GPRASP1 Safeguards Endothelial Aspartate Metabolism to Prevent Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Abstract

BACKGROUND: Pulmonary hypertension (PH) is a serious complication of heart failure with preserved ejection fraction (HFpEF), for which no targeted therapies are currently available. Endothelial dysfunction plays a crucial role in PH associated with HFpEF (PH-HFpEF), yet its molecular drivers remain poorly defined. METHODS: Transcriptome profiling uncovered endothelial characteristics of PH-HFpEF. Endothelial-specific GPRASP1 (GPCR [G protein–coupled receptor]–associated sorting protein 1 deletion in mice was conducted to investigate its participation in PH-HFpEF pathology. Multimodal metabolomics, isotope tracing, proteomics, and mechanistic biochemical assays were used to map downstream pathways and identify druggable mediators. RESULTS: GPRASP1 was significantly lowered in pulmonary endothelial cells of PH-HFpEF models. Endothelial Gprasp1 knockout mice exhibited major PH-HFpEF features, including pulmonary vascular remodeling, elevated pulmonary pressure, diastolic dysfunction, and abnormal glucose/lipid metabolism. GPRASP1 loss impaired tricarboxylic acid cycle activity by stabilizing ASNS (asparagine synthetase), preferentially shifting aspartate toward asparagine synthesis over oxaloacetate production. This metabolic reprogramming led to adenosine triphosphate depletion, reactive oxygen species accumulation, endothelial nitric oxide synthase uncoupling, and nitric oxide deficiency. We discovered that, beyond its classic role in GPCR sorting, GPRASP1 functioned as a noncanonical adaptor protein that scaffolded the E3 ubiquitin ligase PRKN (Parkin) and ASNS, promoting PRKN-dependent K48-linked ubiquitination and proteasomal degradation of ASNS via its C-terminal domain. In parallel, under mitochondrial stress, GPRASP1 strengthened PRKN interactions with MFN1/2, enhanced their K63-linked ubiquitination, and facilitated PRKN-mediated mitophagy. Restoration of GPRASP1 expression or pharmacological inhibition of ASNS activity with olopatadine normalized aspartate utilization, improved mitochondrial bioenergetics, rescued endothelial function, and attenuated cardiopulmonary pathology in PH-HFpEF models. CONCLUSIONS: Our findings unlocked a noncanonical role of GPRASP1 in preserving pulmonary endothelial homeostasis and delineated a novel GPRASP1-PRKN-ASNS axis that connected proteostasis with endothelial metabolic integrity, highlighting aspartate metabolism as a targetable vulnerability in cardiopulmonary disease.