A look at MERCs as UPR^mt regulatory hubs in age-associated heart diseases

INTRODUCTION

Proteotoxic stress is a common challenge for all organisms, and several mechanisms are involved in the defense against stress. Unfolded protein responses (UPRs) are triggered by the accumulation of unfolded or misfolded proteins in cells and occur in response to an imbalance between local protein biosynthesis, loading, and folding in different subcellular compartments, including the endoplasmic reticulum (UPR^ER), mitochondria (UPR^mt), and cytoplasm (UPR^cyto) through distinct mechanisms^[1]. Maintenance of proteostasis requires a close association with energy conversion to support proper protein folding. UPR identifies misfolded proteins within the ER but is triggered by cytosolic protein aggregation, while the integrated stress response (ISR) is activated by stress signals originating from both the ER and the cytosol^[2].

The ER senses and responds to cellular stress triggered by metabolic imbalances or protein folding during the progression of various diseases^[3]. UPR^RE is activated upon alteration of the ER chaperone-protease transcription network to promote the recovery of organelle homeostasis^[4] through two phases: (a) reducing protein synthesis and increasing degradation of misfolded proteins; and (b) initiating transcriptional regulation of target genes responsible for global control of proteostasis^[2]. In general, UPR^ER is controlled by three protein sensors, inositol-requiring enzyme 1 (IRE), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6), which modulate a selective response (adaptive or signaling pathway) to preserve cellular physiology^[3]. Meanwhile, UPR^cyto is induced by misfolded cytosolic proteins (e.g., VHL, tumor suppressor gene) or non-glycosylated viral protein precursors that do not enter the secretory pathway at all^[5,6]; this response is characterized by the action and regulation of multiple chaperone proteins, such as heat shock proteins (Hsp70 and Hsp90) and a subset of J-domain chaperone proteins in the cytosol^[1].

Mitochondria are involved in several cellular functions, including energy generation, programmed cell death, regulation of calcium (Ca²⁺) levels, and lipid metabolism^[7]. Yet, these mechanisms are impaired in the presence of reactive oxygen species (ROS), electron transport chain (ETC) uncoupling, and aberrant protein expression that promotes mitochondrial dysfunction^[8]. This is more evident with advancing age and governs the establishment and progression of age-related diseases^[9]. In addition, mitochondrial homeostasis is maintained by distinct mechanisms of mitochondrial quality control, and one activated response to resolve stress within the mitochondria is UPR^mt. Conversely, when organelle damage is severe, signals for mitochondrial destruction by mitophagy are sent, thus preventing further damage to the mitochondrial network and allowing the cell to recover its homeostasis^[10].

UPR^mt is a nuclear transcriptional response that maintains or restores mitochondrial proteostasis via a mitohormetic response in the mitochondria. This response upregulates glycolysis and downregulates the expression of respiratory chain subunits to balance cellular energy and unload mitochondrial translation and folding machinery^[11]. Notably, in aged organisms with multimorbidity, the UPR^mt is activated, extending life expectancy. For example, in Caenorhabditis elegans, it promotes protein homeostasis by alleviating protein aggregation in neurons and increasing nematode longevity^[12]. However, partly because of UPR^mt activation, OSCP (a subunit of Complex V) loss promotes the opening of the mitochondrial permeability transition pore (mPTP) and shortens the lifespan of nematodes^[13]. Derived from this double-edged sword, in this review, we describe the effects of UPR^mt in cardiac aging models, owing to the enormous impact that cardiovascular diseases (CVDs) have on patients' quality of life and disability, coupled with the close connection between cellular senescence and fatal cardiac outcomes in most CVDs in elderly individuals^[14], making early detection of cardiac degeneration, preventive measures, and treatment of these diseases imperative to promote healthy aging^[15].

Through different disciplines, we have been able to obtain clues regarding the existence of interorganellar contacts. Using biochemistry, we have obtained the co-purification of membranes of different organelles; cell biology provides visualization of close associations and interorganellar links, whereas cell physiology describes cellular functions that require local interactions of adjacent organelles^[16]. Thus, the harmonization of these findings reveals a diversity of nanoscale structures with distinctive functions but with shared characteristics among organelle contacts^[17]. So, based on these observations, and after employing specialized, high-resolution detection methods, we have been able to visualize the different contacts, envisioning a new field of study that we call contactology^[16].

In particular, the connections between mitochondria and ER allow efficient communication that promotes cellular homeostasis through mitochondria-ER contact sites (MERCs) at different levels. For example, loss of MERCs is linked to decreased mitochondrial membrane potential and increased ROS in senescent vascular smooth muscle cells^[18]. In neurons, decreased MERCs interaction induces mitophagy, Ca²⁺ dysregulation, and respiratory capacity, leading to neurodegeneration^[19]. In skeletal muscle, depletion of MERCs leads to alterations in mitochondrial lipidomics and Ca⁺ influx, which are associated with instability of the mitochondrial-RE junction due to loss of tethering proteins^[20,21]. Therefore, these studies highlight the structure and regulation of MERCs as potential targets to improve mitochondrial function. Moreover, in different pathological situations, such as diabetes and obesity, as well as in the field of cellular senescence^[22,23], there has been growing interest in the study of MERCs. However, the relationship between MERCs, UPR^mt, and aging, especially in the development of CVDs, remains unclear. Therefore, in this review, we link UPR^mt changes to communication through MERCs during aging, recalling that these microdomains harbor UPR^ER (as recently reviewed^[24,25]), with an outstanding debt on what occurs in the aging heart.

