Identification of Metabolic Changes in Ileum, Jejunum, Skeletal Muscle, Liver, and Lung in a Continuous I.V. Pseudomonas aeruginosa Model of Sepsis Using Nontargeted Metabolomics Analysis.

Sepsis is a multiorgan disease affecting the ileum and jejunum (small intestine), liver, skeletal muscle, and lung clinically. The specific metabolic changes in the ileum, jejunum, liver, skeletal muscle, and lung have not previously been investigated. Live Pseudomonas aeruginosa, isolated from a patient, was given via i.v. catheter to pigs to induce severe sepsis. Eighteen hours later, ileum, jejunum, medial gastrocnemius skeletal muscle, liver, and lung were analyzed by nontargeted metabolomics analysis using gas chromatography/mass spectrometry. The ileum and the liver demonstrated significant changes in metabolites involved in linoleic acid metabolism: the ileum and lung had significant changes in the metabolism of valine/leucine/isoleucine; the jejunum, skeletal muscle, and liver had significant changes in arginine/proline metabolism; and the skeletal muscle and lung had significant changes in aminoacyl-tRNA biosynthesis, as analyzed by pathway analysis. Pathway analysis also identified changes in metabolic pathways unique for different tissues, including changes in the citric acid cycle (jejunum), ß-alanine metabolism (skeletal muscle), and purine metabolism (liver). These findings demonstrate both overlapping metabolic pathways affected in different tissues and those that are unique to others and provide insight into the metabolic changes in sepsis leading to organ dysfunction. This may allow therapeutic interventions that focus on multiple tissues or single tissues once the relationship of the altered metabolites/metabolism to the underlying pathogenesis of sepsis is determined.

Increasing Cardiomyocyte Atrogin-1 Reduces Aging-Associated Fibrosis and Regulates Remodeling in Vivo.

The muscle-specific ubiquitin ligase atrogin-1 (MAFbx) has been identified as a critical regulator of pathologic and physiological cardiac hypertrophy; it regulates these processes by ubiquitinating transcription factors [nuclear factor of activated T-cells and forkhead box O (FoxO) 1/3]. However, the role of atrogin-1 in regulating transcription factors in aging has not previously been described. Atrogin-1 cardiomyocyte-specific transgenic (Tg+) adult mice (α-major histocompatibility complex promoter driven) have normal cardiac function and size. Herein, we demonstrate that 18-month-old atrogin-1 Tg+ hearts exhibit significantly increased anterior wall thickness without functional impairment versus wild-type mice. Histologic analysis at 18 months revealed atrogin-1 Tg+ mice had significantly less fibrosis and significantly greater nuclei and cardiomyocyte cross-sectional analysis. Furthermore, by real-time quantitative PCR, atrogin-1 Tg+ had increased Col 6a4, 6a5, 6a6, matrix metalloproteinase 8 (Mmp8), and Mmp9 mRNA, suggesting a role for atrogin-1 in regulating collagen deposits and MMP-8 and MMP-9. Because atrogin-1 Tg+ mice exhibited significantly less collagen deposition and protein levels, enhanced Mmp8 and Mmp9 mRNA may offer one mechanism by which collagen levels are kept in check in the aged atrogin-1 Tg+ heart. In addition, atrogin-1 Tg+ hearts showed enhanced FoxO1/3 activity. The present study shows a novel link between atrogin-1-mediated regulation of FoxO1/3 activity and reduced collagen deposition and fibrosis in the aged heart. Therefore, targeting FoxO1/3 activity via the muscle-specific atrogin-1 ubiquitin ligase may offer a muscle-specific method to modulate aging-related cardiac fibrosis.

Muscle-specific regulation of right ventricular transcriptional responses to chronic hypoxia-induced hypertrophy by the muscle ring finger-1 (MuRF1) ubiquitin ligase in mice.

