Abstract Body: Introduction: Macrophages (MF) play major roles in myocardial ischemia/reperfusion (I/R) injury, both beneficial and detrimental. The mechanistic target of rapamycin (mTOR) plays an important role in MF polarization and functionality. It is prominently regulated by tuberous sclerosis complex 2 (TSC2) which constitutively inhibits and regulates mTORC1. Little is known about how such mTORC1 regulation influences the effect of MFs in I/R injured hearts.
Hypothesis: We tested if gene knockdown of TSC2 to constitutively enhance MF mTORC1 activity by TSC2 knockdown impairs or benefits the I/R heart.
Methods/Results: MF-specific TSC2 knock-out mice (MF^TSC2-/-) were generated by crossing flox’d TSC2 mice with those expressing LysM-Cre. MTORC1 was constitutively active in bone marrow-derived MF^TSC2-/-, reflected by increased S6K1 and 4E-BP1 phosphorylation. MF^TSC2-/- exposed to liposaccharide had greater proinflammatory responses, e.g. increased TNF-a secretion, supporting an M1-like phenotype. MF^TSC2-/- also exhibited less M2-like features (reduced Arg1, Mrc1, and Rentla expression). To test the impact of MF^TSC2-/- on infiltrating myocardial MF subtypes post I/R, MF were isolated from the myocardium and sorted by flow-cytometry based on CD45⁺, CD11b⁺, and CD64⁺ and then parsed as ±CCR2 and ±MHC-II. We found MF^TSC2-/- resulted in an increase in infiltrating CCR2^- MHC-II^- phenotypes, which are thought to be anti-inflammatory and to support healing. Consistent with this, we found MF^TSC2-/- hearts exposed to I/R had higher ejection fractions (74.2±10.3%) vs controls (47.2±14.3%, P=0.002). MF^TSC2-/- hearts also had smaller LV internal end-systolic diameter (P=0.009). As LysM is also expressed in neutrophils, we also performed this study in mice where neutrophils were first depleted using an anti-Ly6G antibody. Similar cardioprotective effects were observed with MF^TSC2-/- after I/R: (EF: 56.9±12.3 vs 36.1±9.8, P=0.01). This supports the impact being conferred by MF.
Conclusion: TSC2-deleted MF with constitutive mTORC1 activation confer cardio-protection to I/R injury. These findings help identify a novel signaling mechanism coupling mTORC1/TSC2 in MF to myocyte recovery, highlight novel avenues to improve post ischemic cardiac repair.