Anisha A. Gupte

Rosiglitazone attenuates age- and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein receptor knockout mice†

Nonalcoholic fatty liver disease (NAFLD) is a common complication of obesity that can progress to nonalcoholic steatohepatitis (NASH), a serious liver pathology that can advance to cirrhosis. The mechanisms responsible for NAFLD progression to NASH remain unclear. Lack of a suitable animal model that faithfully recapitulates the pathophysiology of human NASH is a major obstacle in delineating mechanisms responsible for progression of NAFLD to NASH and, thus, development of better treatment strategies. We identified and characterized a novel mouse model, middle‐aged male low‐density lipoprotein receptor (LDLR)−/− mice fed a high‐fat diet (HFD), which developed NASH associated with four of five metabolic syndrome (MS) components. In these mice, as observed in humans, liver steatosis and oxidative stress promoted NASH development. Aging exacerbated the HFD‐induced NASH such that liver steatosis, inflammation, fibrosis, oxidative stress, and liver injury markers were greatly enhanced in middle‐aged versus young LDLR−/− mice. Although expression of genes mediating fatty acid oxidation and antioxidant responses were up‐regulated in young LDLR−/− mice fed HFD, they were drastically reduced in MS mice. However, similar to recent human trials, NASH was partially attenuated by an insulin‐sensitizing peroxisome proliferator‐activated receptor‐gamma (PPARγ) ligand, rosiglitazone. In addition to expected improvements in MS, newly identified mechanisms of PPARγ ligand effects included stimulation of antioxidant gene expression and mitochondrial β‐oxidation, and suppression of inflammation and fibrosis. LDLR‐deficiency promoted NASH, because middle‐aged C57BL/6 mice fed HFD did not develop severe inflammation and fibrosis, despite increased steatosis. Conclusion: MS mice represent an ideal model to investigate NASH in the context of MS, as commonly occurs in human disease, and NASH development can be substantially attenuated by PPARγ activation, which enhances β‐oxidation. (HEPATOLOGY 2010.)

Nuclear Factor (Erythroid-Derived 2)-Like-2 Factor (Nrf2), a Key Regulator of the Antioxidant Response to Protect Against Atherosclerosis and Nonalcoholic Steatohepatitis

Tissue oxidative stress is a common hallmark of atherosclerosis and non-alcoholic steatohepatitis (NASH), 2 conditions linked epidemiologically and pathophysiologically. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the master regulator of inducible antioxidant responses, that can attenuate cellular injury from oxidative stress induced by obesity and other redox insults. Nrf2 expression and activation is reduced in mouse and human vessels that harbor accelerated atherosclerosis and in livers with histologic criteria of NASH. Systemic antioxidants have thus been attractive therapeutic targets, but clinical trials have been largely unsuccessful in improving cardiovascular health. Macrophage-selective Nrf2 activation may, however, provide an approach to reduce vascular and hepatocyte injury without the complications of systemic antioxidants, since macrophages play key roles in the development and progression of both atherosclerosis and NASH. In this article, we review the common mechanisms of oxidative stress and inflammation in atherosclerosis and NASH, and discuss the role of Nrf2 in vascular and hepatocyte protection.

Freshly isolated mitochondria from failing human hearts exhibit preserved respiratory function

In heart failure mitochondrial dysfunction is thought to be responsible for energy depletion and contractile dysfunction. The difficulties in procuring fresh left ventricular (LV) myocardium from humans for assessment of mitochondrial function have resulted in the reliance on surrogate markers of mitochondrial function and limited our understanding of cardiac energetics. We isolated mitochondria from fresh LV wall tissue of patients with heart failure and reduced systolic function undergoing heart transplant or left ventricular assist device placement, and compared their function to mitochondria isolated from the non-failing LV (NFLV) wall tissue with normal systolic function from patients with pulmonary hypertension undergoing heart-lung transplant. We performed detailed mitochondrial functional analyses using 4 substrates: glutamate-malate (GM), pyruvate-malate (PM) palmitoyl carnitine-malate (PC) and succinate. NFLV mitochondria showed preserved respiratory control ratios and electron chain integrity with only few differences for the 4 substrates. In contrast, HF mitochondria had greater respiration with GM, PM and PC substrates and higher electron chain capacity for PM than for PC. Surprisingly, HF mitochondria had greater respiratory control ratios and lower ADP-independent state 4 rates than NFLV mitochondria for GM, PM and PC substrates demonstrating that HF mitochondria are capable of coupled respiration ex vivo. Gene expression studies revealed decreased expression of key genes in pathways for oxidation of both fatty acids and glucose. Our results suggest that mitochondria from the failing LV myocardium are capable of tightly coupled respiration when isolated and supplied with ample substrates. Thus energy starvation in the failing heart may be the result of dysregulation of metabolic pathways, impaired substrate supply or reduced mitochondrial number but not the result of reduced mitochondrial electron transport capacity.

