EPA, not DHA, prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4.

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor ß1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.

Omega-3 fatty acids prevent pressure overload-induced cardiac fibrosis through activation of cyclic GMP/protein kinase G signaling in cardiac fibroblasts.

BACKGROUND: Omega-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) from fish oil ameliorate cardiovascular diseases. However, little is known about the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis, a major cause of diastolic dysfunction and heart failure. The present study assessed the effects of ω-3 polyunsaturated fatty acids on cardiac fibrosis. METHODS AND RESULTS: We assessed left ventricular fibrosis and pathology in mice subjected to transverse aortic constriction after the consumption of a fish oil or a control diet. In control mice, 4 weeks of transverse aortic constriction induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation into myofibroblasts). Dietary supplementation with fish oil prevented transverse aortic constriction-induced cardiac dysfunction and cardiac fibrosis and blocked cardiac fibroblast activation. In heart tissue, transverse aortic constriction increased active transforming growth factor-ß1 levels and phosphorylation of Smad2. In isolated adult mouse cardiac fibroblasts, transforming growth factor-ß1 induced cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid increased cyclic GMP levels and blocked cardiac fibroblast transformation, proliferation, and collagen synthesis. Eicosapentaenoic acid and docosahexaenoic acid blocked phospho-Smad2/3 nuclear translocation. DT3, a protein kinase G inhibitor, blocked the antifibrotic effects of eicosapentaenoic acid and docosahexaenoic acid. Eicosapentaenoic acid and docosahexaenoic acid increased phosphorylated endothelial nitric oxide synthase and endothelial nitric oxide synthase protein levels and nitric oxide production. CONCLUSION: Omega-3 fatty acids prevent cardiac fibrosis and cardiac dysfunction by blocking transforming growth factor-ß1-induced phospho-Smad2/3 nuclear translocation through activation of the cyclic GMP/protein kinase G pathway in cardiac fibroblasts.

An association between gene expression and better survival in female mice following myocardial infarction.

Following myocardial infarction, the prognosis for females is better than males. Estrogen is thought to be protective, but clinical trials with hormone replacement failed to show protection. Here, we sought to identify novel mechanisms that might explain this sex-based difference. By diverging from the traditional focus on sex hormones, we employed a conceptually novel approach to this question by using a non-biased approach to measure global changes in gene expression following infarction. We hypothesized that specific gene programs are initiated in the heart following infarction that might account for this sex-based difference. We induced small, medium, and large infarcts in male and female mice and measured changes in gene expression by microarray following infarction. Regardless of infarct size, survival was better in females, while mortality occurred 3-10 days following infarction in males. Two days following infarction, males developed significant ventricular dilation, the best predictor of mortality in humans. Three days following infarction, we measured gene expression by microarray, comparing male versus female and sham versus surgery/infarction. In general, our results indicate a higher relative level of gene induction in females versus males and identified programs for angiogenesis, extracellular matrix remodeling, and immune response. This pattern of gene expression was linked to less pathologic remodeling in female hearts, including increased capillary density and decreased fibrosis. In summary, our results suggest an association between improved survival and less pathologic remodeling and the relative induction of gene expression in females following myocardial infarction.

Nuclear alpha1-adrenergic receptors signal activated ERK localization to caveolae in adult cardiac myocytes.

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.

