Deletion of Kvβ2 (AKR6) Attenuates Isoproterenol Induced Cardiac Injury with Links to Solute Carrier Transporter SLC41a3 and Circadian Clock Genes

Abstract

Kvβ subunits belong to the aldo-keto reductase superfamily, which plays a significant role in ion channel regulation and modulates the physiological responses. However, the role of Kvβ2 in cardiac pathophysiology was not studied, and therefore, in the present study, we hypothesized that Kvβ2 plays a significant role in cardiovascular pathophysiology by modulating the cardiac excitability and gene responses. We utilized an isoproterenol-infused mouse model to investigate the role of Kvβ2 and the cardiac function, biochemical changes, and molecular responses. The deletion of Kvβ2 attenuated the QTc (corrected QT interval) prolongation at the electrocardiographic (ECG) level after a 14-day isoproterenol infusion, whereas the QTc was significantly prolonged in the littermate wildtype group. Monophasic action potentials verified the ECG changes, suggesting that cardiac changes and responses due to isoproterenol infusion are mediated similarly at both the in vivo and ex vivo levels. Moreover, the echocardiographic function showed no further decrease in the ejection fraction in the isoproterenol-stimulated Kvβ2 knockout (KO) group, whereas the wildtype mice showed significantly decreased function. These experiments revealed that Kvβ2 plays a significant role in cardiovascular pathophysiology. Furthermore, the present study revealed SLC41a3, a major solute carrier transporter affected with a significantly decreased expression in KO vs. wildtype hearts. The electrical function showed that the decreased expression of SLC41a3 in Kvβ2 KO hearts led to decreased Mg²⁺ responses, whereas, in the wildtype hearts, Mg²⁺ caused action potential duration (APD) shortening. Based on the in vivo, ex vivo, and molecular evaluations, we identified that the deletion of Kvβ2 altered the cardiac pathophysiology mediated by SLC41a3 and altered the NAD (nicotinamide adenine dinucleotide)-dependent gene responses.

Keywords: Kvβ subunit; action potential; aldo-keto reductase; heart; magnesium; potassium channel; pyridine nucleotides; redox.

Figure 1

Isoproterenol-induced cardiac hypertrophy and functional alterations. Echocardiography changes in wildtype (WT) and Kvβ2 knockout (KO) saline (SAL) and isoproterenol (ISO)-treated mice. (A) Representative M mode images of the left ventricular chamber. (B) Ejection fraction (EF) determined by the M mode measurements in wildtype mice. The EF values are normalized to wildtype saline. (C) Ejection fraction (EF) determined by M mode measurements in knockout mice. The EF values are normalized to knockout saline. (D) Heart weight measurements normalized with the tibia length. The data represented are the mean ± SEM. ** represents, p < 0.01, * represents p < 0.05.

Figure 2

Isoproterenol-induced electrical prolongation ablated in Kvβ2 KO mice. (A) P interval. (B) QTc interval. (C) JTc interval. The data represented are the mean ± SEM. * represents p < 0.05.

Figure 3

Isoproterenol-induced monophasic action potential (MAP) prolongation was ablated in Kvβ2 KO mice. (A) Representative MAP traces from WT (black lines) and Kvβ2 KO (red lines) hearts (14-16 weeks of age). Solid line indicates saline (SAL) treatment, while dotted line indicates isoproterenol (ISO) treatment for 14 days; scale bar represents 10 milliseconds. (B) Left ventricular (LV) surface MAPs at 20% repolarization (APD 20%). (C) LV surface MAPs at 50% repolarization (APD 50%). (D) LV surface MAPs at 70% repolarization (APD 70%). The data represented are the mean ± SEM. * represents p < 0.05.

Figure 4

Kvβ2 deletion increases the susceptibility to ventricular arrhythmias. (A,B) Programmed electrical stimulation in wildtype (WT) and Kvβ2 knockout (KO) mice. Onset of ventricular tachycardia (VT) after premature stimulus (small asterisk) is shown in Kvβ2 KO mice (none of the six wildtype mice were inducible, but three of the seven KO mice were inducible: * p < 0.05). (C). LV surface MAP triangulation at the baseline measurements. (D) LV surface MAPs at 50% repolarization (APD 50%) during paced stimulations at 5 Hz and 8 Hz. Scale bar represents 100 ms. The data represented are the mean ± SEM. * represents p < 0.05, *** represents p < 0.01, and **** represents p < 0.001.

Figure 5

Heatmap of the differentially expressed genes identified (log2-transformed). Dendrograms were also added to the rows (genes) and columns (samples) to show the clustering results. The color palette “blue–red” was used.

Figure 6

Circadian gene alterations induced in Kvβ2 (voltage gated potassium channel subunit β2) deletion. (A) Protein expression of Kvβ2 in wild type (WT) and knockout (KO) hearts treated with saline (SAL) and isoproterenol (ISO). (B) Quantitative Kvβ2 protein expression from wildtype (WT) hearts, (KO hearts demonstrated no measurable protein expression for Kvβ2). The data represented are the mean ± SEM. (C) PCR (polymerase chain reaction) expression of the key circadian genes selected from the microarray. The data represented the mean ± SEM. * represents p ≤ 0.05.

Figure 7

SLC41 expression and Mg²⁺ concentration alters the monophasic action potentials (MAPs). (A) Protein expression of SLC41a3 in WT and KO hearts. (B) Quantitative SLC41a3 protein expression from WT and KO hearts are the mean ± SEM. * represents p≤0.05. (C) Serum magnesium concentrations from WT and KO mice are the mean ± SEM. (D) LV surface MAPs at 50% repolarization (APD 50%) in WT hearts (blue) and KO hearts (red) normalized to baseline measurements, followed by exposure to MgSO₄ (Mg²⁺) following a recovery period with the original buffer. The data represented are the mean ± SD. * represents p < 0.05.