BACKGROUND: We recently identified a role for the muscle-specific ubiquitin ligase MuRF1 in right-sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1-/- mice exposed to CH are resistant to right ventricular (RV) dysfunction whereas MuRF1 Tg + mice exhibit impaired function indicative of heart failure. The present study was undertaken to understand the underlying transcriptional alterations in the RV of MuRF1-/- and MuRF1 Tg + mice. METHODS: Microarray analysis was performed on RNA isolated from the RV of MuRF1-/-, MuRF1 Tg+, and wild-type control mice exposed to CH. RESULTS: MuRF1-/- RV differentially expressed 590 genes in response to CH. Analysis of the top 66 genes (> 2-fold or < - 2-fold) revealed significant associations with oxidoreductase, transcription regulation, and transmembrane component annotations. The significant genes had promoters enriched for HOXD12, HOXC13, and RREB-1 protein transcription factor binding sites. MuRF1 Tg + RV differentially expressed 150 genes in response to CH. Analysis of the top 45 genes (> 3-fold or < - 3-fold) revealed significant associations with oxidoreductase-metabolic, glycoprotein-transmembrane-integral proteins, and alternative splicing/splice variant annotations. The significant genes were enriched for promoters with ZIC1 protein transcription factor binding sites. CONCLUSIONS: The differentially expressed genes in MuRF1-/- and MuRF1 Tg + RV after CH have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally-enhanced MuRF1-/- hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects of MuRF1 through regulating Rreb-1, and they reveal mechanisms by which MuRF1 may transcriptionally regulate anti-oxidant systems in the face of right heart failure.

Untargeted metabolomics analysis of ischemia-reperfusion-injured hearts ex vivo from sedentary and exercise-trained rats.

Introduction: The effects of exercise on the heart and its resistance to disease are well-documented. Recent studies have identified that exercise-induced resistance to arrhythmia is due to the preservation of mitochondrial membrane potential. Objectives: To identify novel metabolic changes that occur parallel to these mitochondrial alterations, we performed non-targeted metabolomics analysis on hearts from sedentary and exercise-trained rats challenged with isolated heart ischemia-reperfusion injury (I/R). Methods: Eight-week old Sprague-Dawley rats were treadmill trained 5 days/week for 6 weeks (exercise duration and intensity progressively increased to 1 h at 30 m/min up a 10.5% incline, 75-80% VO2max). The recovery of pre-ischemic function for sedentary rat hearts was 28.8 ± 5.4% (N = 12) compared to exercise trained hearts, which recovered 51.9% ± 5.7 (N = 14) (p < 0.001). Results: Non-targeted GC-MS metabolomics analysis of (1) sedentary rat hearts; (2) exercise-trained rat hearts; (3) sedentary rat hearts challenged with global ischemia-reperfusion (I/R) injury; and (4) exercise-trained rat hearts challenged with global I/R (10/group) revealed 15 statistically significant metabolites between groups by ANOVA using Metaboanalyst (p < 0.001). Enrichment analysis of these metabolites for pathway-associated metabolic sets indicated a > 10-fold enrichment for ammonia recycling and protein biosynthesis. Subsequent comparison of the sedentary hearts post-I/R and exercise-trained hearts post-I/R further identified significant differences in three metabolites (oleic acid, pantothenic acid, and campesterol) related to pantothenate and CoA biosynthesis (p ≤ 1.24E-05, FDR ≤ 5.07E-4). Conclusions: These studies shed light on novel mechanisms in which exercise-induced cardioprotection occurs in I/R that complement both the mitochondrial stabilization and antioxidant mechanisms recently described. These findings also link protein synthesis and protein degradation (protein quality control mechanisms) with exercise-linked cardioprotection and mitochondrial susceptibility for the first time in cardiac I/R.

Cardiac ubiquitin ligases: Their role in cardiac metabolism, autophagy, cardioprotection and therapeutic potential.

Both the ubiquitin-proteasome system (UPS) and the lysosomal autophagy system have emerged as complementary key players responsible for the turnover of cellular proteins. The regulation of protein turnover is critical to cardiomyocytes as post-mitotic cells with very limited regenerative capacity. In this focused review, we describe the emerging interface between the UPS and autophagy, with E3's regulating autophagy at two critical points through multiple mechanisms. Moreover, we discuss recent insights in how both the UPS and autophagy can alter metabolism at various levels, to present new ways to think about therapeutically regulating autophagy in a focused manner to optimize disease-specific cardioprotection, without harming the overall homeostasis of protein quality control. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling.