Protein misfolding and aberrant aggregation are associated with many severe disorders, such as neural degenerative diseases, desmin-related myopathy (DRM), and congestive heart failure. Intrasarcoplasmic amyloidosis and increased ubiquitinated proteins are observed in human failing hearts. The pathogenic roles of these derangements in the heart remain unknown. The ubiquitin-proteasome system (UPS) plays a central role in intracellular proteolysis and regulates critical cellular processes. In cultured cells, aberrant aggregation by a mutant (MT) or misfolded protein impairs the UPS. However, this has not been demonstrated in intact animals, and it is unclear how the UPS is impaired. Cross-breeding UPS reporter mice with a transgenic mouse model of DRM featured by aberrant protein aggregation in cardiomyocytes, we found that overexpression of MT-desmin but not normal desmin protein impairs UPS proteolytic function in the heart. The primary defect does not appear to be in the ubiquitination or the proteolytic activity of the 20S proteasome, because ubiquitinated proteins and the peptidase activities of 20S proteasomes were significantly increased rather than decreased in the DRM heart. Therefore, the defect resides apparently in the entry of ubiquitinated proteins into the 20S proteasome. Consistent with this notion, key components (Rpt3 and Rpt5) of 19S proteasomes were markedly decreased, while major components of 20S proteasomes were increased. Additional experiments with HEK cells suggest that proteasomal malfunction observed in MT-desmin hearts is not secondary to cardiac malfunction or to disruption of desmin filaments. Thus, UPS impairment may represent an important pathogenic mechanism underlying cardiac disorders with abnormal protein aggregation.

Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake.

The presence of increased ubiquitinated proteins and amyloid oligomers in failing human hearts strikingly resembles the characteristic pathology in the brain of many neurodegenerative diseases. The ubiquitin-proteasome system (UPS) is responsible for degradation of most cellular proteins and plays essential roles in virtually all cellular processes. UPS impairment by aberrant protein aggregation was previously shown in cell culture but remains to be demonstrated in intact animals. Mechanisms underlying the impairment are poorly understood. We report here that UPS proteolytic function is severely impaired in the heart of a mouse model of intrasarcoplasmic amyloidosis caused by cardiac-restricted expression of a human desmin-related myopathy-linked missense mutation of alphaB-crystallin (CryAB(R120G)). The UPS impairment was detected before cardiac hypertrophy, and failure became discernible, suggesting that defective protein turnover likely contributes to cardiac remodeling and failure in this model. Further analyses reveal that the impairment is likely attributable to insufficient delivery of substrate proteins into the 20S proteasomes, and depletion of key components of the 19S subcomplex may be responsible. The derangement is likely caused by aberrant protein aggregation rather than loss of function of the CryAB gene because UPS malfunction was not evident in CryAB-null hearts and inhibition of aberrant protein aggregation by Congo red or a heat shock protein significantly attenuated CryAB(R120G)-induced UPS malfunction in cultured cardiomyocytes. Because of the central role of the UPS in cell regulation and the high intrasarcoplasmic amyloidosis prevalence in failing human hearts, our data suggest a novel pathogenic process in cardiac disorders with abnormal protein aggregation.

A novel transgenic mouse model reveals deregulation of the ubiquitin-proteasome system in the heart by doxorubicin.

Ubiquitin-proteasome system (UPS) mediated proteolysis is responsible for the degradation of majority of cellular proteins, thereby playing essential roles in maintaining cellular homeostasis and regulating a number of cellular functions. UPS dysfunction was implicated in the pathogenesis of numerous disorders, including neurodegenerative disease, muscular dystrophy, and a subset of cardiomyopathies. However, monitoring in vivo functional changes of the UPS remains a challenge, which hinders the elucidation of UPS pathophysiology. We have recently created a novel transgenic mouse model that ubiquitously expresses a surrogate protein substrate for the UPS. The present study validates its suitability to monitor in vivo changes of UPS proteolytic function in virtually all major organs. Primary culture of cells derived from the adult transgenic mice was also developed and tested for their applications in probing UPS involvement in pathogenesis. Applying these newly established in vivo and in vitro approaches, we have proven in the present study that doxorubicin enhances UPS function in the heart and in cultured cardiomyocytes, suggesting that UPS hyper-function may play an important role in the acute cardiotoxicity of doxorubicin therapy.

Activation of GPR119 Stimulates Human ß-Cell Replication and Neogenesis in Humanized Mice with Functional Human Islets.

