Abstract

Background

   Age‐related heart diseases are significant contributors to increased
   morbidity and mortality. Emerging evidence indicates that mitochondria
   within cardiomyocytes contribute to age‐related increased reactive
   oxygen species (ROS) generation that plays an essential role in
   aging‐associated cardiac diseases.

Methods and Results

   The present study investigated differences between ROS production in
   cardiomyocytes isolated from adult (6 months) and aged (24 months)
   Fischer 344 rats, and in cardiac tissue of adult (18–65 years) and
   elderly (>65 years) patients with preserved cardiac function.
   Superoxide dismutase inhibitable ferricytochrome c reduction assay
   (1.32±0.63 versus 0.76±0.31 nMol/mg per minute; P=0.001) superoxide and
   H[2]O[2] production, measured as dichlorofluorescein diacetate
   fluorescence (1646±428 versus 699±329, P=0.04), were significantly
   higher in the aged versus adult cardiomyocytes. Similarity in
   age‐related alteration between rats and humans was identified in
   mitochondrial‐electron transport chain‐complex‐I‐associated increased
   oxidative‐stress by MitoSOX fluorescence (53.66±18.58 versus
   22.81±12.60; P=0.03) and in 4‐HNE adduct levels (187.54±54.8 versus
   47.83±16.7 ng/mg protein, P=0.0063), indicative of increased
   peroxidation in the elderly. These differences correlated with changes
   in functional enrichment of genes regulating ROS homeostasis pathways
   in aged human and rat hearts. Functional merged collective network and
   pathway enrichment analysis revealed common genes prioritized in human
   and rat aging‐associated networks that underlay enriched functional
   terms of mitochondrial complex I and common pathways in the aging human
   and rat heart.

Conclusions

   Aging sensitizes mitochondrial and extramitochondrial mechanisms of ROS
   buildup within the heart. Network analysis of the transcriptome
   highlights the critical elements involved with aging‐related ROS
   homeostasis pathways common in rat and human hearts as targets.

   Keywords: cardiac aging, electron transport chain, gene expression,
   oxidative stress, reactive oxygen species

   Subject Categories: Oxidant Stress, Aging, Physiology, Myocardial
   Biology, Metabolism
     __________________________________________________________________

Nonstandard Abbreviations and Acronyms

   AU
          arbitrary units

   ETC
          electron transport chain

   IPA
          Ingenuity Pathway Analysis

   LCM
          low‐calcium medium

   ROS
          Reactive oxygen species

Clinical Perspective

What Is New?

     * Selective downregulation in the activity of electron transport
       chain complex I involved in superoxide generation is the common
       link in reactive oxygen species production in aging rat and human
       myocardium.
     * Network analysis of the transcriptome highlights similar mechanisms
       and pathways involved in aging rats and humans with ERK1/2 linking
       to electron transport chain complex I as a common signaling pathway
       in the aging heart.

What Are the Clinical Implications?

     * Recognizing regulators of reactive oxygen species homeostasis and
       its downstream players altered by aging may identify targets that
       can be selectively intervened on to prevent aging‐associated
       electrical or mechanical myocardial dysfunction, such as atrial
       fibrillation and heart failure, prevalent in the elderly.

   Reactive oxygen species (ROS) such as superoxide anion (
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   ) and hydrogen peroxide (H[2]O[2]) are continuously generated at low
   levels within the cell and play an essential role in various
   physiological processes; during stress they may contribute to cell
   injury and death.[42]^1, [43]^2, [44]^3, [45]^4 An increased ROS
   production rate has been implicated in aging and age‐related changes in
   the mammalian heart, leading to gradual functional impairment,[46]^5,
   [47]^6 including reduced tolerance to stress and cellular energetic
   reserves, and predisposing cells to injury.[48]^7, [49]^8, [50]^9,
   [51]^10 The rate of aging‐associated decline in function varies by
   species as well as by organ within the same species and is influenced
   by genetic and environmental factors[52]^11, [53]^12, [54]^13, [55]^14
   with deleterious consequences, increasing predisposition to ischemic
   heart disease, cardiomyopathy, and cardiac arrhythmias.[56]^6, [57]^12,
   [58]^14 The relative contribution of enhanced production versus reduced
   clearance of ROS within the cytoplasm and mitochondria is not fully
   characterized, and the impact of aging versus disease on these pathways
   in humans is not well defined. To address this, we compared age‐related
   alteration in the sensitivity of myocardial tissue to stress‐related
   alteration in oxidant production in cardiomyocytes from disease‐free
   young and aged Fischer 344 rats, an established model of aging, and in
   human cardiac tissue from adult and aged patients undergoing cardiac
   surgery. We also assessed overall change in the expression of genes
   encoding for pathways of ROS production and clearance in young and aged
   rat hearts and in young and aged patients undergoing cardiac surgery.
   From aged rat and human hearts, we identified shared networks and nodes
   that are important for regulating oxidative stress and that are altered
   with aging in both rats and humans. This information provides novel
   insight into potential targets involved in the responsiveness of the
   elderly heart to stress that could be manipulated to prevent
   aging‐associated deterioration in heart function.[59]^15

METHODS

Human Sample Selection

   Atrial appendage tissue was harvested from adult (18–65 years) and aged
   (>65 years) patients undergoing elective open‐heart surgery for
   coronary artery disease, and tissue from those who met inclusion and
   exclusion criteria was used for the analysis of the study. The baseline
   clinical characteristics of these patients is summarized in
   Table [60]S1. Patients with congenital, structural, or functional
   atrial disease—including persistent or permanent atrial fibrillation—or
   class III/IV heart failure, those requiring inotropic support, and
   those with moderate/severe mitral valve disease were excluded. The
   research study was conducted following the Declaration of Helsinki for
   experiments relating to humans. The study was approved by the Aurora
   Health Care Institutional Review Board for human subject research,
   written informed consent was obtained from all subjects, and all
   patients' privacy rights were observed. The data that support the
   findings of this study are available from the corresponding author upon
   reasonable request.

Processing of Human Cardiac Samples

   After clamping and removal of the atrial appendage, the tissue was
   immediately transferred into ice‐cold Dulbecco's phosphate‐buffered
   saline. Fat and connective tissue were removed from the biospecimen,
   and the muscle tissue was freshly used for myofiber isolation or frozen
   in liquid nitrogen (N2) and stored at −80°C for future use. A total of
   47 patients' samples were used in this research for the various
   expressional and functional studies (OXPHOS functional analysis, 4‐HNE
   protein adducts, western blot, or genetic analysis). Tissue was
   collected immediately after removal from the patient and processed
   within 10 to 15 minutes. If any delay occurred in collection or
   storage, the tissue was not used for the study.

Experimental Animals and Design

   Animal experiments were approved by the Institutional Animal Care and
   Use Committee of the Mayo Clinic College of Medicine and conformed to
   the National Institutes of Health Guide for the Care and Use of
   Laboratory Animals (NIH Pub. No. 85‐23, 1996). The young adult
   (6 months old) and aged (24 months old) male Fischer 344 rats were used
   for our aging‐related studies.[61]^16, [62]^17, [63]^18 Young and aged
   rats were acquired from the National Institute on Aging (NIA)
   colonies[64]^19 and were housed in a temperature‐controlled room
   (22–23°C) with a 12:12‐hour light‐dark cycle and fed with tap water and
   Purina Laboratory Chow (No. 5001) ad libitum. Rats were euthanized with
   intraperitoneal sodium pentobarbital (50 mg/kg) before thoracotomy and
   heart dissection. In a set of parallel experiments in young adult and
   aged rats, we determined (1)
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   generation by the mitochondria isolated from ventricles and (2)
   steady‐state and ouabain‐stressed H[2]O[2] production in isolated
   cardiomyocytes.

