Living without creatine: unchanged exercise capacity and response to chronic myocardial infarction in creatine-deficient mice

Abstract

Rationale: Creatine is thought to be involved in the spatial and temporal buffering of ATP in energetic organs such as heart and skeletal muscle. Creatine depletion affects force generation during maximal stimulation, while reduced levels of myocardial creatine are a hallmark of the failing heart, leading to the widely held view that creatine is important at high workloads and under conditions of pathological stress.

Objective: We therefore hypothesised that the consequences of creatine-deficiency in mice would be impaired running capacity, and exacerbation of heart failure following myocardial infarction.

Methods and results: Surprisingly, mice with whole-body creatine deficiency due to knockout of the biosynthetic enzyme (guanidinoacetate N-methyltransferase [GAMT]) voluntarily ran just as fast and as far as controls (>10 km/night) and performed the same level of work when tested to exhaustion on a treadmill. Furthermore, survival following myocardial infarction was not altered, nor was subsequent left ventricular (LV) remodelling and development of chronic heart failure exacerbated, as measured by 3D-echocardiography and invasive hemodynamics. These findings could not be accounted for by compensatory adaptations, with no differences detected between WT and GAMT(-/-) proteomes. Alternative phosphotransfer mechanisms were explored; adenylate kinase activity was unaltered, and although GAMT(-/-) hearts accumulated the creatine precursor guanidinoacetate, this had negligible energy-transfer activity, while mitochondria retained near normal function.

Conclusions: Creatine-deficient mice show unaltered maximal exercise capacity and response to chronic myocardial infarction, and no obvious metabolic adaptations. Our results question the paradigm that creatine is essential for high workload and chronic stress responses in heart and skeletal muscle.

Figure 1. Assessment of exercise capacity by voluntary wheel running

Each point represents mean ± s.e.m. from 8 wild-type (WT), 6 GAMT^−/−, and 6 creatine-supplemented GAMT^−/− female mice (0.75% creatine in chow started 7 days before the running protocol). Maximum speed was not significantly different between groups (A). There was a non-significant trend for GAMT^−/− mice to run less distance per night, however this was not directly related to creatine depletion since creatine supplementation did not alter distance run (B). Tissue creatine levels were undetectable in left ventricle and skeletal muscle of GAMT^−/− mice using HPLC, whereas creatine supplementation returned tissue creatine to normal WT levels (C).

Figure 2. Assessment of maximal exercise capacity by forced treadmill running

Eight wild-type (WT) and GAMT^−/− mice were forced to run to exhaustion using a standardised protocol of escalating speeds and 5° incline. Creatine-free GAMT^−/− mice ran further than control mice covering more vertical distance (panel A). However, there was no difference between genotypes when vertical work was calculated, which takes into account the lower body weight of GAMT^−/− mice. GAMT^−/− mice were then changed to chow containing 0.75% creatine and the running test repeated after 7 days (panel B). Both groups improved running capacity suggesting a learning/training effect, however there was no additional benefit from replenishing tissue creatine levels in GAMT^−/− mice.

Figure 3. Response to chronic myocardial infarction (MI) in WT and GAMT KO mice

(A) Kaplan-Meier survival curves were not significantly different for post-MI survival. (B–C) 3D-echocardiography at 1 and 6 weeks post-MI showing significant dilatation (enlarged end-diastolic volume, EDV) and impaired ejection fraction in both infarct groups compared to sham, but with no differences between genotypes. (D) Post-mortem LV weights (% of sham control) indicate no difference in hypertrophic response. (E–F) LV systolic pressure (LVSP) and end-diastolic pressure (LVEDP) respectively. (G) Inotropic response to β-adrenergic stimulation was significantly lower in all groups compared to WT sham (2-way ANOVA; P<0.001), but was not different between infarct WT and KO groups. (H) Isovolumetric constant of relaxation (tau) was significantly prolonged in both MI groups. *** denotes P<0.001, and ** denotes P<0.01 compared to WT sham group.

Figure 4. Utilization of phosphogens in the heart

(A) Myocardial creatine concentration is reduced in post-MI heart failure in WT hearts. (B) Analogous reduction in guanidinoacetate (GA) in GAMT KO mice. (C) activity of total CK and constituent isoenzymes; * denotes P<0.01 compared to WT sham; † denotes P<0.05 for WT MI versus KO MI; and § denotes P<0.05 for KO sham versus KO MI. (D–G) Representative ³¹P-MRS spectra in Langendorff perfused hearts. Panels D and F are from WT and KO hearts and indicate the large phosphocreatine (PCr) and phosphoguanidinoacetate (PGA) peaks respectively, with three smaller peaks representing the phosphoryl groups of ATP. The arrow shows frequency of selective saturation in magnetization transfer protocol. When this is applied at the specific frequency of the [γ-P]ATP peak, a reduction in PCr peak is observed, indicating phosphoryl flux between ATP and PCr in WT (E). In KO hearts there is no change in PGA peak (G), indicating no measurable flux of [γ-P]-between ATP and GA. (H) Quantification of change in PCr/PGA signal intensity as a function of saturation pulse duration (n=4/group).

Figure 5. Proteomics analysis

(A) Example of 2-D difference in-gel electrophoresis from age-matched WT and GAMT KO hearts. Expansion of several protein subunits from Complex I (B), Complex II (C), Complex III (D), Complex IV (E), and Complex V (F). WT proteins were labelled with Cy3 (red) and GAMT KO proteins were labelled with Cy5 (green) for n=4 paired samples. Proteins were separated in the horizontal direction by isoelectric focusing point (~pH 3 to pH 10) and in the vertical direction by molecular weight (~150 kDa to 10 kDa). Protein subunits were identified by mass spectrometry. Western blots for respiratory chain enzymes (G), with corresponding densitometric analysis (H) confirming no difference in protein expression (n=7 WT and n=8 KO).

Figure 6. Cardiac energetic profile in LV homogenates from 6 month old WT and GAMT KO mice

(A) enzyme activites for total adenylate kinase (AK) and the major glycolytic enzymes: glyceraldehyde- 3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and pyruvate kinase (PK). (B) Activity of total and active fractions of pyruvate dehydrogenase (PDHt and PDHa respectively). (C) Phosphorylated fraction of AMP-activated protein kinase (AMPK) protein. (D) Protein expression of uncoupling protein-3 (UCP3) and superoxide production (E) is not significantly different. (F–G) Oxygen consumption from permealised LV fibres reflecting mitochondrial respiration in response to different substrates. State 2 is basal unstimulated respiration, state 3 is maximal ADP-stimulated respiration, Oligo is uncoupled respiration (state 4o) in presence of oligomycin to inhibit ATP synthase. RCR is the respiratory control ratio (i.e. state 3/4). All data are mean±sem, sample numbers in parenthesis. * denotes P<0.05, ** P<0.01 for WT versus KO.