Cellular remodeling in heart failure disrupts K(ATP) channel-dependent stress tolerance

Abstract

ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, K(ATP) channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.

Fig. 1. Heart failure in TNFα transgenic mice. (A) The 1.2 kB band TNFα transgene in tail-cut PCR of transgenic (TNFα-TG) but not WT mice. (B) Exercise intolerance of TNFα-TG, compared with WT, with lower tolerated workload (inset) and earlier treadmill drop-out (p < 0.05). (C) Left ventricular fractional shortening, by echocardiography, was significantly less in TNFα-TG than WT (p < 0.05). In mice challenged with isoproterenol (0.5 µg i.p.), augmentation of fractional shortening was greater in WT compared with TNFα-TG (p < 0.05). (D) Mortality was greater in TNFα-TG (initial n = 135, 85% censored by 53 weeks) compared with WT (initial n = 175, 99% censored by 53 weeks) mice (p < 0.05). (E) Remodeling in 8-week-old TNFα-TG (TG) mice. Top: chamber dilation and reduced wall thickness at the base of TG versus WT hearts. Middle: distortion of architecture in TG versus rod-shaped WT ventricular cardiomyocytes on scanning microscopy. Bottom: myofibrillar disorganization in TG versus WT ventricular tissue by transmission electron microscopy.

Fig. 2. Intact K_ATP channels in TNFα-TG cardiomyocytes receive altered ATP signal. (A) Intraburst K_ATP channel kinetics were indistinguishable in excised WT and TNFα-TG patches, with characteristic open and closed times (τ_o and τ_c), derived from the best-fit of corresponding distributions, not significantly different (n = 3). (B) K_ATP channel current–voltage relationships (n = 4) with identical channel conductance and rectification in WT and TNFα-TG patches. (C) K_ATP channel activity in excised membrane patches calculated relative to activity in the absence of ATP, and fitted by the Hill equation 1/[1 = x/IC₅₀]^k (solid curves), where x is ATP concentration, h the Hill coefficient and IC₅₀ the half-maximal inhibition concentration. (D and E) K_ATP channels, in open cell-attached mode, show altered effect of ATP in TNFα-TG compared with WT.

Fig. 3. Depressed bioenergetic components create conditions impeding signaling to K_ATP channels in TNFα-TG hearts. (A) Mitochondrial ADP-stimulated respiration is significantly depressed in isolated mitochondria from TNFα-TG compared with WT hearts. (B) While in WT glycogen granules are abundant in electron micrography, in TNFα-TG they are sparse. (C) Creatine kinase (CK) flux, by ¹⁸O-assisted NMR spectroscopy, was significantly reduced in TNFα-TG compared with WT hearts. (D) In the open cell-attached mode, the creatine phosphate (CrP)/creatine kinase system effectively regulated K_ATP channel activity in the WT (upper), but not TNFα-TG (lower). (E) Concentration-response of CrP-stimulated K_ATP channel inhibition in open cell-attached patches. Data fitted by the Hill equation (solid curves) show a significant increase in the IC₅₀ for CrP-induced channel inhibition in TNFα-TG versus WT. The asterisk in (A) and (C) indicates p < 0.05.

Fig. 4. K_ATP channel-dependent membrane control under stress defective in TNFα-TG hearts. (A) In the open cell-attached mode, with spontaneous channel opening suppressed by ATP, dinitrophenol (DNP) induced a vigorous K_ATP channel response in WT, but not TNFα-TG. (B) DNP-induced K_ATP channel activity in WT versus TNFα-TG (p < 0.05). (C) Monophasic action potentials under normoxia (O₂ content = 32 mg/l) and hypoxia (O₂ content = 3.1 mg/l). (D) Under hypoxia, APD₉₀ markedly shortened in WT, but not TNFα-TG hearts.

Fig. 5. Vulnerability to stress in TNFα-TG hearts attenuated by potassium channel openers. (A) Fluo-3-loaded and paced WT cardiomyocytes (upper) tolerated isoproterenol (1 µM) challenge without significant change in maximal systolic and diastolic Ca²⁺ levels. TNFα-TG cardiomyocytes (lower), under isoproterenol stress, developed diastolic Ca²⁺ overload with cell contracture. Ca²⁺-induced fluorescence in a transverse cellular plane versus time is shown in green. Orange and white traces are deconvoluted fluorescent frames, and represent average Ca²⁺ maxima (systole, sys) and minima (diastole, dias). (B) Upper: photomicrographs (40×) of phosphotungstic acid hematoxylin-stained left ventricle 45 min after isoproterenol (2 mg i.p.) with contraction bands (arrows) in TNFα-TG, but not WT. Lower: electron microscopy of a contraction band in TNFα-TG (right) compared with normal sarcomeric pattern in WT (left). (C and D) In TNFα-TG, nicorandil (500 µM) activated K_ATP channels in excised patches (C), and shortened action potential duration (D). (E) Nicorandil (2 mg i.p; n = 4) or pinacidil (0.1 mg i.p; n = 3) versus vehicle (n = 5), 30 s prior to isoproterenol (isoproter.) challenge (0.8–2 mg i.p.), significantly reduced contraction bands in TNFα-TG mice (p < 0.05). Dotted line: average bands in vehicle-treated, isoproterenol-stressed, WT (n = 3). (F) Treatment of TNFα-TG mice twice daily for 1 week with nicorandil (0.5 mg/kg, s.c.; n = 3) or pinacidil (1 mg/kg, s.c.; n = 3) improved glycogen storage (p < 0.05) expressed relative to vehicle-treated TNFα-TG mice (n = 3; dotted line).