Isolation of Cardiomyocytes From Rat Hearts

   Cardiomyocytes were isolated from the hearts of young adult (n=5) and
   aged (n=5) rats, as described previously.[65]^20 In brief, the
   procedure comprises three main steps: (1) dissection and sequential
   retrograde perfusion of the heart at 37°C, firstly, with Tyrode's
   buffer (137 mmol/L NaCl, 10 mmol/L glucose, 10 mmol/L HEPES, 5.4 mmol/L
   KCl, 1 mmol/L MgCl[2], and 2 mmol/L CaCl[2]) for 10 minutes; secondly,
   with a “low‐calcium” medium (LCM) containing 100 mmol/L NaCl, 50 mmol/L
   taurine, 20 mmol/L glucose, 10 mmol/L KCl, 10 mmol/L HEPES, 5 mmol/L
   MgSO[4], and 1.2 mmol/L KH[2]PO[4] and supplemented with 0.13 mmol/L
   CaCl[2] and 2.1 mmol/L EGTA for 2 minutes; and finally, with LCM
   supplemented with 1% bovine serum albumin (BSA), 0.2 mmol/L CaCl[2],
   collagenase (type II, 22 U/mL; Worthington) and pronase (100 µg/mL;
   Serva) for 15 minutes; (2) removal of atria and mincing of the
   ventricular tissue into small pieces before incubation in the enzyme
   solution with gentle stirring at 37°C for 15 minutes; (3) isolation of
   intact cardiomyocytes by serial centrifugation and washing of the
   pellet in LCM supplemented with 0.2 mmol/L CaCl[2] (wash) followed by
   suspension in the wash solution.[66]^21

Isolation of Mitochondria From the Heart

   Mitochondria were isolated from the myocardium of young adult (n=5) and
   aged (n=5) rats as previously described.[67]^22 In summary, ventricles
   were excised from the rat heart, sliced into fine pieces, suspended in
   an isolation buffer containing (in millimoles) sucrose 50, mannitol 20,
   KH[2]PO[4] 5, EGTA 1, and Mops 5 (pH 7.3, adjusted with KOH) with 0.2%
   BSA, and then homogenized to isolate mitochondria. The mitochondrial
   fraction was obtained by differential centrifugation, washed, and
   suspended in isolation buffer free of EGTA and BSA and kept on ice for
   the experiment.[68]^6

Measurement of
[MATH: <msubsup><mi
mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow></msubs
up> :MATH]
Generation by the Mitochondria

   The rate of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   generation was measured as superoxide dismutase (SOD)‐inhibitable
   reduction of acetylated ferricytochrome c. The reaction mixture
   contained 0.1 mol/L potassium phosphate buffer (pH 7.4), 7.2 µmol/L
   acetylated cytochrome c, 2 µmol/L antimycin A, 6 µmol/L rotenone,
   10 mmol/L succinate, and 20 to 30 µg/mL mitochondrial protein with
   glutamate/malate as substrates; 100 units of SOD/mL was added to the
   reference cuvette. The cytochrome c reduction was recorded at 37°C for
   10 minutes by monitoring absorbance at 550 to 540 nm in the presence or
   absence of SOD. The increase in absorbance at 550 to 540 nm is caused
   by the formation of stoichiometric ferrous cytochrome c. The production
   of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   was estimated with the extinction coefficient of 19.0 mM^−1·cm^−1 and
   expressed as a mean of multiple experiments.[69]^23, [70]^24, [71]^25

H[2]O[2] Production Within Isolated Cardiomyocytes

   H[2]O[2] production was estimated using ROS‐sensitive fluorophore, 2′,
   7′‐dichlorofluorescein diacetate (DCFDA) (Molecular Probes, Eugene,
   OR). The freshly isolated cardiomyocytes were placed in collagen‐coated
   glass Petri dishes (Mat Tek Corp., Ashland, MA) at room temperature.
   The cells were incubated for 45 minutes with DCF at 37°C. The DCFDA
   solution was removed, and the cells were washed with serum‐free medium.
   Fluorescence intensity of DCF was measured at 585 nm emission when
   excited at 488 nm by an LSM 510 laser‐scanning confocal microscope.
   Confocal microscopic images were acquired every minute from 0 to
   10 minutes using incorporated software (Carl Zeiss Inc., Thornwood,
   NY). Cardiomyocytes were then stressed with 30, 100, and 300 μmol/L
   ouabain (Sigma) or vehicle (DMSO 1%) for 15 minutes at room
   temperature. Following initial ouabain exposure, cardiomyocytes were
   loaded with the H[2]O[2]‐selective dye DCFDA (4 μmol/L; Molecular
   Probes) for 15 minutes to detect ROS generation. Confocal images were
   acquired with an LSM 510 laser scanning confocal microscope using
   incorporated software. The single‐excitation, single‐emission,
   fluorescent probe DCFDA was excited using the 568 nm line of the Ar/Kr
   laser, and emitted fluorescence was filtered through long‐pass filter
   settings (LP 585). Confocal microscopic images were acquired every
   minute from 0 to 10 minutes after treatment with ouabain, and data were
   analyzed using LSM 400 software (Zeiss).

Determination of 4‐Hydroxynonenal Protein Adducts

   An OxiSelect HNE adduct competitive ELISA commercial kit (Cat #STA‐838,
   Cell Biolabs, INC, San Diego, CA) was used to measure the level of
   4‐HNE in cardiac tissue homogenate. Human cardiac tissue (n=41)
   homogenate prepared in ice‐cold phosphate‐buffered saline with 0.1% BSA
   was centrifuged for 10 minutes at 10 000g, and the 4‐hydroxynonenal
   (4‐HNE) level was assessed in the supernatant as per the kit's
   guidelines.

Mitochondrial Superoxide Production in Permeabilized Human Cardiac Myofibers

   Superoxide production in the cardiac myofibers was determined as
   previously described.[72]^6 Atrial appendage tissue soaked in ice‐cold
   buffer containing (in mmol/L) 7.23 K[2]EGTA, 2.77 CaK[2]EGTA, 20
   imidazole, 20 taurine, 5.7 ATP, 14.3 phosphocreatine, 6.56
   MgCl[2]·6H[2]O, and 50 MES (pH 7.1) was cut into small strips and
   incubated with collagenase type I for 30 to 45 minutes at 4°C.
   Connective tissue and fat were then removed, and the fibers
   mechanically separated along the longitudinal axis and permeabilized
   with saponin (30–50 μg/mL) for 30 minutes at 4°C. Following
   permeabilization, myofibers were washed in an ice‐cold buffer
   containing (in mmol/L) 110 K‐MES (pH 7.4), 35 KCl, 1 EGTA, 5 KH[2]PO4,
   3 MgCl[2]·6H[2]O, and 0.02 blebbistatin with 5 mg/mL BSA. The myofibers
   remained in the ice‐cold buffer, supplemented with 100 μmol/L ADP,
   5 mmol/L glucose, and 1 U/mL hexokinase to keep mitochondria in an
   energized and phosphorylating state,[73]^26 until used for measuring
   mitochondrial superoxide production. The level of superoxide production
   in the myofibers was determined as a change in fluorescence intensity
   of MitoSOX Red (λex/λem=510/580; Cat #[74]M36008, Thermo Fisher
   Scientific, Waltham, MA), a mitochondrial superoxide‐sensitive
   indicator, in response to 10 μmol/L antimycin A (Cat #A8674,
   Sigma‐Aldrich, St. Louis, MO)[75]^6, [76]^27 exposure by time‐lapse
   laser scanning confocal fluorescence microscopy. Images were detected
   in an Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan). The
   change in MitoSOX Red fluorescence intensity before and after antimycin
   A addition was quantified to measure the difference in the superoxide
   production.[77]^6, [78]^27

Mitochondrial Complex (I–V) Enzymatic Activity

   Frozen human cardiac tissue sample (30–50 mg) was homogenized in an
   ice‐cold buffer (1:20 w/v) containing (in mmol/L) 100 KCl, 5 MgCl[2], 2
   EGTA, and 50 Tris/HCl (pH 7.5), using an OMNI GLH‐115 Polytron
   homogenizer (OMNI International). The supernatant collected after
   centrifuging the homogenates at 1000g for 15 minutes at 4°C was used to
   measure the functional activity of mitochondrial electron transport
   chain (ETC) complexes as reported earlier.[79]^6, [80]^14

RNA Extraction

   Freshly isolated human or rat cardiac tissue was snap‐frozen in liquid
   nitrogen before storage at −80°C until subsequent processing for gene
   expression analysis by microarray or by reverse transcription (q)
   polymerase chain reaction (PCR). Frozen human or rat tissue was
   homogenized in TRIzol (Invitrogen Corp., Carlsbad, CA) using a PELLET
   PESTLE motor homogenizer (Kimble‐Kontes, Vineland, NJ). Total RNA was
   extracted using a TRIzol Reagent kit according to the manufacturer's
   specifications.[81]^6, [82]^8 Further purification was carried out
   using RNeasy Mini Kit (QIAGEN Inc., Valencia, CA). RNA concentrations
   were determined using the Infinite 200 NanoQuant (Tecan Group Ltd.,
   Männedorf, Switzerland), and quality/integrity was assessed by Agilent
   2100 Bioanalyzer (Affymetrix, Santa Clara, CA).

