Electrolyte Blood Press.  2018 Jun;16(1):1-10. 10.5049/EBP.2018.16.1.1.

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

• Sung JH
• Song A
• Park T
• Kim E
• Lee S

• KMID: 2416373
• DOI: http://doi.org/10.5049/EBP.2018.16.1.1

Abstract

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.

Keyword

Cardiac hypertrophy; Late embryogenesis abundant protein; HSP22 protein; Homeostasis

MeSH Terms

Angiotensin II*
Angiotensins*
Artemia
Cardiomegaly
Consensus Sequence
Diet
Embryonic Development*
Female
Flow Cytometry
Homeostasis
Hypernatremia
Hypertrophy
In Vitro Techniques
Microscopy, Confocal
Pregnancy
Real-Time Polymerase Chain Reaction
RNA, Messenger
Angiotensin II
Angiotensins
RNA, Messenger

Figure

• 

  Fig. 1 Size of the in vitro hypernatremic model. (A) Cell size was increased by supplementation with 30 mM NaCl for 48 h (*p<0.05). (B) A right shift in the 30 mM NaCl for 48 h group was found.

• 

  Fig. 2 Ang II model was confirmed. (A) Hypertrophy was induced by supplementation of 1 µM Ang II for 48 h (**p<0.01). (B) Right shift in the Ang II model was found.

• 

  Fig. 3 Abnormal granule was confirmed in two different hypertrophic models. (A) No changes, (B) No shifts in SSC-H were found in two models and each control group.

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  Fig. 4 Cytotoxic effects of NaCl and Ang II were compared. Cell viabilities were not statistically different from the value of the control.

• 

  Fig. 5 mRNA expression of HSP22 and NPPa in two hypertrophic models. (A) In the NaCl group, mRNA expression of HSP22 was significantly decreased in a time-dependent manner. (B) HSP22 in the 1 µM Ang II for 48 h group (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 6 Average intracellular free Ca2+ concentration in two hypertrophic models. (A) FSC-H increased in the Ang II model and further increased in the in vitro hypernatremic model, and fluorescent intensity increased in the Ang II model and decreased in the in vitro hypernatremic model. To sum up, the level of free Ca2+ in the region on the cell decreased in the Ang II model and further decreased in the in vitro hypernatremic model when fluorescent intensity was corrected to compensate the difference of cell size. (B) The two hypertrophic models shifted to the right and down. In in vitro hypernatremic model, it was more evident (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 7 Analysis of the single cell to show the intracellular free Ca2+ level. Ca2+ concentration decreased in the Ang II model and decreased further in the in vitro hypernatremic model.

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  Supplement Data 1 The size of cells changed. Cell sizes were measured and compared with the FSC-H values.

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  Supplement Data 2 Morphology of cells. Abnormal cell and nuclear morphologies were not observed among the Control and two hypertrophic models.

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  Supplement Data 3 To validate the primers, amplified cDNA of the Control group was confirmed by DNA gel electrophoresis. The product sizes were 129bp (HSP22), and 135bp (16s rRNA). Lane.1 is Marker, Lane.2, 3, 4 are 18s rRNA, and Lane. 5, 6, 7 are HSP22.

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