Low-Magnitude Mechanical Signals Combined with Zoledronic Acid Reduce Musculoskeletal Weakness and Adiposity in Estrogen-Deprived Mice.

Combination treatment of Low-Intensity Vibration (LIV) with zoledronic acid (ZA) was hypothesized to preserve bone mass and muscle strength while reducing adipose tissue accrual associated with complete estrogen (E 2 )-deprivation in young and skeletally mature mice. Complete E 2 -deprivation (surgical-ovariectomy (OVX) and daily injection of aromatase inhibitor (AI) letrozole) were performed on 8-week-old C57BL/6 female mice for 4 weeks following commencement of LIV administration or control (no LIV), for 28 weeks. Additionally, 16-week-old C57BL/6 female E 2 -deprived mice were administered ±LIV twice daily and supplemented with ±ZA (2.5 ng/kg/week). By week 28, lean tissue mass quantified by dual-energy X-ray absorptiometry was increased in younger OVX/AI+LIV(y) mice, with increased myofiber cross-sectional area of quadratus femorii. Grip strength was greater in OVX/AI+LIV(y) mice than OVX/AI(y) mice. Fat mass remained lower in OVX/AI+LIV(y) mice throughout the experiment compared with OVX/AI(y) mice. OVX/AI+LIV(y) mice exhibited increased glucose tolerance and reduced leptin and free fatty acids than OVX/AI(y) mice. Trabecular bone volume fraction and connectivity density increased in the vertebrae of OVX/AI+LIV(y) mice compared to OVX/AI(y) mice; however, this effect was attenuated in the older cohort of E 2 -deprived mice, specifically in OVX/AI+ZA mice, requiring combined LIV with ZA to increase trabecular bone volume and strength. Similar improvements in cortical bone thickness and cross-sectional area of the femoral mid-diaphysis were observed in OVX/AI+LIV+ZA mice, resulting in greater fracture resistance. Our findings demonstrate that the combination of mechanical signals in the form of LIV and anti-resorptive therapy via ZA improve vertebral trabecular bone and femoral cortical bone, increase lean mass, and reduce adiposity in mice undergoing complete E 2 -deprivation. One Sentence Summary: Low-magnitude mechanical signals with zoledronic acid suppressed bone and muscle loss and adiposity in mice undergoing complete estrogen deprivation. Translational Relevance: Postmenopausal patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors to reduce tumor progression experience deleterious effects to bone and muscle subsequently develop muscle weakness, bone fragility, and adipose tissue accrual. Bisphosphonates (i.e., zoledronic acid) prescribed to inhibit osteoclast-mediated bone resorption are effective in preventing bone loss but may not address the non-skeletal effects of muscle weakness and fat accumulation that contribute to patient morbidity. Mechanical signals, typically delivered to the musculoskeletal system during exercise/physical activity, are integral for maintaining bone and muscle health; however, patients undergoing treatments for breast cancer often experience decreased physical activity which further accelerates musculoskeletal degeneration. Low-magnitude mechanical signals, in the form of low-intensity vibrations, generate dynamic loading forces similar to those derived from skeletal muscle contractility. As an adjuvant to existing treatment strategies, low-intensity vibrations may preserve or rescue diminished bone and muscle degraded by breast cancer treatment.

ACVR2B antagonism as a countermeasure to multi-organ perturbations in metastatic colorectal cancer cachexia.

