Essential role of caveolin-3 in adiponectin signalsome formation and adiponectin cardioprotection

Abstract

Objective: Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection.

Methods and results: Compared with wild-type mice, caveolin-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery; P<0.05). Although the expression level of key APN signaling molecules was normal in Cav-3KO, the cardioprotective effects of APN observed in wild-type were either markedly reduced or completely lost in Cav-3KO. Molecular and cellular experiments revealed that APN receptor 1 (AdipoR1) colocalized with Cav-3, forming AdipoR1/Cav-3 complex via specific Cav-3 scaffolding domain binding motifs. AdipoR1/Cav-3 interaction was required for APN-initiated AMP-activated protein kinase (AMPK)-dependent and AMPK-independent intracellular cardioprotective signalings. More importantly, APPL1 and adenylate cyclase, 2 immediately downstream molecules required for AMPK-dependent and AMPK-independent signaling, respectively, formed a protein complex with AdipoR1 in a Cav-3 dependent fashion. Finally, pharmacological activation of both AMPK plus protein kinase A significantly reduced myocardial infarct size and improved cardiac function in Cav-3KO animals.

Conclusions: Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.

Figure 1

Knockout of Cav-3 abolished APN’s infarct size sparing effect (A), markedly blunted APN’s anti-apoptotic effect (B), and abolished APN’s cardiac functional improvement effect as determined by LVEF (C,D) and ±dP/dtmax (E,F). N=12–15/group. *P<0.05, **P<0.01 vs. respective vehicle group; ^&P<0.05, ^&&P<0.01 vs. WT mice with the same treatment. in ischemic/reperfused heart. N=12–15/group. *P<0.05, **P<0.01 vs. respective vehicle group; ^&P<0.05, ^&&P<0.01 vs. WT mice with the same treatment.

Figure 2

AdipoR1 co-localizes (A) and interacts with Cav-3 (B). AdipoR1 is distributed in caveolae-rich fraction in detergent-resistant (caveolae-rich) fraction (fraction 5–6), but not in detergent-soluble fractions (C). AdipoR1/Cav-3 interaction was detected in cells co-transfected with full length Cav-3 plus full length AdipoR1 (FL), truncated AdipoR1^1–230, or truncated AdipoR1^316–375, but not in cell lysates expressing truncated AdipoR1^1–215 or truncated AdipoR1^337–375 (D). AdipoR1/Cav-3 interaction was detected in cells co-transfected with full length AdipoR1 plus full length Cav-3 (FL) or truncated Cav-3^1–74, but not in cell lysates expressing truncated Cav-3^1–55 or truncated Cav-3^74–151 (E). For confocal microscopic examination, >100 cells were inspected per experiment, and cells with typical morphology are presented. For Western and co-immunoprecipitation, at least 6 samples from different tissue/cell culture dishes were examined and typical blots are presented.

Figure 3

Knockout of Cav-3 abolished APN’s AMPK (A) and ACC (B) activation, and reduced its anti-nitrative effect (C). N=12–15/group. **P<0.01 vs. respective vehicle group; ^&P<0.05, ^&&P<0.01 vs. WT mice with the same treatment.

Figure 4

Knockout of Cav-3 abolished APN’s anti-nitrative (determined by total NO production, A) and anti-oxidative (determined by superoxide production, B; and NOX expression, D) and markedly blunted APN’s anti-iNOS effect (C) in ischemic/reperfused heart determined 3 hours after reperfusion. N=12–15/group. *P<0.05, **P<0.01 vs. respective vehicle group; ^&P<0.05, ^&&P<0.01 vs. WT mice with the same treatment.

Figure 5

Cav-3 interacts with APPL1 (left) and AC (right) to form protein complex (A). AdipoR1/APPL1 and AdipoR1/AC complex formation were detected in tissue samples from WT (B, Lane 1). These interactions were markedly reduced in Cav-3KO cardiac tissue (Lane 2: >80% reduction in 5 repeated experiments). Administration of AICAR (an AMPK activator) plus db-cAMP (a cAMP mimic) significantly reduced infarct size (C) and improved cardiac function (D) in Cav-3KO mice.

Figure 6

Comparison of T-cadherin expression in WT and Cav-3KO heart, and determination of T-cadherin distribution and its interaction with AdipoR1 or Cav-3 (A). Schematic illustration revealing the role of Cav-3 in APN signalsome formation and APN cardioprotective signaling (B). Solid lines: established signaling pathways; dashed lines: pathways requiring additional investigation; blue question mark: other intracellular APN signaling molecules that may also be “tethered” by Cav-3.