Reverse Transcription (q) PCR Assay of Target mRNA

   Equal amounts of RNA isolated from each human cardiac tissue sample,
   n=7 adults (age ≤65 years) and n=10 aged (age >65 years), were
   reverse‐transcribed to cDNA using a miScript RT II kit (Qiagen).
   Quantitative PCRs were performed in an LC480 real‐time PCR system
   (Roche, Risch‐Rotkreuz, Switzerland) using SYBR Green PCR Master Mix
   (Thermo Fisher Scientific). Details of gene‐specific primer sequences
   used in this study are listed in Table [83]S2. The qPCR reactions were
   performed in triplicate for all samples. Relative mRNA expression (∆Cq)
   normalized to reference B2M and 18s RNA expression were determined
   using LightCycler 480 Software.

Western Blot Analysis

   Frozen human cardiac tissue (n=5 adult and n=5 aged) was homogenized in
   RIPA lysis buffer (Abcam, Cambridge, MA) with protease inhibitor
   cocktail freshly added (Millipore Sigma). Protein lysates (15 µg) were
   separated on NuPAGE precast 4% to 12% Bis‐Tris gel (Thermo Fisher
   Scientific) and transferred to polyvinylidene difluoride membranes
   (Thermo Fisher Scientific). Western blotting was performed using
   primary anti‐SOD1 (Cat #ab13498; Abcam), anti‐SOD2 (Cat #ab13194;
   Abcam), and anti‐β‐tubulin (Cat #2148; Cell Signaling).

Bioinformatics: Functional Enrichment Analysis and Gene Target Prioritization

   The Gene Ontology (GO) consortium website
   ([84]http://geneontology.org), via its AmiGO 2 search browser, was used
   to determine all genes associated with ROS metabolic processes in
   humans (website accessed February 29, 2020). Venn diagrams were used to
   intersect this ROS list with our previously published datasets from
   human ([85]GSE173608) and rat ([86]GSE173360) aged hearts to identify
   ROS‐associated candidates.

   Functional deconvolution of mitochondrial‐related and
   extramitochondrial genes involved in ROS metabolism from human and rat
   aged hearts was performed via Ingenuity Pathway Analysis (IPA; Qiagen,
   Germantown, MD; last accessed for analysis June 16, 2020). IPA was used
   to determine prioritized canonical pathways, pathway activity analysis
   (prediction of functional pathways), and mapping of functional gene
   networks defined by the differentially expressed genes in aged human
   and rat hearts. The significance of canonical functional pathways,
   calculated by Benjamini‐Hochberg multiple testing correction (corrected
   P value=P [Corr]), indicates the probability of association between a
   gene/target and a specific functional pathway. Pathway activity
   analysis determines whether a canonical pathway is activated
   (increased) or inhibited (decreased), predicted by a z‐score algorithm
   used to compare the uploaded gene list with the canonical pathway
   patterns and displayed by colored bar charts indicating their
   functional pathway activation z‐scores. The highest priority network
   scores were determined, and the Molecule Activity Predictor Analysis
   (IPA module) was used to predict activation or inhibition of
   non‐focused neighboring molecules within the prioritized functional
   network in human and rat datasets. All gene interactions from networks,
   prioritized, and merged networks were exported from IPA for use in
   Cytoscape (v3.7.1; [87]https://cytoscape.org/) for further network
   analysis (last accessed for analysis June 15, 2020). Prioritization of
   gene targets was achieved through graph theory analysis tools (network
   metrics: neighborhood connectivity, betweenness, and closeness
   centralities) within Cytoscape.

Statistical Analysis

   The statistical analysis was performed using SAS software (version 9.2,
   SAS Institute, Cary, NC) and GraphPad Prism (version 8, GraphPad
   Software, San Diego, CA). The differences between adult and aged rats
   were assessed by Student t test. Comparison of the aged and adult
   humans was performed using the t test for normally distributed
   variables or the nonparametric Kruskal‐Wallis test for the variables
   that deviate from the normal distribution. Mean±standard deviation was
   applied to describe quantitative variables, and percentages were used
   for qualitative variables. The Kolmogorov‐Smirnov test was used to
   evaluate the normality of continuous variable distribution. Simple
   linear regression was performed to investigate the relationship between
   variables. The number of patient samples used for each experiment is
   detailed in Table [88]S3. Bioinformatics analysis for canonical
   functional pathways significance was calculated using false discovery
   rate, Benjamini‐Hochberg multiple testing correction (corrected P
   value=P [Corr]). Probability value (P) and P (Corr) <0.05 were
   considered significant. The rates of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   and H[2]O[2] production were expressed as the mean and SEM.

RESULTS

Aging Alters the Functional Enrichment of Genes Involved in ROS Metabolism
and Depicts Mitochondrial Function and Oxidative Phosphorylation as the Most
Prioritized Pathways

   Our previous work showed gene expression changes in
   mitochondrial‐related genes in both human and rat aging hearts compared
   to adult hearts.[89]^8, [90]^14 Some of these genes coding for
   mitochondrial localized proteins are involved in ROS metabolism,
   including ROS production and clearance. In both rat and human heart
   aging, associated changes in mitochondrial and extramitochondrial
   pathways for ROS production and clearance were observed. These changes
   were predominantly involved in oxidative stress pathways and
   significantly reduced expression of subunits involved in electron
   transport chain complex I. The species‐independent alteration in genes
   common to both rats and humans with aging is summarized in
   Table [91]S4.

   Statistically significant genes from this dataset were intersected with
   ROS metabolism genes (n=285) extracted from the GO consortium website
   ([92]www.geneontology.org/docs/go‐consortium) to identify the
   ROS‐associated changes in the aging heart (Figure [93]1A and 1B,
   Table [94]S4). In human myocardium, out of the 832 differentially
   expressed genes in the aged versus adult heart, 35 genes (4.2%) were
   involved in ROS homeostasis, with 27 (77%) downregulated and 8 (23%)
   upregulated. These 35 differentially expressed genes in human
   myocardium are 14% of the total ROS‐associated genes present within the
   Gene Ontology consortium (Figure [95]1A). Similarly, in the aged rat
   heart, there were 916 differentially expressed genes, out of which 47
   (5.1%) were involved in ROS metabolism; 39 of these genes (83%) were
   downregulated and 8 (17%) upregulated. Out of the total 250
   ROS‐metabolism‐associated genes reported in the Gene Ontology
   consortium, 47 (18.8%) genes were differentially expressed in the aged
   rat heart (Figure [96]1B). In both aged human and rat datasets, the
   majority of genes involved in ROS homeostasis were downregulated.
   Functional pathway enrichment analysis from IPA revealed that
   “mitochondrial dysfunction,” “oxidative phosphorylation,” and “sirtuin
   signaling pathway” were the three prioritized functions in the human
   and rat aging heart datasets (Figure [97]1C and 1D). The human dataset
   exposed three significant canonical pathways (“mitochondrial
   dysfunction,” “oxidative phosphorylation,” and “sirtuin signaling
   pathway”; Figure [98]1C). The pathway activity analysis module from IPA
   predicted a decrease in the activity of the “oxidative phosphorylation”
   pathway (−log P [Corr]=9.85; Figure [99]1C), whereas the “sirtuin
   signaling pathway” was predicted to be increased (−log P [Corr]=9.54;
   Figure [100]1C). However, IPA was not able to discern a significant
   predictive pattern for “mitochondrial dysfunction” (−log P
   [Corr]=13.16; Figure [101]1C). In the aging rat heart,
   ROS‐pathway‐associated genes enriched distinctive and significant
   functional categories (Figure [102]1D). The top three canonical
   pathways, and the pathway activity analysis, matched those identified
   in the aged human heart, however, with much higher statistical
   significance: “mitochondrial dysfunction” (−log P [Corr]=61.50;
   Figure [103]1D), “oxidative phosphorylation” (−log P [Corr]=58.14;
   Figure [104]1D), and “sirtuin signaling pathway” (−log P [Corr]=20.04;
   Figure [105]1D). Furthermore, “glutathione‐mediated detox” and “apelin
   adipocyte signaling pathway” showed a predicted decrease in pathway
   activity (−log P [Corr]=5.25, and −log P [Corr]=5.23, respectively;
   Figure [106]1D). Six other functional pathways, namely “NRF2‐mediated
   oxidative stress response,” “glutathione redox reaction 1,” “superoxide
   radical degradation,” “aryl hydrocarbon receptor signaling,”
   “LPS/IL‐1‐mediated inhibitor RXR function,” and “TCA cycle II” were
   significantly altered but showed a neutral z‐score of zero or close to
   zero, meaning the activity of the pathways was predicted to neither be
   increased nor decreased.