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype in vivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Exercise training does not affect anthracycline antitumor efficacy while attenuating cardiac dysfunction.

Highly effective anthracyclines, like doxorubicin (DOX), have limited clinical use due to protracted cardiotoxic effects. While exercise is known to be cardioprotective, it is unclear whether exercise compromises chemotherapy treatment efficacy. To determine the effect of exercise training on DOX antitumor efficacy as well as DOX-induced cardiotoxicity, female Fisher 344 rats were randomly assigned to sedentary + saline (SED+SAL), SED+DOX, wheel run exercise training + SAL (WR+SAL), or WR+DOX. On week 11, animals were inoculated with 1×10(6) MatBIII tumor cells. Once tumors reached ∼1 cm in diameter, animals were treated with 12 mg/kg of DOX or SAL. Animals were killed 1, 3, or 5 days following treatment. Tumor growth and cardiac function were measured at each interval. DOX accumulation and multidrug resistance protein (MRP) expression were quantified in tumor and heart tissue. No significant difference (P > 0.05) existed between DOX-treated SED and WR groups for tumor measurements. Exercise preserved cardiac function up to 5 days following DOX treatment. Exercise reduced ventricular DOX accumulation and upregulated ventricular MPR1 and MPR2. In contrast, no differences were observed in DOX accumulation or MRP expression in tumors of SED and WR animals. Endurance exercise had no effect on DOX antitumor efficacy as evidenced by a definitive DOX-induced reduction in tumor growth in both the SED and WR groups. Although exercise did not affect the antitumor efficacy of DOX, it still provided protection against cardiac dysfunction. These effects may be mediated by the degree of DOX tissue accumulation secondary to the regulation of MRP expression.

Endurance exercise attenuates cardiotoxicity induced by androgen deprivation and doxorubicin.

Doxorubicin (DOX) is associated with cardiac dysfunction and irreversible testicular damage. Androgen deprivation therapy (ADT) is administered prior to DOX treatment to preserve testicular function. However, ADT may exacerbate DOX-induced cardiac dysfunction. Exercise is cardioprotective, but the effects of exercise on cardiac function during combined ADT and DOX treatment are currently unknown. In this study, male Sprague-Dawley rats were randomly assigned to experimental groups: control (CON), ADT, DOX, or ADT+DOX. Animals received ADT or control implants on days 1 and 29 of the 56-day protocol. Animals remained sedentary (SED) or engaged in treadmill endurance exercise (TM) beginning on day 1. On day 15, the animals received DOX at 1 mg·(kg body mass)(-1)·d(-1) by intraperitoneal injection for 10 consecutive days, or an equivalent volume of saline. On day 57, cardiac function was assessed in vivo and ex vivo. Animals treated with DOX alone, or with combined ADT+DOX, showed significant (P < 0.05) reductions in left ventricular developed pressure (-21% and -27%), maximal rate of pressure development (-29% and -32%), and maximal rate of pressure decline (25% and 31%), respectively when compared with the sedentary control animals. Endurance exercise training attenuated (P > 0.05) cardiac dysfunction associated with combined ADT+DOX treatment, indicating that exercise during simultaneous ADT+DOX treatment is cardioprotective.

The pathophysiology of cancer-mediated cardiac cachexia and novel treatment strategies: A narrative review.

