Set7 Deletion Prevents Glucose Intolerance and Improves the Recovery of Cardiac Function After Ischemia and Reperfusion in Obese Female Mice.

BACKGROUND/AIMS: An obesogenic diet (high fat and sugar, low fiber) is associated with an increased risk for metabolic and cardiovascular disorders. Previous studies have demonstrated that epigenetic changes can modify gene transcription and protein function, playing a key role in the development of several diseases. The methyltransferase Set7 methylates histone and non-histone proteins, influencing diverse biological and pathological processes. However, the functional role of Set7 in obesity-associated metabolic and cardiovascular complications is unknown. METHODS: Wild type and Set7 knockout female mice were fed a normal diet or an obesogenic diet for 12 weeks. Body weight gain and glucose tolerance were measured. The 3T3-L1 cells were used to determine the role of Set7 in white adipogenic differentiation. Cardiac morphology and function were evaluated by histology and echocardiography. An ex vivo Langendorff perfusion system was used to model cardiac ischemia/reperfusion (I/R). RESULTS: Here, we report that Set7 protein levels were enhanced in the heart and perigonadal adipose tissue (PAT) of female mice fed an obesogenic diet. Significantly, loss of Set7 prevented obesogenic diet-induced glucose intolerance in female mice although it did not affect the obesogenic diet-induced increase in body weight gain and adiposity in these animals, nor did Set7 inhibition change white adipogenic differentiation in vitro. In addition, loss of Set7 prevented the compromised cardiac functional recovery following ischemia and reperfusion (I/R) injury in obesogenic diet-fed female mice; however, deletion of Set7 did not influence obesogenic diet-induced cardiac hypertrophy nor the hemodynamic and echocardiographic parameters. CONCLUSION: These data indicate that Set7 plays a key role in obesogenic diet-induced glucose intolerance and compromised myocardial functional recovery after I/R in obese female mice.

Set7 deletion attenuates isoproterenol-induced cardiac fibrosis and delays cardiac dysfunction.

Cardiovascular diseases are the main cause of death worldwide. Recent studies have revealed the influence of histone-modifying enzymes in cardiac remodeling and heart dysfunction. The Set7 methyltransferase regulates the expression of several genes through the methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in cardiac remodeling and heart dysfunction remains unknown. To address this question, wild-type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol or saline. WT mice injected with isoproterenol displayed a decrease in Set7 activity in the heart. In addition, WT and Set7 KO mice injected with isoproterenol exhibited cardiac hypertrophy. Interestingly, Set7 deletion exacerbated cardiac hypertrophy in response to isoproterenol but attenuated myocardial fibrosis. Echocardiograms revealed that WT mice injected with isoproterenol had lowered ejection fractions and fractional shortening, and increased E'-wave deceleration time and E/A ratio compared with their controls. Conversely, Set7 KO mice did not show alteration in these parameters in response to isoproterenol. However, prolonged exposure to isoproterenol induced cardiac dysfunction both in WT and Set7 KO mice. Both isoproterenol and Set7 deletion changed the transcriptional profile of the heart. Moreover, Set7 deletion increased the expression of Pgc1α and mitochondrial DNA content in the heart, and reduced the expression of cellular senescence and inflammation markers in response to isoproterenol. Taken together, our data suggest that Set7 deletion attenuates isoproterenol-induced myocardial fibrosis and delays heart dysfunction, suggesting that Set7 plays an important role in cardiac remodeling and dysfunction in response to stress.

The miRNA-143-3p-Sox6-Myh7 pathway is altered in obesogenic diet-induced cardiac hypertrophy.

