have identified two novel mt-DNA variants exhibiting high mutant heteroplasmy in muscle homogenate inherited maternally. These include a de novo frameshift variant in MT-ND6 (m.14512_14513del) and a transverse mutation in MT-ND1 [87].

Novel approaches to sustain mutation-related CI dysfunction

Mitochondria produce approximately 95% of cellular energy, and abnormalities related to diabetes mellitus, neurological diseases, and CVD can disrupt mitochondrial function. Using mouse models, studies investigated the modulatory effects of mt-DNA variants ND6P25L and COIV421A on cardiomyopathy pathogenesis induced by autosomal mutations in nuclear genes. These mt-DNA variants have significantly impacted mitochondria’s structure, morphology, size, dynamicity, and biochemical output. For instance, the COIV421A variant leads to mitochondrial enlargement, while the ND6P25L variant causes mitochondrial fragmentation. These alterations influence the regulation of mitochondrial dynamics through OPA1, particularly by the loss of long OPA1 isoforms (l-OPA1). Consequently, these events result in occluded inter-mt-DNA complementation, CI inhibition, increased ROS production, and damage to mt-DNA. Notably, the ND6P25L mutation exhibits more deleterious effects than the COIV421A variant [88].

Recent studies have explored utilizing functional complementary approaches to treat disorders arising from mt-DNA mutations and CI dysfunction. Here, researchers examine the efficacy of supplementing functional compounds or specific modifier genes whose activity or expression can complement CI dysfunction-related disorders. A group of researchers explored the potential of anthocyanins, antioxidants known to reduce the risk of chronic diseases, in restoring mitochondrial function against CI and rotenone inhibition caused by amyloid production in an in vitro model [89]. Their findings suggest that Medox anthocyanins can limit the fragmentation of mitochondria triggered by rotenone and mutations in the amyloid precursor protein, restoring their network by upregulating Drp1 and downregulating Mfn2. Under the influence of mitochondrial toxins, this restoration contributes to the organization of mitochondrial components, leading to the recovery of energy transduction and cell viability [89]. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes are syndromes associated with CI deficiency caused by mutations in mitochondria-encoded CI structural genes. These mutations lead to increased production of ROS and inflammation due to electron leakage to oxygen. Researchers have reported elevated levels of prostaglandin E2 (PGE2) in mitochondrial disease patients with MELAS-associated CI deficiency and investigated the use of the redox modulator KH176m to reduce PGE2 release in fibroblast cell lines carrying specific mutations. The mutations studied include V112M in NdufS7, R228Q in NdufS2, and R59X/T423M in NdufV1. Treatment with KH176m inhibited PGE2 formation selectively through PGE2-driven positive feedback inhibition of membranous PGE2. Promising results from these studies have paved the way for further clinical trials to combat mitochondrial diseases [90]. Mechanism of action and potential applications of selective inhibition of microsomal prostaglandin E synthase-1-mediated PGE2 biosynthesis by sonlicromanol’s metabolite KH176m [90].

Another study that utilizes a functional complementation approach relates to the treatment of epileptic encephalopathy. Here, fibroblasts from patients were identified to have mutations in the MT-ND1 gene (3946G > A), resulting in the alteration of the E214K protein, leading to epileptic encephalopathy progression. Overexpression of a complementary gene MRPS18C encoding the Bs18M protein was found to rescue mutational defects by sustaining CI expression and reducing ROS levels. This finding suggests that overexpression of MRPS18C may serve as a potential complementary therapeutic approach for treating MT-ND1 mutations and epileptic encephalopathy [91].

CI dysfunction in CVD

Mitochondrial CI dysfunction is critical in CVD and other pathological conditions (Fig. 5).

Fig. 5.

Heart diseases associated with Complex-I dysfunction. Heart diseases associated with mitochondrial Complex I dysfunction include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), ischemic heart disease (IHD), heart failure with preserved ejection fraction (HFpEF), congenital heart defects, and arrhythmias

The cellular and metabolic functionality of mitochondria, including biogenesis, maintenance, and coordination at the organelle level, underscores their importance in overall cellular homeostasis. Structural and functional abnormalities in mitochondrial CI have been implicated in various diseases, including CVD and neuropathologies. According to the World Health Organization, CVD accounted for approximately 32% of global deaths, with an estimated 17.9 million deaths reported in 2019. Quality control mechanisms, such as mitochondria-associated degradation, the ubiquitin–proteasome system, and mitophagy, have evolved in cells to regulate the turnover of proteins and eliminate dysfunctional mitochondria. These mechanisms respond to and compensate for the nature and severity of mitochondrial dysfunction, often triggering transcriptional or proteomic remodeling within the cell [92].

