Redox homeostasis in cardiac fibrosis: Focus on metal ion metabolism

Abstract

Cardiac fibrosis is a major public health problem worldwide, with high morbidity and mortality, affecting almost all patients with heart disease worldwide. It is characterized by fibroblast activation, abnormal proliferation, excessive deposition, and abnormal distribution of extracellular matrix (ECM) proteins. The maladaptive process of cardiac fibrosis is complex and often involves multiple mechanisms. With the increasing research on cardiac fibrosis, redox has been recognized as an important part of cardiac remodeling, and an imbalance in redox homeostasis can adversely affect the function and structure of the heart. The metabolism of metal ions is essential for life, and abnormal metabolism of metal ions in cells can impair a variety of biochemical processes, especially redox. However, current research on metal ion metabolism is still very limited. This review comprehensively examines the effects of metal ion (iron, copper, calcium, and zinc) metabolism-mediated redox homeostasis on cardiac fibrosis, outlines possible therapeutic interventions, and addresses ongoing challenges in this rapidly evolving field.

Graphical abstract

Highlights

• •The link between iron ions metabolism and cardiac fibrotic disease.
• •Iron ions metabolism play a key role in cardiac fibrosis.
• •Copper Ions Mediate Cell Death in Cardiac Fibrosis.
• •Calcium Ion Homeostasis in Cardiac Fibrosis.
• •Zinc Ion Homeostasis in Cardiac Fibrosis.

1. Overview of cardiac fibrosis

Cardiac fibrosis is a pathological phenotype [1], characterized by the excessive activation of fibroblasts leading to the excessive deposition and abnormal distribution of extracellular matrix (ECM) [2,3]. Cardiac fibrosis can be divided into replacement and reactive types [4]. In patients with myocardial infarction, collagen scarring replaces necrotic cardiomyocytes, often leading to replacement fibrosis. Reactive fibrosis, on the other hand, is usually caused by excessive accumulation of ECM proteins [5], often without significant loss of myocardial cells, and is mainly seen in hypertensive patients, primarily occurring around the cardiac vessels [6]. Almost all heart diseases are accompanied by varying degrees of fibrosis [7], such as pathologic cardiac hypertrophy, mainly characterized by increased cardiomyocyte volume, fibroblast activation, loss of cardiomyocytes, collagen deposition, interstitial and perivascular fibrosis [8,9]; when myocardial infarction occurs, cardiac fibrosis plays an important role in tissue homeostasis and repair [10]. However, long-term fibrosis can alter the structure of the heart, impair heart function, and eventually lead to or worsen heart failure [11].

Imbalances in metal ion homeostasis can lead to severe redox dysregulation, which can lead to the occurrence of cardiac fibrosis, such as when the heart undergoes oxidative stress and the antioxidant system fails to remove excess ceruloplasmin (ROS), ROS accumulates in the cytoplasm and leads to the activation of redox-sensitive protein kinase [12], which in turn promotes cardiomyocyte death and fibroblast proliferation and activation, leading to the occurrence and development of cardiac fibrosis [13,14]. More and more evidence suggest that metal ion metabolism regulates the occurrence and progression of cardiac fibrosis by influencing redox homeostasis, but the role and mechanism of redox homeostasis mediated by various metal ion metabolisms in cardiac fibrosis are not fully understood, so this article mainly reviews the latest progress on the role of iron, copper, calcium, and zinc metabolism in cardiac fibrosis (Fig. 1).

Fig. 1.

Dysregulation of metal ion metabolism can lead to a variety of diseases. There is increasing evidence that the metabolism of metal ions affects the occurrence and development of cardiac fibrosis, and cardiac fibrosis regulates the metabolism of metal ions through various pathways.

