Alterations in ventricular K(ATP) channel properties during aging

Abstract

Coronary heart disease remains the principle cause of mortality in the United States. During aging, the efficiency of the cardiovascular system is decreased and the aged heart is less tolerant to ischemic injury. ATP-sensitive K(+) (K(ATP)) channels protect the myocardium against ischemic damage. We investigated how aging affects cardiac K(ATP) channels in the Fischer 344 rat model. Expression of K(ATP) channel subunit mRNA and protein levels was unchanged in hearts from 26-month-old vs. 4-month-old rats. Interestingly, the mRNA expression of several other ion channels (> 80) was also largely unchanged, suggesting that posttranscriptional regulatory mechanisms occur during aging. The whole-cell K(ATP) channel current density was strongly diminished in ventricular myocytes from aged male rat hearts (also observed in aged C57BL/6 mouse myocytes). Experiments with isolated patches (inside-out configuration) demonstrated that the K(ATP) channel unitary conductance was unchanged, but that the inhibitory effect of cytosolic ATP on channel activity was enhanced in the aged heart. The mean patch current was diminished, consistent with the whole-cell data. We incorporated these findings into an empirical model of the K(ATP) channel and numerically simulated the effects of decreased cytosolic ATP levels on the human action potential. This analysis predicts lesser activation of K(ATP) channels by metabolic impairment in the aged heart and a diminished action potential shortening. This study provides insights into the changes in K(ATP) channels during aging and suggests that the protective role of these channels during ischemia is significantly compromised in the aged individual.

Figure 1. Action potential configuration and whole-cell K_ATP channel density are altered in the aged myocytes compared to young rat ventricular myocytes

A) Representative whole-cell action potentials, evoked by current injection at 1Hz through the patch pipette, in ventricular myocytes isolated from young or aged rats. The horizontal line represents zero voltage. B) Summary data of the action potential duration, recorded at 90% repolarization (APD₉₀), of young (n=14 cells from 3 animals) and aged (n=17 cells from 3 animals) ventricular myocytes. *p<0.0; Mann-Whitney Rank Sum test. C) Current-voltage relationships obtained under normoxic conditions using a ramp voltage protocol (5 to -100 mV; ramp rate of -25 mV·s^-1, applied every 20s from a holding potential of -45 mV). The input resistances were 7.98 ± 0.86 MΩ in aged rats and 6.54 ± 0.71 MΩ in young rats. Data are shown for young (open symbols) and aged (filled symbols) rat ventricular myocytes. D) Current-voltage relationships obtained after application of 2.4-dinitrophenol (DNP, 100 μM). Currents are expressed as current density (pA/pF). The inset shows a representative current trace recorded from a ventricular myocyte. The scale bar represents 4 nA and 20 s. E) Normoxic current-voltage relationships in young and aged mouse ventricular myocytes. F) Current-voltage relationships in mouse ventricular myocytes after application of DNP (100 μM). The cell capacitance was smaller in the young mouse ventricular myocytes (147.6±12.4, n=14) compared to the aged myocytes (197.6±11.7; n=14; p= 0.007, Student's t-test).

Figure 2. Effects of age on nucleotide sensitivity of cardiac K_ATP channels

A) Patch clamp current recordings, made in the inside-out configuration, obtained from isolated ventricular myocytes of young (top trace) and aged (bottom trace) rat hearts. The ATP concentrations were changed as indicated. The dashed line represents zero current. B) The fractional current (mean patch current divided by the current recorded in the absence of ATP) is plotted as a function of the ATP concentrations. The data points are means and SEM for young (open symbols; n=20 patches from 3 animals) and aged (filled symbols; n=16 patches from 3 animals) groups. These data were subjected to curve fitting to a sum of two modified Hill equations (see Methods section for equations). The solid lines represent the result of the curve fitting. The two dotted lines represent the low- and high-sensitivity components of the ATP-sensitivity curve of the aged group. The IC₅₀ values, Hill coefficients and weights of the components are provided in the text.

Figure 3. K_ATP channel subunit mRNA expression in rat aged and young ventricular myocytes

A) semi-quantitative real-time RT-PCR data showing mRNA expression of rat Kir6.1, Kir6.2, SUR1 and SUR2 subunits in total RNA extracted from young (n=5) or aged (n=5) rat ventricles. Data are expressed relative to that of β-actin. B) Conventional RT-PCR was performed to discriminate between SUR2 spice variants expressed in ventricular myocytes isolated from young or aged rat hearts. A primer pair was used that resulted in a 493 bp amplicon for SUR2A and a 317 bp band for SUR2B. The PCR products were sequenced to confirm the identity of the amplicons. C) Bar graph depicting the relative expression of SUR2A or SUR2B (n=4 animals in each group). There were no significant age-dependent differences in these transcripts.

Figure 4. Lack of transcriptional regulation of ion channel genes with aging

Real-time RT-PCR was performed using primers specific to eighty two ion subunits of ion channels, pumps or exchangers, using RNA isolated from young male or female (n=3 each) or aged male or female (n=3 each) hearts. Data were analyzed relative to the average expression of the following reference genes: hydroxymethylbilane synthase, histone H3 and cyclophilin B. Hierarchical clustering was performed after log₂ transformation (no other data adjustments or normalization was performed) and visualized as a heat map using MEV.

Figure 5. K_ATP channel subunit protein expression in young and aged rat hearts

Membrane preparations from young or aged rat hearts were subjected to 10% SDS-PAGE and immunoblotted with antibodies against Kir6.2, SUR1, SUR2A, SUR2B or GAPDH.

Figure 6. Simulating the effects of K_ATP channel opening on action potential duration

The ten Tusscher human ventricular action potential model (ten Tusscher et al. 2004) was used to incorporate a K_ATP channel simulation (Ferrero et al. 1996; Bao et al. 2011b). Action potentials were simulated with 6.3, 5.1, 4.2, 3.4, 2.8, 2.3, 1.8, 1.5, 1.2 and 1 mM ATP using K_ATP channel parameters determined from A) young and B) aged Fischer 344 rat hearts. We simultaneously adjusted ADP to pathophysiologically relevant levels. Since most cellular ADP is bound (e.g. to actin), the free ADP level was estimated to be 5% of the total nucleotide concentration (assumed to be 7mM) minus the ATP concentration (e.g. 35, 95, 140, 180, 210, 235, 260, 275, 290 and 300 μM). C) The action potential duration (measured at -75 mV) is plotted as a function of the ATP:ADP ratio. Note that the action potential properties themselves were not adjusted for age to more accurately examine effects of K_ATP channel opening.