Reduced Na⁺ current density underlies impaired propagation in the diabetic rabbit ventricle

Abstract

Diabetes is associated with an increased risk of sudden cardiac death, but the underlying mechanisms remain unclear. Our goal was to investigate changes occurring in the action potential duration (APD) and conduction velocity (CV) in the diabetic rabbit ventricle, and delineate the principal ionic determinants. A rabbit model of alloxan-induced diabetes was utilized. Optical imaging was used to record electrical activity in isolated Langendorff-perfused hearts in normo-, hypo- and hyper-kalemia ([K(+)]o=4, 2, 12 mM respectively). Patch clamp experiments were conducted to record Na(+) current (I(Na)) in isolated ventricular myocytes. The mRNA/protein expression levels for Nav1.5 (the α-subunit of I(Na)) and connexin-43 (Cx43), as well as fibrosis levels were examined. Computer simulations were performed to interpret experimental data. We found that the APD was not different, but the CV was significantly reduced in diabetic hearts in normo-, hypo-, and, hyper-kalemic conditions (13%, 17% and 33% reduction in diabetic vs. control, respectively). The cell capacitance (Cm) was increased (by ~14%), and the density of INa was reduced by ~32% in diabetic compared to control hearts, but the other biophysical properties of I(Na) were unaltered. The mRNA/protein expression levels for Cx43 were unaltered. For Nav1.5, the mRNA expression was not changed, and though the protein level tended to be less in diabetic hearts, this reduction was not statistically significant. Staining showed no difference in fibrosis levels between the control and diabetic ventricles. Computer simulations showed that the reduced magnitude of I(Na) was a key determinant of impaired propagation in the diabetic ventricle, which may have important implications for arrhythmogenesis.

Keywords: Diabetes rabbit cardiac electrophysiology Na(+)-current.

Figure 1. Quantifying basic electrophysiology parameters via optical mapping in isolated hearts

Comparison of APD₇₀ (left panels) and conduction velocity (CV) (right panels), at BCL=250 ms, in control versus diabetic hearts in (A.) normal [K⁺]_o levels (4mM), (B.) low [K⁺]_o levels (2mM), hypokalemia, and (C.) high [K⁺]_o levels (12mM), hyperkalemia.

Figure 2. Representative activation maps

Comparison of representative activation maps of cardiac propagation in control and diabetic hearts in hyperkalemia ([K⁺]_o=12mM) at BCL=250 ms. “*” = pacing site.

Figure 3. Patch clamp experiments for I_Na in control versus diabetic ventricular cells

(A.) Representative recordings of I_Na. (B.) I–V relationship. (C.) Steady-state activation and inactivation curves. (D.) Recovery from inactivation. [n = 12–17 (N=3)].

Figure 4. mRNA and protein expression of Nav1.5 and Cx43

(A.) mRNA Expression levels of Nav1.5, and Cx43 in control vs. diabetic rabbit LV epicardial tissue. N=5 per group. (B.) Immunoblot showing immunodetection of Nav1.5 (Top), Cx43 expression (Middle), and GAPDH (loading control; Bottom) from epicardial left ventricle samples of control (C) and diabetic (D) rabbits. (C.) Densitometric analysis of Cx43 and Nav1.5 expression normalized to GAPDH levels in control and diabetic rabbits. Values represent data (mean ± SEM) from 4 animals in each condition.

Figure 5. Quantification of interstitial fibrosis in LV rabbit epicardial tissue

(A.) Representative examples of stained sections (red = collagen). (B.) Quantification of % area fibrosis in control (N=7) and diabetic (N=7) rabbit samples.

Figure 6. Computer simulations

(A.) Schematic of 2D model. (B.) Conduction velocity (CV) in simulation vs. experiments in normokalemia, hypokalemia, hyperkalemia in both control and diabetic hearts; diabetes was simulated by reducing the density of I_Na by 30%. (C.) Reduction in CV when Na⁺ current magnitude is reduced by 30%, gap junction coupling is reduced by 20%, Na⁺-K⁺ pump current is reduced by 30%, and C_m is increased by 14% in the reaction diffusion equation.