In CVD, several factors can influence the metabolism, gene expression, and function of CI in mitochondria, leading to different types of CVD, including coronary artery disease, rheumatic heart disease, stroke, and myocardial infarction. One specific factor investigated concerning CVD is hyperhomocysteinemia, which has been shown to alter gene expression and mitochondrial biogenesis. A negative correlation was observed between blood homocysteine levels and mitochondrial DNA content in human white blood cells, suggesting the active inhibition of CI-associated gene expression in mitochondria due to hyperhomocysteinemia [93]. Iron deficiency is also linked to heart failure, with or without anemia. Studies have shown that iron supplementation can partially mitigate the effects of heart failure, but the relationship between iron deficiency, cardiac function, and mitochondrial metabolism remains understudied. Rineau and colleagues developed a mouse model of iron deficiency without anemia to investigate the impact on cardiac function and mitochondrial metabolism in cardiomyocytes; the study revealed reduced activity of mitochondrial CI and impaired left ventricular function, and iron treatment was found to reverse these dysfunctions in heart failure [94].

Among the proposed mechanisms for mitochondrial CI dysfunction associated with coronary artery disease (CAD) is a significant reduction in NDUFC2 gene expression [95]. This reduction is associated with an increased risk of early-onset ischemic stroke and acute coronary syndrome as a genetic risk factor for CVD. In individuals with a deficiency of the NDUFC2 subunit from mitochondrial complex-I, juvenile ischemic stroke, and hypertension have been observed, accompanied by reduced NDUFC2 mRNA levels in peripheral blood mononuclear cells of acute coronary syndrome patients [95, 96]. Furthermore, a specific variant, the T allele at NDUFC2/rs23117379, is prevalent in Caucasians and has been implicated in reduced NDUFC2 gene expression [95].

CI-related metabolic alterations in heart disease

Mitochondria are abundant in cardiomyocytes to meet the high-energy demands of the continuously active heart. In CVD, mitochondrial dysfunction is observed in nearly all cases, as mitochondrial oxidative metabolism supplies approximately 90% of the ATP the heart requires [97] Heart failure (HF), a common condition in CVD, affects around 50% of patients and is characterized by abnormal relaxation or preserved ejection fraction (HFpEF) during end-diastolic pressure, leading to metabolic risk factors, metabolic alterations, and mitochondrial dysfunction. Miranda Silva et al. utilized a ZSF1-obese rat model compared to the control model ZSF1-lean to investigate the underlying mechanisms. They discovered several differences, such as elevated resting Ca⁺² concentrations in the mitochondria and cytosol, reduced Complex-I-associated mitochondrial respiration, and increased mitochondrial swelling in ZSF1-obese rats. These results were attributed to altered calcium handling in the mitochondria and cytosol, which may compensate for the impaired mitochondrial dysfunction associated with CI and maintain the optimal contractile function of the heart in hFpEF in vivo [98].

Systemic inflammation severity and low exercise efficiency in skeletal muscle have been linked to upregulated proteolysis and disturbed energy metabolism in hFpEF [99]. A healthy heart demonstrates metabolic flexibility, utilizing fatty acids, lactate, ketone bodies, glucose, and branched-chain amino acids (BCAAs) to produce ATP in adults. Disruption in cardiac energy metabolism, including amino acid oxidation, can lead to heart failure since amino acids have the potential to generate ATP. BCAA oxidation has been observed in the heart, involving mitochondrial transaminases in the transamination process to produce branched-chain α-keto-acids. In vitro measurements of the accumulation of these molecules with altered metabolites have suggested that BCAA oxidation products inhibit CI and increase superoxide production, resulting in mitochondrial dysfunction [100].

Overall, heart failure and other CVDs are frequently accompanied by a decline in mitochondrial respiration due to metabolic abnormalities [97]. These findings shed light on the importance of understanding mitochondrial CI-related metabolic alterations in the context of heart disease and provide potential targets for therapeutic interventions.

