An open sarcolemmal adenosine triphosphate-sensitive potassium channel is necessary for detrimental myocyte swelling secondary to stress

Abstract

Background: Stress (exposure to hyperkalemic cardioplegia, metabolic inhibition, or osmotic) results in significant myocyte swelling and reduced contractility. In contrast to wild-type mice, these detrimental consequences are not observed in mice lacking the Kir6.2 subunit of the sarcolemmal ATP-sensitive potassium (sK(ATP)) channel after exposure to hyperkalemic cardioplegia. The hypothesis for this study was that an open sK(ATP) channel (Kir6.2 and SUR2A subunits) is necessary for detrimental myocyte swelling to occur in response to stress.

Methods and results: To investigate the role of the sK(ATP) channel in stress-induced myocyte swelling, high-dose pharmacological sK(ATP) channel blockade and genetic deletion (knockout of Kir6.2 subunit) were used. Myocytes were exposed sequentially to Tyrode control (20 minutes), test (stress) solution (20 minutes), and Tyrode control (20 minutes). To evaluate pharmacological channel blockade, myocytes were exposed to hyperkalemic cardioplegia (stress) with and without a K(ATP) channel blocker. To evaluate the effects of genetic deletion, wild-type and sK(ATP) knockout [Kir6.2(-/-)] myocytes were exposed to metabolic inhibition (stress). Myocyte volume was recorded using image-grabbing software. Detrimental myocyte swelling was prevented by high-dose sK(ATP) channel blockade (glibenclamide or HMR 1098) but not mitochondrial K(ATP) channel blockade (5-hydroxydecanoate) during exposure to hyperkalemic cardioplegia. Genetic deletion of the sK(ATP) channel prevented significant myocyte swelling in response to metabolic inhibition.

Conclusions: K(ATP) channel openers prevent detrimental myocyte swelling and reduce contractility in response to stress through an unknown mechanism. Paradoxically, the present data support a role for sK(ATP) channel activation in myocyte volume derangement in response to stress.

Figure 1. Myocytes exposed to cardioplegic stress exhibited significant swelling that was unaltered by the addition of low dose pharmacologic sarcolemmal or mitochondrial K_ATP channel blockade

Myocytes were exposed to control Tyrode's solution for baseline volume measurement (20 min), followed by test solution (cardioplegia, cardioplegia + 10μM HMR 1098, cardioplegia + 20μM HMR 1098, cardioplegia + 10μM glibenclamide, cardioplegia + 20μM glibenclamide, or cardioplegia + 50μM 5-HD) (20 min), and reexposure to Tyrode's (20 min). The x-axis represents time in minutes and the y-axis represents percent volume change relative to baseline. 5 - HD is 5 hydroxydecanoate.

Figure 2. Myocytes exposed to cardioplegic stress exhibited significant swelling that was prevented by high dose sarcolemmal K_ATP channel blockade (HMR 1098 or glibenclamide) but not mitochondrial K_ATP channel blockade (5-HD)

Myocytes were exposed to control Tyrode's solution (20 min) for baseline volume measurement, followed by test solution (cardioplegia, cardioplegia + 40μM HMR 1098, cardioplegia + 40μM glibenclamide, or cardioplegia + 100μM 5-HD) (20 min), and reexposure to Tyrode's (20 min). The x-axis represents time in minutes and the y-axis represents percent volume change relative to baseline. 5 - HD is 5 hydroxydecanoate.

Figure 3A. Wild type (WT) myocytes demonstrate significant swelling during exposure to metabolic inhibition (MI)

Cells were exposed to normal Tyrode's solution for baseline volume measurement (20 min), followed by test solution (20 min), and followed by reexposure to Tyrode's (20 min). The y-axis represents percent volume change relative to baseline and the x-axis represents test solution. Wild Type (WT) myocyte volume remained unchanged when exposed to control Tyrode's solution (NT). Wild Type (WT ) myocyte volume significantly (p= 0.004) increased when exposed to metabolic inhibition (MI).

Figure 3B. Kir 6.2 Knockout (KO) myocytes do not swell during exposure to metabolic inhibition (MI)

Cells were exposed to normal Tyrode's solution for baseline volume measurement (20 min), followed by test solution (20 min), followed by reexposure to Tyrode's (20 min). The y-axis represents percent volume change relative to baseline and the x-axis represents test solution. Kir6.2 knockout (KO) myocyte volume remained unchanged when exposed to control Tyrode's (NT). Kir6.2 knockout (KO) myocyte volume also remained unchanged when exposed to metabolic inhibition (MI).