OVERVIEW ON AGING

Aging is an accumulation of life's consequences, such as molecular and cellular damage, leading to functional decline, chronic diseases, and ultimately mortality^[26]. This process is constantly evolving in numerical terms; by 2030, one in six people in the world will be 60 years old or older. In 2050, the global population of people aged 60 years and over will be 2.1 billion, double by 1.4 billion in 2020. Notably, 426 million people will constitute the population aged ≥ 80 years over a 30-year period (between 2020 and 2050). Owing to their exponential growth, there are major challenges for healthcare and long-term care that require the training of medical professionals and the need for physical and social environments that are better adapted to the elderly.

Throughout history, there have been many questions about the aging process; however, to date, we do not have clear and definitive answers. In light of this, the Aging Biomarker Consortium (ABC) argues that if we can accurately determine our biological age (rather than chronological age), identify exactly why some people are more susceptible to certain age-related diseases (such as heart disease), and assess whether the efficacy of geroprotective treatments is universal, we would have the opportunity to establish biomarkers for aging and predict the transition to a pathological state^[27]. However, even if we were close to achieving this, multiple aspects of aging would be involved, and attention to only one aspect would counteract the progress of others.

As we have described, the increase in the elderly population has been fruitful thanks to medical interventions that have helped prevent the death of elders from age-related diseases; however, simultaneously, the number of older adults suffering from diseases has increased, so the ratio of quality of life to longevity is decreasing. Undoubtedly, the strategy that has set the standard for moving toward healthy aging is intervention with an anti-aging approach that slows the onset and progression of age-related diseases that (theoretically) would enable the life quality vs. longevity balance to be restored. However, it is very difficult to achieve a balance between both factors - living many years without deterioration while maintaining good physical and mental health.

Traditionally, age-related diseases have been investigated separately; however, this approach must be modified to slow the deterioration associated with aging. Geroscience is an interdisciplinary field dedicated to understanding the complex relationship between aging and age-related diseases. It has a boom and greater scope as an integrative scientific field, whose goals can be summarized as follows: (1) to identify biological processes and pathways that contribute to, influence, and/or regulate the deterioration of aging; and (2) to have an integrated view of potential targets to develop therapeutic strategies that help limit disease progression with age^[28]. To achieve these goals, we started with different markers that helped to lay the molecular foundation for aging.

In their most recent publication, López-Otín et al. added three new features (deactivated macroautophagy, chronic inflammation, and dysbiosis)^[29] to those already enunciated ten years ago^[30]. These hallmarks are grouped into the following categories: (1) primary, which is the main cause of age-associated molecular damage; (2) antagonistic, which determines the beneficial and harmful effects on the health of the organism; and (3) integrative, which involves the alteration of intercellular communication^[31]. Each of these categories has specific study targets that have helped corroborate the premise of interdependence in these hallmarks; that is, the experimental attenuation or accentuation of a specific marker often affects other traits, underscoring that aging is a complex process that must be conceived as a whole^[29].

One of the main hallmarks of aging, perhaps the most widely studied one, is mitochondrial dysfunction. This is because mitochondria are especially necessary in tissues with high energy demand, such as the heart, which continuously requires adenosine triphosphate (ATP) to maintain its activity. Conversely, malfunction is considered a key factor in CVDs, leading to decreased ATP production, increased ROS levels, apoptosis, and mitochondrial dynamic imbalance^[32]. In addition to its undeniable involvement in impaired proteostasis, it leads to destabilization of respiratory complexes and reduced organelle turnover (see below).

Significant strides have been made in the field of aging to the point that aging is an unequivocal risk factor for the development of many chronic diseases. This arduous research has facilitated the identification of therapeutic targets for preventing and treating aging and its diseases, as well as the development of useful pharmacological agents for clinical approval in the future^[33], as has been the case with senotherapy for various age-related chronic diseases, including heart disease^[34-36].

MITOCHONDRION AND CARDIAC AGING

Mitochondria play an important role in aging, but the link between mitochondrial dysfunction and age-associated diseases remains unclear^[37]. Cardiac aging is associated with the maladaptation of cellular metabolism, cardiomyocyte senescence, decreased angiogenesis, and increased fibrosis, which together induce cardiac remodeling and dysfunction^[38]. Structural and functional changes in the aging heart have been documented in the literature (please review^[39-41]). Therefore, we focused on describing mitochondrial changes related to their function, morphology, and dynamics in aging organisms [Figure 1].