CI-mediated ROS generation, oxidative stress, and cardiac dysfunction

Environmental factors, such as exposure to toxins and pollutants and genetic predisposition, significantly induce oxidative stress and mitochondrial DNA damage in CVDs [101, 102]. In the heart, cardiomyocytes, endothelial cells, and neutrophils are significant sites of ROS generation, primarily attributed to the uncoupled electron transport between CI and III in mitochondria [103, 104]. The excessive production of ROS leads to a depletion of antioxidants, cellular injury, altered gene expression, and mitochondrial DNA damage, ultimately resulting in impaired oxidative capacity in mitochondria, contributing to the progression of cardiac dysfunction and heart failure [105, 106].

Furthermore, ROS actively participate in the pathophysiology of CVDs by modulating various signaling pathways, dysregulating transcription factors, activating enzymes, and influencing contractile function [107, 108]. Disruption of mitochondrial supercomplexes, notably CI or IV activity, has been identified as a critical trigger for ROS generation in heart failure models and individuals [109]. The coenzyme Q (CoQ) pool, acting as a redox cycling coenzyme, is subjected to substrate feed into complexes I and II for two-electron reduction and electron transfer into complex-III. Depending on the redox potential, incomplete or one-electron reduction of CoQ can produce an extremely reactive radical called semiquinone [110]. ROS production will likely occur if the electrons reverse transfer toward Complex-I. Consequently, these events and mechanisms contribute to decreased CoQ content in plasma among heart failure survivors, and this reduction is inversely proportional to mortality [111].

Humanin, an endogenous peptide encoded by the mitochondrial genome, plays a crucial role in regulating metabolic processes and mitigating ROS generation and peroxide-induced oxidative stress by inhibiting the activity of mitochondrial complexes I and III [112]. The role of humanin has also been discussed in the context of recovery from oxidative stress and its cardioprotective effects. In terms of protection against oxidative stress, humanin blocks caspases and upregulates antioxidant enzymes against ROS and reactive nitrogen species [112]. Additionally, for cardioprotection, humanin induces the activation of protein kinase B and glycogen synthase kinase three pathways, which improve the ratio of cardiomyocytes to fibroblasts in aging hearts [113]. When measured in serum samples, humanin may be a potential diagnostic marker for assessing mitochondrial functionality in CVDs [114].

CI in ischemic heart disease (IHD)

According to the 2019 Global Burden of Disease estimation, there were 197 million reported cases of IHD worldwide, with an increase attributed to population growth and aging [114]. The rising incidence of IHD necessitates attention to preventive measures and therapeutic developments. Ischemic heart failure (IHF) is one of the main etiologies associated with mitochondrial complex syndrome and CAD-dependent cardiomyopathies [115]. In IHF, there is a shift from CI + II state 3 to CI state 2 respiration, leading to mitochondrial dysfunction. As opposed to non-IHF, this shift leads to inefficient oxidative phosphorylation (OXPHOS), high proton leak, and increased production of free radicals [116]. Mammalian ischemic hearts exhibit high levels of ROS accompanied by an A/D transition. The catalytic reaction of CI can be reversed, allowing the transfer of electrons upstream, which is compromised by the proton motive force. The regulation of mitochondrial CI is characterized by a switch between the active (A) enzyme and a de-active (D) or dormant form, known as the A/D transition, in metabolically active hearts under ischemic conditions [117]. The ND6 subunit is essential in maintaining the D-form of CI during the A/D transition in ischemic injury [118]. During the A/D transition, the absence of oxygen as a reactant during ischemia results in the inefficient performance of the D-form of CI to produce superoxide, which may protect against mitochondrial dysfunction and heart failure by lowering the rate of Complex-I-mediated ROS production. Further studies are needed to elucidate the precise mechanisms underlying the A/D transition and the role of CI in IHD, as it holds significant potential for therapeutic interventions targeting mitochondrial dysfunction and oxidative stress in IHF.

Impaired CI function during ischemia exacerbates energy deficits by reducing ATP production. This dysfunction also leads to increased production of reactive oxygen species (ROS), contributing to oxidative stress and damage to cellular components. Additionally, CI dysfunction can cause mitochondrial permeability transition pore (mPTP) opening and impaired calcium handling, both of which further exacerbate ischemic injury and lead to cell death [119]. Therapeutic strategies targeting CI, such as pharmacological agents like metformin, have shown promise in reducing oxidative stress and protecting against ischemic injury [120]. Gene therapy approaches aim to restore functional Complex-I subunits, offering another potential avenue for treatment [121, 122]. Furthermore, mitochondria-targeted antioxidants, such as MitoQ, have been developed to reduce mitochondrial ROS and mitigate ischemia–reperfusion injury [123]. Collectively, these therapeutic interventions underscore the importance of mitochondrial Complex-I in managing IHD and highlight its potential as a therapeutic target. However, many of these strategies failed in human clinical trials, highlighting the importance of understanding the contribution of CI in IHD. One of the pitfalls could be delivering the drug to the CI site at an optimal concentration, which is challenging.