SIGNIFICANCE: Two of the leading causes of death worldwide are cancer and cardiovascular diseases. Most cancer patients suffer from a metabolic wasting syndrome known as cancer-induced cardiac cachexia, resulting in death in up to 30% of cancer patients. Main symptoms of this disease are severe cardiac muscle wasting, cardiac remodeling, and cardiac dysfunction. Metabolic alterations, increased inflammation, and imbalance of protein homeostasis contribute to the progression of this multifactorial syndrome, ultimately resulting in heart failure and death. Cancer-induced cardiac cachexia is associated with decreased quality of life, increased fatiguability, and decreased tolerance to therapeutic interventions. RECENT ADVANCES: While molecular mechanisms of this disease are not fully understood, researchers have identified different stages of progression of this disease, as well as potential biomarkers to detect and monitor the development. Preclinical and clinical studies have shown positive results when implementing certain pharmacological and non-pharmacological therapy interventions. CRITICAL ISSUES: There are still no clear diagnostic criteria for cancer-mediated cardiac cachexia and the condition remains untreated, leaving cancer patients with irreversible effects of this syndrome. While traditional cardiovascular therapy interventions, such as beta-blockers, have shown some positive results in preclinical and clinical research studies, recent preclinical studies have shown more successful results with certain non-traditional treatment options that have not been further evaluated yet. There is still no clinical standard of care or approved FDA drug to aid in the prevention or treatment of cancer-induced cardiac cachexia. This review aims to revisit the still not fully understood pathophysiological mechanisms of cancer-induced cardiac cachexia and explore recent studies using novel treatment strategies. FUTURE DIRECTIONS: While research has progressed, further investigations might provide novel diagnostic techniques, potential biomarkers to monitor the progression of the disease, as well as viable pharmacological and non-pharmacological treatment options to increase quality of life and reduce cancer-induced cardiac cachexia-related mortality.

Skeletal muscle omics signatures in cancer cachexia: perspectives and opportunities.

Cachexia is a life-threatening complication of cancer that occurs in up to 80% of patients with advanced cancer. Cachexia reflects the systemic consequences of cancer and prominently features unintended weight loss and skeletal muscle wasting. Cachexia impairs cancer treatment tolerance, lowers quality of life, and contributes to cancer-related mortality. Effective treatments for cancer cachexia are lacking despite decades of research. High-throughput omics technologies are increasingly implemented in many fields including cancer cachexia to stimulate discovery of disease biology and inform therapy choice. In this paper, we present selected applications of omics technologies as tools to study skeletal muscle alterations in cancer cachexia. We discuss how comprehensive, omics-derived molecular profiles were used to discern muscle loss in cancer cachexia compared with other muscle-wasting conditions, to distinguish cancer cachexia from treatment-related muscle alterations, and to reveal severity-specific mechanisms during the progression of cancer cachexia from early toward severe disease.

Updated systematic review of the effects of exercise on understudied health outcomes in cancer survivors.

INTRODUCTION: The American College of Sports Medicine provided guidelines for exercise prescriptions in cancer survivors for specific cancer- and treatment-related health outcomes. However, there was insufficient evidence to generate exercise prescriptions for 10 health outcomes of cancer treatment. We sought to update the state of evidence. METHODS: We conducted a systematic review of these 10 understudied health outcomes (bone health, sleep, cardiovascular function, chemotherapy-induced peripheral neuropathy (CIPN), cognitive function, falls and balance, nausea, pain, sexual function, and treatment tolerance) and provided an update of evidence. RESULTS: While the evidence base for each outcome has increased, there remains insufficient evidence to generate exercise prescriptions. Common limitations observed across outcomes included: variability in type and quality of outcome measurement tools, variability in definitions of the health outcomes, a lack of phase III trials, and a majority of trials investigating breast or prostate cancer survivors only. CONCLUSION: We identified progress in the field of exercise oncology for several understudied cancer- and treatment-related health outcomes. However, we were not able to generate exercise prescriptions due to continued insufficient evidence base. More work is needed to prescribe exercise as medicine for these understudied health outcomes, and our review highlights several strategies to aid in research acceleration within these areas of exercise oncology.

Cardioprotective effects of preconditioning exercise in the female tumor bearing mouse.

