Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p<0.05; **p<0.01, compared to control. DOI: http://dx.doi.org/10.7554/eLife.17304.003

Article Snippet: Cells were incubated overnight with primary antibody lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:100) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) in PBS with 2% normal goat serum and 1% BSA.

Techniques: Microarray, Expressing, Hybridization, Sequencing, Control, Transfection, Membrane, Staining

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p<0.05; **p<0.01, as indicated or compared to control. DOI: http://dx.doi.org/10.7554/eLife.17304.004

Article Snippet: Cells were incubated overnight with primary antibody lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:100) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) in PBS with 2% normal goat serum and 1% BSA.

Techniques: Clone Assay, Luciferase, Construct, Transfection, Control, Activity Assay, Plasmid Preparation, Activation Assay

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p<0.05, **p<0.01, as indicated or compared to control, #p<0.05, compared to PE+Ad.GFP. DOI: http://dx.doi.org/10.7554/eLife.17304.007

Article Snippet: Cells were incubated overnight with primary antibody lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:100) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) in PBS with 2% normal goat serum and 1% BSA.

Techniques: Expressing, Control, Functional Assay

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p<0.05; **p<0.01, as indicated or compared to control. #p<0.05, ## PP <0.01 compared to PE+Ad.GFP. DOI: http://dx.doi.org/10.7554/eLife.17304.008

Article Snippet: Cells were incubated overnight with primary antibody lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:100) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) in PBS with 2% normal goat serum and 1% BSA.

Techniques: RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization, Control