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The cardiac extracellular matrix is a complex network of proteins and plays a key role in cardiac remodeling after myocardial infarction. One of the extracellular matrix proteins, asporin, is a member of the SLRP family and inhibits TGFβ signaling. However, very little is known about the function and regulation of asporin in cardiac remodeling. Using bioinformatics approach, microarray data analysis from publicly available datasets identified asporin as a top candidate showing an increase in left ventricle tissue with ischemic cardiomyopathy as compared to control donor hearts. To confirm, we detected an increase in asporin expression in paired heart biopsies taken before and after cardiopulmonary bypass surgery by western blot analysis. Our group showed that glucagon like receptor 1 agonist (GLP-1Ra) is beneficial for cardiac remodeling and prevents fibrosis. In a permanent coronary artery ligation model, we found increased extracellular asporin expression after 30 days in border zone and scar area with GLP-1Ra treatment compared to vehicle-treatment. To further elucidate the mechanism of asporin secretion, we used H9c2 cells as well as left ventricle tissue from mice and found that asporin was predominantly present in the heavy membrane enriched fraction (8,000xg pellet from postnuclear supernatant). Sequence analysis of asporin protein using SecretomeP 2.0 server predicted asporin might use the non-conventional form of secretion (secretory autophagy). Differentiation of H9c2 cells is accompanied by autophagy and mitochondrial biogenesis, and we found that the abundance of asporin in total lysate and heavy membrane fraction decreased suggesting it could be released via secretory autophagy. Using recombinant human asporin, we demonstrated that asporin stimulates an increase in LC3 and p62 in parallel to a decrease in COX IV and SMAD2 phosphorylation, indicating mitophagy induction and a decrease in TGFβ signaling. Based on our preliminary data and sequence comparison to a previously reported human peptide, we developed a rodent asporin peptide which is predicted to induce mitophagy while decreasing TGFβ signaling to prevent fibrosis. Studies are ongoing to establish the clinical relevance of asporin in animal models of ischemic injury.

Pioglitazone (PIO) and GLP-1R receptor agonist (GLP1Ra) have been shown to be cardioprotective against ischemic cardiac injury. Much less is known about the effects of these agents on adverse post-MI LV remodeling. Based on our earlier findings that PIO and a GLP1Ra stimulate mitochondrial turnover, we evaluated the effects of these agents on post-MI remodeling. Lean mice underwent permanent coronary artery ligation (PCAL) to ensure that remodeling would be independent of changes in infarct size. Vehicle, PIO and GLP1Ra (Sigma) were administered i.p. 2h after the infarct and then every other day for a total of 6 doses. Echo and histology (Masson’s trichrome) were used to assess LV remodeling 30 days post-MI (late phase); immune cell infiltration was assessed 7 days after PCAL (early phase) via hematoxylin and eosin (H&E) staining. Both PIO and GLP1Ra were associated with significant improvements in ejection fraction and fractional shortening (A); however, at the dose used, neither drug restored function to that of the sham-operated mice (EF 60% and FS 30%, data not shown). GLP1Ra-treated mice exhibited a marked reduction in immune cell infiltration at 7d after PCAL (B), and fibrosis at 30d (C) compared to vehicle or PIO. While PIO had no effect on immune-cell infiltration and fibrosis, it was effective in attenuating post-MI remodeling. While sharing a common endpoint of attenuating adverse remodeling, the finding that PIO stimulates mitochondrial biogenesis and GLP1Ra induces mitophagy may help explain their disparate findings with respect to early phase remodeling.

MicroRNAs (miRs) regulate post-transcriptional gene expression and protein translation. There is now increasing evidence that miRs regulate mitochondrial turnover. miR-33 regulates cholesterol metabolism and mitochondrial function in cardiac fibroblasts and macrophages and has been implicated in promoting fibrosis. Excessive fibrosis contributes to the development of adverse LV remodeling. Given our recent findings that a glucagon-like peptide-1 receptor agonist (GLP1Ra) [Sigma] induces mitophagy and mitigates post-MI LV remodeling, the purpose of this study was to determine its effects on miR-33 expression after MI. We analyzed miR-33 expression in the viable regions of hearts in lean mice after permanent coronary arterial ligation (PCAL) and subsequent treatment with GLP1Ra administered 2h and 48h after the infarction (total of 2 doses). PCAL was associated with a marked increase in miR-33 that peaked at day 3 (data not shown). In the presence of GLP1Ra, miR-33 expression was suppressed 3 days post-PCAL ( A ). Polysome profiling revealed that miR-33 was decreased in the translating fractions of GLP1Ra-treated mice and increased in the non-translating fraction (80S free ribosomes) compared to vehicle ( B and C ). Decreased association of miR-33 with polysomes in GLP1Ra-treated mice would allow for increased target translation. Given that suppression of miR-33 is implicated in promoting fibrosis, a suppression of this miR by acute GLP1Ra treatment could explain the beneficial effects of this drug in limiting adverse cardiac remodeling.