NEW FINDINGS: What is the central question of this study? What is the effect of an obesogenic diet on the expression of microRNAs (miRNAs) involved in cardiac hypertrophy in female mice? What is the main finding and its importance? Female mice fed an obesogenic diet exhibited cardiac hypertrophy associated with increased levels of miRNA-143-3p, decreased mRNA levels of Sox6 and increased mRNA levels of Myh7. Inhibition of miRNA-143-3p increased Sox6 mRNA levels and reduced Myh7 expression in cardiomyocytes, and prevented angiotensin II-induced cardiomyocyte hypertrophy. The results indicate that the miRNA-143-3p-Sox6-Myh7 pathway may play a key role in obesity-induced cardiac hypertrophy. ABSTRACT: Obesity induces cardiometabolic disorders associated with a high risk of mortality. We have previously shown that the microRNA (miRNA) expression profile is changed in obesity-induced cardiac hypertrophy in male mice. Here, we investigated the effect of an obesogenic diet on the expression of miRNAs involved in cardiac hypertrophy in female mice. Female mice fed an obesogenic diet displayed an increased body weight gain, glucose intolerance, insulin resistance and dyslipidaemia. In addition, obese female mice exhibited cardiac hypertrophy associated with increased levels of several miRNAs, including miR-143-3p. Bioinformatic analysis identified Sox6, regulator of Myh7 gene transcription, as a predicted target of miR-143-3p. Female mice fed an obesogenic diet exhibited decreased mRNA levels of Sox6 and increased expression of Myh7 in the heart. Loss-of-function studies in cardiomyocytes revealed that inhibition of miR-143-3p increased Sox6 mRNA levels and reduced Myh7 expression. Collectively, our results indicate that obesity-associated cardiac hypertrophy in female mice is accompanied by alterations in diverse miRNAs, and suggest that the miR-143-3p-Sox6-Myh7 pathway may play a key role in obesity-induced cardiac hypertrophy.

Deletion of miRNA-22 Induces Cardiac Hypertrophy in Females but Attenuates Obesogenic Diet-Mediated Metabolic Disorders.

BACKGROUND/AIMS: Obesity is a risk factor associated with cardiometabolic complications. Recently, we reported that miRNA-22 deletion attenuated high-fat diet-induced adiposity and prevented dyslipidemia without affecting cardiac hypertrophy in male mice. In this study, we examined the impact of miRNA-22 in obesogenic diet-induced cardiovascular and metabolic disorders in females. METHODS: Wild type (WT) and miRNA-22 knockout (miRNA-22 KO) females were fed a control or an obesogenic diet. Body weight gain, adiposity, glucose tolerance, insulin tolerance, and plasma levels of total cholesterol and triglycerides were measured. Cardiac and white adipose tissue remodeling was assessed by histological analyses. Echocardiography was used to evaluate cardiac function and morphology. RNA-sequencing analysis was employed to characterize mRNA expression profiles in female hearts. RESULTS: Loss of miRNA-22 attenuated body weight gain, adiposity, and prevented obesogenic diet-induced insulin resistance and dyslipidemia in females. WT obese females developed cardiac hypertrophy. Interestingly, miRNA-22 KO females displayed cardiac hypertrophy without left ventricular dysfunction and myocardial fibrosis. Both miRNA-22 deletion and obesogenic diet changed mRNA expression profiles in female hearts. Enrichment analysis revealed that genes associated with regulation of the force of heart contraction, protein folding and fatty acid oxidation were enriched in hearts of WT obese females. In addition, genes related to thyroid hormone responses, heart growth and PI3K signaling were enriched in hearts of miRNA-22 KO females. Interestingly, miRNA-22 KO obese females exhibited reduced mRNA levels of Yap1, Egfr and Tgfbr1 compared to their respective controls. CONCLUSION: This study reveals that miRNA-22 deletion induces cardiac hypertrophy in females without affecting myocardial function. In addition, our findings suggest miRNA-22 as a potential therapeutic target to treat obesity-related metabolic disorders in females.

Mutations of Cys and Ser residues in the α5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan.

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.

INTERPLAY BETWEEN BRAIN OXYGENATION AND THE DEVELOPMENT OF HYPOTHERMIA IN ENDOTOXIC SHOCK.

ABSTRACT: There is evidence to suggest that the hypothermia observed in the most severe cases of systemic inflammation or sepsis is a regulated response with potential adaptive value, but the mechanisms involved are poorly understood. Here, we investigated the interplay between brain oxygenation (assessed by tissue P o2 ) and the development of hypothermia in unanesthetized rats challenged with a hypotension-inducing dose of bacterial LPS (1 mg/kg i.v.). At an ambient temperature of 22°C, oxygen consumption (V̇O 2 ) began to fall only a few minutes after the LPS injection, and this suppression in metabolic rate preceded the decrease in core temperature. No reduction in brain P o2 was observed prior to the development of the hypometabolic, hypothermic response, ruling out the possibility that brain hypoxia served as a trigger for hypothermia in this model. Brain P o2 was even increased. Such an improvement in brain oxygenation could reflect either an increased O 2 delivery or a decreased O 2 consumption. The former explanation seems unlikely because blood flow (cardiac output) was being progressively decreased during the recording period. On the other hand, the decrease in V̇O 2 usually preceded the rise in P o2 , and an inverse correlation between V̇O 2 and brain P o2 was consistently observed. These findings do not support the existence of a closed-loop feedback relationship between brain oxygenation and hypothermia in systemic inflammation. The data are consistent with a feedforward mechanism in which hypothermia is triggered (possibly by cryogenic inflammatory mediators) in anticipation of changes in brain oxygenation to prevent the development of tissue hypoxia.

miRNA-22 is involved in the aortic reactivity in physiological conditions and mediates obesity-induced perivascular adipose tissue dysfunction.

