MON-433 - A TR-TNFR2-Β2Ar Signaling Complex Enables T3 to Prevent Tnfalpha Induced, GRK-Mediated Β2Ar Desensitization and Preserve Cardiac Beta-Adrenergic Signaling: Implications for T3 Replacement in Septic Shock Cardiomyopathy

Objective:
Septic cardiomyopathy is an early, potentially reversible complication of septic shock associated with increased mortality. A key mechanism is beta-adrenergic receptor (betaAR) desensitization, promoted in part by pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha), which reduce ventricular contractility. Nonthyroidal illness syndrome is highly prevalent in septic shock and contributes to myocardial hypothyroidism. We previously showed that triiodothyronine (T3) prevents TNFalpha-induced betaAR desensitization in vitro. Here, we defined the molecular mechanism underlying this protective effect.

Methods:
Adult ventricular myocytes (AVMs) were isolated from C57BL/6J wild-type (WT), beta2AR-knockout (beta2AR KO), and beta2AR-GRK knock-in (beta2AR-GRK KI; beta2AR lacking GRK phosphorylation sites) mice. AVM contraction was assessed, and cAMP and PKA activities were measured using compartmentalized FRET biosensors (H187 for cAMP; AKAR3 for PKA). TR-deficient CV-1 cells were used to test T3 dependence on TRalpha or TRbeta. beta2AR-overexpressing HEK293 cells were used to investigate the roles of T3 and TNFalpha in beta2AR desensitization. HEK293 cells were used for exogenous protein overexpression. Protein expression, phosphorylation, and protein-protein interactions were evaluated by Western blotting and immunoprecipitation.

Results:
TNFalpha reduced isoproterenol (ISO)-stimulated AVM contraction and cAMP/PKA activation in WT AVMs; T3 pretreatment prevented these inhibitory effects. In CV-1 cells, selective TR re-expression showed that both TRalpha and TRbeta mediate T3-dependent preservation of cAMP/PKA signaling during TNFalpha exposure. TNFalpha-induced impairment of beta-adrenergic responsiveness was abolished in AVMs from beta2AR KO and beta2AR-GRK KI mice, indicating a GRK-beta2AR-dependent mechanism. Consistent with this, TNFalpha increased beta2AR phosphorylation at GRK sites in beta2AR-overexpressing HEK293 cells, an effect that was prevented by T3 pretreatment. Immunoprecipitation further revealed that TNF receptor 2 (TNFR2) forms a complex with beta2AR and TRs, supporting a direct molecular interaction linking T3-TR signaling to TNFalpha-mediated regulation of beta2AR.

Conclusion:
T3 prevents TNFalpha-induced beta2AR desensitization by suppressing GRK-dependent beta2AR phosphorylation, thereby preserving betaAR-cAMP/PKA signaling and AVM contractile function. This cardioprotective effect requires TR activation and identifies a novel TR-TNFR2-beta2AR crosstalk with potential therapeutic relevance for septic cardiomyopathy.

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