Figure 1. ROS‐associated functional pathways changes in aging human and
Fischer 344 rat hearts.

   Figure 1
   [107]Open in a new tab

   A, Out of the 832 differentially expressed genes (DEG) in aged compared
   to adult hearts, 35 genes (4.2%) were associated with ROS metabolism,
   with 27 genes downregulated and 8 upregulated in the human aged heart.
   B, In the aging rat heart, out of 916 DEGs we found 47 (5.1%)
   ROS‐metabolism‐associated genes (39 downregulated, 8 upregulated). C,
   Functional and pathway analysis exposed three significant canonical
   pathways enriched in the human dataset: “mitochondrial dysfunction”
   (white/gray pattern=ineligible of pathway prediction), “oxidative
   phosphorylation” (blue=predicted decrease in activity of pathway), and
   “sirtuin signaling pathways” (orange=predicted increase in activity).
   Threshold was set at P [Corr] <0.05 (green line). D,
   ROS‐pathway‐associated genes from aged rats enriched distinctive and
   significant functional categories that were similar in rats and humans
   for the top three canonical pathways, with stronger significance
   reported for the former. Furthermore, “glutathione‐mediated detox” and
   “apelin adipocyte signaling pathway” also predicted decrease in
   activity (blue bars). Six other functional pathways were significantly
   enriched; however, they showed neutral predicted activity. (For
   details, see [108]Methods and Materials). IPA indicates Ingenuity
   Pathways Analysis; P [Corr], Corrected P value including multiple
   testing correction (Benjamini Hochberg false discovery rate); and ROS,
   reactive oxygen species.

   Further functional pathway activity analysis was used to determine
   predictive activity patterns of categorical functions associated with
   the “free radical scavenging” function in both the human and rat
   datasets ([109]Table). The human dataset identified four functions
   associated with metabolism, synthesis, and generation of ROS that
   showed a positive z‐score trend but no significant predictive activity
   (P [Corr] <0.01, z‐score >0.5; [110]Table). In comparison to the human
   dataset, the rat dataset did elicit predictive activity on some
   functions. The following functions were predicted to have a significant
   increase in activity within the rat dataset: “production of reactive
   oxygen species” (COX8A, CYBB, NDUFS1, SOD1, SOD2, TXN2, UQCRFS1, and
   XDH), “production of superoxide,” with five genes associated with this
   functional term (CYBB, SOD1, SOD2, TXN2, and XDH), and “synthesis of
   reactive oxygen species” (COX5B, COX6A2, COX8A, CYBB, NDUFS1, SDHC,
   SOD1, SOD2, TXN2, UQCRFS1, and XDH; P [Corr] <0.0001, z‐score >2.0;
   [111]Table). Moreover, the other five terms related to metabolism and
   generation of ROS elicited z‐scores above 1 but did not reach the
   threshold for activity prediction ([112]Table).

Table 1.

   Predicted Activation and Inhibition of Categorical Annotations Within
   the “Free Radical Scavenging” Function
   Free Radical Scavenging Function P [Corr] z‐Score Gene Associated With
   Function
   Human ROS
   Metabolism of ROS <0.0001 0.864 ATG5, ATP7A, BNIP3, CAV1, CYB5R4,
   FOXO3, PINK1, UQCRC2
   Synthesis of ROS 0.0006 0.864 ATG5, BNIP3, CAV1, FOXO3, PINK1, UQCRC2
   Generation of ROS 0.0007 0.555 BNIP3, CAV1, PINK1, UQCRC2
   Production of ROS 0.0075 0.507 ATG5, CAV1, FOXO3, PINK1
   Generation of hydrogen peroxide 0.0014 … CAV1, PINK1
   Scavenging of hydrogen peroxide 0.0030 … FOXO3
   Quantity of ROS 0.0069 … ATG5, FOXO3, NNT
   Rat ROS
   Production of ROS <0.0001 2.205* COX8A, CYBB, NDUFS1, SOD1, SOD2, TXN2,
   UQCRFS1, XDH
   Production of superoxide <0.0001 2.190* CYBB, SOD1, SOD2, TXN2, XDH
   Synthesis of ROS <0.0001 2.128* COX5B, COX6A2, COX8A, CYBB, NDUFS1,
   SDHC, SOD1, SOD2, TXN2, UQCRFS1, XDH
   Formation of ROS <0.0001 1.969 COX6A2, CYBB, SOD2, XDH
   Quantity of ROS <0.0001 1.948 CYBB, GSTA1, PRDX5, SOD1, SOD2, TXN2, XDH
   Quantity of superoxide <0.0001 1.447 CYBB, SOD1, SOD2, TXN2, XDH
   Metabolism of ROS <0.0001 1.186 COX5B, COX6A2, COX8A, CYBB, GPX3,
   GSTA4, NDUFS1, PRDX5, SDHC, SOD1, SOD2, TXN2, UQCRFS1, XDH
   Generation of superoxide <0.0001 1.109 CYBB, SOD1, SOD2, XDH
   Generation of ROS <0.0001 0.906 COX5B, CYBB, SDHC, SOD1, SOD2, UQCRFS1,
   XDH
   Quantity of hydrogen peroxide <0.0001 0.762 PRDX5, SOD1, SOD2, TXN2
   Accumulation of ROS <0.0001 0.600 CYBB, NDUFS1, PRDX5, SDHC, SOD1, SOD2
   Metabolism of hydrogen peroxide <0.0001 −1.955 CYBB, GPX3, PRDX5, SOD1,
   SOD2, UQCRFS1
   Modification of ROS <0.0001 −1.966 GSTA4, PRDX5, SOD1, SOD2
   Accumulation of hydrogen peroxide <0.0001 … NDUFS1, PRDX5, SOD1, SOD2
   Detoxification of ROS <0.0001 … GSTA4, SOD1, SOD2
   Modification of hydrogen peroxide <0.0001 … PRDX5, SOD1, SOD2
   Conversion of superoxide <0.0001 … SOD1, SOD2
   Metabolism of superoxide <0.0001 … CYBB, SOD1, SOD2
   Accumulation of superoxide <0.0001 … NDUFS1, SOD2
   Reduction of superoxide <0.0001 … SOD1, SOD2
   Degradation of hydrogen peroxide <0.0001 … GPX3, PRDX5, SOD1
   Conversion of hydrogen peroxide <0.0001 … SOD1, SOD2
   Biosynthesis of hydrogen peroxide 0.0001 … CYBB, SOD1, SOD2, UQCRFS1
   Removal of superoxide 0.0003 … SOD1, SOD2
   Formation of superoxide 0.0005 … CYBB, SOD2
   Quantity of lipid peroxide 0.0007 … SOD1, XDH
   Catabolism of hydrogen peroxide 0.0009 … GPX3, PRDX5
   Peroxidation of superoxide 0.0020 … SOD1
   Hyperproduction of superoxide 0.0020 … CYBB
   Generation of hydrogen peroxide 0.0026 … SOD1, UQCRFS1
   Breakdown of hydrogen peroxide 0.0040 … SOD1
   Detoxification of monohydroperoxy‐linoleic acid 0.0040 … GSTA4
   Reduction of monohydroperoxy‐linoleic acid 0.0061 … GSTA4
   Clearance of ROS 0.0121 … SOD2
   Production of hydrogen peroxide 0.0128 … SOD1, SOD2
   Reduction of hydrogen peroxide 0.0201 … PRDX5
   [113]Open in a new tab

   Using the Pathway Activity Analysis module from Ingenuity Pathways
   Analysis (IPA), we depict here the functional annotations with inferred
   activation (z‐score >0) or inhibition (z‐score <0) metrics along with
   corrected P value (P [Corr]) and gene names of molecules associated
   with each functional annotation. Both rat and human cardiac
   transcriptomes were used for this analysis. The human ROS dataset
   depicted four functions associated with generation and/or accumulation
   of ROS which showed a positive z‐score trend, however, no significant
   predictive activity. In the rat ROS dataset, three functions were
   predicted to be activated in the rat ROS list: production of ROS,
   production of superoxide and synthesis of ROS (bold and z‐scores
   highlighted with *). Moreover, eight other functions associated with
   generation and/or accumulation of ROS also had a positive z‐score and
   two functions associated with ROS clearance and metabolism had a
   negative z‐score, but they did not reach significant predictive
   threshold. P (Corr)=corrected P value with Benjamini‐Hochberg false
   discovery rate. ROS indicates reactive oxygen species.