CI in myocardial infarction (MI)

Recent studies shed light on the role of mitochondrial CI in MI. During ischemic injury, the D-form of CI becomes predominant and is more susceptible to oxidative and nitrosative damage. However, upon reperfusion, it restricts oxidative burst [117]. Posttranslational modifications, including S-nitrosylation of CI, altered cardiolipin content, and lipid binding, destabilize subunit interactions during ischemic reperfusion and reduce CI activity in cardiac mitochondria [124]. Reperfusion-based preventive therapy offers various pathways and molecular interventions to explore against conditions such as cardiac infarction and ischemic reperfusion injury. During an MI, the occlusion of coronary arteries severely reduces oxygen supply to the myocardial tissue, causing a significant shift in cellular metabolism. The ischemic condition disrupts the normal function of CI, leading to decreased ATP production, which is essential for maintaining myocardial contractility and cellular homeostasis. Additionally, impaired complex-I activity increases the production of ROS, which exacerbates oxidative stress and causes extensive damage to mitochondrial and cellular structures. This oxidative damage further aggravates mitochondrial dysfunction, creating a vicious cycle that exacerbates the ischemic injury. Moreover, the dysregulation of CI contributes to the opening of the mitochondrial permeability transition pore (mPTP), a critical event that leads to loss of mitochondrial membrane potential, further ATP depletion, and initiation of apoptotic and necrotic cell death pathways [125]. These findings highlight the central role of mitochondrial CI in the pathophysiology of MI and underscore its potential as a therapeutic target for reducing myocardial injury and improving clinical outcomes. Noncoding RNAs are essential regulators of mitochondrial function in MI. For example, mitochondrial miR-762 and miR-34a induce cardiomyocyte apoptosis, leading to MI through apoptosis targeting ND2 and inhibition of CI, respectively [6, 126]. MiR-28 enhances cardiomyocyte apoptosis by downregulating mitochondrial ALDH2 under hypoxic conditions. Upregulation of miR-1 in the muscle also targets various ETC proteins in patients with acute MI [127].

CI in myocardial ischemia–reperfusion (IR) injury

CI dysfunction is also prominently involved in the pathophysiology of myocardial IR injury, which arises when blood supply returns to the heart after ischemia or lack of oxygen. Oxygen deprivation leads to reduced ATP production and altered mitochondrial function during ischemia. Reperfusion, while essential for tissue survival, paradoxically exacerbates injury through oxidative stress and calcium overload. Dysfunctional Complex-I is a major contributor to these processes, as it becomes a significant source of ROS during reperfusion, exacerbating oxidative damage to cellular components [128]. The opening of the mitochondrial permeability transition pore (mPTP), often triggered by ROS and calcium overload, leads to loss of mitochondrial membrane potential, further ATP depletion, and activation of cell death pathways, including apoptosis and necrosis [129]. Therapeutic strategies to stabilize Complex I and reduce ROS production have shown promise in mitigating IR injury. Pharmacological agents such as metformin have demonstrated cardioprotective effects by modulating Complex I activity and reducing oxidative stress [130]. Additionally, mitochondria-targeted antioxidants, like MitoQ, have effectively reduced mitochondrial ROS and protected against myocardial IR injury in experimental models [123].