Figure 1. Structural and functional changes in aging mitochondria. With age, both mitochondrial populations undergo alterations in metabolism, which contributes to the development of cardiovascular diseases (CVDs). Subsarcolemmal mitochondria (SSM, located below the membrane) also show lamelliform, swollen cristae, and an increased presence of calcium (Ca²⁺). Interfibrillar mitochondria (IFM, which resides between myofibrils) have tubular cristae, reduced bioenergetic capacity, and less respiration. Additionally, both mitochondrial types showed higher capacity for mitochondrial fission. Created using BioRender (www.app.biorender.com).

UPRmt SIGNALING AND ITS ROLE IN MITOCHONDRIAL FUNCTION AND STRESS RESPONSE

UPR^mt is a transcriptional response triggered by mitochondrial defects during OxPhos, ROS production, ETC dysfunction, imbalance between mitochondrial and nuclear proteins, unfolded proteins, and exogenous stress (e.g., hypoxia, pathogen infections, and xenobiotics)^[67,68]. This response was initially investigated in depth in Caenorhabditis elegans^[69,70] with relevant implications for nematode longevity^[71,72]. However, in the early 2000s, it was described that COS-7 cells transfected with a misfolded mutant of ornithine trans-carbamylase, involved in the urea cycle, accumulated this protein and thus overexpressed nuclear-encoded mitochondrial chaperones and proteases in response to mitochondrial stress^[73].

Different mechanisms promote UPR^mt activation, with the canonical ISR axis being the most studied, involving four protein kinases that uniquely respond to stress signals, which phosphorylate eukaryotic translational initiation factor 2α (eIF2α) at one of the three subunits of the eIF2 complex. These kinases include PERK, heme-regulated inhibitor (HRI), general control non-derepressible 2 kinase (GCN2), and double-stranded RNA-dependent protein kinase (PKR)^[2,74]. Broadly speaking, the goal of UPR^mt is to reduce the global translation rate by favoring the translation of mitochondrial proteases and chaperones to alleviate and promote recovery from mitochondrial stress [Figure 2]. In Caenorhabditis elegans, the UPR^mt is orchestrated by the nuclear accumulation of activating transcription factor associated with stress-1 (ATFS-1) under mitochondrial dysfunction conditions^[75,76]. Briefly, ATFS-1 has an N-terminal mitochondrial targeting sequence that is essential for UPR^mt repression. Normally, ATFS-1 is imported into the mitochondria and degraded; however, ATFS-1 accumulates in the cytosol and migrates to the nucleus because of a nuclear localization signal, where it forms a complex with Hsp10/Hsp60 and coordinates the level of mitochondrial dysfunction with a protective transcriptional response during mitochondrial stress^[70,77].

Figure 2. UPR^mt signaling pathway and its role in preserving mitochondrial integrity in mammals. UPR^mt acts in the defense against mitochondrial stress through mitochondrial stress-nucleus communication. The unfolded proteins in the matrix and/or IMS initiate the activation of different proteins involved in UPR^mt. Following this signal, the development of this response is initiated through the activation of proteins such as JNK, SIRT3, eIF2α, and ERα, which, in turn send signals to the nucleus to induce UPR^mt via other proteins (e.g., CHOP) and transcription factors (e.g., c-Jun, FOXO3, NRF1, OMI/HTRA2) to promote the transcription of genes involved in halting mitochondrial damage and preserving mitochondria in their healthy state. IMS: Intermembrane space; JNK: Jun N-terminal kinase; SIRT3: Sirtuin 3; eIF2α: eukaryotic translational initiation factor 2α; ER-α: Estrogen receptor α; CHOP: CCAAT/enhancer binding proteins (C/EBP) homologous protein; FOXO3: Forkhead box O3; NRF1: Nuclear respiratory factor 1; OMI/HTRA2: High-temperature protein A2. Created using BioRender (www.app.biorender.com).

When the efficiency of the mitochondrial machinery is reduced in mammals, the activation of eIF2α/ATF4/CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) signaling is initiated [Figure 2]. This signaling pathway encodes chaperones (e.g., Hsp10 and Hsp60), antioxidant enzymes, ClpP proteases, and the mitochondrial Lon protease (LonP1). Mechanistically, when misfolded proteins accumulate in the mitochondrial matrix, they are cleaved by ClpP or LonP1 and converted into small peptides^[8,69,78], which in turn activate Jun N-terminal kinase (JNK) while PKR activates CHOP, and subsequently induce the transcription factor c-Jun^[79]. Oddly, the CHOP element (GC/ATTGCA) is the mitochondrial stress response element that, in association with C/EBPβ, upregulates the expression of mitochondrial stress genes, such as YME1L, in response to the accumulation of unfolded proteins in the mitochondrial matrix. In contrast, mitochondrial protein genes (such as LonP1) do not have a CHOP element and are therefore not upregulated^[73,80]. Furthermore, when specific stress occurs in the matrix, eIF2α is phosphorylated by the GCN2 kinase, increasing the translation of CHOP and activating transcription factor 5 (ATF5) [Figure 2].

