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. 2020 Jan 21;10:1535.
doi: 10.3389/fphys.2019.01535. eCollection 2019.

Age-Dependent Decline in Cardiac Function in Guanidinoacetate- N-Methyltransferase Knockout Mice

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Free PMC article

Age-Dependent Decline in Cardiac Function in Guanidinoacetate- N-Methyltransferase Knockout Mice

Dunja Aksentijević et al. Front Physiol. .
Free PMC article

Abstract

Aim: Guanidinoacetate N-methyltransferase (GAMT) is the second essential enzyme in creatine (Cr) biosynthesis. Short-term Cr deficiency is metabolically well tolerated as GAMT-/- mice exhibit normal exercise capacity and response to ischemic heart failure. However, we hypothesized long-term consequences of Cr deficiency and/or accumulation of the Cr precursor guanidinoacetate (GA).

Methods: Cardiac function and metabolic profile were studied in GAMT-/- mice >1 year.

Results: In vivo LV catheterization revealed lower heart rate and developed pressure in aging GAMT-/- but normal lung weight and survival versus age-matched controls. Electron microscopy indicated reduced mitochondrial volume density in GAMT-/- hearts (P < 0.001), corroborated by lower mtDNA copy number (P < 0.004), and citrate synthase activity (P < 0.05), however, without impaired mitochondrial respiration. Furthermore, myocardial energy stores and key ATP homeostatic enzymes were barely altered, while pathology was unrelated to oxidative stress since superoxide production and protein carbonylation were unaffected. Gene expression of PGC-1α was 2.5-fold higher in GAMT-/- hearts while downstream genes were not activated, implicating a dysfunction in mitochondrial biogenesis signaling. This was normalized by 10 days of dietary Cr supplementation, as were all in vivo functional parameters, however, it was not possible to differentiate whether relief from Cr deficiency or GA toxicity was causative.

Conclusion: Long-term Cr deficiency in GAMT-/- mice reduces mitochondrial volume without affecting respiratory function, most likely due to impaired biogenesis. This is associated with hemodynamic changes without evidence of heart failure, which may represent an acceptable functional compromise in return for reduced energy demand in aging mice.

Keywords: cardiac energetics; creatine; energy metabolism; mitochondrial respiration; ventricular function.

