Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during postdiapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/ E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.
Background
Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vascular system characterized by sustained elevation in resting mean pulmonary arterial pressure (>25 mmHg) with presence of occlusive vascular remodeling and progressive loss of microvasculature. PAH patients have a poor five‐year survival rate of only ~50% and the primary cause of death for PAH patients is the right (‐sided) heart failure. Better understanding of pathophysiology of PAH and RHF is required to find novel therapies. In this project, using a rat model of severe PAH, we explored the alterations in expression of genes involved in bioactive lipid pathways in the lungs and the right ventricle (RV) during development of PAH.
Methods
Adult male Sprague Dawley rats were administered a single injection of SU5416 (SU; 20 mg/kg, subcutaneous) and subjected to 3 weeks of hypoxia (10% O2) followed by 4 weeks of normoxia (room air), SUHx model of sever PAH. Right ventricular systolic pressure (RVSP) was measured, and heart and lung samples were collected at 1, 3, 7, 21 and 49‐days post SU injection. Histological analysis was conducted to confirm vascular remodeling in the lungs and development of RV hypertrophy (RVH; RV/LV+S). RTqPCR was performed to evaluate gene expression changes in the genes related to bioactive lipids pathways.
Results
In response to SUHx, progressive increase in RVSP and RVH was observed. Histology of lungs and heart samples confirmed pulmonary vascular and RV remodeling at 7 weeks post SU in the SUHx model. We observed increase in expression of ALOX‐5 in the RV and lungs of SUHx rats, compared to controls. Furthermore, alterations in various cytochrome P450 (CYP) isozymes were observed in the RV and lungs of SUHx rats compared to controls.
Conclusions
Together, we have identified unique changes in bioactive lipid pathway genes in the lungs and the RV that were associated with development of PAH and RV remodeling in SUHx model. Further studies are ongoing to confirm the role of the candidate genes in pathophysiology of PAH and development of RHF.
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