Overproduction of ROS and the accumulation of unfolded proteins in the intermembrane space (IMS) activate estrogen receptor-α (ER-α), which triggers the activation of high-temperature protein A2 (HtrA2/Omi), a nuclear-encoded serine protease located in the IMS. HtrA2/Omi is released from the mitochondria into the cytosol and nucleus in response to stress, inducing caspase-independent and caspase-dependent apoptosis. In contrast, under normal conditions, Omi1/HtrA2 is restricted to IMS, where protease activity is involved in the degradation of undesirable proteins to maintain mitochondrial proteostasis^[81]. In addition, protein quality control depends on HtrA2/Omi and proteasome upregulation, which increases the transcription of nuclear respiratory factor 1 (NRF-1), thereby protecting the mitochondria by preventing mitochondrial potential loss [Figure 2]^[78]. However, a low mitochondrial membrane potential can activate UPR^mt to help stress-resilient cells restore mitochondrial function^[82,83]. For instance, treatment with auraptene increases the expression of junctional proteins (e.g., occludin and claudin-5), which prevent pathogens from crossing into the brain. This protective response is accompanied by an increase in ATF5, LonP1, Hsp60, CHOP, and antioxidant enzymes regulated by NF-E2-related factor 2 (Nrf2)^[84].

UPRmt signaling and aging

It is widely reported that in Caenorhabditis elegans, ATFS-1 acts as the master regulator of UPR^mt; however, its activation alone is not sufficient to extend lifespan^[72]. In contrast, the first studies linking UPR^mt to aging dated back over 10 years ago in Surf1 knockout mice. Specifically, LonP1 levels were found to be increased, Hsp60 and ClpP levels were not modified, and CHOP levels tended to increase in the heart, although not significantly. However, all of these proteins were higher in the skeletal muscle of Surf1 knockout mice^[85]. In fact, forced expression of UPR^mt genes in muscles was sufficient to preserve mitochondrial function and delay age-related locomotor impairment^[86]. LonP1 deficiency induces altered cardiac ventricular development and cardiomyocytes are characterized by mitochondrial swelling, loss of cristae, and protein aggregates^[87]. In the kidneys of aged humans and mice, reduced LonP1 expression is linked to renal fibrosis and mitochondrial dysregulation, which is alleviated after LonP1 is overexpressed^[88].

In the Surf1 knockout mice, the expression of genes involved in UPR^mt in skeletal muscle, as well as the expression of Nrf2 and heme-oxygenase-1 (HO-1), does not differ from that of the wild-type^[85]. In this regard, in a nematode model of the CL2006 strain expressing β-amyloid, Lycium barbarum treatment induced less follicle-stimulating hormone receptor-1-mediated activation of UPR^mt and inhibited ROS production via upregulation of the antioxidant system, thereby ameliorating mitochondrial damage and reducing the progression of Alzheimer’s disease^[89]. These data indicate that loading of misfolded proteins into the mitochondrial matrix also activates the antioxidant-UPR^mt axis through activation of sirtuin 3 (SIRT-3). SIRT3 deacetylates the transcription factor forkhead box O3a (FOXO3a), causing its nuclear transport to the nucleus, where it activates the transcription of antioxidant enzymes such as catalase and superoxide dismutase 2. However, it remains unclear how SIRT3 is associated with the expression of UPR^mt-related genes^[76,90]. In Figure 3, we summarize the signaling molecules involved in UPR^mt activation and the differences between damaged young cardiomyocytes and injured aged cardiomyocytes. UPR^mt proteins are common in cardiomyocytes of both age groups, and their impact on mitochondrial function is also represented. Given a brief glimpse of the signaling pathways involved and best documented in UPR^mt, in the following paragraphs, we describe their linkage to CVDs and aging.

Figure 3. Proteins that induce UPR^mt activation in young and aged damaged cardiomyocytes and their role in mitochondrial function. Proteins that promote UPR^mt activation in young, damaged cardiomyocytes are illustrated in blue, and aged cardiomyocytes in yellow. Proteins common to both age groups, which activate UPR^mt, are represented in green. In young, damaged myocytes, mitochondrial respiration and biogenesis are decreased, while mitophagy is increased (pink squares). In contrast, in aged cardiac cells, mitochondrial bioenergetics improve and mitophagy decreases following UPR^mt activation by the Lon1 peptidase (LonP1), yeast mitochondrial escape 1-like-1 (YME1L), and fibroblast growth factor 21/sirtuin-3 (FGF21/SIRT3, purple squares). HSP: Heat shock protein; ATF5: transcription factor-5; FUNDC1: FUN14 domain containing 1; DIO2: type II deiodinase; ClpP: mitochondrial caseinolytic protease P; Oma1: stress-activated protease 1 m-AAA; CHOP: C/EBP homologous protein; HtrA2: high-temperature requirement protein A2; DDIT3: DNA damage-inducible transcript 3; UBL5: ubiquitin-like protein 5; OPA1: optic atrophy protein 1; Fis1: mitochondrial fission protein 1. Created using BioRender (www.app.biorender.com).