BACKGROUND: Advanced colorectal cancer (CRC) is often accompanied by the development of liver metastases, as well as cachexia, a multi-organ co-morbidity primarily affecting skeletal (SKM) and cardiac muscles. Activin receptor type 2B (ACVR2B) signalling is known to cause SKM wasting, and its inhibition restores SKM mass and prolongs survival in cancer. Using a recently generated mouse model, here we tested whether ACVR2B blockade could preserve multiple organs, including skeletal and cardiac muscle, in the presence of metastatic CRC. METHODS: NSG male mice (8 weeks old) were injected intrasplenically with HCT116 human CRC cells (mHCT116), while sham-operated animals received saline (n = 5-10 per group). Sham and tumour-bearing mice received weekly injections of ACVR2B/Fc, a synthetic peptide inhibitor of ACVR2B. RESULTS: mHCT116 hosts displayed losses in fat mass ( - 79%, P < 0.0001), bone mass ( - 39%, P < 0.05), and SKM mass (quadriceps: - 22%, P < 0.001), in line with reduced muscle cross-sectional area ( - 24%, P < 0.01) and plantarflexion force ( - 28%, P < 0.05). Further, despite only moderately affected heart size, cardiac function was significantly impaired (ejection fraction %: - 16%, P < 0.0001; fractional shortening %: - 25%, P < 0.0001) in the mHCT116 hosts. Conversely, ACVR2B/Fc preserved fat mass ( + 238%, P < 0.001), bone mass ( + 124%, P < 0.0001), SKM mass (quadriceps: + 31%, P < 0.0001), size (cross-sectional area: + 43%, P < 0.0001) and plantarflexion force ( + 28%, P < 0.05) in tumour hosts. Cardiac function was also completely preserved in tumour hosts receiving ACVR2B/Fc (ejection fraction %: + 19%, P < 0.0001), despite no effect on heart size. RNA sequencing analysis of heart muscle revealed rescue of genes related to cardiac development and contraction in tumour hosts treated with ACVR2B/Fc. CONCLUSIONS: Our metastatic CRC model recapitulates the multi-systemic derangements of cachexia by displaying loss of fat, bone, and SKM along with decreased muscle strength in mHCT116 hosts. Additionally, with evidence of severe cardiac dysfunction, our data support the development of cardiac cachexia in the occurrence of metastatic CRC. Notably, ACVR2B antagonism preserved adipose tissue, bone, and SKM, whereas muscle and cardiac functions were completely maintained upon treatment. Altogether, our observations implicate ACVR2B signalling in the development of multi-organ perturbations in metastatic CRC and further dictate that ACVR2B represents a promising therapeutic target to preserve body composition and functionality in 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.

F-box protein-32 down-regulates small-conductance calcium-activated potassium channel 2 in diabetic mouse atria.

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation.

Tumor necrosis factor receptor-associated factor 6 as a nuclear factor kappa B-modulating therapeutic target in cardiovascular diseases: at the heart of it all.

Inflammatory and immune signaling has been documented as a root cause of many cardiovascular pathologies. In this review, we explore the emerging role of tumor necrosis factor receptor-associated factor 6 (TRAF6)-nuclear factor kappa B (NF-κB) signaling axis in atherosclerosis, ischemic heart disease, pathologic cardiac hypertrophy or heart failure, myocarditis, and sepsis-induced cardiomyopathy. We discuss the current understanding of cardiac inflammation in heart disease, present the TRAF6 signaling axis in the heart, then summarize what is known about TRAF6 in pathophysiology of heart disease including proof-of-concept studies that identify the utility of blocking TRAF6 to attenuate cardiac dysfunction, which suggests that TRAF6 is a novel, druggable target in treating cardiovascular disease incurred by inflammatory processes.

Clinical Evidence Supports a Protective Role for CXCL5 in Coronary Artery Disease.

Our goal was to measure the association of CXCL5 and molecular phenotypes associated with coronary atherosclerosis severity in patients at least 65 years old. CXCL5 is classically defined as a proinflammatory chemokine, but its role in chronic inflammatory diseases, such as coronary atherosclerosis, is not well defined. We enrolled individuals who were at least 65 years old and undergoing diagnostic cardiac catheterization. Coronary artery disease (CAD) severity was quantified in each subject via coronary angiography by calculating a CAD score. Circulating CXCL5 levels were measured from plasma, and both DNA genotyping and mRNA expression levels in peripheral blood mononuclear cells were quantified via microarray gene chips. We observed a negative association of CXCL5 levels with CAD at an odds ratio (OR) of 0.46 (95% CI, 0.27-0.75). Controlling for covariates, including sex, statin use, hypertension, hyperlipidemia, obesity, self-reported race, smoking, and diabetes, the OR was not significantly affected [OR, 0.54 (95% CI, 0.31-0.96)], consistent with a protective role for CXCL5 in coronary atherosclerosis. We also identified 18 genomic regions with expression quantitative trait loci of genes correlated with both CAD severity and circulating CXCL5 levels. Our clinical findings are consistent with the emerging link between chemokines and atherosclerosis and suggest new therapeutic targets for CAD.

Kinome and Transcriptome Profiling Reveal Broad and Distinct Activities of Erlotinib, Sunitinib, and Sorafenib in the Mouse Heart and Suggest Cardiotoxicity From Combined Signal Transducer and Activator of Transcription and Epidermal Growth Factor Receptor Inhibition.

