Vascular KATP channels protect from cardiac dysfunction and preserve cardiac metabolism during endotoxemia

Abstract

K_ATP channels in the vasculature composed of Kir6.1 regulate vascular tone and may contribute to the pathogenesis of endotoxemia. We used mice with cell-specific deletion of Kir6.1 in smooth muscle (smKO) and endothelium (eKO) to investigate this question. We found that smKO mice had a significant survival disadvantage compared with their littermate controls when treated with a sub-lethal dose of lipopolysaccharide (LPS). All cohorts of mice became hypotensive following bacterial LPS administration; however, mean arterial pressure in WT mice recovered to normal levels, whereas smKO struggled to overcome LPS-induced hypotension. In vivo and ex vivo investigations revealed pronounced cardiac dysfunction in LPS-treated smKO, but not in eKO mice. Similar results were observed in a cecal slurry injection model. Metabolomic profiling of hearts revealed significantly reduced levels of metabolites involved in redox/energetics, TCA cycle, lipid/fatty acid and amino acid metabolism. Vascular smooth muscle-localised K_ATP channels have a critical role in the response to systemic infection by normalising cardiac function and haemodynamics through metabolic homeostasis. KEY MESSAGES: • Mice lacking vascular K_ATP channels are more susceptible to death from infection. • Absence of smooth muscle K_ATP channels depresses cardiac function during infection. • Cardiac dysfunction is accompanied by profound changes in cellular metabolites. • Findings from this study suggest a protective role for vascular K_ATP channels in response to systemic infection.

Keywords: Cardiac metabolism; Endotoxemia; KATP; Kir6.1; Vascular smooth muscle.

Fig. 1

Mice lacking Kir6.1 are predisposed to an increase risk of LPS-induced death. WT, smKO and gKO mice were dosed with 10 mg/kg LPS (I.P injection) and monitored for 24–48 h. a Kaplan-Meier survival curves for mice (WT-black line, smKO-grey line and gKO-dotted line) treated with LPS. Differences between curves was assessed using the log-rank (Mantel-Cox) test, *P < 0.05, ***P < 0.001 compared with WT, ^#P < 0.01 compared with smKO. b Proportion of death/survival after LPS administration for each genotype over a 48-h period. n = 12–16 mice per group. Fisher’s exact test was used to for statistical analysis, *P < 0.05, ***P < 0.001

Fig. 2

WT and Kir6.1 KO mice are susceptible to severe hypotension following LPS treatment. a Representative mean arterial pressure (MAP) traces from blood pressure telemetry recordings from WT, smKO and gKO mice following I.P injection of 10 mg/kg LPS. b Representative heart rate traces derived from blood pressure telemetry recordings from WT, smKO and gKO mice injected with LPS. c Mean relative MAP (left), mean MAP at baseline (middle) and % drop in MAP at 15 and 18 h post-LPS (right) (n = 5–8 mice for each group). d Mean relative heart rate (left), mean heart rate at baseline (middle) and % change in heart rate at 15 and 18 h post-LPS (n = 5–8 mice for each group). In c and d, data is shown as mean ± SEM. *P < 0.05, **P0.01 (unpaired Student’s t test and 2-way ANOVA). ^#P < 0.05, ^##P < 0.01 compared with baseline (unpaired Student’s t test)

Fig. 3

Cardiac function is substantially reduced in smKO mice following LPS administration. a Mean body temperature and echocardiography parameter measurements from smKO mice and their littermate controls 18 h post-LPS injection (2 mg/kg I.P). Ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (LVEDP) and left ventricular internal diameter (LVID) were derived from short-axis M-mode images. Fractional area change (FAC) was measured from short-axis B-mode images (n = 5–6 mice per group). b Levels of circulating markers for kidney (urea and creatinine) and liver (ALT-alanine acetyltransferase) damage in the blood of smKO mice and their littermate controls (n = 5–6 mice per group). c Representative images (left) and mean cell death (right) from a TUNEL assay on sections from smKO (n = 4) and littermate control (n = 4) hearts. Data is shown as mean ± S.E.M, *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t test)

Fig. 4

Cardiac function is reduced in smKO mice following sepsis induced by cecal slurry administration. a Mean body temperature and echocardiography parameter measurements from smKO mice and their littermate controls 20 h post-cecal slurry injection. Ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (LVEDP) and left ventricular internal diameter (LVID) were derived from short-axis M-mode images. Fractional area change (FAC) was measured from short-axis B-mode images (n = 6 mice for each group). b Levels of circulating markers for kidney (urea and creatinine) and liver (ALT-alanine acetyltransferase) damage in the blood of smKO mice and their littermate controls (n = 6 mice for each group). Data is shown as mean ± S.E.M, *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t test)

Fig. 5

Cardiac function is severely perturbed in isolated LPS-treated smKO mouse hearts. Hearts were isolated from mice 18 h post-LPS administration (2 mg/kg I.P) and mounted via the aorta on to a Langendorff apparatus and retrogradely perfused to investigate coronary function and LV function. Mean HR (a), coronary perfusion pressure (CPP) (b), left ventricular end-diastolic pressure (LVEDP) (c) and left ventricular developed potential (LVDP) (d) measured from isolated hearts of smKO and littermate control mice 18 h post-LPS administration. Starling curves for LVDP (e) and LVEDP (f). Data is shown as mean ± S.E.M, n = 5 mice for each group, **P < 0.01, ***P < 0.001 (unpaired Student’s t test and 2-way ANOVA)

Fig. 6

Cardiac function is preserved in eKO mice following LPS administration. a Mean body temperature and echocardiography parameter measurements from eKO mice and their littermate controls 18 h post-LPS injection (2 mg/kg I.P). Ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (LVEDP) and left ventricular internal diameter (LVID) were derived from short-axis M-mode images. Fractional area change (FAC) was measured from short-axis B-mode images (n = 4–6 mice for each group). b Levels of circulating markers for kidney (urea and creatinine) and liver (ALT-alanine acetyltransferase) damage in the blood of eKO mice and their littermate controls (n = 6 mice for each group). Data is shown as mean ± S.E.M, P > 0.05 (unpaired Student’s t test)

Fig. 7

LPS-induced changes in myocardial metabolomic profile in smKO mice. Metabolomic analysis was carried out using high-resolution ¹H NMR spectroscopy. a Representative spectra from ¹H NMR analysis of WT (top panel) and smKO (lower panel) hearts. b Mean fold change in metabolite levels in smKO hearts compared with WT littermate controls (n = 5–7 mice for each group). Data is shown as mean ± SEM, **P < 0.01, ***P < 0.001 (unpaired Student’s t test)

Fig. 8

Schematic of the proposed protective mechanism of the vascular K_ATP channel during endotoxemia. Opening of vascular K_ATP channels in the coronary circulation in response to LPS-induced stress or indirectly via vasomediators such as CGRP protects myocardial function and prevents myocyte apoptosis by maintaining adequate myocardial perfusion and metabolite provision to meet demand. An increase in metabolism and hence ATP production in the tissues of other vascular beds ultimately restores blood pressure homeostasis and perfusion of vital organs. The absence of vascular K_ATP channels in the coronary circulation leads to a scenario where myocardial metabolism is significantly impacted by failure of substrate delivery to the heart leading to myocyte apoptosis and attenuated myocardial function. Blood pressure fails to recover because of the drop in cardiac output ultimately leading to death