A study focusing on suppressed mitochondrial functionality during early-stage myocardial infarction/reperfusion injury identified S100a8/a9 as a transcriptional regulator associated with cardiomyocyte death, CI inhibition, and reduced expression of the NDUF gene. Targeting the signaling pathways initiated by S100a8/a9 may yield positive results against myocardial injury-induced mitochondrial respiratory dysfunction [131]. An essential mitochondrial protease called LonP1 is necessary for preserving the proteostasis of the mitochondria and reducing cellular stress. By minimizing oxidative damage to proteins and lipids, maintaining mitochondrial redox equilibrium, and reprogramming bioenergetics by lowering CI content and activity, LonP1 overexpression reduces heart injury and limits IR injury [30]. Similarly, in a model of Ndufs4 knockout, decreased mitochondrial respiration chain supercomplexes and CI activity at ages 3 to 4 months protected against IR injury in the heart [132]. These findings underscore the critical role of mitochondrial CI in myocardial IR injury and highlight the potential of targeting this enzyme to develop effective therapeutic interventions. Similar to targeting CI in IHD, compounds targeting CI in the clinical trials failed. IR injury involves complex biochemical processes, including ROS generation, calcium overload, and mitochondrial permeability transition pore (mPTP) opening. These processes are intricately linked and not fully addressed by targeting CI alone. The multidimensional nature of IRI necessitates a broader therapeutic approach that addresses various aspects of the injury simultaneously.

CI in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by the dilation and impaired contraction of the ventricles, leading to systolic dysfunction and heart failure. Mitochondrial dysfunction, particularly involving CI of the electron transport chain, plays a significant role among various contributing factors. Complex I dysfunction impairs ATP production, leading to an energy deficit that compromises the contractility of cardiac myocytes, resulting in ventricular dilation and impaired contraction [133]. Studies have shown that deficiencies in CI reduce ATP synthesis, failing to meet the high-energy demands of the heart muscle [133]. Additionally, dysfunctional CI increases oxidative stress by producing excessive ROS, further damaging mitochondrial DNA, proteins, and lipids, exacerbating mitochondrial dysfunction [134]. This oxidative stress impairs cardiac myocyte function and survival, contributing to DCM [134]. Moreover, impaired mitochondrial calcium handling disrupts essential intracellular signaling for cardiac muscle contraction and relaxation, leading to arrhythmias [135]. Persistent oxidative stress and mitochondrial damage also trigger apoptotic pathways, increasing cardiomyocyte death and fibrotic remodeling, which stiffens the heart and worsens systolic function [136]. Genetic mutations in CI subunits and assembly factors have been identified in DCM patients, highlighting the genetic basis of mitochondrial dysfunction in this condition [137]. Therapeutic strategies targeting oxidative stress, enhancing mitochondrial biogenesis, and correcting genetic defects have shown potential in managing DCM. These approaches aim to restore mitochondrial function, reduce oxidative damage, and improve cardiac performance, offering hope for better clinical outcomes in DCM patients [138, 139].

CI in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal heart muscle thickening, often due to genetic mutations affecting sarcomeric proteins, leading to impaired myocardial energetics and mitochondrial dysfunction. Studies have shown that CI activity is significantly reduced in HCM, contributing to decreased ATP production and energy deficiency in hypertrophic cardiac cells [140]. This energy deficit is particularly detrimental in HCM, where the energy demand is already elevated due to increased cardiac muscle mass and workload. Furthermore, impaired CI function leads to elevated production of ROS, which causes oxidative damage to mitochondrial and cellular structures, exacerbating the pathological remodeling and fibrosis characteristic of HCM.

The accumulation of oxidative stress and mitochondrial damage activates maladaptive signaling pathways that further promote hypertrophy and progression of cardiomyopathy. Recent therapeutic approaches targeting mitochondrial dysfunction in HCM have focused on enhancing CI activity and reducing oxidative stress. For instance, pharmacological agents like perhexiline, which modulates mitochondrial energy metabolism, have shown promise in improving cardiac function and reducing symptoms in HCM patients [141]. Additionally, antioxidants targeting mitochondrial ROS have been explored to mitigate oxidative damage and improve mitochondrial function. These findings highlight the central role of mitochondrial Complex-I dysfunction in the pathophysiology of HCM and underscore the potential of targeting mitochondrial pathways to develop effective treatments for this debilitating condition.

CI in cardiac arrhythmia

Mitochondrial CI, a crucial component of the mitochondrial ETC, is fundamental to cardiac energy metabolism and has been increasingly implicated in the pathogenesis of cardiac arrhythmias. CI dysfunction leads to impaired ATP production and elevated ROS generation, which could play pivotal roles in developing arrhythmogenic substrates [142]. The heart relies heavily on ATP to maintain ionic gradients across cell membranes, which is essential for proper electrical activity and contractility. Reduced ATP levels due to CI impairment can disrupt these gradients, leading to altered ion channel function and increased susceptibility to arrhythmias [143]. Additionally, excessive ROS production can cause oxidative modification of cardiac ion channels and connexins, proteins critical for electrical coupling between cardiomyocytes, further promoting arrhythmic events [144].