BACKGROUND: Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. METHODS AND RESULTS: We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. CONCLUSIONS: Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment.

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.

Effects of the kinase inhibitor sorafenib on heart, muscle, liver and plasma metabolism in vivo using non-targeted metabolomics analysis.

BACKGROUND AND PURPOSE: The human kinome consists of roughly 500 kinases, including 150 that have been proposed as therapeutic targets. Protein kinases regulate an array of signalling pathways that control metabolism, cell cycle progression, cell death, differentiation and survival. It is not surprising, then, that new kinase inhibitors developed to treat cancer, including sorafenib, also exhibit cardiotoxicity. We hypothesized that sorafenib cardiotoxicity is related to its deleterious effects on specific cardiac metabolic pathways given the critical roles of protein kinases in cardiac metabolism. EXPERIMENTAL APPROACH: FVB/N mice (10 per group) were challenged with sorafenib or vehicle control daily for 2 weeks. Echocardiographic assessment of the heart identified systolic dysfunction consistent with cardiotoxicity in sorafenib-treated mice compared to vehicle-treated controls. Heart, skeletal muscle, liver and plasma were flash frozen and prepped for non-targeted GC-MS metabolomics analysis. KEY RESULTS: Compared to vehicle-treated controls, sorafenib-treated hearts exhibited significant alterations in 11 metabolites, including markedly altered taurine/hypotaurine metabolism (25-fold enrichment), identified by pathway enrichment analysis. CONCLUSIONS AND IMPLICATIONS: These studies identified alterations in taurine/hypotaurine metabolism in the hearts and skeletal muscles of mice treated with sorafenib. Interventions that rescue or prevent these sorafenib-induced changes, such as taurine supplementation, may be helpful in attenuating sorafenib-induced cardiac injury.

Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes.

Endogenous formaldehyde is abundantly present in our bodies, at around 100 µM under normal conditions. While such high steady state levels of formaldehyde may be derived by enzymatic reactions including oxidative demethylation/deamination and myeloperoxidation, it is unclear whether endogenous formaldehyde can initiate and/or promote diseases in humans. Here, we show that fluorescent malondialdehyde-formaldehyde (M2FA)-lysine adducts are immunogenic without adjuvants in mice. Natural antibody titers against M2FA are elevated in atherosclerosis-prone mice. Staining with an antibody against M2FA demonstrated that M2FA is present in plaque found on the aortic valve of ApoE -/- mice. To mimic inflammation during atherogenesis, human myeloperoxidase was incubated with glycine, H2O2, malondialdehyde, and a lysine analog in PBS at a physiological temperature, which resulted in M2FA generation. These results strongly suggest that the 1,4-dihydropyridine-type of lysine adducts observed in atherosclerosis lesions are likely produced by endogenous formaldehyde and malondialdehyde with lysine. These highly fluorescent M2FA adducts may play important roles in human inflammatory and degenerative diseases.

MMI-0100 Inhibits Cardiac Fibrosis in a Mouse Model Overexpressing Cardiac Myosin Binding Protein C.

BACKGROUND: Cardiac stress can trigger production of a 40-kDa peptide fragment derived from the amino terminus of the cardiac myosin-binding protein C. Cardiac stress, as well as cMyBP-C mutations, can trigger production of 1 such truncated protein fragment, a 40-kDa peptide fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40-kDa fragment in mouse cardiomyocytes (cMyBP-C40k) leads to cardiac disease, fibrosis, and death within the first year. Fibrosis can occur in many cardiovascular diseases, and mitogen-activated protein kinase--activated protein kinase-2 signaling has been implicated in a variety of fibrotic processes. Recent studies demonstrated that mitogen-activated protein kinase--activated protein kinase-2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C40k cardiomyocyte expression. METHODS AND RESULTS: Nontransgenic and cMyBP-C40k inducible transgenic mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long-term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy, or probability of survival. In the inducible transgenic group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy, and prolonged survival. CONCLUSIONS: Pharmaceutical inhibition of mitogen-activated protein kinase--activated protein kinase-2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic cMyBPC40k disease.

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.

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.

BRG1 and BRM function antagonistically with c-MYC in adult cardiomyocytes to regulate conduction and contractility.

