This study sought to characterize the time course, extent and mechanism whereby increased myocardial Cr content leads to cardiac dysfunction. First, we demonstrated that 1H-MRS can be used as a tool to non-invasively monitor myocardial metabolism, in this case myocardial Cr content, over one year. Second, we showed that Cr levels are elevated and constant in CrT-OE hearts over one year of age. Third, we demonstrated that the development of hypertrophy and heart failure was progressive in the CrT-OE model. That is, at 6 weeks of age, cardiac function and LV mass were normal in CrT-OE mice despite elevated Cr levels, with cardiac dysfunction developing and worsening between 6 and 52 weeks of age. Fourth, we showed that enolase content was selectively decreased in CrT-OE hearts, which resulted in suppressed enolase activity and impaired glycolytic capacity. Finally, we propose that increased expression of the β-enolase repressor factor 1 may lead to the down-regulation of enolase content in CrT-OE hearts, which may play a role in the development of heart failure and LV hypertrophy in this model.
The current study used a longitudinal MR protocol to investigate the temporal relationship between increased total intracellular Cr content and cardiac function and mass. We showed that an elevation in myocardial Cr levels of 2-fold was attained by 6 weeks of age and remained constant over the 52 week protocol. Our initial phenotypic study on CrT-OE mice suggested that the development of LV hypertrophy and heart failure were due to augmented free ADP levels and subsequent drop in the heart’s free energy change levels, resulting from the heart’s inability to keep the increased Cr-pool adequately phosphorylated as PCr [24
]. However, the current study showed that, at the early 6 week time-point, function was still normal despite elevated total Cr content, with contractile dysfunction not developing until 16 weeks of age. One potential explanation for this may be that the phosphorylated fraction of Cr (i.e., PCr) initially keeps up with the increase in total Cr (i.e., PCr increases proportionally to total Cr), thus keeping free ADP levels normal, and only with progressive age do hearts become incapable of keeping the augmented Cr pool adequately phosphorylated. In other words, after 6 weeks of age, the phosphorylated Cr fraction drops and free ADP increases, leading to contractile dysfunction, as shown in our earlier work using isolated heart 31
P-MRS to measure PCr in ~32 week old mice [24
]. In principle, this hypothesis could be tested by measuring PCr and total Cr contents in the 6 week old mice. Unfortunately, due to their miniature size, we were unable to obtain spectra with sufficient signal-to-noise ratio in 6 week old perfused mouse hearts that would allow us to detect a 20–30% change in PCr. With further technological development in MRS measurements this may ultimately become feasible. Also, in our experience, HPLC measurements of PCr are not accurate or stable enough to allow assessment of the anticipated difference in levels. However; here we demonstrate an additional explanation for the progressive development of heart failure related to substrate utilization.
Using proteomic approaches we screened for potential molecular changes leading to contractile dysfunction in CrT-OE mice. It is important to note that since contractile dysfunction in the CrT-OE model developed between 6 and 16 weeks of age, animals aged 12–14 weeks were used for the proteomic and biochemical analyses. We felt that this relatively young age provided the best opportunity to observe the potential molecular mechanisms contributing to heart failure and hypertrophy in CrT-OE mice, as opposed to the biochemical changes associated with heart failure. Although hundreds of cardiac proteins were detected in the 2D DIGE analysis, only 7 proteins were differentially expressed in WT and CrT-OE mice with more than a 1.3-fold change. The detection of relatively few protein differences in CrT-OE hearts was advantageous because it implied that increased myocardial Cr content resulted in rather specific modifications.