Cancer cachexia, a metabolic wasting syndrome, affects up to 80% of cancer patients and leads to the death in up to 20% of cancer patients. While research is growing in the field, there are still no clear diagnostic criteria and cancer cachexia remains an untreated condition. Aerobic exercise has been shown to positively impact cachexia by slowing its development and attenuating muscle loss. The most effective timing, duration, and intensity of exercise as a preventative and protective measure against cancer cachexia remains questionable. Therefore, the purpose of this study was to examine the effects of preconditioning exercise as a protective measure for tumor-mediated muscle wasting. Female LC3 Tg+ and wildtype mice were randomly separated into four groups, sedentary non-tumor bearing (SED + NT), sedentary tumor bearing (SED + T), treadmill exercise non-tumor bearing (TM + NT), and treadmill exercise tumor bearing (TM + T). Mice underwent an 8-week treadmill exercise training protocol (TM) or remained sedentary (SED). Next, mice were implanted with tumor cells (T group; 5 × 105 Lewis Lung Carcinoma cells in flank) or remained non-tumor (NT) for 4 weeks. Tumor bearing resulted in a significant decline in cardiac function. SED + T showed a significant decrease in fractional shortening (p < 0.05) when compared to the other groups. This coincided with an increase in beclin-1 and MyD88 protein expression and decrease in p-FOXO1 (inactivated) protein expression in SED + T mice. Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to preserve cardiac function (TM + T not significantly different than SED + NT). Exercise-mediated cardioprotection also coincided with abolished beclin-1 and MyD88 signaling that was not significantly elevated in TM + T mice. Additionally, TM resulted in a 22-fold decrease in estimated tumor volume (p < 0.05) and a 45% decrease in tumor mass (p < 0.05) compared to SED tumors. The data indicate potential cardioprotective effects of preconditioning exercise on preserving cardiac structure and function, as well as regulating autophagic (beclin-1), inflammatory (TGF-ß and MyD88), and atrophy (p-FOXO1) pathways during tumor bearing. Preconditioning exercise may be an effective and accessible treatment intervention for early-stage cancer survivors. This data is crucial in identifying the significance of exercise and the timing of exercise as a protective measure against the detrimental effects of cancer cachexia.

Doxorubicin Exposure Causes Subacute Cardiac Atrophy Dependent on the Striated Muscle-Specific Ubiquitin Ligase MuRF1.

Background Anthracycline chemotherapeutics, such as doxorubicin, are used widely in the treatment of numerous malignancies. The primary dose-limiting adverse effect of anthracyclines is cardiotoxicity that often presents as heart failure due to dilated cardiomyopathy years after anthracycline exposure. Recent data from animal studies indicate that anthracyclines cause cardiac atrophy. The timing of onset and underlying mechanisms are not well defined, and the relevance of these findings to human disease is unclear. Methods and Results Wild-type mice were sacrificed 1 week after intraperitoneal administration of doxorubicin (1-25 mg/kg), revealing a dose-dependent decrease in cardiac mass ( R2=0.64; P<0.0001) and a significant decrease in cardiomyocyte cross-sectional area (336±29 versus 188±14 µm2; P<0.0001). Myocardial tissue analysis identified a dose-dependent upregulation of the ubiquitin ligase, MuRF1 (muscle ring finger-1; R2=0.91; P=0.003) and a molecular profile of muscle atrophy. To investigate the determinants of doxorubicin-induced cardiac atrophy, we administered doxorubicin 20 mg/kg to mice lacking MuRF1 (MuRF1-/-) and wild-type littermates. MuRF1-/- mice were protected from cardiac atrophy and exhibited no reduction in contractile function. To explore the clinical relevance of these findings, we analyzed cardiac magnetic resonance imaging data from 70 patients in the DETECT-1 cohort and found that anthracycline exposure was associated with decreased cardiac mass evident within 1 month and persisting to 6 months after initiation. Conclusions Doxorubicin causes a subacute decrease in cardiac mass in both mice and humans. In mice, doxorubicin-induced cardiac atrophy is dependent on MuRF1. These findings suggest that therapies directed at preventing or reversing cardiac atrophy might preserve the cardiac function of cancer patients receiving anthracyclines.

Effects of voluntary wheel running on goserelin acetate-induced bone degeneration.

