Single cell regulatory architecture of human pancreatic islets suggests sex differences in ß cell function and the pathogenesis of type 2 diabetes.

Biological sex affects the pathogenesis of type 2 and type 1 diabetes (T2D, T1D) including the development of ß cell failure observed more often in males. The mechanisms that drive sex differences in ß cell failure is unknown. Studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in ß cell failure in diabetes. Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D (including 37 donors from the Human Pancreas Analysis Program [HPAP] dataset) using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. In cultured islets from nondiabetic (ND) donors, in the absence of the in vivo hormonal environment, sex differences in islet cell type gene accessibility and expression predominantly involved sex chromosomes. Of particular interest were sex differences in the X-linked KDM6A and Y-linked KDM5D chromatin remodelers in female and male islet cells respectively. Islets from T2D donors exhibited similar sex differences in differentially expressed genes (DEGs) from sex chromosomes. However, in contrast to islets from ND donors, islets from T2D donors exhibited major sex differences in DEGs from autosomes. Comparing ß cells from T2D and ND donors revealed that females had more DEGs from autosomes compared to male ß cells. Gene set enrichment analysis of female ß cell DEGs showed a suppression of oxidative phosphorylation and electron transport chain pathways, while male ß cell had suppressed insulin secretion pathways. Thus, although sex-specific differences in gene accessibility and expression of cultured ND human islets predominantly affect sex chromosome genes, major differences in autosomal gene expression between sexes appear during the transition to T2D and which highlight mitochondrial failure in female ß cells.

Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice.

Risk factors contributing to dementia are multifactorial. Accumulating evidence suggests a role for pathogens as risk factors, but data is largely correlative with few causal relationships. Here, we demonstrate that intermittent murine cytomegalovirus (MCMV) infection of mice, alters blood brain barrier (BBB) permeability and metabolic pathways. Increased basal mitochondrial function is observed in brain microvessels cells (BMV) exposed to intermittent MCMV infection and is accompanied by elevated levels of superoxide. Further, mice score lower in cognitive assays compared to age-matched controls who were never administered MCMV. Our data show that repeated systemic infection with MCMV, increases markers of neuroinflammation, alters mitochondrial function, increases markers of oxidative stress and impacts cognition. Together, this suggests that viral burden may be a risk factor for dementia. These observations provide possible mechanistic insights through which pathogens may contribute to the progression or exacerbation of dementia.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

High-plasma soluble prorenin receptor is associated with vascular damage in male, but not female, mice fed a high-fat diet.

Plasma soluble prorenin receptor (sPRR) displays sexual dimorphism and is higher in women with type 2 diabetes mellitus (T2DM). However, the contribution of plasma sPRR to the development of vascular complications in T2DM remains unclear. We investigated if plasma sPRR contributes to sex differences in the activation of the systemic renin-angiotensin-aldosterone system (RAAS) and vascular damage in a model of high-fat diet (HFD)-induced T2DM. Male and female C57BL/6J mice were fed either a normal fat diet (NFD) or an HFD for 28 wk to assess changes in blood pressure, cardiometabolic phenotype, plasma prorenin/renin, sPRR, and ANG II. After completing dietary protocols, tissues were collected from males to assess vascular reactivity and aortic reactive oxygen species (ROS). A cohort of male mice was used to determine the direct contribution of increased systemic sPRR by infusion. To investigate the role of ovarian hormones, ovariectomy (OVX) was performed at 32 wk in females fed either an NFD or HFD. Significant sex differences were found after 28 wk of HFD, where only males developed T2DM and increased plasma prorenin/renin, sPRR, and ANG II. T2DM in males was accompanied by nondipping hypertension, carotid artery stiffening, and aortic ROS. sPRR infusion in males induced vascular thickening instead of material stiffening caused by HFD-induced T2DM. While intact females were less prone to T2DM, OVX increased plasma prorenin/renin, sPRR, and systolic blood pressure. These data suggest that sPRR is a novel indicator of systemic RAAS activation and reflects the onset of vascular complications during T2DM regulated by sex.NEW & NOTEWORTHY High-fat diet (HFD) for 28 wk leads to type 2 diabetes mellitus (T2DM) phenotype, concomitant with increased plasma soluble prorenin receptor (sPRR), nondipping blood pressure, and vascular stiffness in male mice. HFD-fed female mice exhibiting a preserved cardiometabolic phenotype until ovariectomy revealed increased plasma sPRR and blood pressure. Plasma sPRR may indicate the status of systemic renin-angiotensin-aldosterone system (RAAS) activation and the onset of vascular complications during T2DM in a sex-dependent manner.

Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.

Amyloid [Formula: see text] (1-42) peptide impairs mitochondrial respiration in primary human brain microvascular endothelial cells: impact of dysglycemia and pre-senescence.

Diabetes increases the risk of Alzheimer's disease (AD). We investigated the impact of glucose concentrations on the ß-amyloid (Aß)-induced alteration of mitochondrial/cellular energetics in primary human brain microvascular endothelial cells (HBMECs). HBMECs were grown and passaged in media containing 15 mmol/l glucose (normal) based on which the glucose levels in the media were designated as high (25 mmol/L) or low (5 mmol/L). HBMECs were treated with Aß (1-42) (5 µmol/l) or a scrambled peptide for 24 h and mitochondrial respiratory parameters were measured using Seahorse Mito Stress Test. Aß (1-42) decreased the mitochondrial ATP production at normal glucose levels and decreased spare respiratory capacity at high glucose levels. Aß (1-42) diminished all mitochondrial respiratory parameters markedly at low glucose levels that were not completely recovered by restoring normal glucose levels in the media. The addition of mannitol (10 mmol/l) to low and normal glucose-containing media altered the Aß (1-42)-induced bioenergetic defects. Even at normal glucose levels, pre-senescent HMBECs (passage 15) displayed greater Aß (1-42)-induced mitochondrial respiratory impairments than young cells (passages 7-9). Thus, hypoglycemia, osmolarity changes, and senescence are stronger instigators of Aß (1-42)-induced mitochondrial respiration and energetics in HBMECs and contributors to diabetes-related increased AD risk than hyperglycemia.

Glycolytic and Oxidative Phosphorylation Defects Precede the Development of Senescence in Primary Human Brain Microvascular Endothelial Cells.

Alterations of mitochondrial and glycolytic energy pathways related to aging could contribute to cerebrovascular dysfunction. We studied the impact of aging on energetics of primary human brain microvascular endothelial cells (HBMECs) by comparing the young (passages 7-9), pre-senescent (passages 13-15), and senescent (passages 20-21) cells. Pre-senescent HBMECs displayed decreased telomere length and undetectable telomerase activity although markers of senescence were unaffected. Bioenergetics in HBMECs were determined by measuring the oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Cellular ATP production in young HBMECs was predominantly dependent on glycolysis with glutamine as the preferred fuel for mitochondrial oxidative phosphorylation (OXPHOS). In contrast, pre-senescent HBMECs displayed equal contribution to ATP production rate from glycolysis and OXPHOS with equal utilization of glutamine, glucose, and fatty acids as mitofuels. Compared to young, pre-senescent HBMECs showed a lower overall ATP production rate that was characterized by diminished contribution from glycolysis. Impairments of glycolysis displayed by pre-senescent cells included reduced basal glycolysis, compensatory glycolysis, and non-glycolytic acidification. Furthermore, impairments of mitochondrial respiration in pre-senescent cells involved the reduction of maximal respiration and spare respiratory capacity but intact basal and ATP production-related OCR. Proton leak and non-mitochondrial respiration, however, were unchanged in the pre-senescent HBMECs. HBMECS at passages 20-21 displayed expression of senescence markers and continued similar defects in glycolysis and worsened OXPHOS. Thus, for the first time, we characterized the bioenergetics of pre-senescent HBMECs comprehensively to identify the alterations of the energy pathways that could contribute to aging.

Peroxynitrite decomposition catalyst enhances respiratory function in isolated brain mitochondria.

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.

Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.

Diets with low n-6:n-3 PUFA ratio protects rats from fructose-induced dyslipidemia and associated hepatic changes: Comparison between 18:3 n-3 and long-chain n-3 PUFA.

In the present study, we investigated the impact of substituting alpha-linolenic acid (ALA) or long-chain n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) for linoleic acid and hence decreasing n-6:n-3 PUFA ratio on high-fructose diet-induced hypertriglyceridemia and associated hepatic changes. Weanling male Wistar rats were divided into four groups and fed with starch-diet (n-6:n-3 PUFA ratio 215:1) and high-fructose diets with different n-6:n-3 PUFA ratio (215:1, 2:1 with ALA and 5:1 with long-chain n-3 PUFA) for twenty-four weeks. Substitution of linoleic acid with ALA (n-6:n-3 PUFA ratio of 2) or long-chain n-3 PUFA (n-6:n-3 PUFA ratio of 5) protected the rats from fructose-induced dyslipidemia, hepatic oxidative stress and corrected lipogenic and proinflammatory gene expression. Both ALA and long-chain n-3 PUFA supplementation also reversed the fructose-induced upregulation of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) gene, which is involved in the generation of active glucocorticoids in tissues. Although both ALA and LC n-3 PUFA prevented fructose-induced dyslipidemia to a similar extent, compared to ALA, LC n-3 PUFA is more effective in preventing hepatic oxidative stress and inflammation.