2. Iron ions

2.1. Imbalances in iron ion metabolism mediate oxidative stress damage in the heart

Iron metabolism is crucial for maintaining a balance in the body, and disorders related to it are prevalent worldwide. These conditions include iron deficiency and iron overload, but regardless of the type, an imbalance in the homeostasis of iron ions in the heart can lead to adverse damage. Inflammation, liver disease, tumors, blood transfusion dependence, and ineffective hematopoiesis will all lead to an increase in the amount of iron ions in the body, while insufficient intake, malabsorption, and excessive loss will lead to iron deficiency in the body [15,16]. The heart has a high demand for energy, and since iron plays an important role in the function of mitochondria and enzymes, a deficiency of iron can lead to metabolic dysfunction in the mitochondria, ultimately worsening or causing heart failure [17]. This, in turn, can result in fibrosis of the heart. Non-transferrin-bound iron (NTBI) binds primarily to albumin and low molecular weight molecules, resulting in the deposition of iron in various tissues, especially in excitatory tissues like the heart, which contain Ca²⁺ channels [18]. In cardiac tissue, functional voltage-gated Ca²⁺ channels are highly expressed and are therefore sensitive to iron overload. The accumulation of NTBI in the heart cells can lead to oxidative stress, primarily through the production of ROS via specific chemical reactions. When the body's antioxidant system is not strong enough to remove excess ROS, these harmful compounds can gradually accumulate and adversely affect lipids, nucleic acids, and proteins [19]. In cardiomyocytes, oxidative stress can lead to impaired excitation-contraction coupling, inhibition of the function of SERCA2 (a protein involved in calcium uptake), elevated cytoplasmic calcium levels, impaired relaxation and later contractility, membrane damage (including mitochondrial membranes), lipid peroxidation, ATP deficiency, inhibition of oxidative phosphorylation, and direct damage to mitochondrial DNA and DNA. Moreover, NTBI can directly stimulate fibroblast activation, leading to increased proliferation and differentiation into myofibroblasts, ultimately contributing to increased fibrosis in the cardiac tissue [20]. Iron deposition in subepicardial cells and granulation cells was reported to be evident in mouse models of cardiac fibrosis induced by angiotensin II infusion, and treatment with deferoxamine (an iron chelator) reduced the degree of cardiac fibrosis in rats, suggesting that iron deposition can exacerbate angiotensin II-induced cardiac fibrosis [21]. Iron deficiency (ID) is one of the common and serious complications of chronic heart failure (CHF). The mechanism may be that heart failure activates neurohormones in cardiomyocytes, altering key molecular regulation in iron homeostasis, leading to decreased intracellular iron levels and impaired mitochondrial function [22]. Intravenous ferric carboxymaltose has been found to significantly improve cardiac function, improve prognosis, reduce mortality, and reduce the risk of recurrence [23].

2.2. Redox dysregulation leads to ferroptosis in cardiomyocytes

Upon binding to Fe³⁺, the plasma protein transferrin (TF) is recognized by transferrin receptor-1 (TFR1) in the cell membrane, and then the complex of Fe³⁺ and TFR1 is internalized into the cell as an endogen, after which the metalloreductase STEAP3 reduces Fe³⁺ to Fe²⁺ and releases it into the cytosol by the divalent metal transporter 1 (DMT1) [24]. Ferroportin (FPN) mediates intracellular Fe²⁺ transport to the extracellular space and is the only known mammalian iron export protein [25]. Under aerobic conditions, intracellular Fe²⁺ is converted to Fe³⁺ by the Fenton reaction, which in turn produces hydroxyl radicals or peroxide radicals, which further promote lipid peroxidation [26]. Mutations in iron overload or iron transport-related genes lead to an increase in labile Fe²⁺ content, which then drives overwhelming lipid peroxidation.

