Phosphoinositide 3-kinaseγ (PI3Kγ) is activated by G-protein coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the β-adrenergic receptor (βAR, a GPCR) complex altering G-protein coupling. PI3Kγ inhibition results in significant increase of βAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 & 93 resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of β2ARs is observed with phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in β2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.

Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.

Back ground Pro-inflammatory cytokine tumor necrosis factor α (TNFα) induces β-adrenergic receptor (βAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results Two different pro-inflammatory transgenic mouse models with cardiac overexpression of Myotrophin (a pro-hypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate βAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling βAR desensitization independent of sympathetic overdrive. TNFα-mediated βAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both the mouse models. In vitro studies in β2 AR overexpressing HEK 293 cells showed significant βAR desensitization, GRK2 upregulation and recruitment to the βAR complex following TNFα. Interestingly, inhibition of PI3K abolished GRK2-mediated βAR phosphorylation and GRK2 recruitment upon TNFα. Furthermore, TNFα-mediated βAR phosphorylation was not blocked with βAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gβγ sequestering peptide βARK-ct could not prevent βAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the βAR is Gβγ independent. siRNA knock down of GRK2 resulted in loss of TNFα-mediated βAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility showing that TNFα-induced βAR desensitization is GRK2 dependent. Conclusions TNFα-induced βAR desensitization is mediated by GRK2 and is independent of Gβγ uncovering a hitherto unknown cross-talk between TNFα and βAR function providing the underpinnings of inflammation-mediated cardiac dysfunction.