Nitric oxide synthase inhibitors negatively regulate respiration in isolated rodent cardiac and brain mitochondria.

Nitric oxide (NO) is known to exert inhibitory control on mitochondrial respiration in the heart and brain. Evidence supports the presence of NO synthase (NOS) in the mitochondria (mtNOS) of cells; however, the functional role of mtNOS in the regulation of mitochondrial respiration is unclear. Our objective was to examine the effect of NOS inhibitors on mitochondrial respiration and protein S-nitrosylation. Freshly isolated cardiac and brain nonsynaptosomal mitochondria were incubated with selective inhibitors of neuronal (nNOS; ARL-17477, 1 µmol/L) or endothelial [eNOS; N5-(1-iminoethyl)-l-ornithine, NIO, 1 µmol/L] NOS isoforms. Mitochondrial respiratory parameters were calculated from the oxygen consumption rates measured using Agilent Seahorse XFe24 analyzer. Expression of NOS isoforms in the mitochondria was confirmed by immunoprecipitation and Western blot analysis. In addition, we determined the protein S-nitrosylation by biotin-switch method followed by immunoblotting. nNOS inhibitor decreased the state IIIu respiration in cardiac mitochondria and both state III and state IIIu respiration in brain mitochondria. In contrast, eNOS inhibitor had no effect on the respiration in the mitochondria from both heart and brain. Interestingly, NOS inhibitors reduced the levels of protein S-nitrosylation only in brain mitochondria, but nNOS and eNOS immunoreactivity was observed in the cardiac and brain mitochondrial lysates. Thus, the effects of NOS inhibitors on S-nitrosylation of mitochondrial proteins and mitochondrial respiration confirm the existence of functionally active NOS isoforms in the mitochondria. Notably, our study presents first evidence of the positive regulation of mitochondrial respiration by mitochondrial nNOS contrary to the current dogma representing the inhibitory role attributed to NOS isoforms.NEW & NOTEWORTHY Existence and the role of nitric oxide synthases in the mitochondria are controversial. We report for the first time that mitochondrial nNOS positively regulates respiration in isolated heart and brain mitochondria, thus challenging the existing dogma that NO is inhibitory to mitochondrial respiration. We have also demonstrated reduced protein S-nitrosylation by NOS inhibition in isolated mitochondria, supporting the presence of functional mitochondrial NOS.

Measuring Respiration in Isolated Murine Brain Mitochondria: Implications for Mechanistic Stroke Studies.

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice.

Cerebral blood flow (CBF) is uniquely regulated by the anatomical design of the cerebral vasculature as well as through neurovascular coupling. The process of directing the CBF to meet the energy demands of neuronal activity is referred to as neurovascular coupling. Microvasculature in the brain constitutes the critical component of the neurovascular coupling. Mitochondria provide the majority of ATP to meet the high-energy demand of the brain. Impairment of mitochondrial function plays a central role in several age-related diseases such as hypertension, ischemic brain injury, Alzheimer's disease, and Parkinson disease. Interestingly, microvessels and small arteries of the brain have been the focus of the studies implicating the vascular mechanisms in several age-related neurological diseases. However, the role of microvascular mitochondrial dysfunction in age-related diseases remains unexplored. To date, high-throughput assay for measuring mitochondrial respiration in microvessels is lacking. The current study presents a novel method to measure mitochondrial respiratory parameters in freshly isolated microvessels from mouse brain ex vivo using Seahorse XFe24 Analyzer. We validated the method by demonstrating impairments of mitochondrial respiration in cerebral microvessels isolated from old mice compared to the young mice. Thus, application of mitochondrial respiration studies in microvessels will help identify novel vascular mechanisms underlying a variety of age-related neurological diseases.

Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research.

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.

Absence of Tissue Inhibitor of Metalloproteinase-4 (TIMP4) ameliorates high fat diet-induced obesity in mice due to defective lipid absorption.