Mitochondrial ROS can also activate various signaling pathways that contribute to structural remodeling of the heart, such as fibrosis and hypertrophy, creating a proarrhythmic environment. For instance, studies have shown that mitochondrial-targeted antioxidants, such as Mito-TEMPO, can reduce arrhythmic episodes by mitigating oxidative stress and preserving mitochondrial function [145]. Furthermore, genetic models with CI deficiencies have demonstrated increased arrhythmia susceptibility, underscoring the enzyme’s role in maintaining cardiac electrical stability [146]. These findings suggest that therapies to enhance Complex-I function and reduce mitochondrial ROS may offer new avenues for preventing and treating cardiac arrhythmias. Current research explores pharmacological agents and gene therapy approaches to stabilize CI activity, with promising results in preclinical models. This highlights the potential of targeting mitochondrial pathways to improve outcomes for patients with arrhythmias linked to mitochondrial dysfunction.

CI in diabetes-induced heart failure with preserved ejection fraction (HFpEF)

CI is also critically involved in the pathogenesis of diabetes-induced HFpEF. In diabetes, chronic hyperglycemia leads to increased fatty acid oxidation and thus reduces substrate flexibility between glucose and fatty acid in the heart [147]. This phenomenon may lead to dysfunctional CI and increased ROS generation, which causes oxidative damage to mitochondrial DNA, proteins, and lipids, exacerbating mitochondrial dysfunction [148]. The oxidative stress and impaired energy production associated with CI dysfunction contribute to the pathological remodeling observed in HFpEF, including myocardial fibrosis and hypertrophy. These structural changes increase myocardial stiffness and impair diastolic function, hallmark features of HFpEF. Moreover, ROS generated by CI dysfunction activates various pro-inflammatory signaling pathways, further promoting myocardial fibrosis and inflammation, creating a vicious cycle that deteriorates cardiac function [149].

Therapeutic strategies targeting mitochondrial CI in diabetic HFpEF aim to enhance mitochondrial function and reduce oxidative stress. Pharmacological agents such as metformin and mitochondria-targeted antioxidants like MitoQ have shown promise in experimental models by improving Complex-I activity, reducing ROS production, and ameliorating cardiac dysfunction [123, 144]. Additionally, lifestyle interventions like exercise and dietary modifications have positively impacted mitochondrial function and reduced oxidative stress in diabetic patients, suggesting a multifaceted approach to managing HFpEF. A recent study highlighted the role of A-kinase anchoring protein 121 (AKAP1) as a therapeutic target against DC and related cardiac diseases. AKAP1, known for its regulatory role in mitochondrial function, has been implicated in DC pathogenesis. Downregulation of AKAP1 has been observed in a streptozotocin-induced diabetic mouse model mimicking type 1 diabetes. AKAP1 interacts with NDUFS1, a subunit of mitochondrial CI involved in maintenance, and regulates CI activity through mitochondrial translocation. AKAP1 deficiency leads to mitochondrial dysfunction and cardiomyocyte apoptosis in diabetic cardiomyopathy. Thus, the upregulation of Akap1 may offer a potential therapeutic approach for patients with myocardial injury and diabetes [150]. Similar to the role of CI in other heart diseases, these findings underscore the critical role of mitochondrial Complex-I in developing diabetes-induced HFpEF and highlight the potential of targeting mitochondrial pathways to develop effective treatments for this condition.

CI in congenital heart disease

In congenital heart disease (CHD), which encompasses a range of structural heart defects present at birth, mitochondrial dysfunction, particularly involving CI, has been increasingly recognized as a significant contributor to the pathophysiology. Studies have shown that mutations in genes encoding CI subunits can lead to congenital defects in cardiac development and function, highlighting the importance of mitochondrial integrity in early cardiac morphogenesis [36]. CI dysfunction in CHD results in impaired oxidative phosphorylation, leading to reduced ATP production, which is critical for the high-energy demands of the developing heart. This energy deficit can disrupt normal cardiac growth and function, contributing to the structural abnormalities seen in CHD. Furthermore, defective CI activity increases the production of ROS, causing oxidative stress and further damaging mitochondrial and cellular components. The resultant oxidative damage can impair cell signaling pathways crucial for cardiac development and exacerbate the pathological remodeling observed in CHD [151].

