This study examined how G-protein coupled receptor kinase 2 (GRK2)–mediated phosphorylation of adiponectin receptor 1 (AdipoR1) at serine 205 (Ser205) contributes to myocardial injury in diabetic cardiomyopathy (DCM). The investigators also tested whether preventing Ser205 phosphorylation by site-directed mutagenesis (S205A) restores adiponectin (APN) signaling and improves cardiac structure and function.
Young C57BL/6J mice (4 weeks old) were randomized into normal control (NC) and DCM model groups (n=10 per group). DCM was induced by a high-fat diet (HFD) plus intraperitoneal streptozotocin (STZ). After 21 weeks, subsets of animals were used for biochemical, functional, histologic, and molecular analyses.
Complementary experiments used AdipoR1-knockout neonatal mice. Neonatal cardiomyocytes were used for plasmid co-transfection and to test rescue by wild-type versus Ser205-mutant AdipoR1. Additional AdipoR1-knockout pups received AAV9 vectors encoding either AdipoR1WT or AdipoR1S205A shortly after birth; these mice were later studied in vitro and in vivo after DCM induction.
Compared with NC mice, DCM-model C57BL/6J mice showed significantly higher blood glucose, triglycerides, low-density lipoprotein cholesterol, and plasma APN (all P <0.001). Echocardiographic or strain-derived indices of diastolic function were impaired in the DCM group: E/A ratio, radial strain rate, reverse radial strain rate (rRSR), longitudinal strain rate (LSR), and reverse longitudinal strain rate (rLSR) differed between groups (all P <0.05).
Histologically, myocardial fibrosis measured by Masson’s trichrome staining was greater in DCM mice (fibrotic area fraction 9.20±1.66) than in NC mice (1.24±0.51; P <0.001).
Myocardial protein analysis showed elevated GRK2 levels in DCM mice (4.81±0.46 vs 1.03±0.09 in NC; P <0.001) and higher overall phosphorylated serine (p-Ser) levels (1.75±0.21 vs 0.98±0.05; P <0.001).
Co-immunoprecipitation revealed a reduced binding ratio of adaptor protein APPL1 to AdipoR1 in myocardial tissue from DCM mice (0.398±0.085) relative to NC (0.978±0.088; P <0.001), consistent with functional decoupling of AdipoR1 from its downstream adaptor.
Primary adult cardiomyocytes isolated from DCM-model C57BL/6J mice exhibited blunted APN-stimulated phosphorylation of the downstream AMPK/Akt signaling pathway compared with cells from controls.
In vitro studies used AdipoR1-knockout neonatal cardiomyocytes with controlled expression of AdipoR1 constructs and GRK2. These experiments tested whether Ser205 phosphorylation is the molecular event by which GRK2 blocks APN signaling.
Plasmid co-transfection experiments in AdipoR1-knockout neonatal cardiomyocytes compared four conditions: Ad-empty+AdipoR1WT, Ad-empty+AdipoR1S205A, Ad-GRK2+AdipoR1WT, and Ad-GRK2+AdipoR1S205A. Results showed that the Ser205-to-alanine mutation (AdipoR1S205A) markedly increased APN-stimulated downstream signaling: the p-AMPK/AMPK ratio was 4.025±0.767 in the 3×Flag-AdipoR1S205A+APN group versus 1.003±0.087 in the PBS-treated counterpart (P <0.001). The p-Akt/Akt ratio similarly increased (4.125±0.544 vs 0.990±0.034; P <0.001).
When GRK2 was overexpressed, the AdipoR1S205A construct preserved the AdipoR1–APPL1 interaction in primary adult cardiomyocytes from AdipoR1-knockout mice: the AAV9-AdipoR1S205A+Ad-GRK2 group restored AdipoR1–APPL1 interaction compared with AAV9-AdipoR1WT+Ad-GRK2 (P =0.014). This indicates that preventing Ser205 phosphorylation prevents GRK2-dependent uncoupling of AdipoR1 from its adaptor.
AdipoR1-knockout neonatal mice received AAV9-AdipoR1WT or AAV9-AdipoR1S205A on postnatal day 3. After maturation and DCM induction (HFD+STZ), animals received continuous APN infusion via osmotic minipumps. In the in vivo DCM setting, the AAV9-AdipoR1S205A+DCM group exhibited improved diastolic function (higher E/A ratio, rRSR, LSR, and rLSR) and a lower myocardial fibrotic area fraction compared with the AAV9-AdipoR1WT+DCM group (all P <0.05).
The data support a model in which elevated myocardial GRK2 in DCM promotes phosphorylation of AdipoR1 at Ser205, leading to reduced binding of APPL1 and impaired transmission of APN signals through AMPK/Akt. This decoupling associates with worsened diastolic dysfunction and increased myocardial fibrosis in the mouse DCM model.
Targeted inhibition of AdipoR1 Ser205 phosphorylation via site-directed mutagenesis (S205A) restored AdipoR1–APPL1 coupling, rescued APN-stimulated AMPK/Akt phosphorylation, and produced measurable improvements in cardiac function and fibrosis in both in vitro and in vivo experiments.
These findings identify Ser205 phosphorylation as a critical molecular event by which GRK2 impairs APN–AdipoR1 metabolic signaling in DCM and demonstrate that preventing this phosphorylation can ameliorate cardiac dysfunction in the experimental models used.
(Note: full methodological details, raw datasets, and additional experimental parameters were reported in the original publication.)