Figure 6. Hypoxia decreases PLN protein levels and suppresses Pln transcription.

Neonatal rat ventricular myocytes were treated with hypoxia (1% O₂) as indicated. Cells were then harvested for protein or mRNA analysis. (A and B) Western blot analyses were performed with the indicated antibodies (A). Densitometric analysis from 3 experiments is shown in B. ^*P < 0.05; ^**P < 0.01, compared with 0 hours, 1-way ANOVA. (C) Real-time PCR was performed to analyze relative Pln mRNA levels. n = 3, ^*P < 0.05; ^**P < 0.01, compared with 0 hours, 1-way ANOVA. (D) HL-1 cardiomyocytes were transfected with a pGL3-Pln promoter (–156 to +64) luciferase construct, together with pcDNA3, or with a construct expressing TR-α or NCOR2, as indicated. Cells were also cotransfected with a construct expressing Renilla luciferase as the internal control. Twenty-four hours after transfection, cells were cultured under 21% or 1% O₂ conditions for 24 hours. Cells were then harvested and luciferase activity determined with a luminometer. Relative luciferase activity was calculated from 3 separate experiments. ^*P < 0.05; ^#P < 0.01, 2-way ANOVA.