Ferroptosis is an iron-dependent programmed cell death characterized by unique morphological changes in mitochondria (loss of mitochondrial crests). ROS due to iron deposition in cardiomyocytes leads to excessive intracellular production of lipid peroxides, which consume large amounts of plasma membrane polyunsaturated fatty acids, thereby increasing plasma membrane permeability and disrupting its integrity and functionality, ultimately mediating ferroptosis in cells [27]. The massive loss of cardiac cells stimulates the activation and proliferation of fibroblasts and secretes an excess of extracellular matrix, leading to the development of alternative cardiac fibrosis [4]. Prominin 2 (PROM 2) is localized to the protrusions of the cell membrane and is a pentaspanin protein involved in the dynamic regulation of lipids. Mechanistically, prominin2 can promote the formation of exosomes and ferritin-containing multivesicular bodies (MVBs), transporting iron out of cells, thus fighting ferroptosis [28] (Fig. 2). Doxorubicin (Dox) has limited its clinical application in various malignancies because it causes irreversible heart disease. The occurrence and progression of Dox cardiomyopathy has been linked to ferroptosis. Sorting Nexin 3 (SNX3), a reverse transcriptase-related cargo binding protein and one of the exact targets capable of effectively treating cardiac hypertrophy, is able to directly interact with the transferrin receptor protein 1 (TFRC1) and promote its recycling to increase iron deposition [29], and Dox treatment induces Parkinsonism associated deglycase (Park7) [30], leading to an imbalance in iron homeostasis, triggering ferroptosis, which ultimately causes the occurrence and progression of Dox-induced cardiomyopathy. Hemochromatosis is a classic iron overload disorder characterized by excessive absorption and accumulation of iron in the body. Iron overload in hemochromatosis can significantly affect the cardiovascular system, leading to the occurrence of cardiac fibrosis and even heart failure, increasing the mortality rate of patients [31].

Fig. 2.

ROS triggered by iron deposition in cardiomyocytes triggers the production of lipid peroxides, which deplete excess plasma membrane polyunsaturated fatty acids, increasing membrane permeability, leading to impaired integrity and function, and ultimately mediating ferroptosis.

3. Copper ions

3.1. Imbalance in copper ion metabolism affects oxidative phosphorylation in mitochondria

Copper plays a significant role as a structural or catalytic cofactor in various proteins and enzymes associated with the heart. These copper-related binding proteins are involved in regulating mitochondrial respiration, iron metabolism, antioxidant defense, connective tissue cross-linking, and other essential functions within the body. The intricate involvement of copper in these biological processes highlights its importance in maintaining proper cardiac function and overall health [32]. Patients with liver and kidney insufficiency are unable to transport the ingested copper ions in the body in time, resulting in the deposition of copper ions in the body. In addition, some inherited or acquired disorders can cause copper ion homeostasis [33,34]. Since copper is essential for the proper functioning of cells, and copper overload is cytotoxic, the amount and distribution of available copper in the body must be tightly controlled to minimize the potential toxicity of copper while meeting metabolic needs. Copper-dependent enzyme deficiencies, copper transporter dysfunction, and copper deficiency can all contribute to heart disease. Therefore, maintaining a proper copper balance is essential for overall health [[35], [36], [37], [38], [39], [40]]. Mitochondrial respiratory chain complex IV contains copper-dependent cytochrome c oxidase (CCO), which uses heme and copper ions as required cofactors, essential components of oxidative phosphorylation [41]. Copper deficiency greatly reduces CCO activity and significantly affects the ability of mitochondrial respiration in the heart [[42], [43], [44], [45], [46], [47]]. Copper deficiency impairs mitochondrial function and energy production, leading to compensatory cardiac hypertrophy and fibrosis, manifested by compensatory mitochondrial biogenesis and increased size, deterioration of mitochondrial ultrastructure, and decreased or absent cristae [48,49]. In addition, copper deficiency can also affect the protein expression of MT-CO, the subunit of CCO, thereby further reducing the activity of CCO. Copper and zinc are also present in superoxide dismutase 1 (SOD1), which converts superoxide radicals (O²⁻) into molecular oxygen and hydrogen peroxide [50]. Ceruloplasmin (CP) is a type of iron oxidase that not only mobilizes iron, but also carries more than 90% of the copper in plasma, so it both maintains copper ion homeostasis and plays an important role in antioxidant defense [51].