Using humanized mice with functional human islets, we investigated whether activating GPR119 by PSN632408, a small molecular agonist, can stimulate human ß-cell regeneration in vivo. Human islets were transplanted under the left kidney capsule of immunodeficient mice with streptozotocin- (STZ-) induced diabetes. The recipient mice were treated with PSN632408 or vehicle and BrdU daily. Human islet graft function in the mice was evaluated by nonfasting glucose levels, oral glucose tolerance, and removal of the grafts. Immunostaining for insulin, glucagon, and BrdU or Ki67 was performed in islet grafts to evaluate α- and ß-cell replication. Insulin and CK19 immunostaining was performed to evaluate ß-cell neogenesis. Four weeks after human islet transplantation, 71% of PSN632408-treated mice achieved normoglycaemia compared with 24% of vehicle-treated mice. Also, oral glucose tolerance was significantly improved in the PSN632408-treated mice. PSN632408 treatment significantly increased both human α- and ß-cell areas in islet grafts and stimulated α- and ß-cell replication. In addition, ß-cell neogenesis was induced from pancreatic duct cells in the islet grafts. Our results demonstrated that activation of GPR119 increases ß-cell mass by stimulating human ß-cell replication and neogenesis. Therefore, GPR119 activators may qualify as therapeutic agents to increase human ß-cell mass in patients with diabetes.

Involvement of S6 kinase and p38 mitogen activated protein kinase pathways in strain-induced alignment and proliferation of bovine aortic smooth muscle cells.

Bovine aortic smooth muscle cell (SMC) phenotype can be altered by physical forces. This has been demonstrated by cyclic strain-induced changes in proliferation and alignment. However, the intracellular coupling pathways remain ill defined. In the present study, we examined whether the p38 and S6 kinase pathway were involved in the mitogenic and morphological changes seen in SMCs exposed to cyclic strain. We seeded bovine aortic SMCs on silastic membranes that were deformed with 150-mmHg vacuum. Cyclic strain induced both alignment and proliferation of SMCs. SB202190, a specific inhibitor of p38, hindered SMC alignment, but not proliferation. Rapamycin, a specific inhibitor of the mTOR-S6 kinase pathway, attenuated strain-induced proliferation, but not alignment. Peak activation of p38 and S6 kinase was 351 +/- 76.9% at 5 min and 363 +/- 56.2% at 60 min compared with static control, respectively (P < 0.05). The results suggest that strain-induced SMC alignment is dependent on activation of p38, but not S6 kinase. Strain induced SMC proliferation is S6 kinase, but not p38 activation, dependent.

Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase.

PURPOSE: The aim of this study was to investigate the molecular targets of reactive oxygen species (ROS) and to determine whether cyclic strain induces smooth muscle cell (SMC) alignment via the ROS system. We assessed stretch-induced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activation and the redox sensitivity of cyclic strain-stimulated activation of the mitogen-activated protein kinase (MAPK) family. METHODS: SMCs were seeded on flexible collagen I-coated plates and exposed to cyclic strain. NAD(P)H oxidase activation was measured with lucigenin-enhanced chemiluminescent detection of superoxide. Activation of MAPK was detected by determining phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK1/2), and p38 MAPK with immunoblotting. In other experiments, SMCs were exposed to diphenylene iodonium (DPI), an NAD(P)H inhibitor, 30 minutes before stretch. MAPK activation and cell orientation were then assessed. RESULTS: Cyclic strain elicits a rapid increase in intracellular NADH/NADPH oxidase in SMCs. There was also a rapid and robust phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Cyclic strain-induced intracellular NAD(P)H generation was almost completely blocked with DPI. DPI also inhibited the strain-induced phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Both the p38 MAPK specific inhibitor, SB 202190, and DPI blocked cyclic strain-induced cell alignment, but PD98059, an ERK1/2-specific inhibitor, and SP600125, an anthrazolone inhibitor of JNK, did not. CONCLUSION: Our results provide evidence that p38 MAPK is a critical component of the oxidant stress ROS-sensitive signaling pathway and plays a crucial role in vascular alignment induced by cyclic stain.