Aging Is Associated With Alteration of Gene Expression Encoding for
Mitochondrial ROS Production and Clearance Pathways

   Figure [114]2 summarizes the statistically significant alteration in
   the expression of genes encoding for proteins involved in ROS
   production and clearance pathways in human (Figure [115]2A) and rat
   hearts (Figure [116]2B). The significant affect associated with the
   aged human heart was seen on complex I of the ETC, involved in the
   production of ROS, with downregulation of five subunits (NDUFA6,
   NDUFA9, NDUFB5, NDUFAB8, and NDUFS2) and upregulation of two complex
   assembly factors (NDUFAF5 and NDUFAF6) (Figure [117]2A). GLRX2, GLRX3,
   and PRDX4, which are involved in the clearance of ROS, were
   downregulated in the human aged heart, whereas TXNDC15 was upregulated.
   A similar change was seen in the aged rat hearts, with complex I having
   the majority of changes (13 genes downregulated; Figure [118]2B). Of
   note, some common genes were altered in both humans and rats, while
   other genes were unique to the rat myocardium (Figure [119]2A and 2B;
   Table [120]S4). Both NOX2 (1.6‐fold; P=0.016) and XDH (1.4‐fold;
   P=0.027; Table [121]S4), two major enzymes involved in
   extramitochondrial superoxide production, were significantly
   upregulated in the rat heart but not significantly different between
   the adult and aged human myocardium. Table [122]S4 summarizes the
   information on the differentially expressed subunits of the
   mitochondrial ETC as well as extramitochondrial ROS production and
   clearance and other ROS metabolism pathways along with fold change and
   their P values. In the mitochondrial ROS clearance pathway, significant
   differences were present between rat and human aging myocardium: in rat
   myocardium, GPX3 (1.6‐fold; P<0.005), GTSA1 (2.6‐fold; P<0.001), PRDX4
   (1.3‐fold; P=0.028), and TXNRD3 (2.2‐fold; P<0.001) were upregulated
   and others (PRDX5, SOD1, SOD2, TXN2) were downregulated; in human
   myocardium, only downregulation of the genes involved in mitochondrial
   ROS clearance was observed (Figure [123]2A and 2B; Table [124]S4).
   Differences also were present between rat and human hearts in their
   extramitochondrial ROS generation and clearance pathways.

Figure 2. Differentially expressed genes associated with mitochondrial and
extramitochondrial ROS metabolism pathways.

   Figure 2
   [125]Open in a new tab

   Transcriptomic changes in genes associated with ROS metabolism (ROS
   production and clearance) in aged human and rat hearts compared to
   adults (detail annotations found in Table [126]S4). Shown here is a
   schematic representation of ROS production and clearance pathways,
   highlighting the genes in (A) aged human atria and (B) aged rat
   myocardium that are significantly upregulated (red) and downregulated
   (green) when compared to adult hearts. Genes that show no change in
   expression are depicted in gray. C, Functional activities of
   mitochondrial complexes as an indicator of oxidative phosphorylation in
   human atrial tissue of adult (age 18–65 years) and elderly (aged
   >65 years) patients with well‐preserved atrial and ventricular function
   who underwent open heart surgery. Data are expressed as nmol/min/mg
   protein and presented as mean±standard deviation. Complex I (n=11 for
   adults and n=25 for elderly), Complex II (n=9 for adults and n=25 for
   elderly), Complex III (n=5 for adults and n=10 for elderly), Complex IV
   (n=12 for adults and n=10 for elderly), and Complex V (n=11 for adults
   and n=25 for elderly) comparison of the adult and elderly groups was
   performed using t test, with significance set at P<0.05. ROS indicates
   reactive oxygen species.

   To correlate changes in the gene expression of proteins involved in
   mitochondrial ROS production, activities of individual ETC complexes
   were determined in myocardial tissue from adult and aged patients
   (Figure [127]2C). ETC complex I activity, as determined by
   rotenone‐sensitive reduction of ubiquinone‐1, was significantly reduced
   in the elderly compared to adult (73.61±33.02 versus
   111.4±42.76 nmol/min per mg protein, respectively, P=0.039;
   Figure [128]2C), similar to previously reported findings.[129]^8,
   [130]^14 The functional activities of the remaining four OXPHOS
   complexes in humans were not significantly altered by age (complex II,
   P=0.2672; complex III, P=0.6216; complex IV, P=0.5216; complex V,
   P=0.3912; Figure [131]2C), a finding similar to that in aged rat and
   human myocardium.[132]^8, [133]^14

Age‐Dependent Changes in Expression of Selected Genes Involved in ROS
Metabolism in the Human Heart

   ROS metabolism‐associated genes in the aged versus adult human heart
   showed that most of the genes that are differentially expressed with
   aging are localized to the mitochondria (Table [134]S4). To define the
   effect of age on the mRNA expression of selected ROS production and
   clearance pathway genes in the human heart, qPCR was used to analyze
   myocardial tissue from patients ages 45 to 83 years who underwent
   open‐heart surgery. The mitochondrial complex I subunit NDUFA6, which
   was significantly reduced in both aged human and rat hearts in
   microarray analysis, demonstrated a highly significant inverse
   correlation (R ^2=0.4059; P=0.008) in expression with advancing age
   (Figure [135]3A). In individuals older than 65 years, the total mRNA
   expression of mitochondrial complex I subunit NDUFA6 was reduced
   −0.6‐fold compared to the younger age group (Figure [136]3A inset).
   NADPH oxidase 2 (NOX2), a major extramitochondrial source for ROS
   production, showed no significant alteration between those older or
   younger than 65 years (Figure [137]3B). For the ROS clearance pathway,
   the expression of TXN2, a redox‐active protein that regulates ROS
   levels, showed an inverse relationship with advancing age (R ^2=0.3041;
   P=0.03) (Figure [138]3C) with the levels reduced −0.82‐fold in those
   65 years or older compared to younger individuals (Figure [139]3C
   inset). In contrast, TXNRD3, which was significantly downregulated in
   rat hearts (Table [140]S4), was not significantly altered in the hearts
   of young and old humans (Figure [141]3D).

Figure 3. Assessment of expression of selected genes in human adult and aging
myocardium.

   Figure 3
   [142]Open in a new tab

   Depicted are mRNA expression levels of selected genes associated with
   ROS metabolism in human adult (age 18–65 years, n=7) and aging (age
   >65 years, n=10) myocardium. A, Scatter plot examining age‐dependent
   correlation of age and mRNA gene expression reveals significant inverse
   correlation in age vs NDUFA6 (R ^2=0.41), with total mRNA expression
   values from adult and elderly patients. Bars highlighting distribution
   of individual gene expression values along with mean±standard deviation
   values for each age group (inset). B, Inverse correlation in age vs
   NOX2 mRNA expression (R ^2=0.005). Total mRNA expression shown in
   inset. C, TXN2 (R ^2=0.304) shows significant inverse correlation with
   age, with significant total mRNA downregulation in elderly patients
   compared to adults (inset). D, No significant correlation was seen in
   age vs TXNRD3 (R ^2=0.08); however, an upward trend is observed. Data
   were analyzed by regression analysis of the sample population (n=17),
   *P<0.05.