It was found that proper supplementation of copper content in the hearts of fibrotic mice with chelating agents was able to reduce the level of collagen III in heart tissue and reduce the degree of cardiac fibrosis. While fibroblasts in the hearts of mice supplemented with copper remained expanded, levels of matrix metalloproteinase-2 (MMP-2), the main enzyme that degrades collagen in the heart, were also significantly increased. In addition, mRNA and protein levels of tissue inhibitors of matrix metalloproteinase-1 and -2 (TIMP-1 and TIMP-2) due to fibrosis also decreased to normal with copper supplementation. Thus, the reduction of cardiac fibrosis by supplementation with cardiac copper ions is at least partly associated with the increased activity of MMP-2, which leads to collagen degradation [52].

In addition, cardiac delivery of copper ions has been found to alleviate fibrosis levels, restore vascular density, and improve cardiac contractility by increasing the concentration of copper ions in the infarcted heart. The number of myofibroblasts in the heart decreased significantly after copper supplementation, while the total number of fibroblasts did not change. At the same time, the products of collagen I and III, as well as collagenase inhibitors, were also significantly reduced. However, levels of MMP-1-producing fibroblasts and metalloproteinase-1 (MMP-1) are markedly elevated. Thus, copper ions may lead to fibrinolytic conversion and improve cardiac function by inhibiting the conversion of fibroblasts to myofibroblasts [53].

3.2. Copper ions mediate cell death in cardiac fibrosis

When copper ion metabolism is disordered, it can damage mitochondrial function through inflammatory pathways and oxidative stress responses, impairing lipid metabolism pathways, leading to autophagy and cell death [[54], [55], [56], [57], [58]]. Copper induces different forms of programmed cell death, including autophagy and apoptosis, through a variety of mechanisms, including inhibition of proteasomes, accumulation of ROS, and anti-angiogenic factors [59]. Apoptosis occurs mainly in the mitochondria, endoplasmic reticulum, and nucleus. Autophagy caused by copper signal-mediated activation of the mTOR-ULK1/2 pathway is a self-protective mechanism to remove damaged organelles, but excessive autophagy can lead to cellular damage [60]. Copper ions cause pyroptosis of cells through the Fenton/Haber Weiss reactions, and these reactions involve ROS-mediated destructive cell death [61,62]. Cuproptosis occurs mainly in cells that are rich in mitochondria or have a high need for energy. Excess copper ions are mediated by ferredoxin 1 (FDX1) and participate in the tricarboxylic acid (TCA) process, induce the aggregation of dihydrolipoyl transacetylase (DLAT), cause abnormal folding of mitochondrial proteins, lead to the loss of Fe–S protein, and eventually die due to defective cellular energy metabolism [63]. In addition, although glutathione (GSH) and SOD have certain antioxidant capacity, they are generally difficult to resist the damaging effects of ROS produced by oxidative stress mediated by copper ion imbalance [64] (Fig. 3). It is worth mentioning that Wilson's disease (WD) is an inherited treatable disorder of copper metabolism characterized by pathological deposition of copper. Patients with this condition usually have a pathologic phenotype of congenital heart disease, and an overload of copper in the heart may be a potential factor [65].

Fig. 3.

Copper ions mediate programmed death of cardiomyocytes. Copper can mediate various types of cell death in vivo, mainly including apoptosis, autophagy, pyroptosis, and cuproptosis recently discovered via copper-mediated.