A common treatment option for many breast and prostate cancer patients is the use of a luteinizing hormone-releasing hormone agonist such as goserelin acetate (GA) which reduces sex hormone levels. This treatment, however, is associated with bone degeneration, and exercise has been suggested as a means of preventing this side effect. Little is known about the effects of low intensity, low volume exercise on GA-induced bone loss. The purpose of this study, therefore, was to investigate the effects of voluntary wheel running on bone architecture in growing male (M) and female (F) rats receiving GA treatment. Rats received an 8-week GA treatment or placebo (CON) and were either housed in cages equipped with voluntary running wheels (WR) or remained sedentary (SED) in standard cages throughout the experimental period. Following treatments, tibiae were excised and analyzed for cortical bone (cross-sectional volume, cortical volume, marrow volume, cortical thickness) and cancellous bone (bone volume/total volume, trabecular number, trabecular thickness, trabecular spacing) using micro-computed tomography. Treatment with GA resulted in a significant reduction in running wheel distances in both sexes throughout the study period (P<0.05). GA treatment had no effect on cortical bone architecture in neither sex (P>0.05). Cancellous bone degeneration, however, was observed in M and F SED+GA (P<0.05). No significant differences were observed in M WR+GA animals in bone volume/total volume, trabecular number and trabecular spacing when compared to M SED+CON (P>0.05). In F WR+GA, trabecular thickness did not differ from that of F SED+CON (P>0.05), and trabecular spacing was found to be significantly lower than F SED+GA (P<0.05). The current report indicates that 8 weeks of GA treatment promotes cancellous bone degeneration, and voluntary wheel running provides no clear osteoprotection in growing male and female rats.

Exercise Protects against Cancer-induced Cardiac Cachexia.

Cancer has been shown to negatively stimulate autophagy, leading to a decline in cardiac function. Although exercise is cardioprotective, its influence over autophagy-mediated tumor growth and cardiac function are not well defined. PURPOSE: This study aimed to determine the effect of exercise on tumor morphology and cardiac function. METHODS: Fisher 344 rats (n = 28) were assigned to one of four groups: 1) sedentary non-tumor bearing (SED), 2) sedentary tumor bearing (SED + T), 3) wheel run non-tumor bearing (WR), or 4) wheel run tumor bearing (WR + T). Rats remained sedentary or exercised for 6 wk. At week 4, rats in tumor groups were inoculated with MatBIII tumor cells. At week 6, cardiac function was measured. RESULTS: SED + T animals exhibited significantly lower left ventricular developed pressure when compared with SED, WR, and WR + T (P < 0.05). This coincided with a significant increase in cardiac autophagy (increased LC3-II) in SED + T animals when compared with SED, WR, and WR + T (P < 0.05). Furthermore, SED + T hearts showed a significant increase in ß-myosin heavy chain expression versus nontumor groups (P < 0.05). Tumor mass was significantly larger (P < 0.001) in SED + T animals when compared with WR + T animals, which was accompanied by a significant increase in tumor LC3-II protein expression (P < 0.05). CONCLUSION: Nonexercised tumor-bearing rats showed severe cardiac dysfunction and excessive, maladaptive autophagy in the heart and tumors. Voluntary exercise preserved cardiac function and attenuated the autophagic response in heart and tumor tissues. This preservation may be related to the reduced tumor growth in aerobically exercised rats, to the improved regulation of autophagy by exercise, or both.

Adverse Effects of Fenofibrate in Mice Deficient in the Protein Quality Control Regulator, CHIP.

