Myc regulates cardiac hypertrophy and also affects intermediate metabolism in non-cardiac tissue. Our objective was to test the hypothesis that Myc-induced substrate utilization changes to the citric acid cycle signal for hypertrophy development and promote compensated hypertrophy. Echocardiogram and ex vivo working heart experiments confirmed that this was compensated hypertrophy as function was preserved or slightly improved with Myc-induction. Myc affected myocardial substrate utilization patterns at established hypertrophy (7dMyc), with a marked increase in free fatty acid oxidation with concurrently decreased lactate oxidation.
We chose the 3dMyc group to determine whether substrate utilization changes precede hypertrophy and may therefore signal for its development. Demonstrating a metabolic shift prior to hypertrophic growth is necessary (but not sufficient) to establish a signaling role. The current study justified this time point for pre-hypertrophy metabolic evaluation since the heart weight to body weight ratios are similar to Cont and there was no change in serial LV posterior wall thickness. Metabolic changes are considered part of the hypertrophic process; however altered utilization patterns may also drive hypertrophy development. For example, cardiac specific PPARα overexpression promotes myocardial fatty acid oxidation through transcription of genes involved in this process. These changes lead to hypertrophy, demonstrating that alterations in substrate citric acid cycle contribution can promote hypertrophy development [25
]. Given Myc’s ability to regulate substrate metabolism in non-cardiac tissue, we hypothesized that changes in substrate utilization may lead to hypertrophy development in this model. Ahuja et al previously reported that Myc activation for 3 days increased the unlabeled Fc to acetyl-CoA and ultimately the citric acid cycle [14
]. However, those experiments included only a small number of mice and were considered preliminary. Our current study with greater numbers (n=6) showed only a modest trend towards increased unlabeled Fc in 3dMyc, which disappeared in follow up studies with insulin-containing perfusate. As no other changes in substrate utilization occurred prior to hypertrophy (i.e. in the 3dMyc versus Cont), oxidative shifts are not the primary signal for cardiac growth in this model. However, oxidative changes may still be required to provide energy and substrates for rapid protein synthesis and growth.
Unlike the 3dMyc, 7dMyc significantly altered substrate contribution to the citric acid cycle. Free fatty acid contribution increased nearly 50% corresponding with decreased exogenous glucose contribution. Absolute flux of free fatty acids and lactate were also changed in established hypertrophy representing oxidative metabolism. Myc-induced hypertrophy maintained compensated function while increasing fatty acid oxidation. This is notable as many investigators are exploring pharmacologic modulation of substrate utilization for heart failure treatment. Typically, however, the therapeutic goal is to inhibit fatty acid oxidation for reasons including that this theoretically requires 11–12% more oxygen for a given amount of ATP produced than glucose oxidation [3
]. Trimetazidine is a partial mitochondrial fatty acid β-oxidation inhibitor used to treat chronic stable angina in Europe and Asia. Multiple small, short term studies utilizing trimetazidine to treat heart failure demonstrated improved LV ejection fraction, exercise performance and plasma heart failure biomarkers [26
]. Similarly, the cardiac specific PPARα overexpressing mice discussed above had decreased cardiac function [25
]. Our results do not contradict these studies, but it demonstrates that increased fatty acid oxidation does not uniformly cause functional abnormalities in the hypertrophied myocardium. Future studies are planned to determine the necessity of these metabolic changes for maintaining compensated function in our model. Additionally, understanding the mechanisms whereby fatty acid oxidation is not functionally detrimental in this model would provide useful information for developing metabolic therapies to treat heart failure and diabetic cardiomyopathy which is associated with increased fatty acid oxidation.
Although Myc is upregulated in nearly all types of hypertrophy, the metabolic phenotype in the Myc-inducible mice did not fully recapitulate changes described in physiologic hypertrophy models such pressure overload. As such, our findings are due to Myc-induction and not secondary to hypertrophy. Fatty acid oxidation levels are typically unchanged or mildly diminished in compensated physiologic hypertrophy and decreased in heart failure with physiologic hypertrophy [3
]. One potential reason for this difference could be that Myc levels are lower in physiologic hypertrophy thereby affecting intermediate metabolism to a lesser degree. Xiao et al [11
] showed that Myc mRNA levels in this transgenic mouse are similar to non-transgenic mice after 6 hours of transverse aortic constriction. They did not report protein levels or temporal data, so differences cannot be completely ruled out. Another more likely possibility is that additional metabolic regulation counterbalances Myc. The etiology of decreased fatty acid oxidation in physiologic hypertrophy and heart failure is incompletely understood but it is likely at least partially due to reduced transcriptional activation of genes regulated by peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1α) and retinoic acid X receptor α (RXRα) [1
]. These proteins form heterodimers that transcriptionally control fatty acid oxidation encoding genes. We did not find any differences in PPARα or PGC-1 protein levels. In physiologic hypertrophy, changes in PPARα, PGC-1and/or RXRα activity may counteract Myc’s effect on fatty acid oxidation. Myc may prevent a larger reduction in fatty acid oxidation. Future studies are planned using the cardiac specific inducibly inactivate Myc mice (described by Zhong et al [12
])to elucidate Myc’s role in metabolic changes from hypertrophy secondary to pressure overload.
