Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-ß signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.

Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction.

Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48+/-3 in wt and 45+/-3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.

In vivo matrix metalloproteinase-7 substrates identified in the left ventricle post-myocardial infarction using proteomics.

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

Cardiorenal effects of adenosine subtype 1 (A1) receptor inhibition in an experimental model of heart failure.

BACKGROUND: Elaboration of a number of bioactive substances, including adenosine, occurs in heart failure (HF). Adenosine, through the adenosine subtype 1 (A1) receptor, can reduce renal perfusion pressure and glomerular filtration rate and increase tubular sodium reabsorption, which can affect natriuresis and aquaresis. Accordingly, the present study examined the acute effects of selective A1 receptor blockade on hemodynamics and renal function in a model of HF. STUDY DESIGN: HF was induced in adult pigs (n = 19) by chronic pacing (240 beats/min for 3 weeks). The pigs were then instrumented for hemodynamic and renal function measurements. After baseline measurements were taken, pigs received either A1 block [ 1 mg/kg BG9719 (1,3-dipropyl-8-[2(5,6-epoxynorbornyl)]xanthine; n = 9)] or infusion of vehicle (n = 10), and measurements were repeated at intervals for up to 2 hours. Normal controls (n = 7) were included for comparison. RESULTS: Cardiac output remained unchanged between the A1 block and vehicle groups throughout the study. Pulmonary vascular resistance fell 38% from baseline at 10 minutes post-A1 block in the HF group (p < 0.05) with no change in the vehicle group. At 10 minutes post-A1 block, urine flow increased sixfold and sodium excretion increased over 10-fold (for both, p < 0.05) with no change in the vehicle group. At 10 minutes post-A1 block, creatinine clearance increased with no change in the vehicle group. At 10 minutes post-A1 block, plasma renin activity had increased over threefold (p <0.05), and it returned to baseline levels by 30 minutes post-A1 block. CONCLUSIONS: The unique findings from this study were threefold. First, increased A1 receptor activation contributes to renal mediated fluid retention in HF. Second, selective A1 blockade can induce diuresis without hemodynamic compromise and with possible benefit to pulmonary resistance in a model of HF. A1 blockade transiently increased plasma renin activity with no change in hemodynamics. These unique results suggest that selective A1 blockade can be a useful adjunctive diuretic in the setting of HF.