We previously reported how the loss of CHIP expression (Carboxyl terminus of Hsc70-Interacting Protein) during pressure overload resulted in robust cardiac dysfunction, which was accompanied by a failure to maintain ATP levels in the face of increased energy demand. In this study, we analyzed the cardiac metabolome after seven days of pressure overload and found an increase in long-chain and medium-chain fatty acid metabolites in wild-type hearts. This response was attenuated in mice that lack expression of CHIP (CHIP-/-). These findings suggest that CHIP may play an essential role in regulating oxidative metabolism pathways that are regulated, in part, by the nuclear receptor PPARα (Peroxisome Proliferator-Activated Receptor alpha). Next, we challenged CHIP-/- mice with the PPARα agonist called fenofibrate. We found that treating CHIP-/- mice with fenofibrate for five weeks under non-pressure overload conditions resulted in decreased skeletal muscle mass, compared to wild-type mice, and a marked increase in cardiac fibrosis accompanied by a decrease in cardiac function. Fenofibrate resulted in decreased mitochondrial cristae density in CHIP-/- hearts as well as decreased expression of genes involved in the initiation of autophagy and mitophagy, which suggests that a metabolic challenge, in the absence of CHIP expression, impacts pathways that contribute to mitochondrial quality control. In conclusion, in the absence of functional CHIP expression, fenofibrate results in unexpected skeletal muscle and cardiac pathologies. These findings are particularly relevant to patients harboring loss-of-function mutations in CHIP and are consistent with a prominent role for CHIP in regulating cardiac metabolism.

Ubiquitin Ligases and Posttranslational Regulation of Energy in the Heart: The Hand that Feeds.

Heart failure (HF) is a costly and deadly syndrome characterized by the reduced capacity of the heart to adequately provide systemic blood flow. Mounting evidence implicates pathological changes in cardiac energy metabolism as a contributing factor in the development of HF. While the main source of fuel in the healthy heart is the oxidation of fatty acids, in the failing heart the less energy efficient glucose and glycogen metabolism are upregulated. The ubiquitin proteasome system plays a key role in regulating metabolism via protein-degradation/regulation of autophagy and regulating metabolism-related transcription and cell signaling processes. In this review, we discuss recent research that describes the role of the ubiquitin-proteasome system (UPS) in regulating metabolism in the context of HF. We focus on ubiquitin ligases (E3s), the component of the UPS that confers substrate specificity, and detail the current understanding of how these E3s contribute to cardiac pathology and metabolism. © 2017 American Physiological Society. Compr Physiol 7:841-862, 2017.

Exercise-Induced Alterations in Skeletal Muscle, Heart, Liver, and Serum Metabolome Identified by Non-Targeted Metabolomics Analysis.

BACKGROUND: The metabolic and physiologic responses to exercise are increasingly interesting, given that regular physical activity enhances antioxidant capacity, improves cardiac function, and protects against type 2 diabetes. The metabolic interactions between tissues and the heart illustrate a critical cross-talk we know little about. METHODS: To better understand the metabolic changes induced by exercise, we investigated skeletal muscle (plantaris, soleus), liver, serum, and heart from exercise trained (or sedentary control) animals in an established rat model of exercise-induced aerobic training via non-targeted GC-MS metabolomics. RESULTS: Exercise-induced alterations in metabolites varied across tissues, with the soleus and serum affected the least. The alterations in the plantaris muscle and liver were most alike, with two metabolites increased in each (citric acid/isocitric acid and linoleic acid). Exercise training additionally altered nine other metabolites in the plantaris (C13 hydrocarbon, inosine/adenosine, fructose-6-phosphate, glucose-6-phosphate, 2-aminoadipic acid, heptadecanoic acid, stearic acid, alpha-tocopherol, and oleic acid). In the serum, we identified significantly decreased alpha-tocopherol levels, paralleling the increases identified in plantaris muscle. Eleven unique metabolites were increased in the heart, which were not affected in the other compartments (malic acid, serine, aspartic acid, myoinositol, glutamine, gluconic acid-6-phosphate, glutamic acid, pyrophosphate, campesterol, phosphoric acid, creatinine). These findings complement prior studies using targeted metabolomics approaches to determine the metabolic changes in exercise-trained human skeletal muscle. Specifically, exercise trained vastus lateralus biopsies had significantly increased linoleic acid, oleic acid, and stearic acid compared to the inactive groups, which were significantly increased in plantaris muscle in the present study. CONCLUSIONS: While increases in alpha-tocopherol have not been identified in muscle after exercise to our knowledge, the benefits of vitamin E (alpha-tocopherol) supplementation in attenuating exercise-induced muscle damage has been studied extensively. Skeletal muscle, liver, and the heart have primarily different metabolic changes, with few similar alterations and rare complementary alterations (alpha-tocopherol), which may illustrate the complexity of understanding exercise at the organismal level.

