Deletion of Kvβ1.1 subunit leads to electrical and haemodynamic changes causing cardiac hypertrophy in female murine hearts

Abstract

What is the central question of this study? The goal of this study was to evaluate sex differences and the role of the potassium channel β1 (Kvβ1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvβ1.1 in females led to cardiac hypertrophy characterized by increased heart size, prolonged monophasic action potentials, elevated blood pressure and increased myosin heavy chain α (MHCα) expression. In contrast, male mice showed only electrical changes. Kvβ1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvβ1.1 upregulated MHCα. Cardiovascular disease is the leading cause of death and debility in women in the USA, and cardiac arrhythmias are a major concern. Voltage-gated potassium (Kv) channels along with the binding partners; Kvβ subunits are major regulators of the action potential (AP) shape and duration (APD). The regulation of Kv channels by the Kvβ1 subunit is unknown in female hearts. In the present study, we hypothesized that the Kvβ1 subunit is an important regulator of female cardiac physiology. To test this hypothesis, we ablated (knocked out; KO) the KCNAB1 isoform 1 (Kvβ1.1) subunit in mice and evaluated cardiac function and electrical activity by using ECG, monophasic action potential recordings and echocardiography. Our results showed that the female Kvβ1.1 KO mice developed cardiac hypertrophy, and the hearts were structurally different, with enlargement and increased area. The electrical derangements caused by Kvβ1.1 KO in female mice included long QTc and QRS intervals along with increased APD (APD20-90% repolarization). The male Kvβ1.1 KO mice did not develop cardiac hypertrophy, but they showed long QTc and prolonged APD. Molecular analysis showed that several genes that support cardiac hypertrophy were significantly altered in Kvβ1.1 KO female hearts. In particular, myosin heavy chain α expression was significantly elevated in Kvβ1.1 KO mouse heart. Using a small interfering RNA strategy, we identified that knockdown of Kvβ1 increases myosin heavy chain α expression in H9C2 cells. Collectively, changes in molecular and cell signalling pathways clearly point towards a distinct electrical and structural remodelling consistent with cardiac hypertrophy in the Kvβ1.1 KO female mice.

Figure 1. Cardiac structural analysis by morphometry

A, female heart weight (HW) normalized with tibia length (TL) from wild-type (WT) and Kvβ1.1 knockout (KO) hearts; bar graph shows means ± SEM (n = 10), and ^*P < 0.05. B, male HW normalized with TL from WT and KO hearts; bar graph shows means ± SEM (n = 15). C, cross-sectional image of the heart from WT and KO female mice. Heart sections were 25 μm thick and stained with Haematoxylin and Eosin; left ventricle (Lv) and right ventricle (Rv) are labelled. D, cross-sectional area of hearts measured with ImageJ software, and means ± SEM plotted using a bar graph (n = 4), and ^*P < 0.05.

Figure 2. Female cardiac measurements by echocardiography

A, B-mode short-axis image (left panel) of the left ventricle (LV) with papillary muscles (P) visible, and M-mode image (right panel) of the interior of the LV with left ventricular internal dimension at systole and diastole (LVIDs and LVIDd) along with left ventricular anterior and posterior wall at diastole (LVAWd and LVPWd). B, LVIDs and LVIDd dimensions measured in WT and KO hearts. Bar graph shows means ± SEM (n = 13), and ^*P < 0.05. C, LV mass estimated using M-mode images; bar graph represents means ± SEM (n = 13), and ^*P < 0.05. D, stroke volume (SV) per beat obtained from M-mode images; bar graph shows means ± SEM (n = 13), and ^*P < 0.05.

Figure 3. Blood pressure measurements

A–C, female systolic blood pressure (SBP; A), female diastolic blood pressure (DBP; B) and female heart rate (HR; C) measurements from WT and KO hearts; bar graphs show means ± SEM (n = 8 mice), and ^*P < 0.05. D–F, male SBP (D), male DBP (E) and male HR (F) measurements from WT and KO hearts; bar graphs show means ± SEM (n = 8 mice).

Figure 4. Female haemodynamic changes

A, B-mode image of the ascending aorta (Ao Arch) and pulse wave (PW) Doppler image of the blood flow in the ascending aorta (top panel), and B-mode image of the pulmonary artery (Pul Artery) and PW Doppler image of the blood flow in the pulmonary artery (bottom panel). B–D, Aortic mean gradient pressure (in millimetres of mercury (B), aortic velocity–time integral (VTI) taken from PW Doppler imaging (C) and pulmonary VTI taken from PW Doppler imaging (D), in WT and KO mice; bar graphs show means ± SEM (n = 8), and ^*P < 0.05.