Mechanical Unloading Promotes Myocardial Energy Recovery in Human Heart Failure

Background—Impaired bioenergetics is a prominent feature of the failing heart, but the underlying metabolic perturbations are poorly understood. Methods and Results—We compared metabolomic, gene transcript, and protein data from 6 paired samples of failing human left ventricular tissue obtained during left ventricular assist device insertion (heart failure samples) and at heart transplant (post-left ventricular assist device samples). Nonfailing left ventricular wall samples procured from explanted hearts of patients with right heart failure served as novel comparison samples. Metabolomic analyses uncovered a distinct pattern in heart failure tissue: 2.6-fold increased pyruvate concentrations coupled with reduced Krebs cycle intermediates and short-chain acylcarnitines, suggesting a global reduction in substrate oxidation. These findings were associated with decreased transcript levels for enzymes that catalyze fatty acid oxidation and pyruvate metabolism and for key transcriptional regulators of mitochondrial metabolism and biogenesis, peroxisome proliferator-activated receptor &ggr; coactivator 1&agr; (PGC1A, 1.3-fold) and estrogen-related receptor &agr; (ERRA, 1.2-fold) and &ggr; (ERRG, 2.2-fold). Thus, parallel decreases in key transcription factors and their target metabolic enzyme genes can explain the decreases in associated metabolic intermediates. Mechanical support with left ventricular assist device improved all of these metabolic and transcriptional defects. Conclusions—These observations underscore an important pathophysiologic role for severely defective metabolism in heart failure, while the reversibility of these defects by left ventricular assist device suggests metabolic resilience of the human heart.

High-Fat Feeding-Induced Hyperinsulinemia Increases Cardiac Glucose Uptake and Mitochondrial Function Despite Peripheral Insulin Resistance

In obesity, reduced cardiac glucose uptake and mitochondrial abnormalities are putative causes of cardiac dysfunction. However, high-fat diet (HFD) does not consistently induce cardiac insulin resistance and mitochondrial damage, and recent studies suggest HFD may be cardioprotective. To determine cardiac responses to HFD, we investigated cardiac function, glucose uptake, and mitochondrial respiration in young (3-month-old) and middle-aged (MA) (12-month-old) male Ldlr(-/-) mice fed chow or 3 months HFD to induce obesity, systemic insulin resistance, and hyperinsulinemia. In MA Ldlr(-/-) mice, HFD induced accelerated atherosclerosis and nonalcoholic steatohepatitis, common complications of human obesity. Surprisingly, HFD-fed mice demonstrated increased cardiac glucose uptake, which was most prominent in MA mice, in the absence of cardiac contractile dysfunction or hypertrophy. Moreover, hearts of HFD-fed mice had enhanced mitochondrial oxidation of palmitoyl carnitine, glutamate, and succinate and greater basal insulin signaling compared with those of chow-fed mice, suggesting cardiac insulin sensitivity was maintained, despite systemic insulin resistance. Streptozotocin-induced ablation of insulin production markedly reduced cardiac glucose uptake and mitochondrial dysfunction in HFD-fed, but not in chow-fed, mice. Insulin injection reversed these effects, suggesting that insulin may protect cardiac mitochondria during HFD. These results have implications for cardiac metabolism and preservation of mitochondrial function in obesity.

Combination of angiotensin II and l-NG-nitroarginine methyl ester exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.

Estrogen receptor alpha activation enhances mitochondrial function and systemic metabolism in high-fat-fed ovariectomized mice

Estrogen impacts insulin action and cardiac metabolism, and menopause dramatically increases cardiometabolic risk in women. However, the mechanism(s) of cardiometabolic protection by estrogen remain incompletely understood. Here, we tested the effects of selective activation of E2 receptor alpha (ERα) on systemic metabolism, insulin action, and cardiac mitochondrial function in a mouse model of metabolic dysfunction (ovariectomy [OVX], insulin resistance, hyperlipidemia, and advanced age). Middle‐aged (12‐month‐old) female low‐density lipoprotein receptor (Ldlr)−/− mice were subjected to OVX or sham surgery and fed “western” high‐fat diet (WHFD) for 3 months. Selective ERα activation with 4,4′,4″‐(4‐Propyl‐[1H]‐pyrazole‐1,3,5‐triyl) (PPT), prevented weight gain, improved insulin action, and reduced visceral fat accumulation in WHFD‐fed OVX mice. PPT treatment also elevated systemic metabolism, increasing oxygen consumption and core body temperature, induced expression of several metabolic genes such as peroxisome proliferator‐activated receptor gamma, coactivator 1 alpha, and nuclear respiratory factor 1 in heart, liver, skeletal muscle, and adipose tissue, and increased cardiac mitochondrial function. Taken together, selective activation of ERα with PPT enhances metabolic effects including insulin resistance, whole body energy metabolism, and mitochondrial function in OVX mice with metabolic syndrome.