Post-translationally modified muscle-specific ubiquitin ligases as circulating biomarkers in experimental cancer cachexia.

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Up to 80% of cancer patients experience cachexia, with 20-30% of cancer-related deaths directly linked to cachexia. Despite efforts to identify early cachexia and cancer relapse, clinically useful markers are lacking. Recently, we identified the role of muscle-specific ubiquitin ligases Atrogin-1 (MAFbx, FBXO32) and Muscle Ring Finger-1 in the pathogenesis of cardiac atrophy and hypertrophy. We hypothesized that during cachexia, the Atrogin-1 and MuRF1 ubiquitin ligases are released from muscle and migrate to the circulation where they could be detected and serve as a cachexia biomarker. To test this, we induced cachexia in mice using the C26 adenocarcinoma cells or vehicle (control). Body weight, tumor volume, and food consumption were measured from inoculation until ~day 14 to document cachexia. Western blot analysis of serum identified the presence of Atrogin-1 and MuRF1 with unique post-translational modifications consistent with mono- and poly- ubiquitination of Atrogin-1 and MuRF1 found only in cachectic serum. These findings suggest that both increased Atrogin-1 and the presence of unique post-translational modifications may serve as a surrogate marker specific for cachexia.

Non-Targeted Metabolomics Analysis of the Effects of Tyrosine Kinase Inhibitors Sunitinib and Erlotinib on Heart, Muscle, Liver and Serum Metabolism In Vivo.

Background: More than 90 tyrosine kinases have been implicated in the pathogenesis of malignant transformation and tumor angiogenesis. Tyrosine kinase inhibitors (TKIs) have emerged as effective therapies in treating cancer by exploiting this kinase dependency. The TKI erlotinib targets the epidermal growth factor receptor (EGFR), whereas sunitinib targets primarily vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR).TKIs that impact the function of non-malignant cells and have on- and off-target toxicities, including cardiotoxicities. Cardiotoxicity is very rare in patients treated with erlotinib, but considerably more common after sunitinib treatment. We hypothesized that the deleterious effects of TKIs on the heart were related to their impact on cardiac metabolism. Methods: Female FVB/N mice (10/group) were treated with therapeutic doses of sunitinib (40 mg/kg), erlotinib (50 mg/kg), or vehicle daily for two weeks. Echocardiographic assessment of the heart in vivo was performed at baseline and on Day 14. Heart, skeletal muscle, liver and serum were flash frozen and prepped for non-targeted GC-MS metabolomics analysis. Results: Compared to vehicle-treated controls, sunitinib-treated mice had significant decreases in systolic function, whereas erlotinib-treated mice did not. Non-targeted metabolomics analysis of heart identified significant decreases in docosahexaenoic acid (DHA), arachidonic acid (AA)/ eicosapentaenoic acid (EPA), O-phosphocolamine, and 6-hydroxynicotinic acid after sunitinib treatment. DHA was significantly decreased in skeletal muscle (quadriceps femoris), while elevated cholesterol was identified in liver and elevated ethanolamine identified in serum. In contrast, erlotinib affected only one metabolite (spermidine significantly increased). Conclusions: Mice treated with sunitinib exhibited systolic dysfunction within two weeks, with significantly lower heart and skeletal muscle levels of long chain omega-3 fatty acids docosahexaenoic acid (DHA), arachidonic acid (AA)/eicosapentaenoic acid (EPA) and increased serum O-phosphocholine phospholipid. This is the first link between sunitinib-induced cardiotoxicity and depletion of the polyunsaturated fatty acids (PUFAs) and inflammatory mediators DHA and AA/EPA in the heart. These compounds have important roles in maintaining mitochondrial function, and their loss may contribute to cardiac dysfunction.