Figure 5. Electrocardiographic recordings

A, averaged trace of lead II ECG recording from WT (black) and KO (red) hearts showing QTc duration from female mice. B–E, female QTc interval (B) female QRS duration (C), male QTc interval (D) and male QRS duration (E), in WT and KO mice; bar graphs represent means ± SEM (n = 10), and ^*P < 0.05.

Figure 6. Ventricular repolarization changes

A, averaged recordings of the monophasic action potential in WT (black) and KO (red) hearts from females. B, female action potential durations (in milliseconds) at 20, 50, 70 and 90% repolarization in WT and KO mice; bar graph shows means ± SEM (n = 6), and ^*P < 0.05. C, averaged recordings of monophasic action potential in WT (black) and KO (red) hearts (n = 3) from males. D, male action potential durations (in milliseconds) at 20, 50, 70 and 90% repolarization in WT and KO mice; bar graph shows means ± SEM (n = 9), and ^*P < 0.05. Monophasic action potential recordings were obtained during perfusion at 37°C.

Figure 7. Female cardiac real-time PCR expression, protein and protein–protein interaction analysis

A, expression of myosin heavy chain (MHC) isoforms α and β, PI3K, GATA4, GATA6 and BMP10 (known cardiac specific hypertrophy markers); bar graph shows means ± SEM (n = 3), and ^*P < 0.05. Genes were normalized with a housekeeping gene (HPRT). B, Western blot images of MHCα and MHCβ from WT and KO left ventricular homogenate. C, bar graph shows means ± SEM (n = 3), and ^*P < 0.05; bands were normalized with Ponceau S-stained full lanes. D, Western blot image of KO left ventricle (Lv) and Cos-7 cells transfected with Kvβ1.1–DDK plasmid (β1.1^DDK; lane 1) and Cos-7 cells transfected with β1.1^DDK alone (lane 2). Lane 1 was incubated overnight with DDK-coated agarose beads. Myosin heavy chain α (MHCα) primary antibody (1:200 dilution) was incubated overnight with blot, and a 225 kDa band was noted in lane 1, with a limited band seen in lane 2. E, PCR expression of MHCα, GATA4 and GATA6 72 h after Kvβ1.1 small interfering RNA treatment in H9C2 cells (rat cardiomyoblasts); bar graph shows means ± SEM (n = 3), and ^*P < 0.05. ^†P < 0.07.

Figure 8. Real-time PCR expression analysis

A, female expression of voltage-gated potassium channels involved in cardiac repolarization, including Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3 and Kv10.2; bar graph shows means ± SEM (n = 3), and ^*P < 0.05. Genes were normalized with a housekeeping gene (18S). B, expression of voltage-gated potassium channel subunits Kvβ1.2, Kvβ2 and KCHIP2; bar graph shows means ± SEM (n = 3), and ^*P < 0.05. C, expression of voltage-gated potassium channel subunit Kvβ1.1 in wild-type male (WTM) versus wild-type female (WTF) mouse heart; bar graph shows means ± SEM (n = 3), and ^*P < 0.05. Genes were normalized with a housekeeping gene (HPRT).

Figure 9. Gene network analysis

The top two networks identified by Ingenuity Pathway Analysis based on qPCR-expression data were merged. Potential interactions between KCNAB1 and MYH6 (MHCα) or GATA factors were incorporated into the analysis and indicated by dashed black lines. Relative gene expression differences between the WT and KO groups are depicted by a colour gradient from green to red; green represents higher expression for WT, whereas red represents KO. ERK1/2, extracellular signal-regulated protein kinases 1 and 2; IL1, Interleukin-1; IL1B, Interleukin 1-Beta; IL6, Interleukin-6; KCND2, Kv4.2; KCNIP2, Kv Channel Interacting Protein 2; KCNAB1, Kv Beta 1.1; MYH6, Myosin heavy chain alpha; NFkB, Nuclear Factor Kappa-B; PI3K, Phosphoinositide-3-Kinase; PIK3R1, Phosphoinositide-3-Kinase Regulatory Subunit 1; Pka, cAMP-dependent Protein Kinase, Pkc, Protein Kinase C; RsK, Ribosomal Protein S6 Kinase.