GRB2 Affects Post-Myocardial Infarction Heart Failure by Regulating the TLR4/NF-κB Pathway-Mediated M1 Macrophage...

Myocardial infarction (MI) and MI-related heart failure (HF) continue to represent predominant causes of disability and death globally. This study aimed to explore the specific involvement of growth factor receptor-bound protein 2 (GRB2) in M1 macrophage polarization and post-MI HF, as well as the underlying molecular mechanisms involved.

Sprague-Dawley rats underwent left anterior descending coronary artery ligation to generate a MI rat model. M1 polarization of RAW264.7 macrophages was induced through treatment with both lipopolysaccharide (LPS) and interferon-γ (IFN-γ). H9C2 cardiomyocytes were exposed to hypoxia to create an in vitro model mimicking MI. Evaluation of cardiac function was conducted using echocardiography, whereas Masson's trichrome staining was applied to examine pathological changes in rat myocardial tissue. The M1/M2 macrophage proportion and cardiomyocyte apoptosis level in rat myocardial tissue and in vitro cell models were assessed by flow cytometry. Immunofluorescence analysis was conducted to quantify GRB2 expression as well as the levels of macrophage polarization marker proteins in rat myocardial tissue and cells. The expression levels of macrophage polarization marker proteins, GRB2, as well as proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and apoptosis in rat myocardial tissues and cells were measured through Western blot.

Rats from the MI group showed significantly decreased ejection fraction and fractional shortening, accompanied by markedly increased left ventricular internal systolic/diastolic diameter, obvious collagen deposition in the myocardial tissue, and aggravated myocardial fibrosis. In addition, a markedly elevated proportion of M1 macrophages in rat myocardial tissue and an upregulation of GRB2 in macrophages were demonstrated in the MI group. In vitro experiments showed that LPS + IFN-γ significantly induced M1 polarization of macrophages and the expression of inflammatory factors and GRB2 protein. After interfering with GRB2 expression, the level of M1 polarization of macrophages decreased, and the expression levels of inflammatory factors and proteins related to TLR4/NF-κB pathway significantly decreased. Treatment of macrophages with RS 09 significantly reversed the inhibitory effect of GRB2 inhibition on M1-type macrophage polarization. In addition, under hypoxic conditions, conditioned medium (CM) from M1-type macrophages could increase the apoptosis level of cardiomyocytes, while CM from macrophages with GRB2 expression interference significantly reduced the apoptosis level of cardiomyocytes.

The expression level of GRB2 was significantly increased in the myocardial tissue of MI rats. Inhibiting the expression of GRB2 would lead to a weakened polarization of M1 macrophages. In HF after MI, GRB2 may promote the polarization of M1 macrophages by regulating the TLR4/NF-κB pathway, thereby aggravating myocardial cell damage.