Chau, Sarah

Background: Dysfunction of systemic vascular endothelial cells (ECs) is common in heart failure with preserved ejection fraction (HFpEF). We sought to determine if organ-specific EC translational heterogeneity is present in HFpEF. Using a transgenic mouse model (RiboTag EC ), we analyzed the translatome (the mRNAs bound with ribosomes) of organ-specific ECs in a mouse HFpEF model induced by hypertensive/metabolic stresses. Methods: Six-month-old male RiboTag EC ( Cdh5 CreERT2+ / Rpl22 HA/+ ) mice, expressing a hemagglutinin tag in EC ribosomal protein Rpl22, were fed a high-fat diet and L-NAME water (N=4) or a normal diet and water (N=5) for 7 weeks. Organs were collected for Translating Ribosome Affinity Purification, RNA isolation, and RNA-seq. RNA-seq data were visualized using uniform manifold approximation and projection (UMAP). Differentially expressed genes (DEGs; absolute fold change ≥ 2 and false discovery rate (FDR) adjusted p-value ≤ 0.05). Pathway enrichment analyses were conducted (Ingenuity Pathways Analysis (IPA)). Results: In UMAP, the EC translatome clustered by organ (heart, lung, skeletal muscle, and liver) highlighting organ-specific EC heterogeneity and by disease (HFpEF) status with cardiac ECs showing the most dramatic changes with HFpEF (Fig A). Cardiac ECs in HFpEF (vs Control) had 283 downregulated and 23 upregulated DEG (Fig B) out of 14,923 genes identified. There were significant differences in 142 canonical pathways (16 upregulated, 126 downregulated), including LXR/RXR activation, DHCR 24 signaling, regulation of insulin-like growth factors, eNOS signaling, acute phrase response (all down regulated) and IL-1b-mediated inhibition of RXR and unfolded protein response (both up regulated) (Fig C). Conclusion: HFpEF induction caused extensive translational changes in EC but particularly cardiac ECs. In cardiac ECs, metabolism, anti-inflammatory, vasodilation, tissue repair, and angiogenesis pathways were inhibited, while pro-inflammatory response, insulin resistance, and proteostasis pathways were activated.

Heart failure with preserved ejection fraction (HFpEF), characterized by cardiac diastolic dysfunction, leads to premature mortality with limited effective treatments. A recent study in HFpEF patients suggested neuropilin 1 (NRP1), an endothelial cell (EC) originated prognostic marker, might contribute to the pathogenesis of HFpEF. However, the functional role of NRP1 in the pathogenesis of HFpEF remains unclear. Purpose: To determine the functional role of NRP1 in the pathogenesis of HFpEF. Hypothesis: NRP-1 contributes to the pathogenesis of HFpEF, potentially mediated through chronic inflammation. Methods: HFpEF was induced by hypertensive and metabolic stresses in control (n=14) and tamoxifen-induced, endothelial cell-specific NRP1 knockout (NRP1 iEC-KO ) mice (n=13). We assessed cardiac diastolic function via echocardiography and pressure-volume loop through invasive catheters. To determine the inflammation level, we tested leukocyte adhesion, inflammatory marker vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) mRNA level in control and NRP1 shRNA knockdown (NRP1 KD) human umbilical vein endothelial cells (HUVECs) with a stimulation of TNF-α. Student t-test (two tailed) was used and the criterion level of significance was set at p<0.05. Results: We found a significantly improved diastolic function in NRP1 iEC-KO mice, evidenced by a significantly reduced end diastolic pressure in NRP1 iEC-KO mice (8±2mmHg; n=7) compared to the controls (17±2mmHg; n=9; p=0.01) after HFpEF induction. E/A ratio was also significantly lower in NRP1 iEC-KO mice (2.0±0.2; n=10) compared to controls (2.7±0.3; n=12; p=0.04). Moreover, the isovolumic relaxation time was also significantly reduced in NRP1 iEC-KO mice (18±0.8ms; n=13) compared to controls (20±0.8ms; n=14; p=0.03). In addition, In HUVECs, NRP1 KD significantly reduced inflammatory marker VCAM-1 mRNA expression (95±16; n=5) compared to controls (293±36; n=5; p<0.01) upon TNF-α treatment. Similarly, NRP1 KD significantly reduced ICAM-1 mRNA expression (44±14; n=5) compared to controls (83±9; n=5; p=0.03) upon TNF-α treatment. Furthermore, leukocyte adhesion was significantly lower in the NRP1 KD group (57.0 ±8.6 cells/field) compared to the control group (90.8 ±9.1 cells/field; p=0.04). Conclusion: Endothelial NRP1 contributes to cardiac diastolic dysfunction and the pathogenesis of HFpEF, potentially mediated through the activation of chronic low-grade inflammation.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle–specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.