Aging Increases Superoxide (
[MATH: <msubsup><mi
mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow></msubs
up> :MATH]
) Production in Mitochondria from the Rat Heart

   Mean
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   production in isolated mitochondria from the myocardium of aged rats
   was 1.7‐fold greater than that in young adult rats. The production of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   was 1.32±0.63 nmol/mg protein/min in aged rat hearts (n=5) compared to
   0.76±0.31 nmol/mg protein/min in young adult rat hearts (n=5, P=0.001;
   Figure [143]4A). Thus, under similar experimental conditions,
   mitochondria from aged hearts produced 70% more
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   as determined by the SOD‐inhibitable cytochrome c reductase assay that
   measures mean
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   production (Figure [144]4A).

Figure 4. Rate of suppressible superoxide (
[MATH: <msubsup><mi
mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow></msubs
up> :MATH]
) production in the presence of glutamate/malate substrate (5 mmol/L) in
isolated mitochondria from adult and aged rat hearts.

   Figure 4
   [145]Open in a new tab

   A, Increased reduction manifests as an increase in
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   production with aging (n=19, P<0.01). B, Left panel: Age‐related
   changes in H[2]O[2] production in unstressed isolated cardiomyocytes
   from adult and aged rats were measured using the DCFDA assay. Shown are
   confocal images at 0 and 10 minutes after 45 minutes of DCFDA
   pre‐incubation. Right Panel: DCFDA quantification exhibits a
   significant increase in fluorescence in unstressed cardiac cells
   isolated from aged animals (n=5 adult vs n=5 aged rats, P=0.003). C,
   Time‐dependent increase in DCFDA fluorescence in aged cardiomyocytes
   compared to that of adult cardiomyocytes with or without reactive
   oxygen species (ROS) production stimulated by 100 µmol/L ouabain
   treatment (n=5; *P<0.001, ^ψ P<0.01; bottom left graph). Differences in
   ouabain‐induced fluorescence at 10 minutes between adult (6 months old)
   and aged (24 months old) ventricular cardiomyocytes isolated from
   Fischer 344 rats demonstrate a significant increase in ROS production
   in aged animals (bottom right).

Aging Increases H[2]O[2] Production in Isolated Cardiomyocytes in the
Unstressed and Stressed States

   H[2]O[2] production from the isolated cardiomyocytes was measured in
   the unstressed state. Following 45 minutes of incubation with DCFDA,
   the change in fluorescence intensity was measured with a confocal
   microscope every minute for 10 minutes (Figure [146]4B). The cumulative
   results of five independent experiments in adult rats and aged rats are
   shown in the right graph in Figure [147]4B. Cardiomyocytes from aged
   rats produced a 2.2‐fold higher level of H[2]O[2] than cardiomyocytes
   from adult animals (1646±428 versus 699±329 arbitrary units [AU] of
   fluorescence, P=0.003) (Figure [148]4B). A significant time‐dependent
   (1–10 minutes) increase in DCF fluorescence was observed in aged
   cardiomyocytes compared to that observed in adult cardiomyocytes
   without ouabain (Figure [149]4C, left graph). Ouabain (100 µmol/L)
   induced a statistically significant increase in DCF fluorescence in
   cardiomyocytes from aged rats (1646±428 to 3222±234, P=0.006) but not
   from adult rats (699±329 to 989±366; P=0.17; Figure [150]4C, left
   graph), demonstrating a 3.7‐fold higher production of H[2]O[2] in aged
   cardiomyocytes than in adult cardiomyocytes following ouabain treatment
   (P=0.00004; Figure [151]4C, right graph). These differences in
   ouabain‐induced H[2]O[2] fluorescent signal at 10 minutes between adult
   and aged cardiomyocytes from rats indicate increased ouabain
   sensitivity and higher oxidative stress in aged myocardium
   (Figure [152]4C).

Aging‐Related Increase in Superoxide Production and 4‐HNE Protein Adducts in
Human Myocardium

   Mitochondrial superoxide production, as assessed by MitoSOX
   fluorescence in response to complex III inhibitor antimycin A, was also
   significantly higher in the human permeabilized myofibers isolated from
   aged (age >65 years) myocardium than in that from adult (age ≤65 years)
   myocardium (Figure [153]5A). The level of 4‐HNE protein adducts, a
   marker of lipid peroxidation and oxidative stress, was also
   significantly increased in elderly (age >65 years) versus adult (age
   ≤65 years) myocardium (187.54±54.8 versus 47.83±16.7 ng/mg protein,
   P=0.0063; Figure [154]5B). The levels of 4‐HNE protein adducts
   significantly correlated with increasing age (R ^2=0.36; P<0.001)
   (Figure [155]5C), indicating an age‐associated increase in oxidative
   stress and lipid peroxidation.

Figure 5. Difference in mitochondrial superoxide level and 4‐HNE protein
adducts in the adult and aged human right atrial appendages.

   Figure 5
   [156]Open in a new tab

   A, Difference in fluorescence intensity of MitoSOX Red, indicative of
   superoxide production in permeabilized myofibers from adult (age
   18–65 years) and aged (age >65 years) right atria after antimycin A
   (10 μmol/L; 15 minutes) stimulation. Fibers were stained with
   2.5 μmol/L MitoSOX Red (λex/λem=510/580), and fluorescence intensity
   was determined using laser scanning confocal fluorescence microscopy
   (Olympus FV1200). Data are presented as mean±standard error; n=6 for
   adult and n=8 for aged. Comparison of the adult and elderly groups was
   performed using t test, and P<0.05 was considered significant. B,
   Increased levels of 4‐HNE were observed in aged tissue following
   normalization to protein concentration in tissue supernatant. Data are
   presented as mean±standard deviation; n=14 for adult, n=27 aged. C,
   Scatter plot demonstrating age‐dependent linear regression of the
   levels of 4‐HNE protein adduct indicative of overall lipid peroxidation
   in human myocardial tissue of the patient sample population (n=41) who
   underwent elective open‐heart surgery with age.

Aging Alters Levels of SOD1 and SOD2 in the Human Heart

   The expression of SOD1 and SOD2 mRNA coding for two major superoxide
   scavengers superoxide dismutase (SOD) 1 and SOD2 present in the heart
   was downregulated in the aging rat myocardium (Table [157]S4). In
   humans, immunoblots of SOD1 and SOD2 and reference protein β‐tubulin in
   myocardium from various age groups (28–79 years) demonstrated a similar
   reduction in mean SOD1 protein expression (P=0.048) with aging but not
   SOD2 (P=0.106; Figure [158]6A through 6C). Overall, an inverse
   relationship in the expression of SOD1 protein level (a statistically
   significant decline) was observed with increasing age (R ^2=0.5725;
   P=0.011; Figure [159]6D), which is in contrast to a higher expression
   of SOD2 level with increasing age (R ^2=0.138; P=0.29; Figure [160]6E).

Figure 6. Age‐associated changes in protein expression level of superoxide
dismutase (SOD) 1 and SOD2 in human myocardium. Immunoblot data from human
atrial appendage tissue lysate normalized to β‐tubulin expression were
analyzed in ≤65 years old and >65 years old groups.

   Figure 6
   [161]Open in a new tab

   A, Immunoblots of human atrial tissue from different‐age patient tissue
   showed specific bands with expected molecular size of SOD1 (≈16 kDa),
   SOD2 (≈26 kDa), and GAPDH (≈36 kDa). B, There were significant
   decreases in SOD1 expression with increasing age, (C) without any
   significant changes observed for SOD2 between ≤65 years (n=5) and
   >65 years (n=5), comparison of the adult and elderly groups was
   performed using t test (P<0.05 was considered significant). D and E,
   Scatter plot analysis of age vs protein expression showed a negative
   correlation for SOD1 (R ^2=0.5725), whereas SOD2 (R ^2=0.138) exhibited
   a positive correlation.

Functional Network Analysis of Prioritized Genes With Aging in Human and Rat
Aging‐Associated Networks

   Functional network analysis of the merged collective network for human
   and rat samples revealed common genes prioritized in human and rat
   aging‐associated networks (Figure [162]7). Specifically, human samples
   prioritized MYC, AKT, ERK1/2, P38 MAPK, and NFKB complex as betweenness
   and closeness centrality network nodes, whereas rat samples exhibited
   priority of TP53, MYC, ERK, and AKT genes (Figure [163]7A and 7B). In
   addition, species‐specific enrichment was observed.