Therapeutic strategies to improve mitochondrial function and reduce oxidative stress in CHD are under investigation. Mitochondria-targeted antioxidants, such as MitoQ, have shown potential in experimental models to mitigate ROS production and protect against mitochondrial damage, thereby improving cardiac function [123]. Additionally, interventions aimed at enhancing CI activity and overall mitochondrial health through gene therapy are also being explored to support normal cardiac development and function in patients with CHD.

CI in cardiac pressure overload

Cardiac pressure overload, characterized by the thickening of the ventricular wall diameter and high myocardial cell density, is an early-stage response in cardiac hypertrophy (CH). Mitochondrial dysfunction has been implicated in CH associated with angiotensin II, and a study by Zou et al. (2021) investigated the transcriptional and bioinformatics aspects of mitochondrial CI in a transverse aortic constriction mouse model [152]. The researchers found downregulated expression of Ndufs1, a component of CI, in hypertrophic heart tissue compared to normal tissue. They also observed a decline in mt-DNA content and elevated ROS in cardiomyocytes, highlighting the association between Ndufs1-associated mitochondrial dysfunction and pressure overload-induced CH. Elevated ROS production in mitochondria triggers the expression of Gαq and angiotensin II, which are associated with heart failure and CH, respectively [105]. Previous studies have explored the effects of antioxidants in mitigating pressure overload, such as superoxide dismutase, which was found to reduce hypertrophy [153], and SS20 or SS31, which prevented contractile reduction [154].

Mitochondrial ROS has been implicated in the pathogenesis of cardiac dysfunction, including diastolic, systolic, and mitochondrial dysfunction. Animal models have shown a mild reduction in CI function between 2 and 10 weeks, with notable impairment at 20 weeks due to pressure overload. The activity of CI was found to contribute to a 25% reduction in mitochondrial respiration activity, and the combined activities of CI and III accounted for approximately 45% of the total activity [155].

CI is a potential therapeutic target in various heart disease

Around 60 compounds belonging to different families are known to inhibit CI function. As we have discussed the working mechanism of CI earlier, the rate of their catalytic efficiency can be regulated based on the effects of inhibitors and activators at various levels, such as type of inhibitor or activators, their binding sites, working mechanisms, and impact on kinetics of enzyme including coordination among subunits. The inhibitors are categorized into three classes (A, B, and C) based on the kinetic behavior of the CI enzyme. Classes A, B, and C comprise the prototype of series piericidin A, rotenone, and capsaicin, respectively. The binding sites for inhibitors are broadly classified as hydrophobic pockets because their categorization based on binding sites has yet to be done cordially with these three classes.

CI is well described for maintaining proton (4H⁺/2e⁻) gradient and redox reaction in the ETC. Hence, electron leakage and ROS production may happen here [156]. In CI, the Q reduction occurs through a dual Q-gated pump, similar to the proton motive role in the N2 cluster. Oxidation–reduction of cluster N2 thoroughly reduces the attached Q molecule at site B to stabilize the ubisemiquinone that reciprocates the rotenone-sensitive radical. Hence, the first electron liberates from NADH in such a way. Rotenone, piericidin A, piericidin B, aureothin, amytal, 4-alkyl-acridones, 4-alkyl-MPP analogs, and phenoxan are a few more semiquinone antagonists or class B inhibitors. Later, the second electron reduces from NADH and passes on to ubiquinone through low-potential cofactors via reach on the hydrophobic site A, triggering proton pumping. All class A inhibitors are quinone antagonists, and therefore, these inhibitors antagonize here at the Q site. A few examples of these inhibitors are Rolliniastatin-2, Piericidin A, and Idebenone. Consecutively, the formation of semiquinone at site A efficiently dismutases the semiquinone stabilized at site B, further liberating the ubiquinol product out of the complex. After dismutation, the oxidized Q remains at site B to re-initiate the catalytic cycle, which is critical for CI function. The quinol antagonists are known for unstable semiquinone and can diverge the dismutation step. These are called class C inhibitors, for example, quinol products, reduced Q-2 b, myxothiazol, stigmatellin, TDS, 2 M-TIO, meperidine, and NP that may amplify these unproductive forms at steady-state [157]. Acute treatment with high doses of metformin given during the first stages of reperfusion reduces cardiac injury by partially inhibiting CI, along with a decrease in ROS production, an increase in mitochondrial calcium retention capacity, and a reduction in the susceptibility of the permeability transition pore (MPTP) opening. In vivo, infarct size is decreased in the cardiac AMPK-knockdown mice when high doses of metformin are administered. Metformin’s defense against early reperfusion likely involves an AMPK-independent regulation of CI [158]. Several studies showed that CI suppression reduced reperfusion ROS generation by pharmacological methods or indirect manipulations of Complex-I, such as metformin [130], amobarbital [159], or an acidic environment [160], stimulating mitochondrial STAT signaling [161].

