The Different Expression Patterns of HSP22, a Late Embryogenesis Abundant-like Protein, in Hypertrophic H9C2 Cells Induced by NaCl and Angiotensin II

BACKGROUND: High-NaCl diet is a contributing factor for cardiac hypertrophy. The role of HSP22 as a protective protein during cardiac hypertrophy due to hypernatremia is unclear. Accordingly, this study aimed to establish a cellular hypernatremic H9C2 model and to compare the expression of HSP22 in Ca2+ homeostasis between a high-NaCl and angiotensin II-induced hypertrophic cellular H9C2 model. METHODS: Real-time PCR was performed to compare the mRNA expression. Flow cytometry and confocal microscopy were used to analyze the cells. RESULTS: The addition of 30 mM NaCl for 48 h was the most effective condition for the induction of hypertrophic H9C2 cells (termed the in vitro hypernatremic model). Cardiac cellular hypertrophy was induced with 30 mM NaCl and 1 µM angiotensin II for 48 h, without causing abnormal morphological changes or cytotoxicity of the culture conditions. HSP22 contains a similar domain to that found in the consensus sequences of the late embryogenesis abundant protein group 3 from Artemia. The expression of HSP22 gradually decreased in the in vitro hypernatremic model. In contrast to the in vitro hypernatremic model, HSP22 increased after exposure to angiotensin II for 48 h. Intracellular Ca2+ decreased in the angiotensin II model and further decreased in the in vitro hypernatremic model. Impaired intracellular Ca2+ homeostasis was more evident in the in vitro hypernatremic model. CONCLUSION: The results showed that NaCl significantly decreased HSP22. Decreased HSP22, due to the hypernatremic condition, affected the Ca2+ homeostasis in the H9C2 cells. Therefore, hypernatremia induces cellular hypertrophy via impaired Ca2+ homeostasis. The additional mechanisms of HSP22 need to be explored further.

Molecular cloning and expression analysis of a late embryogenesis abundant protein gene (SmLEA2) from Salvia miltiorrhiza / 中草药

Objective: To clone and characterize a late embryogenesis abundant protein SmLEA2 with its promoter region from Salvia miltiorrhiza, and to predict its probable function. Methods: SmLEA2 was cloned by PCR and RT-PCR from genomic DNA and cDNA. Protein structure and phylogenetic relationships were carried out by bioinformatic analysis. Gene expression in different organs and different development periods was detected by qPCR. Gene expression was also detected under different treatments. Results: By analyzing the cDNA library for S. miltiorrhiza with BLAST program, one of these sequences showed a high homology with late embryogenesis abundant (LEA) protein and was named as SmLEA2 (GenBank: HQ676610). We obtained 1 961 bp gene sequences of SmLEA2, which contained an intron and a single opening reading frame (ORF) of 960 bp encoding 319 amino acid peptides. Bioinformatic analysis showed that the putative SmLEA2 protein was a hydrophilic protein without signal peptides and transmembrane domains. The SmLEA2 protein was predicted with a molecular weight of 35 340 and a theoretical isoelectric point of 4.77. SmLEA2 was expressed in the roots, stems, and leaves of S. miltiorrhiza, and the most abundant in the stems. With the development of the flowers and seeds, its expression increased gradually. Expression of SmLEA2 could be induced by methyl jasmonate (MeJA) and abscisic acid (ABA). Conclusion: Thus, we speculate that SmLEA2 may be involved in seed development and plant defenses.

Isolation and expression analysis of LEA genes in peanut (Arachis hypogaea L.).

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.