Figures

FIGURE 1
FIGURE 1
In vivo left ventricular hemodynamic function in WT and GAMT–/– mice at 18 months of age (A) GAMT–/– mice have lower body weight throughout their life. (B) Heart rate, (C) LV systolic pressure (LVSP), (D) LV end-diastolic pressure (LVEDP), (E) maximal rate of pressure rise (dP/dtmax) as a measure of contractility, and (F) time constant of isovolumetric relaxation (tau). N = 12 (5F/7M) per genotype, ∗∗denotes P < 0.01, ∗∗∗∗P < 0.0001 for WT versus KO at the same age by unpaired t-test (A–D,F) and Welch’s t-test (E). All values are mean ± SEM.
FIGURE 2
FIGURE 2
Cardiac energetic profile in >1 year GAMT–/– and wild type mice. (A) Representative 1H-NMR spectra showing metabolomic profile of WT and GAMT–/– heart. Highlighted (dotted line) is the complete absence of PCr and Cr but presence of P-GA and GA in GAMT–/– sample versus WT. Enzyme activities for (B) adenylate kinase (AK) (WT n = 9 5F/4M, GAMT–/– n = 5 2F/3M), (C) creatine kinase – Total (WT n = 15 GAMT–/– n = 12), mitochondrial CK (Mito), MM, MB, and BB isoforms (WT n = 6 GAMT–/– n = 7), (D) glycolytic enzymes glycerlaldehyde-3-phosphate dehydrogenase (GAPDH) (WT n = 9 5F/4M GAMT–/– n = 4 2F/2M), 3-phosphoglycerate kinase (PGK) (WT n = 5 2F/3M GAMT–/– n = 5 2F/3M), pyruvate kinase (PK) (WT n = 9 5F/4M GAMT–/– n = 5 2F/3M), (E) F1F0 ATP Synthase (mitochondrial electron transport chain complex V) (WT n = 4 1F/3M GAMT–/– n = 5 3F/2M), (F) pyruvate dehydrogenase total (PDHt), active (PDHa), and ratio of total to active (PDHt/PDHa) as an indicator of the extent of enzyme complex activation (n = 5/group 3F/2M), and (G) AMPK protein expression (n = 4/group) Denotes P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 for GAMT–/– versus wild type by unpaired t-test. Mouse mean age-84 weeks. All values are mean ± SEM.
FIGURE 3
FIGURE 3
Mitochondrial phenotype develops with age in GAMT–/– mice (A,B) Representative electron micrographs at 50 week age; 10000× magnification. (C) Stereological analysis shows lower mitochondrial volume density in GAMT–/– compared to WT (n = 3). (D) Mitochondrial DNA copy number in a separate group of mice (WT n = 6, GAMT–/– n = 5). (E) Citrate synthase activity inversely correlates with age in KO, but not in WT mice (n = 14/group 6F/8M). (F,G) Protein carbonylation in LV homogenates from >1 year old mice was not different (n = 7–8) nor were there differences in superoxide production (n = 8). (H) Activity (AUC) of purified monoamine oxidase (MAO) in presence of preferential substrate, tyramine (Tyr), is inhibited by monoamine oxidase inhibitor (MAOI). Creatine (Cr) and guanidinoacetate (GA) are equally good substrates for MAO, but only in the absence of primary amines (n = 30/group). (I) mRNA expression of PGC-1α and its upstream and downstream regulated genes by real-time quantitative PCR in LV from GAMT–/– and WT mice >1 year age (n = 4/group). Fold change normalized to control concentration (WT levels = 1) with propagated errors (SEM). (J) Uncoupling protein 3 (UCP3) expression and representative western blot (n = 4/group 2F/2M). Denotes P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus WT by unpaired t-test. Pearson’s correlation coefficient was used to assess the relationship between the variables. Mouse mean age 84 weeks (C–J). All values are mean ± SEM.
FIGURE 4
FIGURE 4
Mitochondrial oxygen consumption. Data from aging GAMT–/– and WT permeabilized LV fibers were normalized to citrate synthase activity to correct for the reduced mitochondrial volume density observed in aging GAMT–/– hearts. The metabolic substrates chosen for mitochondrial respiration were physiological in the presence of a functional Krebs’ cycle intermediate (malate) allowing mitochondrial OXPHOS to proceed normally, providing reduced intermediates (NADH + H+ and FADH2) individually to complexes I (pyruvate) (A) and II (succinate) (B), or combined complex I + II (fatty acid metabolism analogs palmitoyl-carnitine; palmitoyl CoA + carnitine) (C,D) of the respiratory chain allowing the thermodynamic cascade through the Q-cycle and complex III to complex IV and O2. State 2 is basal unstimulated respiration, State 3 is maximal ADP-stimulated respiration, State 4 is oligomycin uncoupled respiration (ATP synthase activity inhibited), RCR is respiratory control ratio (State3/State4) (WT n = 6 3F/3M GAMT–/– n = 9 4F/5M). Mouse mean age 72 weeks. All values are mean ± SEM.
FIGURE 5
FIGURE 5
Dietary creatine supplementation for 10 days normalizes the functional and metabolic phenotype of creatine-naïve GAMT–/– hearts Impact of dietary creatine on (A) Body weight; (B) LV systolic pressure, LVSP; (C) contractile reserve, i.e., increase in dP/dtmax with maximal β-adrenergic stimulation. Dietary creatine supplementation normalizes PGC1-α gene expression (D), concomitant with a return to normal tissue concentrations for guanidinoacetate (GA), phosphoguanidinoacteate (PGA) (Ei,F), creatine and PCr (Eii,F). Unsupervised principal component analysis of 1H NMR derived metabolite concentrations show good separation between GAMT–/– and WT and GAMT–/– Cr-fed groups (G). Metabolite concentrations expressed as fold change compared to WT levels in GAMT–/– and GAMT–/– creatine supplemented hearts (H). (WT n = 4 2F/2M GAMT–/– n = 6 3F/3M GAMT–/– + Cr n = 7 3F/4M). P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 GAMT–/– vs. WT, $GAMT–/– vs. GAMT–/– + creatine by one way ANOVA. Mouse age 60 weeks. All values are mean ± SEM.

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