Regulation of intermediate metabolism
Surprisingly, the substrate utilization changes do not appear to be due to transcription and/or translation of fatty acid oxidation genes. Ahuja et al previously demonstrated unchanged gene expression of the fatty acid oxidation transcription factors PGC-1α, PGC-1β, PGC-1 related coactivator, PPARα, estrogen-related receptor α, and nuclear respiratory factor [14
]. Medium chain acyl-CoA dehydrogenase (MCAD) and carnitine palmitoyl-transferase 1 (CPT-1) RNA levels were decreased [14
], although our study demonstrated that this does not reduce fatty acid oxidation. As noted earlier, protein levels of the transcription factors PPARα and PGC-1α were unchanged confirming that global transcription of metabolic genes is unlikely. Thereafter we explored total protein levels of specific enzymes. Fatty acid oxidation regulation occurs at multiple steps from cellular uptake to mitochondrial transport to β-oxidation [3
] but we did not find protein changes that would augment fatty acid oxidation such as in mCPT-I, lCPT-I, FAT/CD36, MCD and ACC. PDK2 and PDK4 inhibit pyruvate dehydrogenase and therefore glucose and lactate oxidation. This could reciprocally promote fatty acid utilization according to the Randle hypothesis. However, we did not find changes in either PDK isoform. Overall, Myc did not augment fatty acid oxidation through transcription or translation of the commonly investigated regulatory enzymes. Further work is necessary to determine whether other enzymes are transcriptionally affected by Myc.
Many metabolic enzymes undergo post-translational modification altering their activity [3
]. Myc is primarily a transcriptional regulator, but recent studies have demonstrated transcription-independent functions [16
]. O-GlcNAc post-translational modification was increased by Myc-induction in our study. Recent work suggests O-GlcNAc promotes myocardial fatty acid oxidation [17
]. Laczy and colleagues perfused isolated rat hearts with glucosamine to acutely increase total protein O-GlyNAcylation under normoxic condition [17
]. Glucosamine caused a dose dependent increase in O-GlcNAc levels, which was associated with increased palmitate oxidation. This metabolic change was attributed to increased FAT/CD36 membrane translocation and fatty acid cellular uptake. FAT/CD36 immunoprecipitation demonstrated evidence of O-GlcNAc modification. We are currently evaluating whether Myc activation increases FAT/CD36 membrane levels in our model.
In addition to regulating cardiac substrate metabolism, O-GlcNAcylation may promote hypertrophy in our model. Facundo and colleagues recently demonstrated that O-GlcNAc signaling is necessary for NFAT-mediated hypertrophy [19
]. NFAT activation from phenylephrine was blunted by O-GlcNAc inhibition in neonatal rat cardiac myocytes. Further, O-GlcNAc augmentation increased NFAT activation and nuclear translocation independent of hypertrophic stimuli. In our study, the O-GlcNAc band near 58 kDa was greater in 3dMyc compared to Cont demonstrating that the increase in O-GlcNAcylation begins prior to hypertrophy. Because of the nonspecific nature of the O-GlcNAc antibodies, additional work is required to identify O-GlcNAcylated proteins within the ~58 kDa band and at other molecular weights that may promote the development of hypertrophy in our model. Interestingly, Morrish et al demonstrated that inhibiting the pathway for producing O-GlcNAc reduced growth in Myc-expressing cultured fibroblast [16
]. We plan future studies to elucidate Myc’s role in myocardial O-GlcNAcylation.
Myc is an important regulator of cardiac hypertrophy. In the current study, we show that Myc affects substrate utilization for the citric acid cycle. This process does not precede hypertrophy and therefore does not signal for hypertrophy development. With established compensated hypertrophy, Myc-induction increased fatty acid utilization suggesting a potential mechanistic role. Based upon our results, further studies are warranted to determine whether fatty acid oxidation promotes functional compensation in the model as well as Myc’s role in metabolic changes from physiologic hypertrophy.