Sympathetic activation causes focal adhesion signaling alteration in early compensated volume overload attributable to isolated mitral regurgitation in the dog

Abstract

We reported that left ventricular (LV) dilatation after 4 weeks of isolated mitral regurgitation (MR) in the dogs is marked by extracellular matrix loss and an increase in adrenergic drive. Given that extracellular matrix proteins and their receptor integrins influence beta-adrenergic receptor (beta-AR) responses in vitro, we tested whether beta1-AR activation modulates focal adhesion (FA) signaling and LV remodeling in these same dogs with isolated MR. Normal dogs were compared with dogs with MR of a 4-week duration and with MR dogs treated with beta(1)-AR blockade (beta(1)-RB) (extended-release metoprolol succinate, 100 mg QD) that was started 24 hours after MR induction. In MR LVs, a decrease in collagen accumulation compared with normal dogs was associated with a decrease in FA kinase tyrosine phosphorylation, along with FA kinase interaction with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively, as determined by immunoprecipitation assays. There was increased phosphorylation of stress related molecules p38 mitogen-activated protein kinase (MAPK) and Hsp27 and survival signaling kinases extracellular signal-regulated kinase 1/2 and AKT, with no evidence of cardiomyocyte apoptosis. beta(1)-RB attenuated FA signaling loss and prevented p38 MAPK, Hsp27, and AKT phosphorylation induced by MR and significantly increased LV epicardial collagen content. However, beta(1)-RB did not improve LV endocardial collagen loss or LV dilatation induced by MR. Isolated myocytes from normal and MR dog hearts treated with beta(1)- or beta(2)-AR agonists demonstrated no difference in FA kinase, p38 MAPK, Hsp27, or AKT phosphorylation. These results showed that chronic stimulation of beta(1)-AR during early compensated MR impairs FA signaling that may affect myocyte/fibroblast-extracellular matrix scaffolding necessary for LV remodeling.

Figure 1

Volume percent collagen of LV endocardium and epicardium in control, 4W-MR, and 4W-MR+β₁-RB dogs. *P<0.05 vs control; #P<0.05, epicardium in 4W-MR vs. 4W-MR+β₁-RB dogs.

Figure 2. β₁-RB prevents FAK tyrosine downregulation induced by MR

(A) Left ventricular extracts from control, 4W-MR, and 4W-MR+β₁-RB dogs were immunoprecipitated (IP) with anti-FAK antibodies and immunoblotted with anti-phosphotyrosine antibodies. Top, representative autoradiogram (with each lane from a single gel exposed for the same duration). Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR. (B) representative immunoblots showing accumulation of phospho-FAK Tyr-397, -576/577, -861, and -925 in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blots were stripped and blotted with anti-FAK antibodies.

Figure 3. Alteration of FAK downstream signaling induced by MR is prevented by β₁-RB

(A) FAK immunoprecipitates from LV lysates of control, 4W-MR, and 4W-MR+β₁-RB dogs were resolved on SDS-PAGE and blotted with anti-phosphotyrosine, p130^Cas, paxillin, or FAK antibodies. (B–C) p130^Cas or paxillin immunoprecipitates from control, 4W-MR, or 4W-MR+β₁-RB LV extracts were resolved on SDS-PAGE and blotted with anti-phosphotyrosine, anti-p130^Cas (B), or anti-paxillin (C) antibodies. Top, representative autoradiograms (with each lane from a single gel exposed for the same duration). Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR. (D) Representative immunoblot showing accumulation of phospho-Pyk2 Tyr-402 in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blot was stripped and blotted with anti-Pyk2 antibodies.

Figure 3. Alteration of FAK downstream signaling induced by MR is prevented by β₁-RB

(A) FAK immunoprecipitates from LV lysates of control, 4W-MR, and 4W-MR+β₁-RB dogs were resolved on SDS-PAGE and blotted with anti-phosphotyrosine, p130^Cas, paxillin, or FAK antibodies. (B–C) p130^Cas or paxillin immunoprecipitates from control, 4W-MR, or 4W-MR+β₁-RB LV extracts were resolved on SDS-PAGE and blotted with anti-phosphotyrosine, anti-p130^Cas (B), or anti-paxillin (C) antibodies. Top, representative autoradiograms (with each lane from a single gel exposed for the same duration). Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR. (D) Representative immunoblot showing accumulation of phospho-Pyk2 Tyr-402 in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blot was stripped and blotted with anti-Pyk2 antibodies.