UPRmt IN AGE-RELATED CARDIOVASCULAR DISEASES

The development of diseases is an inherent and inevitable fact that occurs when organisms age, a scenario that worsens when there is gradual deterioration of mitochondrial function that contributes to reduced cellular function. It is at this time that the UPR^mt is responsible for promoting the repair and recovery of mitochondrial homeostasis through compensatory processes related to proteostasis and the antioxidant system, thus achieving the regulation of mitochondrial dysfunction in age-related diseases^[91,92]. Indeed, it has been suggested that moderate mitochondrial stress and subsequent activation of UPR^mt delay the aging process and thereby is a therapeutic target for the treatment of age-associated diseases^[93].

Specifically, the heart and its specialized cells have high mitochondrial abundance. Consequently, many CVDs such as HF, hypertension, and IR injury are associated with defects in OxPhos and mitochondrial structural changes^[94], thus promoting endothelial dysfunction, inflammation, and cardiomyocyte death^[8]. In the face of these damage events, activation of UPR^mt could improve mitochondrial function and myocardial contractility, reduce cardiomyocyte mortality, and ameliorate the processes of fibrosis and pathological remodeling^[95]. Increased risk of CVDs is associated with metabolic syndrome, and trimethylamine N-oxide (TMAO) is linked to these disorders. Research has shown that TMAO promotes metabolic dysfunction by binding to and directly activating the PERK/FOXO1 pathway^[96]. In healthy humans, plasma TMAO is higher in older individuals (64 ± 7 years) than in younger individuals (22 ± 2 years), which is inversely related to brachial artery dilatation. Indeed, dietary supplementation for six months with TMAO induces an aging-like impairment in endothelium-dependent dilatation of the carotid artery in young mice (8 weeks of age)^[97].

Changes in cardiac metabolism are closely associated with the onset and progression of CVDs. In this light, the increase in mitochondrial bioenergetics reduces apoptosis and tissue damage, suggesting that the rise in UPR^mt in cardiomyopathies is also related to this response. Accordingly, silencing of DIO2 in hiPSC-CMs induces the downregulation of genes involved in mitochondrial biogenesis (e.g., PGC1α and PPARα) and fatty acid metabolism (e.g., acetyl-CoA carboxylase α and β, and fatty acid synthase), which are key factors in the regulation of energy metabolism. Consequently, UPR^mt activation and mitochondrial bioenergetics decrease, inducing ROS production and reducing cell viability^[65]. In addition, the activation of UPR^mt by oligomycin induces glucose and oxygen consumption in neurons under hypoxic stress^[98]. In line with this, it was observed in cardiac IR mice that ATF5 activation significantly induces the transcription of several UPR^mt-linked proteins and upregulation of mitochondrial and glycolysis genes that collectively ameliorate cardiac damage compared to ATF5 knockout mice^[11]. It has also been reported that the LonP1/ATF4 axis activates UPR^mt, which reduces hepatic steatosis and increases oxidative metabolism, leading to reduced fat mass and improved mitochondrial function in skeletal muscle^[99], a phenomenon that could also occur in cardiac muscle. Thus, UPR^mt activation plays a crucial role in the regulation of glucose and fatty acid metabolism in cardiomyocytes, especially under stressful conditions. These data emphasize the possibility of using UPR^mt activation as a treatment for cardiometabolic diseases and even slowing the progression of these age-related diseases. At the time of writing this review, a manuscript on the involvement of UPR^mt in CVDs was published (please review^[100]); however, the influence of age on UPR^mt has not been discussed. In the following sections, we describe the mechanisms of this response in the three most prevalent cardiac diseases in the elderly.

Dilated cardiomyopathy

Left ventricular enlargement and global deterioration of systolic function (EF < 45%) in the absence of coronary artery disease or increased load are the defining features of Dilated cardiomyopathy (DCM)^[125]. In addition, impaired metabolic energy and mitochondrial dysfunction follow systolic dysfunction and eventually HF^[126].

DCM patients aged 59 years have low expression of DNA damage-inducible transcript 3 and UBL-5, the regulatory elements of UPR^mt[113]. Sprague-Dawley rats with neurogenic hypertension-induced DCM exhibit mitochondrial changes at morphological and bioenergetic levels that are associated with an increased presence of ROS. Specifically, mitochondrial fission 1 protein levels increase, whereas OPA1 levels decrease, suggesting lower mitochondrial biogenesis^[127]. Indeed, low OPA1 levels are associated with altered mitochondrial dynamics in the presence of small mitochondria with dilated cristae, inflammation, and epithelial senescence in the skeletal muscles of 18-month-old mice and sedentary older adults (53-82 years old)^[128]. Furthermore, OPA1 deletion promotes ER stress in primary myoblasts and skeletal muscles of 40-week-old mice^[20].

