Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes.

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.

Globular and full-length forms of adiponectin mediate specific changes in glucose and fatty acid uptake and metabolism in cardiomyocytes.

OBJECTIVE: Our aim was to investigate the regulation of glucose and fatty acid metabolism in cardiomyocytes by the globular (gAd) and full-length (fAd) forms of adiponectin. METHODS: We produced fAd (consisting of high, medium and low molecular weight oligomers) in a mammalian expression system and gAd in bacteria. These were used to treat primary neonatal rat cardiomyocytes (up to 48 h), and we employed 3H- or 14C-labeled substrates to monitor glucose uptake and subsequent metabolism via oxidation, glycogen synthesis or lactate production and fatty acid uptake and oxidation. Enzymatic assay for acetyl CoA carboxylase activity was employed, and protein phosphorylation and expression was determined by immunoblotting cell lysates. The role of adiponectin receptor (AdipoR) isoforms was determined via siRNA-mediated knockdown. RESULTS: There was an initial (1 h) increase in glucose uptake and oxidation in response to gAd or fAd. Fatty acid uptake was stimulated by gAd or fAd, and by 24 h a decrease in acetyl CoA carboxylase activity and elevated fatty acid oxidation were observed. After 48 h increased fatty acid oxidation correlated with decreased glucose oxidation and pyruvate dehydrogenase activity, while glycogen synthesis and lactate production increased. Both gAd and fAd elicited phosphorylation of AMP kinase, insulin receptor substrate-1, Akt and glycogen synthase kinase-3beta. Knockdown of AdipoR1 or AdipoR2 attenuated the effect of both gAd and fAd on fatty acid uptake and oxidation. Only AdipoR1 knockdown prevented the ability of gAd (1 h) to increase glucose uptake and oxidation; however, reducing either AdipoR1 or AdipoR2 expression attenuated the long-term (24 h) effects of gAd. CONCLUSIONS: These results clearly demonstrate that gAd and fAd mediate distinct and time-dependent effects on cardiomyocyte energy metabolism via AdipoR1 and AdipoR2.

Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes.

OBJECTIVE: There is a well-documented association between obesity and heart failure although the mechanistic basis for this correlation is unclear. Both extracellular matrix remodeling and left ventricular hypertrophy are well-defined components of remodeling in heart failure, and here we further investigate the role of leptin, the obese gene product, on these parameters. METHODS: We used primary human pediatric ventricular cardiomyocytes combined with gelatin zymography, quantitative PCR analysis, proline and leucine incorporation assays, and investigation of kinase activation by Western blotting. RESULTS: We show using gelatin zymography that leptin dose-dependently (0-60 nM) increased proteolytic activity at approximately 72 kDa. Accordingly, upon quantitative PCR analysis we found that leptin increased expression of matrix metalloproteinase-2 (MMP-2). Leptin also caused an increase in collagen type III and IV mRNA expression and a decrease in collagen type I mRNA expression. This was reflected in no significant change in total collagen synthesis, measured by [3H]proline incorporation, in response to leptin. A statistically significant increase in cell size, [3H]leucine incorporation, and expression of well-characterized markers of cardiac hypertrophy, namely cardiac alpha-actin and myosin light chain, were observed in response to leptin. We demonstrate activation of Janus-activated kinase and mitogen-activated protein kinase pathways by leptin, and using pharmacological inhibitors we show that these signaling pathways play a role in mediating the effects of leptin. CONCLUSIONS: Our findings show that leptin regulates cell size, stimulates MMP-2 expression, and alters the profile, but not the total content, of collagen in human cardiomyocytes. This indicates the potential for altered leptin sensitivity to directly regulate cardiac remodeling in obesity.