Figure 4. Differential activation of ERK_1/2, p38 MAPK, and JNK after MR

Top, representative immunoblots showing accumulation of phospho-p38-MAPK, -ERK_1/2, and -JNK in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blots were stripped and blotted with anti-p38-MAPK, -ERK_1/2, or -JNK antibodies. Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR.

Figure 4. Differential activation of ERK_1/2, p38 MAPK, and JNK after MR

Top, representative immunoblots showing accumulation of phospho-p38-MAPK, -ERK_1/2, and -JNK in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blots were stripped and blotted with anti-p38-MAPK, -ERK_1/2, or -JNK antibodies. Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR.

Figure 5. Induction of MR is not associated with myocardial cell apoptosis

LV homogenates from control, 4W-MR, and 4W-MR+β₁-RB groups were assessed for apoptosis. (A) Caspase-3 activity was measured using fluorogenic substrate. Results are expressed as relative fluorescence unit (RFU)/min/mg protein. (B) DNA fragmentation as measured by anti-histone antibody ELISA. Results are expressed as relative OD410-OD500/mg protein. Values are mean ±SE. (C) Morphometry of TUNEL staining of LV sections from control, 4W-MR, and 4W-MR+β₁-RB.

Figure 5. Induction of MR is not associated with myocardial cell apoptosis

LV homogenates from control, 4W-MR, and 4W-MR+β₁-RB groups were assessed for apoptosis. (A) Caspase-3 activity was measured using fluorogenic substrate. Results are expressed as relative fluorescence unit (RFU)/min/mg protein. (B) DNA fragmentation as measured by anti-histone antibody ELISA. Results are expressed as relative OD410-OD500/mg protein. Values are mean ±SE. (C) Morphometry of TUNEL staining of LV sections from control, 4W-MR, and 4W-MR+β₁-RB.

Figure 6. MR-induced AKT and Hsp27 phosphorylation is prevented by β₁-RB

Top, representative immunoblots showing accumulation of phospho-AKT and -Hsp27 in LV extracts from control, 4W-MR, and 4W-MR+β₁-RB. Blots were stripped and reblotted with Anti-AKT or -Hsp27 antibodies. Bottom, fold induction, n=6 each group, *P<0.05 4W-MR vs. control, #P<0.05 4W-MR+β₁-RB vs. 4W-MR.

Figure 7. Preservation of β-AR signaling in MR isolated cardiomyocytes

Cardiac myocytes were isolated from control (A) or 4W-MR (B) LV as described in materials and methods. (A, B) representative immunoblots showing accumulation of phospho-FAK, -p38-MAPK, -Hsp27, and -AKT after treatment with 10µmol/L isoproterenol (Iso) or 1µmol/L Zinterol (Zint) for 5-minutes compared with control (Ctrl): 1µmol/L of CGP20712A was added 30-minutes before isoproterenol stimulation. (C) Summary graphs represent quantitative data from 4 independent experiments from cells derived from 4 different animals. *P<0.05 vs. control.

Figure 7. Preservation of β-AR signaling in MR isolated cardiomyocytes

Cardiac myocytes were isolated from control (A) or 4W-MR (B) LV as described in materials and methods. (A, B) representative immunoblots showing accumulation of phospho-FAK, -p38-MAPK, -Hsp27, and -AKT after treatment with 10µmol/L isoproterenol (Iso) or 1µmol/L Zinterol (Zint) for 5-minutes compared with control (Ctrl): 1µmol/L of CGP20712A was added 30-minutes before isoproterenol stimulation. (C) Summary graphs represent quantitative data from 4 independent experiments from cells derived from 4 different animals. *P<0.05 vs. control.