Notably, following the import of OPA1 from the nucleus into the mitochondria, the maintenance of normal mitochondrial morphology depends on the proteolytic processing of this protein. In particular, protease YME1L mediates the turnover of OPA1, thus limiting its total accumulation^[129]. Deletion of YME1L induces the proteolysis of OPA1 by Oma1, leading to mitochondrial fragmentation, cell death, fibrosis, and ventricular remodeling, thereby promoting DCM^[111]. Moreover, under conditions of metabolic stress, OPA1 processing is dispensable for prolonging lifespan and protecting old OxPhos-deficient Cox^-/- mice against mitochondrial cardiomyopathy. Interestingly, this protective framework is related to enhanced communication between the mitochondria and ER^[129].

Direct evidence for the involvement of UPR^mt in DCM is presented in cardiac-specific DARS2-deficient mice that progressively develop DCM due to increased mitochondrial proteostatic stress and low mitochondrial respiration; however, when the ClpP protease is absent, partial attenuation of the disease and increased longevity of the mice are observed^[130]. In the context of senescence, the palmitic/oleic acid combination induces a senescent phenotype in HepG2 cells characterized by oxidative stress, inflammation, and UPR^mt by increasing hsp90 and hsp60 expression and decreasing clpP, events that are prevented by lipoic acid treatment^[131]. This suggests a relevant role for ClpP in aging, and in the establishment and progression of age-related diseases.

Exercise upregulates fibroblast growth factor 21 (FGF21) in individuals aged 20-60 years, thereby enhancing its cardiometabolic activity. Furthermore, in 26-week-old mice, routine exercise increased Klotho expression through the FGF21/SIRT-3 signaling pathway, thereby reversing mitochondrial dysfunction, oxidative stress, and cardiac fibrosis in mice with diabetic cardiomyopathy^[126]. Indeed, overexpression of SIRT-3 protects mitochondria from post-ischemic brain dysfunction through activation of the UPR^mt via the FOXO3/Sphk1 pathway. Interestingly, inhibition of both pathways alleviates the favorable influence of SIRT-3 on neuronal function and mitochondrial behavior^[132]. Like SIRT-3, SIRT-1 also declines with age; in light of this, 50-68-year-old patients with DCM show significant dysregulation of SIRT-1, but AMP-activated protein kinase (AMPK) phosphorylation is sex-dependent. Oddly, the protein levels of SIRT-3 and HSP60 are lower in male patients with DCM, suggesting that alterations in mitochondrial homeostasis are age-associated and that men with DCM are more susceptible^[133]. Although there are few studies on UPR^mt in aging, it is possible that SIRT-3 and -1 activation may modify this response and positively influence age-associated CVDs^[134]. Figure 4 summarizes the UPR^mt pathways involved in CVDs during aging.

Figure 4. Mechanisms involved in the activation of UPRmt in the development of age-associated CVDs. The progression of cardiovascular disease (CVDs) is accompanied by overproduction of ROS, accumulation of unfolded proteins, changes in mitochondrial dynamics, fibrosis, and cardiomyocyte death. UPR^mt, as a response to mitochondrial damage, has emerged as a therapeutic target for the treatment of age-associated CVDs as it modulates the mitochondrial repair and recovery processes. In heart failure (HF), decreased LonP1, HSP70/60/9, and YME1L proteases have been reported to cause the accumulation of misfolded proteins inside the mitochondria and, thus, the activation of UPR^mt. In acute myocardial infarction (AMI), in addition to being characterized by calcium (Ca²⁺) overload, inflammation, and oxidative stress, a decrease in LonP1 has also been observed, which activates UPR^mt through the p-eIF2α-ATF5 pathway, which induces the latter to enter the nucleus. In dilated cardiomyopathy (DCM), decreased mitochondrial NAD-dependent Sirtuin-3/1 deacetylases (SIRT-3 and -1) and HSP60 activate UPR^mt. In the face of this damage, the implementation of strategies (such as exercise and diet) has successfully mitigated UPR^mt by modulating the response of LonP1, YME1L, and SIRT-3 in elderly patients with CVDs. ROS: Reactive oxygen species; LonP1: Lon1 peptidase; HSP: Heat shock protein; YME1L: yeast mitochondrial escape protein type 1; p-eIF2α: phosphorylation of eukaryotic initiation factor 2 alpha; ATF5: transcription factor-5. Created using BioRender (www.app.biorender.com).

MERCs AS REGULATORY PLATFORMS OF THE UPRmt IN CARDIAC AGING

UPR^mt is a mitochondria-targeted protein modification program that upregulates proteins related to or located in the mitochondria^[120]. However, impaired mitochondrial function and integrity activate UPR^mt, which in turn is regulated by mitochondrial-nucleus communication^[93].

Organelles have a continuous, tight, and functional interaction with each other owing to membrane contact sites (MCSs), a new insight provided by cell biology into organelle function^[17]. Nevertheless, interorganellar contactology is a very complex field, as each of these interactions is facilitated by specific and endogenous tethering and spacer proteins that, at the same time, are multifunctional and not exclusive to MCSs^[135]. They not only create a physical bridge but also serve distinct cellular functions such as lipid transport, Ca²⁺ signaling, and organelle inheritance^[136,137]. Importantly, proteins found within MCSs can exist as single polypeptide chains or complexes of multiple proteins (monomeric, multimeric, or heteromeric). Moreover, they often possess specific organelle membrane-binding or transmembrane domains^[137].