4. Calcium ions

4.1. Calcium mediates mitochondria-dependent oxidative stress damage

Malignant tumors and hyperparathyroidism are common causes of hypercalcemia, while patients with hypoparathyroidism, vitamin D deficiency, and heart failure often lead to hypocalcemia due to long-term use of diuretics [66]. Ca²⁺ not only regulates the excitation-contraction coupling of cardiomyocytes, but also determines cell function and fate by regulating mitochondrial metabolism and oxidative stress signaling, with mitochondria accounting for about one-third of the cell volume in cardiomyocytes [67], and located near the sarcoplasmic reticulum (SR), which facilitates the exchange of metabolites between the two organelles and their connectivity to each other [[68], [69], [70], [71]]. Ca²⁺ and ROS are thought to be the main transduction signals in SR and mitochondria [72,73]. In mitochondria, the presence of Ca²⁺ mediates the tricarboxylic acid cycle to produce NADH and FADH2 to provide electrons for the electron transport chain (ETC) [74]. Approximately one-fifth of the electrons in ETC may leak into molecular oxygen to form superoxide anions [75], after which many superoxide anions are converted to hydrogen peroxide, primarily by superoxide dismutase 2 (SOD2) in mitochondria [76,77], which are then cleared by peroxiredoxin (Prx) and glutathione peroxidase (Gpx) [78,79]. In cardiac fibrosis, the activation of voltage-gated L-type Ca²⁺ channels increase, and excess calcium flows into the cytoplasm [80,81], resulting in increased mitochondrial uptake of Ca²⁺, resulting in the production of large amounts of ROS [82]. In addition, reduced SERCA2a activity in cardiac fibrosis leads to reduced uptake of calcium in the cytoplasm by SR, and increased levels of transforming growth factor (TGF-β) lead to Ca²⁺ leakage in SR, further exacerbating Ca²⁺ overload in the mitochondria, leading to more severe oxidative stress damage [[83], [84], [85]]. In addition, excess Ca²⁺ or ROS can activate mitochondrial permeability transition pores (mPTPs) on the inner membrane of the mitochondria [86,87], when water and all solutes<1500 Da are able to enter the mitochondria through the inner mitochondrial membrane, causing mitochondrial swelling and rupture, ultimately leading to apoptosis [[88], [89], [90]].

4.2. Calcium channels are involved in the regulation of oxidative stress

The excessive production of ROS is observed in most pathophysiological conditions of the heart, which exacerbates cardiac remodeling and dysfunction. The transient receptor potential channel 3 (TRP3) of calcium has been shown to be involved in the production of ROS and can exacerbate cardiac fibrosis [91,92]. Although ROS can be produced by xanthine oxidase, NADPH oxidase (Nox), mitochondrial electron transport chain, and nitric oxide synthase, the ROS produced in the heart is mainly derived from Nox [[93], [94], [95]]. Of the seven members of the Nox proteins, Nox2 and Nox4 are expressed mainly in the heart. Among them, Nox2 can form a relatively stable complex with TRP3, thereby protecting Nox2 from proteasomal degradation and increasing its protein abundance. In the pathological state, the protein level of TRPC3 will increase, resulting in a significant increase in the abundance of Nox2 on the plasma membrane, resulting in an abnormal increase in the production of ROS in cardiomyocytes, which further activates the RhoA pathway in cardiomyocytes and fibroblasts, leading to the occurrence and development of cardiac fibrosis [96]. At the same time, deletion of the TRPC3 gene inhibits cardiac fibrosis with stress overload or angiotensin-II infusion [92,97] (Fig. 4).

Fig. 4.

In cardiac fibrosis, a variety of factors cause excess calcium ions to enter the mitochondria, causing the production of a large amount of ROS and impairing the structure and function of the mitochondria. At the same time, TRP3 stabilizes Nox2, and the ROS produced by Nox2 activates the RhoA pathway in cardiomyocytes and fibroblasts, leading to cardiac fibrosis.

5. Zinc ions

5.1. Abnormal zinc metabolism promotes oxidative stress in cardiomyocytes

As an essential trace element for life activities, zinc can exert antioxidant and anti-inflammatory effects in the body, and also has the role of regulating the structure and function of various metalloproteinases [98]. Inadequate uptake, malabsorption, increased physiological demand, and acute infection are several common causes of zinc deficiency in clinical practice, however, in an unhealthy cellular environment, such as hypoxia, increased intracellular zinc levels and zinc overload may occur [99]. An imbalance in zinc homeostasis in the heart can lead to oxidative stress damage [100]. The study found that myocardial tissue extracted from zinc-deficient mice fed with a zinc-deficient diet was observed to reduce the zinc ion content, while ROS was significantly increased, and a large number of cardiomyocyte deaths were observed. As a result, a deficiency of zinc ions in the heart can cause severe oxidative stress [101].