RATIONALE: The contractile dysfunction that underlies heart failure involves perturbations in multiple biological processes ranging from metabolism to electrophysiology. Yet the epigenetic mechanisms that are altered in this disease state have not been elucidated. SWI/SNF chromatin-remodeling complexes are plausible candidates based on mouse knockout studies demonstrating a combined requirement for the BRG1 and BRM catalytic subunits in adult cardiomyocytes. Brg1/Brm double mutants exhibit metabolic and mitochondrial defects and are not viable although their cause of death has not been ascertained. OBJECTIVE: To determine the cause of death of Brg1/Brm double-mutant mice, to test the hypothesis that BRG1 and BRM are required for cardiac contractility, and to identify relevant downstream target genes. METHODS AND RESULTS: A tamoxifen-inducible gene-targeting strategy utilizing αMHC-Cre-ERT was implemented to delete both SWI/SNF catalytic subunits in adult cardiomyocytes. Brg1/Brm double-mutant mice were monitored by echocardiography and electrocardiography, and they underwent rapidly progressive ventricular dysfunction including conduction defects and arrhythmias that culminated in heart failure and death within 3weeks. Mechanistically, BRG1/BRM repressed c-Myc expression, and enforced expression of a DOX-inducible c-MYC trangene in mouse cardiomyocytes phenocopied the ventricular conduction defects observed in Brg1/Brm double mutants. BRG1/BRM and c-MYC had opposite effects on the expression of cardiac conduction genes, and the directionality was consistent with their respective loss- and gain-of-function phenotypes. To support the clinical relevance of this mechanism, BRG1/BRM occupancy was diminished at the same target genes in human heart failure cases compared to controls, and this correlated with increased c-MYC expression and decreased CX43 and SCN5A expression. CONCLUSION: BRG1/BRM and c-MYC have an antagonistic relationship regulating the expression of cardiac conduction genes that maintain contractility, which is reminiscent of their antagonistic roles as a tumor suppressor and oncogene in cancer.

Corticosteroids Are Essential for Maintaining Cardiovascular Function in Male Mice.

Activation of the hypothalamic-pituitary-adrenal axis results in the release of hormones from the adrenal glands, including glucocorticoids and mineralocorticoids. The physiological association between corticosteroids and cardiac disease is becoming increasingly recognized; however, the mechanisms underlying this association are not well understood. To determine the biological effects of corticosteroids on the heart, we investigated the impact of adrenalectomy in C57BL/6 male mice. Animals were adrenalectomized (ADX) at 1 month of age and maintained for 3-6 months after surgery to evaluate the effects of long-term adrenalectomy on cardiac function. Morphological evaluation suggested that ADX mice showed significantly enlarged hearts compared with age-matched intact controls. These changes in morphology correlated with deficits in left ventricular (LV) function and electrocardiogram (ECG) abnormalities in ADX mice. Correlating with these functional defects, gene expression analysis of ADX hearts revealed aberrant expression of a large cohort of genes associated with cardiac hypertrophy and arrhythmia. Combined corticosterone and aldosterone replacement treatment prevented the emergence of cardiac abnormalities in ADX mice, whereas corticosterone replacement prevented the effects of adrenalectomy on LV function but did not block the emergence of ECG alterations. Aldosterone replacement did not preserve the LV function but prevented ECG abnormalities. Together, the data indicate that adrenal glucocorticoids and mineralocorticoids either directly or indirectly have selective effects in the heart and their signaling pathways are essential in maintaining normal cardiac function.

MuRF1 mono-ubiquitinates TRα to inhibit T3-induced cardiac hypertrophy in vivo.

Thyroid hormone (TH) is recognized for its role in cellular metabolism and growth and participates in homeostasis of the heart. T3 activates pro-survival pathways including Akt and mTOR. Treatment with T3 after myocardial infarction is cardioprotective and promotes elements of physiological hypertrophic response after cardiac injury. Although T3 is known to benefit the heart, very little about its regulation at the molecular level has been described to date. The ubiquitin proteasome system (UPS) regulates nuclear hormone receptors such as estrogen, progesterone, androgen, and glucocorticoid receptors by both degradatory and non-degradatory mechanisms. However, how the UPS regulates T3-mediated activity is not well understood. In this study, we aim to determine the role of the muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) in regulating T3-induced cardiomyocyte growth. An increase in MuRF1 expression inhibits T3-induced physiological cardiac hypertrophy, whereas a decrease in MuRF1 expression enhances T3's activity both in vitro and in cardiomyocytes in vivo MuRF1 interacts directly with TRα to inhibit its activity by posttranslational ubiquitination in a non-canonical manner. We then demonstrated that a nuclear localization apparatus that regulates/inhibits nuclear receptors by sequestering them within a subcompartment of the nucleus was necessary for MuRF1 to inhibit T3 activity. This work implicates a novel mechanism that enhances the beneficial T3 activity specifically within the heart, thereby offering a potential target to enhance cardiac T3 activity in an organ-specific manner.