Given the significance of CI in pressure overload-related conditions, it has been targeted as a preventive measure. R118, an AMPK activator and CI inhibitor developed by Rigel Pharmaceuticals has shown promising effects. In a study, R118 demonstrated various protective effects, including improved functionality of vascular endothelial cells, better blood pressure regulation, enhanced skeletal muscle blood flow in aged obese mice with peripheral arterial disease, and enhanced cardiac metabolism in skeletal muscle in mice with type 2 diabetes [11]. Furthermore, R118 was effective against cardiovascular dysfunction associated with angiotensin II infusion in mice. However, recent phase 1 clinical trials reported severe lactic acidosis as a side effect of R118, emphasizing the need for further research and development in the context of mitochondrial CI inhibition as a therapeutic approach for pressure overload [11].

Conclusion

Our current review highlights the central role of mitochondria, particularly mitochondrial CI, its mechanisms in contributing to various heart diseases (Fig. 6). Mitochondria are vital for energy production in higher organisms, consuming a significant portion of the oxygen we breathe. They are intricately involved in various cellular processes, and their dysfunction is linked to various diseases. In the heart, mitochondrial oxidative metabolism is crucial, and its impairment, often due to environmental and genetic factors causing oxidative stress and mitochondrial DNA damage, is a significant contributor to CVDs. The dysfunction of CI in the mitochondria is especially pivotal in the pathology of these diseases, influencing CH, heart failure, and other conditions. Current research focuses on understanding these mechanisms and identifying therapeutic interventions targeting mitochondrial function, offering hope for effective strategies to prevent and treat CVDs. This comprehensive knowledge of mitochondrial function and dysfunction, particularly in CI, is crucial for developing new approaches to manage and treat CVD effectively.

Fig. 6.

Mitochondrial Complex I and its mechanistic causes of various heart diseases. The schematic representation highlights Complex I dysfunction leading to specific mechanisms that cause various heart diseases. FAO Fatty Acid Oxidation, ROS Reactive Oxygen Species, ATP Adenosine Triphosphate, mPTP mitochondrial Permeability Transition Pore

Future directions

Defects in mitochondrial CI are usually accompanied by secondary defects at the cellular level, including altered morphology of mitochondrial networks, altered membrane potential, changes in intracellular calcium homeostasis, and elevated ROS production. These occur in addition to alterations in NADH oxidoreductase enzyme activity [162]. Notably, the extent of these dysfunctions depends on the metabolic environment in the heart, which varies based on the nature of the pathology. Consequently, therapies directly targeting CI in HFrEF may not be viable for HFpEF and vice versa. Therefore, it is crucial to identify and mitigate secondary damages resulting from CI dysfunction. Studies have observed a quantitative correlation between residual CI activity and ROS levels [163]. Inhibiting ROS production can successfully ameliorate negative consequences of CI dysfunction, such as membrane potential depolarization. However, clinical studies confirming these protective roles in patients with dysfunctional CI have yet to be conducted. Another major secondary defect caused by CI deficiency is altered cellular calcium homeostasis. Reduced CI activity, such as genetic mutations, impairs mitochondrial ATP production, leading to lower ATP levels needed to operate the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). SERCA is the calcium transporter that regulates cardiomyocyte contraction-relaxation cycles [164]. These secondary defects caused by CI deficiencies may precipitate heart failure. Thus, it is vital to identify and target these secondary defects.

Despite promising therapeutic strategies targeting mitochondrial CI, there remains a significant gap in translating these findings from preclinical models to clinical applications. Future directions should focus on optimizing CI modulators’ delivery and efficacy, understanding these interventions’ long-term effects, and conducting robust clinical trials to establish their safety and effectiveness in patients with CI dysfunction. Addressing these gaps will be crucial in advancing mitochondrial-targeted therapies for improving outcomes in CI dysfunction-associated heart diseases.