Tissue inhibitor of metalloproteases (TIMPs) are inhibitors of matrix metalloproteinases (MMPs) that regulate tissue extracellular matrix (ECM) turnover. TIMP4 is highly expressed in adipose tissue, its levels are further elevated following high-fat diet, but its role in obesity is unknown. Eight-week old wild-type (WT) and Timp4-knockout (Timp4 -/-) mice received chow or high fat diet (HFD) for twelve weeks. Timp4 -/- mice exhibited a higher food intake but lower body fat gain. Adipose tissue of Timp4 -/- -HFD mice showed reduced hypertrophy and fibrosis compared to WT-HFD mice. Timp4 -/- -HFD mice were also protected from HFD-induced liver and skeletal muscle triglyceride accumulation and dyslipidemia. Timp4 -/--HFD mice exhibited reduced basic metabolic rate and energy expenditure, but increased respiratory exchange ratio. Increased free fatty acid excretion was detected in Timp4 -/--HFD compared to WT-HFD mice. CD36 protein, the major fatty acid transporter in the small intestine, increased with HFD in WT but not in Timp4 -/- mice, despite a similar rise in Cd36 mRNA in both genotypes. Consistently, HFD increased enterocyte lipid content only in WT but not in Timp4 -/- mice. Our study reveals that absence of TIMP4 can impair lipid absorption and the high fat diet-induced obesity in mice possibly by regulating the proteolytic processing of CD36 protein in the intestinal enterocytes.

Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction.

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPß and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPß, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.

TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis.

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3 Interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2 mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Further, aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. However, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen IIIα1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) mRNA expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease.

Differential impact of mechanical unloading on structural and nonstructural components of the extracellular matrix in advanced human heart failure.

Adverse remodeling of the extracellular matrix (ECM) is a significant characteristic of heart failure. Reverse remodeling of the fibrillar ECM secondary to mechanical unloading of the left ventricle (LV) by left ventricular assist device (LVAD) has been subject of intense investigation; however, little is known about the impacts on nonfibrillar ECM and matricellular proteins that also contribute to disease progression. Explanted failing hearts were procured from patients with nonischemic dilated cardiomyopathy (DCM) with or without LVAD support, and compared to nonfailing control hearts. LV free wall specimens were formalin-fixed, flash-frozen or optimum cutting temperature-mount frozen. Histologic and biochemical assessment of fibrillar ECM showed that LVAD support was associated with lower levels of insoluble collagen, collagen type I mRNA, and collagen I/III ratio compared with no-LVAD hearts. A disintegrin and Metalloproteinase with Thrombospondin Motifs-2 (ADAM-TS2), a procollagen endopeptidase, was reduced in no-LVAD but not in LVAD hearts. The rise in ECM proteolytic activities was significantly lower in LVAD hearts. Matrix metalloproteinases (MMP1, MMP2, MMP8, MMP13, and MT1-MMP/MMP14) were comparable between DCM hearts. Tissue inhibitor of metalloproteinase (TIMP)3 and TIMP4 messenger RNA and protein showed the greatest reduction in no-LVAD hearts. Basement membrane proteins exhibited less severe disarray of laminin and fibronectin-1 in LVAD-supported hearts. The rise in matricellular protein, osteopontin, was suppressed in LVAD hearts, whereas secreted protein, acidic, cysteine-rich (SPARC) levels was unaffected by LVAD. Mechanical unloading of the failing DCM hearts can restore the fibrillar ECM and the basement membrane, contributing toward improved clinical outcomes. However, persistent elevation of matricellular proteins such as SPARC could contribute to the relapse of failing hearts on removal of LVAD support.

Extracellular matrix communication and turnover in cardiac physiology and pathology.

Despite significant advances in treating heart disease, heart failure remains a major cause of morbidity and mortality. Regardless of the initiating cause(s), heart failure is associated with disruptions in the myocardial extracellular matrix (ECM). ECM is a dynamic structure and its physiological turnover is mediated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Research in the past two decades has revealed that the function of ECM extends beyond its role in providing structural support. Similarly, ECM regulatory proteins, MMPs and TIMPs, have been demonstrated to play diverse and ECM-independent roles in tissue remodeling and homeostasis. ECM is a network structure that in addition to providing structural support, serves as an extracellular reservoir for a number of growth factors and cytokines, and plays a central role in interstitial transport of different molecules (hormones, growth factors, drugs, etc.). This is mainly through the action of nonstructural ECM components, proteoglycans and matricellular proteins, which are also critical in cell-ECM interactions and overall ECM remodeling. As such, sustaining the ECM integrity is not only critical in preserving cardiac geometry and function, it is essential in ensuring optimal delivery of different molecules to their site of action. Further, ECM composition and integrity in disease should be considered in designing drugs with a specific site of action. In this review article, we provide an overview of the ECM structure, components, its function in interstitial transport, heart disease-dependent ECM remodeling, and the potential therapeutic approaches in preserving the diseased myocardial ECM and cardiac function.