Figure 7. Cell proliferation and differentiation pathways genes are
prioritized targets in the aging heart in association with reactive oxygen
species (ROS).

   Figure 7
   [164]Open in a new tab

   A, Merged networks from human ROS data in aged hearts show prioritized
   nodes with high betweenness centrality (MYC, AKT, ERK1/2, ERK, FOXO3,
   P38 MAPK, NFKB [complex], and PI3K [complex]) and high closeness
   centrality (MYC, AKT, NFKB [complex], P38 MAPK, PI3K [complex], ERK1/2,
   RNF4, and ERK). B, Rat merged networks identified prioritized nodes
   with high betweenness centrality (TP53, MYC, adenosine triphosphate,
   JNK, ERK, NFKB [complex], AKT, and ERK1/2) and high closeness
   centrality (TP53, MYC, adenosine triphosphate, immunoglobulin, SOD1,
   mitochondrial complex I, ERK, and AKT).

   Analysis of individual subnetworks that comprise the collective network
   for human and rat further revealed that the top prioritized network
   (Network 1) had identical enriched top functions for each dataset,
   namely “metabolic disease,” “developmental disorder,” and “hereditary
   disorder” (Figure [165]8). Using the Molecule Predictor Analysis module
   in IPA, specific genes within each subnetwork were predicted to be
   activated/inhibited with respect to present gene expression data
   (Figure [166]8A and 8B). Although these unique gene
   activation/inhibition patterns were specific for human and rat samples,
   there were genes common to both networks, including ERK1/2 (predicted
   gene activation in both datasets), NDUFA9, and NDUFA6 (Figure [167]8C)
   which underlay enriched functional terms of NADH dehydrogenase and
   mitochondrial complex I.

Figure 8. Prediction of ERK1/2 pathway activation as a response to
downregulation of mitochondrial complex I in the aged heart.

   Figure 8
   [168]Open in a new tab

   Molecule predictor (MP) analysis in IPA was used to analyze the highest
   priority network from reactive oxygen species‐related genes in aging
   hearts. A, MP analysis of IPA Network 1 in the human dataset (Score 53,
   top functions: metabolic disease, developmental disorder, hereditary
   disorder) predicted activation of PP2A, P38 MAPK, ERK1/2, PDGF BB,
   insulin, AP1, NOS, and the 26S proteasome as a consequence of net gene
   expression dynamics. CREB, AMPK, ARDB, and GSK3 were predicted to be
   inhibited. Color and molecule shape indicated in legends. B, MP
   analysis of Network 1 from the rat dataset (Score 66, top functions:
   metabolic disease, developmental disorder, hereditary disorder) showed
   only one molecule that predicted activation, ERK1/2, which predicted
   indirect activation of glutathione peroxidase. C, Venn diagram
   highlights common nodes in both networks (five nodes), namely: ERK1/2,
   NDUFA9, NDUFA6, NADH dehydrogenase, and mitochondrial complex I.

DISCUSSION

   The main findings of our study are that with aging in both human and
   rat hearts there are alterations in the expression of genes coding for
   proteins involved in ROS production and clearance pathways and that
   selective downregulation is seen in genes encoding for mitochondrial
   ETC complex I and III sites of ROS production in rat mitochondria but
   only for complex I in human myocardium. In the extramitochondrial ROS
   generation pathway, mRNA expression of both XDH and NOX2 was
   upregulated in rats but not in the human heart. The gene expression
   changes were correlated with the reduction in functional activities of
   ETC complex I but not with other complexes. These changes were
   associated with enhanced ROS production in the aged myocardium in both
   humans and rats, as demonstrated by enhanced superoxide production and
   lipid peroxidation product in the human heart and increased H[2]O[2]
   production in the aged rat heart at baseline and with metabolic stress
   in isolated cardiac myocytes. Network analysis of the transcriptome
   highlights the critical elements involved with aging and the downstream
   effect of gene alteration on functional pathways, hinting at a possible
   linkage between complex I dysregulation and the ERK1/2 signaling
   pathway in the aging heart.

   Aging‐related mitochondrial changes and reduced myocardial functional
   reserves affect the heart's physiological function and responsiveness
   to stress[169]^14; however, the molecular basis for this is not
   entirely understood. In the present study, we showed that steady‐state
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   and H[2]O[2] production were significantly increased in aged animals
   compared to adult counterparts. In addition, the oxidant responsiveness
   to stress induced by ouabain exposure was well tolerated in the adult
   cardiomyocytes but not in cardiomyocytes from the aged animal. Similar
   differences in susceptibility to superoxide production with antimycin
   exposure were present in permeabilized myofibers from human aged
   myocardium but not adult. This was also reflected in a 3.8‐fold higher
   level of 4‐HNE protein adduct, a marker of lipid peroxidation product,
   in myocardium from humans 65 years and older compared to that from
   younger individuals. The increased susceptibility to oxidant production
   in the aging heart was associated with differential expression of genes
   involved in ROS production and clearance as revealed by transcriptome
   profiling in both a rat model of aging and humans. This suggests that
   aging‐associated enhanced susceptibility to oxidative stress is related
   to changes in ROS production and clearance independent of species or
   organism lifespan. The common link in ROS production in both species
   was the selective downregulation in the activity of ETC complex I
   involved in superoxide generation. NDUFA6 and NDUFA9 ETC subunits, and
   NDUFAF5 and NDUFAF6, the assembly factors of complex I, were identified
   as the prioritized molecules that may underlie the aging‐associated
   reductions in mitochondrial OXPHOS efficiency and an enhanced tendency
   to produce superoxide within mitochondria in both species. Thus, our
   data indicate that similar mechanisms and pathways are involved in
   aging rats and humans, independent of differences in longevity, food
   consumption, and lifestyle.

   Age‐associated increased susceptibility to oxidative stress has been
   shown to underlie myocardial dysfunction under metabolic
   stress.[170]^6, [171]^14 Low levels of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   and H[2]O[2] continuously generated as part of the normal metabolic
   process are essential for the maintenance of physiological cellular
   function; however, excessive oxidant production becomes detrimental,
   resulting in myocardial dysfunction and promoting cell injury after
   metabolic stress such as ischemia/reperfusion.[172]^1, [173]^2,
   [174]^3, [175]^4, [176]^28, [177]^29 Both cytosolic NADPH oxidase 2 and
   mitochondrial ETC are ROS generation sites within the cardiac myocyte,
   but the relative contribution to aging‐associated dysfunction is not
   fully defined.[178]^30 The present study recapitulated the enhanced
   sensitivity of ROS production in myocytes/myofibers from the aging
   heart and identified key elements regulating ROS synthesis and ROS
   clearance pathways that were differentially expressed, providing
   evidence for compounded impairment of ROS metabolism with
   aging.[179]^31, [180]^32, [181]^33 Genes encoding for proteins involved
   in the ROS clearance cluster belong to several enzyme classes,
   including oxide dismutases, glutathione transferases, and
   oxidoreductases, and were significantly downregulated in rat and human
   hearts but with significant variability, which may be due to species
   differences or be the result of other factors related to differences in
   longevity, diet, or intrinsic differences in heart rate and level of
   stress between the two species.[182]^6, [183]^13, [184]^14, [185]^24,
   [186]^28, [187]^29, [188]^31, [189]^34

   Furthermore, the results from our functional network analysis highlight
   a possible downstream effect among dysregulation of complex I of the
   mitochondrial ETC, production of ROS, and the activation of the ERK1/2
   signaling pathway in the aging heart. ROS has been implicated in the
   pathogenesis of heart failure, specifically in the stimulation of
   cardiac hypertrophy, where H[2]O[2]‐promoted cardiac hypertrophy has
   been shown to be attributed to activation of the ERK1/2 signaling
   pathway.[190]^2, [191]^35, [192]^36, [193]^37, [194]^38, [195]^39
   Ischemia‐reperfusion, neurohormones, exercise, comorbidities such as
   diabetes mellitus and others, and metabolic stressors can enhance the
   sensitivity of ROS production in the aging heart.[196]^24, [197]^30,
   [198]^33, [199]^40 In the present study, we used ouabain as a mild
   pharmacologic stressor to identify cardiomyocytes' heightened
   sensitivity to H[2]O[2] production in the aged rat heart and a
   negligible effect in cardiomyocytes from adult rats.