Among the most notable protein modifications in CrT-OE hearts involved the selective down-regulation of α- and β-enolase (Supplemental Figure 1
). A decrease in enolase content has been reported in the diabetic heart [33
] as well as models of cardiomyophathy [39
] and hypertrophy [40
]. In the diabetic heart several glycolytic enzymes were down-regulated [33
] and in the hypertrophied heart a decrease in β-enolase was coupled to a compensatory increase in α-enolase [40
] or phosphofructokinase-1 [41
]. In contrast, α- and β-enolase were both decreased in CrT-OE hearts, with no changes to the additional six glycolytic enzymes detected. Since changes in metabolic flux typically result from alterations to an entire biochemical pathway, as opposed to single rate-limiting steps [42
], it was surprising that the down-regulation of enolase alone could mediate a change in energy-metabolism. In this context, it is interesting that a metabolic myopathy caused by a specific deficiency of β-enolase (glycogenosis type XIII) has been identified [44
]. The patient suffering with this disorder demonstrated a progressive decline in skeletal muscle function, eventually preventing performance of sustained muscle exercises [44
]. In analogy, it is conceivable that a specific decrease in myocardial enolase content may be a contributor to progressive heart failure in CrT-OE mice. Consistent with the decrease in enolase activity, CrT-OE mice showed a significant suppression in enolase enzyme activity and anaerobic lactate production, which suggests reduction in glycolytic flux ( and ). Furthermore, strong correlations were shown between decreased enolase activity/lactate production and increased total Cr content, suggesting a direct link between increased myocardial Cr levels and impaired glycolytic capacity in the CrT-OE model. The contribution of glycolysis to the cell’s overall ATP content is generally thought to be minor under “normal” conditions, making it unlikely that decreased glycolytic capacity alone could contribute to heart failure. However, Aasum et al determined that glucose utilization contributes more to the overall myocardial ATP production in the mouse than in other species [45
]. While fatty acids are the preferred oxidative substrate in human and rat heart [46
], glucose appears to be favored in mouse heart [45
]. Therefore, glycolytically-derived ATP may be preferentially used for contraction in the mouse heart, potentially explaining why decreased glycolytic flux may be detrimental in CrT-OE mice. Collectively, these findings imply that to the development of progressive hypertrophy and heart failure may in part be mediated by a compromise in glycolytic capacity at the level of enolase. [48
There is evidence that β-enolase, through its interaction with other proteins, may play a role in intracellular energy transfer. Previous studies have observed in vitro
binding of β-enolase to pyruvate kinase, phosphoglycerate mutase, muscle creatine kinase (MM-CK), aldolase and troponin [50
]. Additionally, a weak in vivo
interaction has been observed between β-enolase and MM-CK [52
]. Since the location of this “β-enolase protein complex” is favored near the sarcomeric apparatus, down-regulation of β-enolase in CrT-OE hearts may cause contractile dysfunction by impairing delivery of glycolytically-derived ATP to the subcellular sites where energy is needed for contraction. Collectively, these data are consistent with the notion that decreased glycolytic capacity may lead to progressive heart failure in CrT-OE mice.
Metabolic fluxes can be regulated by altering enzyme concentration, post-translational modifications, and the interaction of enzymes with substrates, products or allosteric effectors. A recent study in yeast demonstrated that glycolytic flux is largely controlled by the regulation of protein synthesis and degradation [53
]. Although little is known about the mechanisms of translational regulation of glycolytic proteins, the available reports emphasize the regulatory role of transcription factors [54
]. Thus, we screened WT and CrT-OE hearts for changes in protein content of the β-enolase repressor factor-1 (βERF-1), which is a negative regulator of β-enolase gene transcription. CrT-OE hearts showed more than an 80% increase in βERF-1 content, with respect to WT controls (), suggesting that expression of β-enolase in CrT-OE heart may be regulated at the level of transcription. Since little is known about the expression level of βERF-1 and its activity, further work is required to interpret the biological significance of this finding. In addition to the decrease in enolase content, it is also important to point out that the expanded panel in shows an isoelectric shift for both α- and β-enolase toward the acidic region of the gel, consistent with a post-translational modification such as phosphorylation, glycosylation or oxidation. Importantly, phosphorylation of enolase has previously been demonstrated [55
], and more recently, hyper-phosphorylation of α-enolase was shown to decrease enzymatic activity in the hypertrophied LV [37
]. While further experimentation is required to identify and determine whether enolase is post-translationally modified in CrT-OE hearts, it is important to point out that decreased enolase content may not be the only protein modification regulating enolase activity.
A limitation of our work is that our data do not prove a cause-and-effect relationship between decreased glycolytic flux and heart failure in CrT-OE mice. Further experiments in intact hearts could be performed to confirm that glycolytic flux (in addition to capacity) is indeed decreased in this model; however, even these measurements would not establish a causal relationship. One approach to address this might be to cross our CrT-OE mice with GLUT1 over-expression mice [56
], which show increased glycolytic capacity, to test whether this prevents the development of LV dysfunction.
In summary, this work shows that CrT-OE mice have chronically elevated levels of creatine over their first year of life. In spite of this, cardiac function is initially normal and declines progressively only after 6 weeks of age. The mechanism for this decline may result from impaired glycolysis at the level of enolase, in part mediated by increased βERF-1 expression.