Zhonghua Yi Xue Za Zhi. 2026 Sep 22;106(35):3772-3781. doi: 10.3760/cma.j.cn112137-20260508-01227.
ABSTRACT
Objective: To explore the role and mechanism of G-protein coupled receptor kinase 2 (GRK2)-mediated phosphorylation of adiponectin receptor 1 (AdipoR1) in diabetic cardiomyopathy (DCM), and to verify the therapeutic effect of site-directed mutagenesis targeting serine at position 205 (Ser205) of AdipoR1. Methods: Twenty 4-week-old C57BL/6J mice were randomly assigned to the normal control (NC) group and the DCM model group using a random-number table, with 10 mice per group. The DCM model was established in the DCM group by high-fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). After 21-week intervention, 6 mice from each group were selected using a random-number table for measurements of serum biochemical parameters, adiponectin (APN) levels and cardiac diastolic function. Following euthanasia, myocardial tissues were harvested for Masson's trichrome staining to evaluate the degree of myocardial fibrosis. Co-immunoprecipitation and Western blot (WB) assays were performed to determine GRK2 expression, phosphorylated serine (p-Ser) levels, and the binding capacity between AdipoR1 and adaptor protein, phosphotyrosine-binding domain and leucine zipper motif 1 (APPL1). The remaining 4 mice per group were used for isolation of primary adult cardiomyocytes, which were treated with exogenous APN or phosphate-buffered saline (PBS). Phosphorylation status of the downstream adenosine monophosphate-activated protein kinase (AMPK)/protein kinase B (Akt) signaling pathway was subsequently detected. Twenty-eight 3-day-old AdipoR1-knockout neonatal mice were included in further experiments. Four of these AdipoR1-knockout neonatal mice were randomly selected by a random-number table to isolate primary cardiomyocytes. Plasmid co-transfection was conducted to assess AdipoR1-APPL1 interaction and downstream AMPK/Akt phosphorylation upon GRK2 overexpression. Four experimental subgroups were set: Adenovirus-empty(Ad-empty)+3×Flag-AdipoR1WT, Ad-empty+3×Flag-AdipoR1S205A, adenovirus-mediated GRK2(Ad-GRK2)+3×Flag-AdipoR1WT, and Ad-GRK2+3×Flag-AdipoR1S205A. This experiment aimed to identify whether Ser205 phosphorylation serves as the key molecular event whereby GRK2 blocks APN signaling. From the remaining 24 AdipoR1-knockout neonatal mice, 12 were randomly allocated into two groups (6 mice per group) using a random-number table. On postnatal day 3, mice received an injection of either adeno-associated virus serotype 9 (AAV9)-AdipoR1WT or AAV9-AdipoR1S205A. Ten weeks later, primary adult cardiomyocytes were isolated and divided into 4 subgroups: AAV9-AdipoR1WT+Ad-empty, AAV9-AdipoR1WT+Ad-GRK2, AAV9-AdipoR1S205A+Ad-empty, and AAV9-AdipoR1S205A+Ad-GRK2. GRK2 was overexpressed in vitro, and immunofluorescence co-localization was applied to quantify the co-localization of AdipoR1 and APPL1. The leftover 12 AdipoR1-knockout neonatal mice were randomized into two groups (6 mice per group) with a random-number table, and injected with the aforementioned viruses on postnatal day 3. Four weeks after virus injection, DCM was induced by HFD plus STZ administration, yielding two in-vivo groups: AAV9-AdipoR1WT+DCM (wild-type group) and AAV9-AdipoR1S205A+DCM (point-mutation group). APN was continuously delivered via osmotic minipumps. Eight weeks after APN intervention, cardiac function and myocardial fibrosis were evaluated. Results: In C57BL/6J mice, the DCM group exhibited significantly higher levels of blood glucose, triglycerides, low-density lipoprotein-cholesteral and adiponectin (APN) compared with the NC group (all P<0.001). Statistically significant differences were observed between the two groups in the E/A ratio, radial strain rate, reverse radial strain rate (rRSR), longitudinal strain rate (LSR), and reverse longitudinal strain rate (rLSR) (all P<0.05). The fraction of myocardial fibrotic area was larger in the DCM group than in the NC group (9.20±1.66 vs 1.24±0.51, P<0.001). Myocardial protein levels of GRK2 (4.81±0.46 vs 1.03±0.09, P<0.001) and p-Ser (1.75±0.21 vs 0.98±0.05, P<0.001) were elevated in the DCM group relative to the NC group. The binding ratio of APPL1 to AdipoR1 in myocardial tissue was decreased in the DCM group versus the NC group (0.398±0.085 vs 0.978±0.088, P<0.001). APN-stimulated AMPK/Akt phosphorylation was blunted in primary adult cardiomyocytes isolated from DCM-model C57BL/6J mice. In-vitro experiments using AdipoR1-knockout neonatal mouse cardiomyocytes demonstrated that following AdipoR1 Ser205 site mutation, the ratios of p-AMPK/AMPK (4.025±0.767 vs 1.003±0.087, P<0.001) and p-Akt/Akt (4.125±0.544 vs 0.990±0.034, P<0.001) were markedly increased in the 3×Flag-AdipoR1S205A+APN group compared with the 3×Flag-AdipoR1S205A+PBS group. In primary adult cardiomyocytes derived from AdipoR1-knockout mice, the AAV9-AdipoR1S205A+Ad-GRK2 group restored the AdipoR1-APPL1 interaction in the presence of GRK2 when compared with the AAV9-AdipoR1WT+Ad-GRK2 group (P=0.014). In the in-vivo DCM model established in AdipoR1-knockout mice, the AAV9-AdipoR1S205A+DCM group displayed higher E/A ratio, rRSR, LSR and rLSR values, together with a lower myocardial fibrotic area fraction (all P<0.05), relative to the AAV9-AdipoR1WT+DCM group. Conclusions: GRK2-mediated phosphorylation of AdipoR1 at serine 205 triggers APN-AdipoR1 metabolic dysfunction, which represents a critical contributor to aggravated cardiac dysfunction in DCM. This mechanism is associated with the decoupling of AdipoR1 from APPL1. Targeted inhibition of AdipoR1 phosphorylation via site-directed mutagenesis restores APN-AdipoR1 signaling and ameliorates cardiac dysfunction in DCM.
PMID:42763212 | DOI:10.3760/cma.j.cn112137-20260508-01227