Traci L. Parry

Functional Amyloid Signaling via the Inflammasome, Necrosome, and Signalosome: New Therapeutic Targets in Heart Failure

As the most common cause of death and disability, globally, heart disease remains an incompletely understood enigma. A growing number of cardiac diseases are being characterized by the presence of misfolded proteins underlying their pathophysiology, including cardiac amyloidosis and dilated cardiomyopathy (DCM). At least nine precursor proteins have been implicated in the development of cardiac amyloidosis, most commonly caused by multiple myeloma light chain disease and disease-causing mutant or wildtype transthyretin (TTR). Similarly, aggregates with PSEN1 and COFILIN-2 have been identified in up to one-third of idiopathic DCM cases studied, indicating the potential predominance of misfolded proteins in heart failure. In this review, we present recent evidence linking misfolded proteins mechanistically with heart failure and present multiple lines of new therapeutic approaches that target the prevention of misfolded proteins in cardiac TTR amyloid disease. These include multiple small molecule pharmacological chaperones now in clinical trials designed specifically to support TTR folding by rational design, such as tafamidis, and chaperones previously developed for other purposes, such as doxycycline and tauroursodeoxycholic acid. Last, we present newly discovered non-pathological “functional” amyloid structures, such as the inflammasome and necrosome signaling complexes, which can be activated directly by amyloid. These may represent future targets to successfully attenuate amyloid-induced proteotoxicity in heart failure, as the inflammasome, for example, is being therapeutically inhibited experimentally in autoimmune disease. Together, these studies demonstrate multiple novel points in which new therapies may be used to primarily prevent misfolded proteins or to inhibit their downstream amyloid-mediated effectors, such as the inflammasome, to prevent proteotoxicity in heart failure.

MuRF2 regulates PPARγ1 activity to protect against diabetic cardiomyopathy and enhance weight gain induced by a high fat diet.

BackgroundIn diabetes mellitus the morbidity and mortality of cardiovascular disease is increased and represents an important independent mechanism by which heart disease is exacerbated. The pathogenesis of diabetic cardiomyopathy involves the enhanced activation of PPAR transcription factors, including PPARα, and to a lesser degree PPARβ and PPARγ1. How these transcription factors are regulated in the heart is largely unknown. Recent studies have described post-translational ubiquitination of PPARs as ways in which PPAR activity is inhibited in cancer. However, specific mechanisms in the heart have not previously been described. Recent studies have implicated the muscle-specific ubiquitin ligase muscle ring finger-2 (MuRF2) in inhibiting the nuclear transcription factor SRF. Initial studies of MuRF2−/− hearts revealed enhanced PPAR activity, leading to the hypothesis that MuRF2 regulates PPAR activity by post-translational ubiquitination.MethodsMuRF2−/− mice were challenged with a 26-week 60% fat diet designed to simulate obesity-mediated insulin resistance and diabetic cardiomyopathy. Mice were followed by conscious echocardiography, blood glucose, tissue triglyceride, glycogen levels, immunoblot analysis of intracellular signaling, heart and skeletal muscle morphometrics, and PPARα, PPARβ, and PPARγ1-regulated mRNA expression.ResultsMuRF2 protein levels increase ~20% during the development of diabetic cardiomyopathy induced by high fat diet. Compared to littermate wildtype hearts, MuRF2−/− hearts exhibit an exaggerated diabetic cardiomyopathy, characterized by an early onset systolic dysfunction, larger left ventricular mass, and higher heart weight. MuRF2−/− hearts had significantly increased PPARα- and PPARγ1-regulated gene expression by RT-qPCR, consistent with MuRF2’s regulation of these transcription factors in vivo. Mechanistically, MuRF2 mono-ubiquitinated PPARα and PPARγ1 in vitro, consistent with its non-degradatory role in diabetic cardiomyopathy. However, increasing MuRF2:PPARγ1 (>5:1) beyond physiological levels drove poly-ubiquitin-mediated degradation of PPARγ1 in vitro, indicating large MuRF2 increases may lead to PPAR degradation if found in other disease states.ConclusionsMutations in MuRF2 have been described to contribute to the severity of familial hypertrophic cardiomyopathy. The present study suggests that the lack of MuRF2, as found in these patients, can result in an exaggerated diabetic cardiomyopathy. These studies also identify MuRF2 as the first ubiquitin ligase to regulate cardiac PPARα and PPARγ1 activities in vivo via post-translational modification without degradation.

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.

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 E3s 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.

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.

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.

Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo

Background: Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production. Methods: We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle. Results: Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10−3), carnosine (0.40-fold of controls, p = 1.88 × 10−2), fumaric acid (0.40-fold of controls, p = 7.40 × 10−4), lactamide (0.33-fold of controls, p = 4.84 × 10−2), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10−2), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10−2), glutamic acid (2.48-fold of controls, p = 2.63 × 10−2), and proline (1.73-fold of controls, p = 3.01 × 10−2). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10−4, FDR 4.7 × 10−2), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction. In contrast, two pathways, inosine-5′-monophosphate (VIP Score 3.91) and 3-phosphoglyceric acid (VIP Score 3.08) mainly contributed to the LDE signature, with two metabolites (phosphoglyceric acid and inosine-5′-monophosphate) being significantly decreased. When the BF and LDE were compared, the most significant metabolite was phosphoric acid, which was significantly less in the GRMD BF compared to control and GRMD LDE groups. Conclusions: The identification of elevated BF oleic acid (a long-chain fatty acid) is consistent with recent microarray studies identifying altered lipid metabolism genes, while alterations in arginine and proline metabolism are consistent with recent studies identifying elevated L-arginine in DMD patient sera as a biomarker of disease. Together, these studies demonstrate muscle-specific alterations in GRMD-affected muscle, which illustrate previously unidentified metabolic changes.

Fenofibrate unexpectedly induces cardiac hypertrophy in mice lacking MuRF1.

The muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) is critical in regulating both pathological and physiological cardiac hypertrophy in vivo. Previous work from our group has identified MuRF1s ability to inhibit serum response factor and insulin-like growth factor-1 signaling pathways (via targeted inhibition of cJun as underlying mechanisms). More recently, we have identified that MuRF1 inhibits fatty acid metabolism by targeting peroxisome proliferator-activated receptor alpha (PPARα) for nuclear export via mono-ubiquitination. Since MuRF1-/- mice have an estimated fivefold increase in PPARα activity, we sought to determine how challenge with the PPARα agonist fenofibrate, a PPARα ligand, would affect the heart physiologically. In as little as 3 weeks, feeding with fenofibrate/chow (0.05% wt/wt) induced unexpected pathological cardiac hypertrophy not present in age-matched sibling wild-type (MuRF1+/+) mice, identified by echocardiography, cardiomyocyte cross-sectional area, and increased beta-myosin heavy chain, brain natriuretic peptide, and skeletal muscle α-actin mRNA. In addition to pathological hypertrophy, MuRF1-/- mice had an unexpected differential expression in genes associated with the pleiotropic effects of fenofibrate involved in the extracellular matrix, protease inhibition, hemostasis, and the sarcomere. At both 3 and 8 weeks of fenofibrate treatment, the differentially expressed MuRF1-/- genes most commonly had SREBP-1 and E2F1/E2F promoter regions by TRANSFAC analysis (54 and 50 genes, respectively, of the 111 of the genes >4 and <-4 log fold change; P ≤ .0004). These studies identify MuRF1s unexpected regulation of fenofibrates pleiotropic effects and bridges, for the first time, MuRF1s regulation of PPARα, cardiac hypertrophy, and hemostasis.

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.

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.