Beta1-adrenergic receptors promote focal adhesion signaling downregulation and myocyte apoptosis in acute volume overload

Abstract

Numerous studies demonstrated increased expression of extracellular matrix (ECM) proteins and activation of focal adhesion (FA) signaling pathways in models of pressure overload-induced cardiac hypertrophy. However, little is known about FA signaling in response to volume overload where cardiac hypertrophy is associated with ECM loss. This study examines the role of beta1-adrenergic receptors (β(1)-ARs) in FA signaling changes and myocyte apoptosis induced during acute hemodynamic stress of volume overload. Rats with eccentric cardiac hypertrophy induced after aorto-caval fistula (ACF) develop reduced interstitial collagen content and decreased tyrosine phosphorylation of key FA signaling molecules FAK, Pyk(2) and paxillin along with an increase in cardiac myocyte apoptosis. ACF also increased activation of PTEN, a dual lipid and protein phosphatase, and its interaction with FA proteins. β(1)-AR blockade (extended-release of metoprolol succinate, 100mg QD) markedly attenuated PTEN activation, restored FA signaling and reduced myocyte apoptosis induced by ACF at 2days, but failed to reduce interstitial collagen loss and left ventricular dilatation. Treating cultured myocytes with β(1)-AR agonists or adenoviral expression of β(1)-ARs caused PTEN activation and interaction with FA proteins, thus leading to FA signaling downregulation and myocyte apoptosis. Adenoviral-mediated expression of a catalytically inactive PTEN mutant or wild-type FAK restored FA signaling downregulation and attenuated myocyte apoptosis induced by β(1)-ARs. Collectively, these data show that β(1)-AR stimulation in response to ACF induces FA signaling downregulation through an ECM-independent mechanism. This effect involves PTEN activation and may contribute to adverse cardiac remodeling and function in the course of volume overload.

Figure 1. Acute ACF induces FA signaling downregulation

(A) Top, Representative picrosirius red staining.(Bar = 40 μM) Bottom, Interstitial collagen accumulation as determined by morphometric analysis. (B–C) LV extracts from control and ACF operated rats were assessed for immunoblot analysis. Top, representative immunoblots (with each lane from a single gel exposed for the same duration). Bottom, fold induction, n=6 each group, *P<0.05 vs. sham. (D) LV lysates from sham or ACF animals were immunoprecipitated (IP) with anti-FAK antibodies and immunoblotted with anti-phosphotyrosine, p130^Cas, paxillin or FAK antibodies.

Figure 2. β₁-RB attenuates FA signaling downregulation induced after acute ACF

Sham (Sh) and ACF animals were treated with vehicle or β₁-RB for 2-days. (A) Ejection fraction. (B) Heart weight to body weight ratio (HW/BW). (C) Top: Picrosirius red stained LV sections. (Bar = 40 μM) Bottom: Interstitial collagen volume quantification. Values are mean ±SEM. *P<0.05 vs. sham (n=6 for each experimental group) (D–E) Representative immunoblot showing accumulation of Y³⁹⁷-FAK and FAK (D) or Y⁵⁸⁰-SHP₂, SHP₂, non-phospho (np)-PTEN, and PTEN (E). 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 vs. sham, †P<0.05 vs. ACF. (F) PTEN immunoprecipitates (IP) were immunoblotted with anti-FAK or -PTEN antibodies.

Figure 3. β₁-RB attenuates myocyte apoptosis induced after acute ACF

(A) Number of TUNEL-positive myocytes was expressed as a percentage of total nuclei detected by DAPI staining. (B–C) LV homogenates from shams or animals subjected to ACF treated with vehicle or β₁-RB were assayed for DNA fragmentation assay using anti-histone antibody ELISA (B) or for caspase-3 activity using specific fluorogenic substrate (C). Results are expressed as relative OD₄₁₀-OD₅₀₀/mg protein (B) or as relative fluorescence unit (RFU)/min/mg protein (C). Values are mean ±SEM. *P<0.05 vs. sham, †P<0.05 vs. ACF. (D) Representative immunoblots in sham and ACF operated animals treated with vehicle or β₁-RB.

Figure 4. Effect of acute versus chronic stimulation of β₁-ARs on FA signaling

(A and C) Lysates from NRCMs untreated or treated with 10 μmol/L isoproterenol (ISO) for the indicated time were assessed for immunoblot analysis. (B and D) NRCMs were pretreated with 1 μmol/L prazosin (Praz) or 1 μmol/L CGP20712A (CGP) for 15-min prior to stimulation with 10 μmol/L ISO, 10 μmol/L norepinephrine (NE), or 100 nmol/L zinterol (Zint) for 5-min (B) or 24-hrs (D). Cell lysates were immunoprecipitated with anti-FAK antibodies and then immunoblotted with anti-phosphotyrosine (P-Tyr), p130^Cas, paxillin or FAK antibodies. (E–F) NRCMs were infected with Lac-Z (10 pfu/cell) or β₁-AR adenoviruses at the indicated titration and assayed for immunoblot analysis (E) or for immunoprecipitation assay using anti-FAK antibodies (F). Left: representative immunoblots. Right: Quantification of experiments expressed as mean ±SE from three separate cultures. *P 0.05 vs. control.

Figure 5. Chronic stimulation of β₁-ARs induces PTEN activation

(A) NRCMs were treated with isoproterenol (ISO), norepinephrine in presence of prazosin (NE+Praz.) and 1 μmol/L forskolin (Forsk) for 24-hrs and processed for immunoblot analysis. (B) PTEN immunoprecipitates from NRCMs untreated or treated with ISO for the indicated time were assayed for FAK and PTEN immunoblotting. (C) NRCMs were infected with Lac-Z (10 pfu/cell) or β₁-AR adenoviruses at the indicated titration and were assayed for immunoblot analysis. (D) NRCMs were pretreated with 5 μmol/L BAPTA-AM or 5 μmol/L KN93 for 45-min prior to ISO treatment for 12- or 24-hrs and then assessed for immunoblot analysis. Left: Representative immunoblots. Right: Quantification of experiments expressed as mean ±SE from three separate cultures. *P 0.05 vs. control; †P 0.05 vs. ISO treated cells.

Figure 6. PTEN mediates FA signaling alteration and myocyte apoptosis induced by β₁-ARs

NRCMs were infected with adenoviruses expressing Lac-Z (20 pfu/cell), WT-PTEN (20 pfu/cell), DN-PTEN (20 pfu/cell), transactivator (TA) (10 pfu/cell), WT-FAK (10 pfu/cell), or Y³⁹⁷ FAK mutant (10 pfu/cell) for 48-hrs. (A) Infected cells were treated with ISO for 24-hrs and then assessed for immunoblot analysis. Left: Representative immunoblots. Right: Quantification of experiments expressed as mean ±SE from three separate cultures. *P 0.05 vs. Lac-Z control; †P 0.05 vs. ISO treated Lac.Z infected cells. (B–E) Infected NRCMs were untreated or treated with ISO for 72-hrs. Lysates were processed for caspase-3 activity (B and D) or DNA fragmentation assay (C and E). Results are for triplicate determinations from a single experiment (mean ±SE). *P 0.05 vs. Lac-Z control; †P 0.05 vs. ISO treated cells.