Mitochondria and ER exchange signals to maintain cellular homeostasis through microdomains known as MERCs, usually in the range of 10-80 nm. These platforms differ from mitochondria-associated membranes (MAMs), which are membranes obtained in the laboratory by ultracentrifugation in which proteins that could be part of their machinery can be explored^[31]. MERCs are also involved in the regulation of mitochondrial function and ER stress^[24]; however, they also depend on features such as size, density, and thickness^[20]. For example, in mice transfected with a linker transgene designed for cardiac muscles, an increased number of MERCs was observed. This resulted in the mitochondrial remodeling of large and dense clusters in cardiomyocytes. In addition, vulnerability to acute adrenergic stress, cell death, and mitochondrial Ca²⁺ overload was lower in adult female mice undergoing IR^[135]. Meanwhile, OPA1 deficiency in skeletal muscle promotes increased communication of MERCs in accordance with the increased expression of proteins involved in Ca²⁺ exchange. Simultaneously, OPA1 deficiency increases ATF4-mediated ER stress, indicating that mitochondrial stress regulates MERC formation through different mechanisms, such as the ISR pathway^[20].

MERC dysfunction is closely related to obesity, type II diabetes, CVDs, and aging, which are chronic illnesses characterized by increased oxidative and ER stress^[20,31]. In this regard, β-nicotinamide mononucleotide ameliorated mitochondrial dysfunction and ER stress in the liver of mice on a chronic high-fat diet, which is related to increased MERCs interaction, helped improve intracellular ATP, and mitigated lipid metabolic alterations in these mice^[138]. In Mfn2 knockout oocytes, the upregulation of genes related to ER is linked to abnormal mitochondrial-ER interactions. Although Mfn2 knockout offspring exhibit hyperglycemia, mitochondrial function and ER alterations do not change in the liver or skeletal muscle^[22].

In the hearts of lipopolysaccharide-treated Beclin-1 knockout mice, the function of MAMs decreases. Remarkably, when mice were treated with a peptide activator of this protein, cardiac function improved through the restoration of MAMs^[139]. In DCM, impaired metabolic function and mitochondria-RE interactions have important implications for diastolic dysfunction^[140]. In contrast, HF mice with marked sinoatrial node dysfunction (16 weeks old) exhibit an abnormal mitochondrial structure and an increased distance from MERCs, leading to Ca²⁺ mishandling and mitochondrial dysfunction^[141].

Collectively, we assert that MAM deficiency and UPR^ER perturbation are coupled with diastolic dysfunction in CVDs^[140]; however, our MERCs-UPR^mt-aging triad remains an unsolved crossword puzzle [Figure 5]. In this respect, a couple of studies have led the way in exploring their study. For instance, SIRT-1 is a well-characterized regulator of longevity, but recent findings have shown its enrichment in MERCs and activation of UPR^mt in adipose tissue. Downstream of this response, lipid regulation is triggered in MERCs through cooperation between clusterin and SIRT-1. Noticeably, the expression of hsp9, lonp1, and Hsf1 were increased in MERCs isolated from adipose tissue with Adipo-SIRT-1, whereas clusterin deficiency reduced the expression of these genes in MERCs isolated from adipose tissue of SIRT-1 knockout^[142].

Figure 5. Proteins involved in mitochondrial unfolded protein response (UPR^mt) activation and regulation by MERCs in cardiac aging. Convergence between the endoplasmic reticulum (ER)-mitochondria-nucleus organelles maintains cellular homeostasis. Thus, dysfunction of this communion is intimately associated with the development of cardiovascular disease (CVDs) during aging. In particular, tie proteins (LonP1 and Sirt1) enhance communication between mitochondria-RE contact sites (MERCs), both of which are nuclear-encoded and subsequently translated, identified, distributed, and inserted as anchor proteins between MERCs. Overexpression of Sirt1 and LonP1 increases MERCs and, in parallel, activates UPR^mt. SIRT1 regulates energy homeostasis, promotes mitohormesis, and prevents ER stress, whereas LonP1 decreases mitochondrial fragmentation, activates the unfolded protein response of endoplasmic reticulum unfolded protein response (UPR^RE), maintains mitochondrial integrity, and decreases oxidative damage. Created using BioRender (www.app.biorender.com).

Nevertheless, it was recently demonstrated that LonP1 deficiency in 10-week-old mouse cardiomyocytes promoted alterations in the integrity of MAMs and induced an increased expression of YME1L. Incredibly, UPR^RE activation is more sensitive than UPR^mt activation following the loss of LonP1 in cardiac cells. In addition, the loss of this UPR^mt protease dramatically shortens life expectancy, with a half-life of 60 weeks, owing to cardiac remodeling and dysfunction, leading to DCM and HF. Thus, the authors indicate that, on the one hand, LonP1 functions as an anchoring protein in MAMs and that, in addition, it is indispensable for cardiac function^[4]. Experimental evidence for the involvement of proteins related to UPR^mt and their regulation by MAMs/MERCs in aging should be gathered, which will open a gap for further in-depth studies.