   The effect of aging on the genomic profile of enzymatic pathways of ROS
   generation and clearance has not been fully characterized in the human
   heart. Mitochondria are the primary source of ROS production in the
   heart and, conversely, are most vulnerable to oxidative damage, leading
   to a vicious cycle of ever‐increasing ROS production and dysfunctional
   mitochondrial damage with age.[200]^41 Several lines of evidence
   support age‐related decline in cardiac mitochondrial oxidative
   phosphorylation, mitochondrial ETC complexes, and ATP synthase.[201]^6,
   [202]^8, [203]^14, [204]^15 Previously, we and others showed
   transcriptional downregulation of genes encoding subunits of
   mitochondrial ETC complexes[205]^8, [206]^14, [207]^42, [208]^43 that
   contribute to the decrease in the activity and efficiency of
   mitochondria under metabolic and oxidative stress[209]^6, [210]^14,
   [211]^44, [212]^45, [213]^46 as well as disease conditions reminiscent
   of aging, such as atrial fibrillation and heart failure in
   humans.[214]^6, [215]^47 In our previous study,[216]^14 we demonstrated
   that among all five OXPHOS complexes, complex I was predominantly
   affected by aging at the gene and protein expression level in human
   cardiac tissue. This was associated with a decline in the functional
   activity of complex I. Similarly, our group has shown an aging‐related
   decline in complex I subunits and activity level in rat
   myocardium.[217]^8 In hearts from aging rats, several complex V
   subunits were also downregulated, with a significant reduction in
   functional activity compared to adults. The main differences between
   the adult and aged hearts that are common between rats and humans were
   in complex I subunit expression and activity (Table [218]S1), with no
   significant change in complex V or other OXPHOS complexes observed in
   adult and aged humans compared to rats. This suggests that distinctions
   exist related to species, overall longevity variances, different
   metabolic demands with marked variances in heart rate, and other
   physiological processes, along with possible confounding effects of
   comorbidities in humans not experienced by animals free of other
   illnesses. Moreover, these changes in mitochondrial respiratory
   capacity reduction could be related to aging‐associated alteration in
   complex I activity or supercomplex destabilization as suggested by
   Gomez et al.[219]^48 The loss of supercomplexes can compromise
   stoichiometries of OXPHOS complexes I, III, and IV as previously
   demonstrated in aged rats,[220]^49 which makes electron flux through
   the ETC inefficient, reducing mitochondrial energetic reserve as
   reported by other studies.[221]^48, [222]^50

   Differences in the expression of genes coding for the main
   extramitochondrial and mitochondrial sites of ROS production between
   the aged and adult rat and human hearts were observed. A 40% to 60%
   upregulation of expression of subunits of NADPH oxidase 2 (NOX2) and
   xanthine oxidoreductase (XOR) genes of extramitochondrial ROS
   production in aged rats was seen compared to adults, along with
   subunits of mitochondrial ETC complexes I and III. The expression of
   NOX2 and XOR in humans remained unchanged between the adult and aged
   myocardium, in which the changes were mainly confined to the major
   mitochondrial site of ROS production within ETC complex I. The ROS
   clearance pathways also showed a differential effect of aging on
   transcripts level, downregulated by 20% to 60% in aged hearts compared
   to adult hearts (P<0.02). These changes were observed specifically in
   genes SOD1, SOD2, GSTA4, GSTM2, GSTM3, TXN2, TXNDC4, TXNDC9, and PRDX5,
   which code for, respectively, SOD1 and SOD2, glutathione S‐transferase
   A4, M2 and M3, thioredoxin 2, thioredoxin domain containing 4 and 9,
   and peroxiredoxin 5. Overall, this suggests an aging‐associated change
   that predisposes to higher ROS levels and a more exaggerated response
   to stress, with attenuation of antioxidant mechanisms. A compensatory
   upregulation (20–260%) of transcripts GPX3, GSTA1, TXNRD3, and
   PRDX4—coding for glutathione peroxidase 3, glutathione S‐transferase
   A1, thioredoxin reductase 3, and peroxiredoxin 4, respectively, all
   involved in ROS clearance—was observed with aging, which may help
   reduce oxidant levels. The downregulated expression of both SOD1
   (predominantly localized in the cytosol but also mitochondria) and SOD2
   (localized within mitochondria) in rat hearts indicates that the
   dismutation of
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   is altered at both of these sites.[223]^51 These changes in SOD1 and
   SOD2 transcripts present in the aging rat model were not observed in
   human aged hearts, in which only protein expression of SOD1 was
   significantly downregulated and inversely correlated with age. The
   level of SOD2 proteins in the human heart was increased with aging and
   may represent a compensatory mechanism for countering the oxidative
   stress caused by increased mitochondrial
   [MATH: <msubsup><mi
   mathvariant="normal">O</mi><mn>2</mn><mrow><mo>·</mo><mo>-</mo></mrow><
   /msubsup> :MATH]
   production. These findings suggest that individual components of
   oxidant production and clearance may be affected differentially with
   aging in humans compared to animal models and may need to be further
   characterized to identify targets to reduce oxidative stress and
   maintain the redox homeostasis with aging. The glutathione peroxidases
   GPx2, GPx3, GPx4‐7 associated with glutathione peroxidase (GPx1), which
   offers protection against myocardial ischemia‐reperfusion
   injury,[224]^52, [225]^53 were upregulated, akin to earlier
   observations.[226]^54, [227]^55 Thioredoxin and thioredoxin reductases
   are important components of the cellular antioxidant system involved in
   ROS scavenging and regulation of cell survival and cell death,[228]^56
   and in this study transcript levels of Txnrd3 were upregulated in aging
   rats. GPx3 and Txnrd3 belong to the group of redox enzymes that contain
   catalytically active selenocysteine (SEC) residues.[229]^57, [230]^58
   The increase in expression of GPX3 and TXNRD3 is thus notable since the
   majority of ROS scavengers were either downregulated or showed no
   significant change in expression. This suggests that enzymes with SEC
   residues may serve as a secondary layer of protection in response to
   increased oxidative stress, activated when the primary line of defense,
   the mitochondrial antioxidant enzyme system against ROS generation,
   becomes compromised with aging. Indeed, the concept of genetic
   regulation of ROS production and clearance with age is strongly
   supported by caloric restriction experiments that demonstrated how
   transcriptional reprogramming of gene sets leads to overall oxidative
   stress reduction.[231]^59 Concisely, the reduced expression of
   important components of these protective pathways in the aging heart in
   both rats and humans highlights a common mechanism underlying the
   reduced capacity of the aged heart to counter oxidative stress that, in
   turn, increases predisposition to myocardial injury under stress, as
   well as promotion of interstitial fibrosis and overall myocardial
   dysfunction.[232]^60, [233]^61

   The main limitation of the study is that the observed changes in the
   levels of individual transcripts were small (20% to 60%); however, the
   cumulative effect of these changes in the ROS production and clearance
   pathways could have a significant impact on the aging‐associated
   susceptibly to oxidative stress and myocardial dysfunction.[234]^60

Clinical Implication

   In summary, our findings suggest that aging influences both
   mitochondrial and extramitochondrial pathways of ROS production and
   clearance within the myocardium, disbalancing the oxidant and
   antioxidant system, promoting a higher level of oxidants under
   steady‐state conditions, and, in response to metabolic stress with
   significant downstream effects, promoting a higher susceptibility to
   injury. ROS‐mediated activation of the ERK1/2 pathway identified in
   network analysis warrants further studies; recognizing components of
   ROS homeostasis regulators and downstream players altered by aging may
   identify targets that can be selectively intervened on to prevent
   aging‐associated electrical or mechanical myocardial dysfunction, such
   as atrial fibrillation and heart failure, prevalent in the elderly.

Sources of Funding

   This work was supported by research grants from the National Institute
   of Aging (RO1‐AG21201) and National Heart, Lung, and Blood Institute,
   NIH (RO1‐HL089542 and RO1 HL101240). None of the funding sources had
   any input into study design; collection, analysis, and interpretation
   of data; writing of the report; or in the decision to submit the
   article for publication.

Disclosures

   None.

Supporting information

   Tables S1–S4
   [235]Click here for additional data file.^ (455.2KB, pdf)

Acknowledgments