Differential Mitochondrial Function in Remodeled Right and Nonremodeled Left Ventricles in Pulmonary Hypertension.

OBJECTIVESnRight ventricular failure is the primary reason for mortality in pulmonary hypertension (PH), but little is understood about the energetics of the failing right myocardium. Our aim was to examine mitochondrial function and proteomic signatures in paired remodeled right (RM-RV) and non-remodeled left (NRM-LV) ventricular tissue samples procured during heart-lung transplantation.nnnMETHODS AND RESULTSnContractile dysfunction in RM-RV and preserved contractile function in NRM-LV were determined clinically and by echocardiography. Mitochondria were isolated from fresh paired RV and LV wall specimens of explanted hearts. Respiratory states in response to 4 substrates and an uncoupler were analyzed. Proteomic analysis on the mitochondrial isolates was performed with the use of liquid chromatography-mass spectrometry. The RM-RV mitochondria exhibited higher succinate state 4 levels with lower respiratory control ratio (RCR) compared with state 4 levels for pyruvate-malate (PM) and glutamate-malate (GM). RM-RV mitochondria also exhibited lower state 3 for palmitoyl-carnitine (PC) and state 4 for all complex I substrates compared with NRM-LV. The mean RCR were greater in RM-RVs than in NRM-LVs for PM and GM, which is consistent with tight coupling (low state 4 rates, higher RCRs); however, low RM-RV state 3 rates suggest concurrent substrate-dependent impairment in respiratory capacity. Mitochondrial proteomics revealed greater levels of mitochondrial ADP-ATP translocase and proteins of ATP synthesis, mitochondrial pyruvate and short branched chain acyl-CoA metabolism in RM-RV.nnnCONCLUSIONSnThe mitochondrial respiration and proteomics in RM-RV are different from NRM-LV. These results have important implications in expanding our understanding of RV metabolism and future management of RV failure.

Mitochondrial Function in Non-ischemic Heart Failure

Provision for the continuous demand for energy from the beating heart relies heavily on efficient mitochondrial activity. Non-ischemic cardiomyopathy in which oxygen supply is not limiting results from etiologies such as pressure overload. It is associated with progressive development of metabolic stress culminating in energy depletion and heart failure. The mitochondria from the ventricular walls undergoing non-ischemic cardiomyopathy are subjected to long periods of adaptation to support the changing metabolic milieu, which has been described as mal-adaptation since it ultimately results in loss of cardiac contractile function. While the chronicity of exposure to metabolic stressors, co-morbidities and thereby adaptive changes in mitochondria maybe different between ischemic and non-ischemic heart failure, the resulting pathology is very similar, especially in late stage heart failure. Understanding of the mitochondrial changes in early-stage heart failure may guide the development of mitochondrial-targeted therapeutic options to prevent progression of non-ischemic heart failure. This chapter reviews findings of mitochondrial functional changes in animal models and humans with non-ischemic heart failure. While most animal models of non-ischemic heart failure exhibit cardiac mitochondrial dysfunction, studies in humans have been inconsistent despite confirmed reduction in ATP production. This chapter also reviews the possibility of impairment of substrate supply processes upstream of the mitochondria in heart failure, and discusses potential metabolism-targeted therapeutic options.

Polymer Functionalization of Isolated Mitochondria for Cellular Transplantation and Metabolic Phenotype Alteration

Abstract Aberrant mitochondrial energy transfer underlies prevalent chronic health conditions, including cancer, cardiovascular, and neurodegenerative diseases. Mitochondrial transplantation represents an innovative strategy aimed at restoring favorable metabolic phenotypes in cells with dysfunctional energy metabolism. While promising, significant barriers to in vivo translation of this approach abound, including limited cellular uptake and recognition of mitochondria as foreign. The objective is to functionalize isolated mitochondria with a biocompatible polymer to enhance cellular transplantation and eventual in vivo applications. Herein, it is demonstrated that grafting of a polymer conjugate composed of dextran with triphenylphosphonium onto isolated mitochondria protects the organelles and facilitates cellular internalization compared with uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells has profound effects on respiration, mediating a shift toward improved oxidative phosphorylation, and reduced glycolysis. These findings represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.