AIMS: Blood vessels are surrounded by perivascular adipose tissue (PVAT), which plays an important role in vascular tonus regulation due to its anticontractile effect; however, this effect is impaired in obesity. We previously demonstrated that miRNA-22 is involved in obesity-related metabolic disorders. However, the impact of miRNA-22 on vascular reactivity and PVAT function is unknown. AIM: To investigate the role of miRNA-22 on vascular reactivity and its impact on obesity-induced PVAT dysfunction. MAIN METHODS: Wild-type and miRNA-22 knockout (KO) mice were fed a control or a high-fat (HF) diet. To characterize the vascular response, concentration-responses curves to noradrenaline were performed in PVAT- or PVAT+ thoracic aortic rings in absence and presence of L-NAME. Expression of adipogenic and thermogenic markers and NOS isoforms were evaluated by western blotting or qPCR. KEY FINDINGS: HF diet and miRNA-22 deletion reduced noradrenaline-induced contraction in PVAT- aortic rings. Additionally, miRNA-22 deletion increased noradrenaline-induced contraction in PVAT+ aortic rings without affecting its sensitivity; however, this effect was not observed in miRNA-22 KO mice fed a HF diet. Interestingly, miRNA-22 deletion reduced the contraction of aortic rings to noradrenaline via a NOS-dependent mechanism. Moreover, HF diet abolished the NOS-mediated anticontractile effect of PVAT, which was attenuated by miRNA-22 deletion. Mechanistically, we found that PVAT from miRNA-22 KO mice fed a HF diet presented increased protein expression of nNOS. SIGNIFICANCE: These results suggest that miRNA-22 is important for aorta reactivity under physiological circumstances and its deletion attenuates the loss of the NOS-mediated anticontractile effect of PVAT in obesity.

Ablation of miRNA-22 protects against obesity-induced adipocyte senescence and ameliorates metabolic disorders in middle-aged mice.

High-fat diet (HFD) promotes obesity-related metabolic complications by activating cellular senescence in white adipose tissue (WAT). Growing evidence supports the importance of microRNA-22 (miR-22) in metabolic disorders and cellular senescence. Recently, we showed that miR-22 deletion attenuates obesity-related metabolic abnormalities. However, whether miR-22 mediates HFD-induced cellular senescence of WAT remains unknown. Here, we uncovered that obese mice displayed increased pri-miR-22 levels and cellular senescence in WAT. However, miR-22 ablation protected mice against HFD-induced WAT senescence. In addition, in vitro studies showed that miR-22 deletion prevented preadipocyte senescence in response to Doxorubicin (Doxo). Loss-of-function studies in vitro and in vivo revealed that miR-22 increases H2ax mRNA and γH2ax levels in preadipocytes and WAT without inducing DNA damage. Intriguingly, miR-22 ablation prevented HFD-induced increase in γH2ax levels and DNA damage in WAT. Similarly, miR-22 deletion prevented Doxo-induced increase in γH2ax levels in preadipocytes. Adipose miR-22 levels were enhanced in middle-aged mice fed a HFD than those found in young mice. Furthermore, miR-22 deletion attenuated fat mass gain and glucose imbalance induced by HFD in middle-aged mice. Overall, our findings indicate that miR-22 is a key regulator of obesity-induced WAT senescence and metabolic disorders in middle-aged mice.

miRNA-22 deletion limits white adipose expansion and activates brown fat to attenuate high-fat diet-induced fat mass accumulation.

