Heart Failure and MEF2 Transcriptome Dynamics in Response to ß-Blockers.

Myocyte Enhancer Factor 2 (MEF2) mediates cardiac remodelling in heart failure (HF) and is also a target of ß-adrenergic signalling, a front-line treatment for HF. We identified global gene transcription networks involved in HF with and without ß-blocker treatment. Experimental HF by transverse aortic constriction (TAC) in a MEF2 "sensor" mouse model (6 weeks) was followed by four weeks of ß-blockade with Atenolol (AT) or Solvent (Sol) treatment. Transcriptome analysis (RNA-seq) from left ventricular RNA samples and MEF2A depleted cardiomyocytes was performed. AT treatment resulted in an overall improvement in cardiac function of TAC mice and repression of MEF2 activity. RNA-seq identified 65 differentially expressed genes (DEGs) due to TAC treatment with enriched GO clusters including the inflammatory system, cell migration and apoptosis. These genes were mapped against DEGs in cardiomyocytes in which MEF2A expression was suppressed. Of the 65 TAC mediated DEGs, AT reversed the expression of 28 mRNAs. Rarres2 was identified as a novel MEF2 target gene that is upregulated with TAC in vivo and isoproterenol treatment in vitro which may have implications in cardiomyocyte apoptosis and hypertrophy. These studies identify a cohort of genes with vast potential for disease diagnosis and therapeutic intervention in heart failure.

Temporal analysis of mechanisms leading to stimulation of glucose uptake in skeletal muscle cells by an adipokine mixture derived from primary rat adipocytes.

OBJECTIVE: The direct effects of adipokines on skeletal muscle metabolism have been well established. As the combinatorial effects of adipokine mixtures are likely to be of more physiological relevance, we used a coculture system of primary rat adipocytes and L6 skeletal muscle cells to examine the effects of adiponectin derived from primary rat adipocytes on rat skeletal muscle cells. RESULTS: We showed that coculture with adipocytes stimulated glucose uptake in L6 cells within 30 min and this correlated with an increase of glucose transporter isoform 4 (GLUT4) localization to the plasma membrane. These effects were dependent on the reorganization of the actin cytoskeleton, demonstrated by rhodamine-labeled phalloidin immunofluorescence, as cytochalasin D attenuated the glucose uptake induced by adipocyte-conditioned media. Temporal analysis revealed that enhanced glucose uptake was maintained after 24 h of coculture, and this was attributed to an increase in both GLUT1 expression and the cell surface content of GLUT4. We established a role for adiponectin in mediating these effects as antibody-mediated neutralization attenuated the metabolic effects of adipocyte-conditioned media. Furthermore, compound C blocked these effects, suggesting an important role for AMPK. Importantly, when we compared the effects of full-length recombinant adiponectin with adipocyte-conditioned media, we confirmed that recombinant adiponectin was unable to stimulate glucose uptake in L6 cells despite having an important role in adipocyte-conditioned media. CONCLUSIONS: Our results demonstrate the importance of examining the effects of adipokines in the context of physiologically relevant mixtures to accurately determine their metabolic effects on skeletal muscle.

Leptin prevents the metabolic effects of adiponectin in L6 myotubes.

AIMS/HYPOTHESIS: Adiponectin and leptin are negatively and positively correlated with human obesity respectively, and have both been shown to regulate energy metabolism in skeletal muscle. However, little is known about their signalling and functional crosstalk. Here we investigated the effects of leptin on metabolic actions of (1) globular adiponectin (gAd) and (2) full-length adiponectin (fAd) in L6 cells. METHODS: Glucose uptake was measured upon gAd and fAd treatment after incubation with different doses (0.3, 0.6, 3, 6, 60 nmol/l) of leptin for 6, 12 and 24 h. We also measured adiponectin receptor (ADIPOR) expression and stimulation of downstream signalling by gAd and fAd using co-immunoprecipitation and western blotting following leptin pretreatment, as well as analysis of fatty acid uptake and oxidation using radiolabelled tracers. RESULTS: Leptin attenuated the stimulation of glucose uptake by gAd and fAd in a dose- and time-dependent manner, a finding correlated with decreased levels of ADIPOR1 and ADIPOR2. gAd and fAd increased palmitate uptake via activation of AMP protein kinase (T172), enhanced expression of the fatty acid transporter CD36, phosphorylated acetyl-CoA carboxylase (S79) and enhanced palmitate oxidation, all of which were attenuated by leptin pretreatment. Adiponectin can also enhance insulin sensitivity via direct signalling crosstalk; here we show that enhanced insulin-stimulated IRS-1 (Y612) and Akt (T308) phosphorylation in response to fAd was attenuated by leptin. APPL1 was recently identified as a critical mediator of adiponectin action in skeletal muscle. We demonstrated that leptin attenuated binding of APPL1 to LKB1, a downstream target leading to AMPK phosphorylation. CONCLUSIONS/INTERPRETATION: The direct metabolic and insulin-sensitising effects of adiponectin were attenuated in the presence of leptin.