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.

Non-targeted metabolomics of Brg1/Brm double-mutant cardiomyocytes reveals a novel role for SWI/SNF complexes in metabolic homeostasis.

Mammalian SWI/SNF chromatin-remodeling complexes utilize either BRG1 or Brm as alternative catalytic subunits to alter the position of nucleosomes and regulate gene expression. Genetic studies have demonstrated that SWI/SNF complexes are required during cardiac development and also protect against cardiovascular disease and cancer. However, Brm constitutive null mutants do not exhibit a cardiomyocyte phenotype and inducible Brg1 conditional mutations in cardiomyocyte do not demonstrate differences until stressed with transverse aortic constriction, where they exhibit a reduction in cardiac hypertrophy. We recently demonstrated the overlapping functions of Brm and Brg1 in vascular endothelial cells and sought here to test if this overlapping function occurred in cardiomyocytes. Brg1/Brm double mutants died within 21 days of severe cardiac dysfunction associated with glycogen accumulation and mitochondrial defects based on histological and ultrastructural analyses. To determine the underlying defects, we performed nontargeted metabolomics analysis of cardiac tissue by GC/MS from a line of Brg1/Brm double-mutant mice, which lack both Brg1 and Brm in cardiomyocytes in an inducible manner, and two groups of controls. Metabolites contributing most significantly to the differences between Brg1/Brm double-mutant and control-group hearts were then determined using the variable importance in projection analysis. Increased cardiac linoleic acid and oleic acid suggest alterations in fatty acid utilization or intake are perturbed in Brg1/Brm double mutants. Conversely, decreased glucose-6-phosphate, fructose-6-phosphate, and myoinositol suggest that glycolysis and glycogen formation are impaired. These novel metabolomics findings provide insight into SWI/SNF-regulated metabolic pathways and will guide mechanistic studies evaluating the role of SWI/SNF complexes in homeostasis and cardiovascular disease prevention.

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

BACKGROUND: In 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 PPARß 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. METHODS: MuRF2-/- 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, PPARß, and PPARγ1-regulated mRNA expression. RESULTS: MuRF2 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. CONCLUSIONS: Mutations 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.

Muscle ring finger-3 protects against diabetic cardiomyopathy induced by a high fat diet.

BACKGROUND: The pathogenesis of diabetic cardiomyopathy (DCM) involves the enhanced activation of peroxisome proliferator activating receptor (PPAR) transcription factors, including the most prominent isoform in the heart, PPARα. In cancer cells and adipocytes, post-translational modification of PPARs have been identified, including ligand-dependent degradation of PPARs by specific ubiquitin ligases. However, the regulation of PPARs in cardiomyocytes and heart have not previously been identified. We recently identified that muscle ring finger-1 (MuRF1) and MuRF2 differentially inhibit PPAR activities by mono-ubiquitination, leading to the hypothesis that MuRF3 may regulate PPAR activity in vivo to regulate DCM. METHODS: MuRF3-/- mice were challenged with 26 weeks 60% high fat diet to induce insulin resistance and DCM. Conscious echocardiography, blood glucose, tissue triglyceride, glycogen levels, immunoblot analysis of intracellular signaling, heart and skeletal muscle morphometrics, and PPARα, PPARß, and PPARγ1 activities were assayed. RESULTS: MuRF3-/- mice exhibited a premature systolic heart failure by 6 weeks high fat diet (vs. 12 weeks in MuRF3+/+). MuRF3-/- mice weighed significantly less than sibling-matched wildtype mice after 26 weeks HFD. These differences may be largely due to resistance to fat accumulation, as MRI analysis revealed MuRF3-/- mice had significantly less fat mass, but not lean body mass. In vitro ubiquitination assays identified MuRF3 mono-ubiquitinated PPARα and PPARγ1, but not PPARß. CONCLUSIONS: These findings suggest that MuRF3 helps stabilize cardiac PPARα and PPARγ1 in vivo to support resistance to the development of DCM. MuRF3 also plays an unexpected role in regulating fat storage despite being found only in striated muscle.