MicroRNA-301a mediated regulation of Kv4.2 in diabetes: identification of key modulators

Abstract

Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3'untranslated region (3'-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.

Figure 1. Physical parameters of the db/db and age matched wild type (Wt) mice.

The averaged body weights of db/db and Wt group normalized to tibia (A), heart weights normalized to tibia (B) and body weight(C), H&E staining of db/db and Wt hearts (D), cross-sectional area of the heart (E), RV thickness (F), Septal wall thickness (G) and LV thickness (H) are presented. All values presented in the bar diagram are mean (±SEM n = 15–16) with *p≤0.05.

Figure 2. Transcriptional regulation in the right ventricle of diabetic (db/db) heart.

Quantitative real-time PCR (qRT-PCR) in right ventricle (RV) of wild type (Wt) and diabetic (db/db) mouse hearts with potassium channels Kv1.4, 4.2, 2.1, 4.3, 1.5, 10.2, and sodium channels Scn1b and Scn5a, along with Kv channel gene chaperon KChIP2 (A), transcriptional factors such as GATA4, GATA6, Irx5, NFkB (B), and Hif1α, along with MHC-α, MHC-β and Gja1 (C). Bars represent mean (±SEM) expression in fold, n = 3 and * represents p≤0.05.

Figure 3. Differential expression of genes in left ventricle.

The qRT-PCR analysis of Kv4.2 and 1.4 in epicardium, endocardium and apex region (A), GATA4, GATA6, Irx5, NFkB (B), Kv2.1, Kv4.3, Scn1b, Scn5a, and KChIP2 (C) in epicardium of left ventricle (LV) from wild type (Wt) and diabetic (db/db) mouse hearts. Normalized fold values were expressed in bar diagrams are mean (±SEM) of n = 3 and * represents p≤0.05.

Figure 4. Protein profile in right ventricle of diabetic hearts.

Comparative Western blot analysis of the key potassium channels along with transcription factors, chaperons and hypertrophic markers are shown (A). Band intensities for Kv4.2, 1.4, KChIP2 (B), Irx5, NFkB, MHC6 and MHC7 (C) were measured and presented as bar diagrams after normalizing with GAPDH band intensities. All the values presented here are mean (±SEM) of n = 5–6, and * represents p≤0.05.

Figure 5. Western analysis of ion channel expression and key proteins.

Protein profiling of differentially expressed genes in left ventricle of db/db group compared with wild type (Wt) controls (A). Band intensities for Kv4.2, 1.4, KChIP2 (B), Irx5, NFkB, MHC6 and MHC7 (C) were measured and presented as bar diagrams after normalizing with GAPDH band intensities. All the values presented here are mean (±SEM) of n = 5–6, and * represents p≤0.05.

Figure 6. Extracellular and intracellular expressions of TNF-α in diabetic hearts.

Plasma and tissue homogenates were used for measurement of TNF-α by using ELISA kit. The right and the left ventricle were evaluated separately for measuring the TNF-α in a regional specific manner. The values are presented as bar diagrams of either TNFα protein expression in pg/ml of plasma (A) or pg of TNFα/µg of total protein (B). All the values presented here are mean (±SEM) of n = 5.

Figure 7. MicroRNA-301a regulated expression of Kv4.2.

The expression of miR-301 in db/db and Wt mouse right and left ventricles were assessed by Taq-man PCR (A). Rat cardiomyocytes (H9C2) were transfected with either scrambled or miR-301 inhibitor (50 nM) for different time points 24, 48 and 72 hours and miR-301a expressions were quantified using TaqMan assay (B). The bars labeled with 72‡ are the cells transfected with inhibitor (50 nM) for 72 h with additional supply of inhibitor at 48 h. The expression of Kv4.2, Kv4.3 KChIP2, Irx5, and NFkB were quantified using qRT-PCR for the cells transfected with miR-301a inhibitor or scrambled inhibitor for 24 h (C), 48 h (D) and 72 h (E). All values are normalized with housekeeping gene (U6 RNA for TaqMan and HPRT for qRT-PCR) and plotted as mean (±SEM) of n = 3–6. * represents p≤0.05, ** with p≤0.005, and *** with p≤0.0005.

Figure 8. Post-transcriptional regulation of Kv4.2 by miR-301a by direct binding.

The in silico analysis showing the direct binding of miR-301a to the ‘seed’ sequence on 3′-UTR region of Kv4.2 gene (A). Quantification of luciferase activity in H9C2 cells showing direct binding of miR-301a to the ‘seed’ sequence of Kv4.2 gene (B). Protein expression profile of Kv4.2 (24, 48, and 72 h) and Irx5 (48 h and 72 h) genes in H9C2 cells transfected with miR-301a inhibitor (50 nM) using Western blotting analysis (C). All the values presented in the bar diagram are mean (±SEM) of n = 3–6 and * represents p≤0.05.

Figure 9. Kv current recording in H9C2 cells treated with miR-301a inhibitor.

H9C2 cells were either transfected with miR-301a inhibitor (50 nM) or scrambled inhibitor and potassium channel currents were measured by whole cell patch clamp technique after 72 h of treatment. (A) Representative whole cell currents recorded from H9C2 cells normalized to I_peak are shown at +50 mV. (B) The tau (τ) calculated from the recordings with scrambled or miR-301a inhibitor is shown as bar graph representing mean ± SEM with p≤0.05, n = 11 for scrambled and n = 22 for miR-301a inhibitor.

Figure 10. Cellular pathway affecting the cardiac arrhythmia, cardiovascular disease and tachycardia in RV of diabetic hearts.

The differentially regulated genes in qRT-PCR analysis in right ventricle of db/db hearts compared to their wild type controls were taken as input data and Ingenuity Pathway Analysis (IPA) software was used to build interactive network(s) based on the relations available from its library. Genes shown in green color were down-regulated and up-regulated genes were shown in red color, where intensity of color is proportional to the fold values. Dotted arrows represent indirect relations and solid lines represent direct relations between the genes. Genes that are present in the network but not present in the input data are shown without any color.