Subtle Role for Adenylate Kinase 1 in Maintaining Normal Basal Contractile Function and Metabolism in the Murine Heart

Figures

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  FIGURE 1. Biochemical characterisation of relevant metabolic pathways in left ventricle from WT and homozygous AK1-OE mice. AK1 protein activity was 31% higher than WT without affecting the activity of other AK isoforms; n = 6 (3M shown by solid symbols/3F) (A). Total activity of creatine kinase was not altered (B; coloured symbols show combined M/F; WT:6M/7F, OE:8M/7F), but there was a subtle shift in isoenzyme activities from MM to MB (C). For CK activities, values for M and F are shown separately, in addition to combined M/F (last 2 columns). For Mt-CK, MM-CK, MB-CK and BB-CK: WT: 7M/7F; OE: 7M/8F. Total creatine levels were slightly elevated in AK1-OE (AK1/AK1) hearts (WT 7M/9F, OE 8M/7F) (D; last 2 columns are combined M/F) but no changes were seen in creatine transporter (CrT) mRNA (E) (n = 4 M). Citrate synthase activity was unchanged (F; last 2 columns are combined M/F); WT n = 14 (7M/7F), OE n = 15 (7M/8F), similarly to total adenine nucleotides (G; last 2 columns are combined M/F) WT; n = 16 (7M/9F), OE n = 15 (8M/7F). There was no difference in the fraction of phosphorylated AMPK protein (H,I; last 2 columns are combined M/F), WT n = 13 (5M/8F), OE n = 15 (5M/10F). Unpaired…

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  FIGURE 2. 1H-MRS metabolomic profile from ventricular myocardium. Principal component analysis (PCA) for aqueous metabolites (A) and lipids (B). Concentrations of all detected metabolites are shown relative to mean wild-type (WT) levels. These are represented by box and whiskers plots for aqueous fraction (C) and lipid fraction (D). The box indicates median values with 25th and 75th percentiles and the whiskers the min and max values. Significant changes are shown in colour according to legend. Only age-matched males were used, n = 8 per genotype, mean age of 25 weeks. Data analysis as described in Online Supplement and by Unpaired Student’s t-test. Combined ventricular weight was higher in AK1-OE (AK1/AK1) mice compared to wild-type (E), comparisons by Unpaired Student’s t-test.

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  FIGURE 3. Left ventricular weight and composition in wild-type (WT/WT) and AK1-OE (AK1/AK1) mice. (A) LV post mortem weight normalised to tibial length in female mice was not different (left hand columns; WT n = 14, OE n = 16; Ages 28 ± 4 and 27 ± 4, respectively), while LV/tibial length was higher in male AK1-OE mice (WT n = 16, OE n = 16 both aged 22 ± 2 weeks). (B) This was not associated with systematic activation of foetal hypertrophy genes for atrial natriuretic peptide (Nppa), brain natriuretic peptide (Nppb), β-myosin heavy chain (Myh7) and alpha-skeletal actin (Acta1) (WT n = 7, OE n = 4 all male aged 16–17 weeks). (C) LV histological sections (Masson-Goldner Staining) showing average myocyte cross-sectional in WT and AK1-OE male hearts (55–157 myocytes per heart from n = 4 hearts) (P = 0.01) (20x objective lens). (D) Fibrosis in WT transgenic mice, as analysed by MassonGoldner Staining (P = 0.8) (10x objective lens). (E) LV wet and dry weights were obtained in a subset of mice from panel (A) and used to calculate tissue water content, which was elevated only in males (P = 0.01). Comparison between two genotypes was by unpaired Student’s t-test using Welch’s correction where…

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  FIGURE 4. In vivo left ventricular haemodynamic measurements. (A) There was a trend for LV endsystolic pressure to be lower in AK1-OE (AK1/AK1) females compared to wild-type (left-hand columns) that was statistically significant in the male mice (right-hand columns). LV end-diastolic pressure (B), heart rate (C) and contractility (assessed as the maximal rate of pressure change, dP/dtmax; (D) were unaffected by genotype for either sex. However, tau, the isovolumetric constant of relaxation was prolonged in male AK1-OE compared to male wild-type (E) and the maximum contractility obtained under β-adrenergic stimulation with dobutamine was lower (F). Females n = 9 and males n = 10 with mean age 17 weeks throughout. Data is mean ± SEM, with same sex comparisons between genotype by Student’s t-test.

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  FIGURE 5. Left ventricular function during global no-flow ischaemia for 20 and 30 min reperfusion in Langendorff perfused hearts. An example pressure trace showing pre-ischaemic baseline function and during terminal reperfusion arrhythmia in an AK1-OE (AK1/AK1) heart (A). Analysis of function in surviving hearts (n = 7 wild-type and n = 3 AK1-OE) (B) showing end-diastolic pressures (C), and developed pressures (D), in addition to heart rate is unaffected (E). Data is mean ± SEM, expressed as a percentage of the average baseline values. Analysis is by two-way ANOVA with Student’s t-tests to compare genotypes at all time-points using the Holm-Sidak method to correct for multiple comparisons. In a separate experiment perfusate was collected during the first 10 min of reperfusion for measurement of adenosine release (F) and of residual myocardial levels of total adenine nucleotide pool (G) and total creatine (H). Data is mean ± SEM (n = 9–10 WT and n = 8 AK1-OE, all male mice), analysis by two-tailed unpaired Student’s t-test.

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  FIGURE 6. Myocardial infarct size following 45 min regional ischaemia and 24 h reperfusion in vivo. (A) Example slices from wild-type (WT/WT) and AK1-OE (AK1/AK1) hearts showing typical tricolour staining pattern of blue for unaffected tissue, red + white for the ischaemic area-at-risk, and white indicating areas of tissue necrosis. (B) The area-at risk, i.e., the area that was made ischaemic, was not significantly different between genotypes, nor did AK1-OE alter the size of necrotic injury (C). Data is mean ± SEM with comparison by unpaired t-test. Wild-type n = 13 (6F/7M) and AK1-OE n = 15 (8F/7M).