BACKGROUND: Obesity, characterized by excessive expansion of white adipose tissue (WAT), is associated with numerous metabolic complications. Conversely, brown adipose tissue (BAT) and beige fat are thermogenic tissues that protect mice against obesity and related metabolic disorders. We recently reported that deletion of miR-22 enhances energy expenditure and attenuates WAT expansion in response to a high-fat diet (HFD). However, the molecular mechanisms involved in these effects mediated by miR-22 loss are unclear. METHODS AND RESULTS: Here, we show that miR-22 expression is induced during white, beige, and brown adipocyte differentiation in vitro. Deletion of miR-22 reduced white adipocyte differentiation in vitro. Loss of miR-22 prevented HFD-induced expression of adipogenic/lipogenic markers and adipocyte hypertrophy in murine WAT. In addition, deletion of miR-22 protected mice against HFD-induced mitochondrial dysfunction in WAT and BAT. Loss of miR-22 induced WAT browning. Gain- and loss-of-function studies revealed that miR-22 did not affect brown adipogenesis in vitro. Interestingly, miR-22 KO mice fed a HFD displayed increased expression of genes involved in thermogenesis and adrenergic signaling in BAT when compared to WT mice fed the same diet. CONCLUSIONS: Collectively, our findings suggest that loss of miR-22 attenuates fat accumulation in response to a HFD by reducing white adipocyte differentiation and increasing BAT activity, reinforcing miR-22 as a potential therapeutic target for obesity-related disorders.

Angiotensin II type 2 receptor mediates high fat diet-induced cardiomyocyte hypertrophy and hypercholesterolemia.

Obesity is the major risk factor for several cardiovascular and metabolic disorders. Previous studies reported that deletion of Angiotensin II type 2 receptor (AT2R) protects against metabolic dysfunctions induced by high fat (HF) diet. However, the role of AT2R in obesity-induced cardiac hypertrophy remains unclear. Male AT2R knockout (AT2RKO) and wild type (AT2RWT) mice were fed with control or HF diet for 10 weeks. HF diet increased cardiac expression of AT2R in obese mice. Deletion of AT2R did not affect body weight gain, glucose intolerance and fat mass gain induced by HF feeding. However, loss of AT2R prevented HF diet-induced hypercholesterolemia and cardiac remodeling. Mechanistically, we found that pharmacological inhibition or knockdown of AT2R prevented leptin-induced cardiomyocyte hypertrophy in vitro. Collectively, our results suggest that AT2R is involved in obesity-induced cardiac hypertrophy.

Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders.

Suboptimal intrauterine conditions as changed hormone levels during critical periods of the development are considered an insult and implicate in physiological adaptations which may result in pathological outcomes in later life. This study evaluated the effect of maternal hyperthyroidism (hyper) on cardiac function in adult offspring and the possible involvement of cardiac Renin-Angiotensin System (RAS) in this process. Wistar dams received orally thyroxin (12 mg/L) from gestational day 9 (GD9) until GD18. Adult offspring at postnatal day 90 (PND90) from hyper dams presented increased SBP evaluated by plethysmography and worse recovery after ischemia-reperfusion (I/R), as evidenced by decreased LVDP, +dP/dT and -dP/dT at 25 min of reperfusion and by increased infarct size. Increased cardiac Angiotensin I/II levels and AT1R in hyper offspring were verified. Herein, we provide evidences that maternal hyperthyroidism leads to altered expression of RAS components in adult offspring, which may be correlated with worse recovery of the cardiac performance after ischemic insults and hypertension.

Maternal hyperthyroidism alters the pattern of expression of cardiac renin-angiotensin system components in rat offspring.

INTRODUCTION: Changes in perinatal environment can lead to physiological, morphological, or metabolic alterations in adult life. It is well known that thyroid hormones (TH) are critical for the development, growth, and maturation of organs and systems. In addition, TH interact with the renin-angiotensin system (RAS), and both play a critical role in adult cardiovascular function. The objective of this study was to evaluate the effect of maternal hyperthyroidism on cardiac RAS components in pups during development. MATERIALS AND METHODS: From gestational day nine (GD9), pregnant Wistar rats received thyroxine (T4, 12 mg/l in tap water; Hyper group) or vehicle (control group). Dams and pups were killed on GD18 and GD20. RESULTS: Serum concentrations of triiodothyronine (T3) and T4 were higher in the Hyper group than in the control group dams. Cardiac hypertrophy was observed in Hyper pups on GD20. Cardiac angiotensin-converting enzyme (ACE) activity was significantly lower in Hyper pups on both GD18 and GD20, but there was no difference in Ang I/Ang II levels. Ang II receptors expression was higher in the Hyper pup heart on GD18. CONCLUSIONS: Maternal hyperthyroidism is associated with alterations in fetal development and altered pattern of expression in RAS components, which in addition to cardiac hypertrophy observed on GD20 may represent an important predisposing factor to cardiovascular diseases in adult life.