5.2. The imbalance of zinc ion metabolism affects the scavenging of free radicals by inhibiting the expression of MT

The main site of ROS production is the mitochondria, and this organelle is also the main site of oxidative stress damage [102]. In normal organisms, ROS levels are in dynamic equilibrium. Metallothionein (MT), which makes up the mitochondrial respiratory chain, is able to scavenge ROS in the body, including peroxides, superoxide, and hydroxyl radicals, either directly or through reactive oxygen species [103,104]. MT-1 and MT-2 are expressed in all mammalian organs [105]. However, studying the heart tissue of zinc-deficient mice found that the levels of MT-1 and MT-2 decreased significantly [106]. As a result, the clearance rate of ROS in cardiomyocytes is severely reduced, the level of ROS is significantly increased, and oxidative stress damage can be observed in most cardiomyocytes. At the same time, myofibroblasts in the heart proliferate and activate on a large scale, producing a large number of collagen I/III, tissue inhibitors of metalloproteinase (TIMPs), matrix metalloproteinases (MMPs) and fibronectin (FN) [107]. MMPs can degrade various components of the extracellular matrix (ECM) and are key enzymes that regulate cardiac remodeling, and their activity is inhibited by TIMPs [108,109]. ECM can affect the normal function of the heart, and the ECM is generally in a dynamic and stable state, which mainly includes collagen fibers I and III, FN and a small amount of collagen fibers IV, V, VI, etc [110]. About more than a quarter of MMPs have been found to be involved in cardiac fibrosis [111]. MMP-2 and MMP-9 can degrade collagen I, IV, V, and MMP-2 can also degrade collagen III. In addition, MMP-1, MMP-3, and MMP-12 also play an important role in cardiac remodeling [112]. The study found that the expression of MMPs in the heart tissue of zinc-deficient mice was reduced [113], while the expression levels of TIMPs, α-SMA and collagen were increased. It can be seen that zinc deficiency can lead to cardiac fibrosis by affecting the levels of MMPs and TIMPs (Fig. 5).

Fig. 5.

Zinc ion homeostasis affects the expression level of MT, which in turn affects the clearance rate of ROS, resulting in oxidative stress damage of cardiomyocytes, activation and proliferation of fibroblasts, and excessive ECM production.

6. Concluding remarks and future perspectives

As described herein, the metabolism of metal ions is tightly controlled by different regulatory modes, and the coordinated regulation of metal ion uptake, efflux, distribution, and storage in cells is important for normal cardiomyocyte function. Imbalances in metal ion homeostasis leads to the development and progression of cardiac fibrosis through various pathways, especially mediated redox. This article focuses on the regulation of metal ion levels in four hearts and the role of their mediated redox in cardiac fibrosis to help broaden the current understanding of the multiple complex roles of metal ions. It can be seen that the control of stable and balanced metal ion metabolism is one of the promising targets for the treatment of cardiac fibrosis, although their exact mechanism of action has not been fully studied. Therefore, further studies and experiments are necessary to determine the potential of metal ion mediated redox reaction as a therapeutic approach in cardiac fibrosis.

Sources of funding

This project was supported by National Natural Science Foundation of China (82170236, 82330048, 81700212), Key research and development projects of Anhui Province (202104j07020037), Excellent Youth Research Project in University of Anhui Province (2023AH030116), Translational medicine research project of Anhui Province (2021zhyx-C61), Excellent Top Talents Program of Anhui Province Universities (gxyqZD2022023) and National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University (2020GMFY02).

CRediT authorship contribution statement

Zhen-Yu Liu: Software, Investigation, Data curation. Zhi-Yan Liu: Software, Resources, Investigation. Li-Chan Lin: Software, Methodology, Investigation. Kai Song: Software, Methodology, Data curation. Bin Tu: Software, Formal analysis. Ye Zhang: Supervision, Validation. Jing-Jing Yang: Writing – review & editing, Software, Formal analysis. Jian-Yuan Zhao: Writing – review & editing, Software, Investigation. Hui Tao: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

TThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.