UPRmt-TARGETED STRATEGIES TO RESTORE MITOCHONDRIAL FUNCTION IN AGE-RELATED CVDs

The search for therapeutic strategies targeting mitochondrial quality control, specifically UPR^mt, has become a topic of current interest. To this end, the implementation of different molecules (natural and/or synthetic) that modulate UPR^mt to improve mitochondrial function and reduce cardiomyocyte damage is a novel tool, but little is known about aging. Table 1 lists some examples of molecules that have been studied in cardiac models, specifying their involvement in aging models.

To describe examples of natural compounds, activation of FUNDC1 by oligomycin in IR activates UPR^mt-related genes, such as hsp60, hsp10, lonp1, and clpP, which improves mitochondrial dynamics and alleviates myocardial damage^[120]. In mice with lipopolysaccharide-induced cardiac injury, the activation of ATF5 by salvianolic acid results in its transcription and translocation to the nucleus, activating UPR^mt[143]. Apigenin increases ATF5 and SIRT-1 expression, thereby reducing doxorubicin-induced cardiomyopathy^[144]. Tetrahydrocurcumin (THC), the main metabolite of curcumin, induces cardioprotection in mice with HF by increasing the ATF5/CHOP signaling cascade and the ClpP and LonP1 proteases. Strikingly, inhibiting PGC-1α abrogates the protective effect of THC and the activation of UPR^mt[95]. Meanwhile, treatment of HF mice with Xingang tablet (XYT) promoted upregulation of ATF5 and LonP1, resulting in improved cardiac function and structure. Simultaneously, FUNDC1-regulated mitophagy is initiated, which ameliorates mitochondrial dysfunction in HF mice^[145].

Although not in the heart, these natural compounds have been considered anti-aging agents. For example, salvianolic acid improves mitochondrial function and NAD⁺ metabolism through SIRT1 activation during skin aging^[146] and dementia^[147]. Apigenin reduces skeletal muscle atrophy by increasing mitochondrial function, reducing apoptosis^[148], and upregulating SIRT1 in aged oocytes^[149]. Moreover, baicalein ameliorates aortic structural deterioration in aged rats induced by D-galactose^[150], age-related Parkinson’s disease^[151], and osteoporosis through FOXO-1 signaling^[152]. Its effect on cardiomyocytes and UPR^mt has only been described in palmitic acid-treated AC16 cells, in which increased Nrf2, inhibition of UPR^mt, and alleviation of mitochondrial damage are related to the positive effect of baicalein^[153]. Taken together, these studies suggest that natural compounds can activate UPR^mt and induce homeostasis of mitochondrial function.

Furthermore, after transfection with lentivirus overexpressing C1q-tumor necrosis factor-related protein 3 (CTRP3) in HF mice, mitochondrial function and cardiomyocyte survival improved through the activation of the ATF5/SIRT-1 pathway^[154]. Conversely, downregulation of UPR^mt improved mitochondrial function and morphology in cardiomyocytes subjected to hypoxia/reoxygenation by enhanced therapy with the Chinese medicine Zishen Tongyang Huoxue (TYHX) decoction with β-tubulin stability^[155]. In the same vein, a recent study indicated that in ischemia, the expression of the DNA-dependent protein kinase (DNA-Pkcs) catalytic subunit increased, but SIRT-5 decreased, leading to cardiac tissue damage. However, in mice treated with quercetin, aberrant UPR^mt is arrested by a decrease in DNA-Pkcs^[156]. Finally, other strategies that have been implemented include the use of metformin, which increases ATF5, LonP1, and Hsp60 in spontaneously hypertensive rats, and is associated with improved cardiac function, less fibrosis, and recovery of cellular proteostasis modulated by HSF-1/UPR^mt[157]. Meanwhile, choline treatment ameliorates mitochondrial dysfunction through UPR^mt in cardiac hypertrophy, although negative regulation of SIRT-3 inhibits the protective role of choline by low AMPK activation and thus impairs UPR^mt[158]. Despite the significant advances in UPR^mt as a possible biomarker of CVDs and the compounds that may have an effect on this pathway, more information is needed on what would occur in aged organisms since, as we have described, the effects may be different with respect to young and adult organisms.

CONCLUSION

The present review highlights UPR^mt as a timely and innovative field of study in aging, whose response would allow the maintenance of mitochondrial homeostasis in aging organisms. In the coming years, we expect that scientific research will be directed toward describing the benefits of interorganellar communication in regulating UPR^mt during aging, especially when suffering from chronic diseases such as heart disease. There is also a need to expand our knowledge about the possible association of changes in interorganellar contactology and UPR^mt in the canonical mechanism of multiple therapeutic interventions that have been shown to help slow down the establishment of aging-related diseases and increase the lifespan of organisms.