Interestingly, in addition to downregulation of mitochondrial and energy metabolic pathways, the pn-csERRα/γ knockdown RNA-seq dataset was significantly enriched (downregulated) for Gene Ontology or KEGG terms related to cardiac structural pathways (Table I in the Data Supplement), including adult contractile protein gene isoforms. For example, expression of Tnni3 was downregulated, whereas the level of the fetal isoform, Tnni1, was upregulated in the pn-ERRα/γ knockdown hearts (Figure 3D; Figure IVC in the Data Supplement). This isoform switch was also reflected at the protein level based on immunoblotting, albeit more modestly (Figure 3E). These changes were also seen in isolated cardiac myocytes (Figure IVD in the Data Supplement). In addition, levels of the transcript encoding myosin regulatory light chain 2 (Myl2), a ventricular/cardiac muscle isoform, were downregulated in the pn-csERRα/γ knockdown hearts (Figure 3D; Figure IVC in the Data Supplement). Expression of other cardiac structural and ion channel/transporter genes, including Myl3, Tnnc1, Mybpc3, Atp1a1, Atp1a2, Cacna2d3, and Kcnq1, were also reduced (Figure 3D; Figure IIIA and IVC in the Data Supplement). Moreover, levels of genes involved in Ca2+ handling functions, such as Pln, Ryr2, and Atp2a2, were downregulated (Figure 3D; Figure IVC in the Data Supplement). The gene expression changes in the pn-csERRα/γ knockdown hearts were similar in males and females (Figure 3D; Figure IVE and IVF in the Data Supplement). Collectively, these data indicate that ERRα and ERRγ are not only necessary for energy metabolic maturation but also participate in the transcriptional control of a broad array of adult cardiac structural genes during postnatal developmental maturation.
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