In this study, to study the pathological role of Myc in cardiomyopathy and heart failure, we developed and characterized a newly established bi-transgenic mouse (MHC-Myc), which expresses Myc specifically in cardiomyocytes in a doxycycline-dependent manner. Since Myc is only expressed temporally during development, not in adult, in cardiomyocytes, the ability to regulate the expression of Myc after development was especially important in this study. In MHC-Myc mice, the expression of Myc is tightly regulated by doxycycline and no significant phenotype is observed until Myc was induced. However, induction of Myc (Myc-ON) for two weeks led to dramatic changes in the heart including an ectopic reactivation and expression of cell cycle markers in cardiomyocytes and mitochondrial perturbations that culminated in severe hypertrophy and, ultimately, death. These results strongly support the notion that increased Myc in adult cardiomyocytes induces cell cycle re-entry that leads to cardiomyopathy and is consistent with a previous report showing that the conditional inactivation of Myc attenuates cardiac hypertrophy induced by pressure overload and other hypertrophic stimuli 
. However, in contrast to a previous Myc inducible mouse model 
, induction of Myc in MHC-Myc animals ultimately causes heart failure resulting in death in a relatively short duration (2–3 weeks). This difference is likely a consequence of mouse strain and expression level of Myc (Robb MacLellan, personal communication). While our MHC-Myc mouse is derived from FVB strain, the transgenic mouse line used by others 
is derived from C57Bl/6 and C3H mixed background. Since the effect of genetic background on the development of cardiomyopathy and heart failure is well documented 
, it is likely that genetic factor(s) affect subsequent phenotypes. In addition, our MHC-Myc mouse studies here appear to have significantly higher levels of Myc expression and, consequently, a more robust phenotype than previous models. Supporting this, the number of cardiomyocytes re-entering the cell cycle in our MHC-Myc animals is much greater than reported previously 
. Therefore, the different phenotypes observed between transgenic mice is likely a consequence of the extent to which cardiomyocytes re-enter the cell cycle and progress into S phase, leading to the development of hypertrophy and consequent heart failure. Supporting this, recent studies have suggested that the adult myocardium is capable of limited cell division in certain pathological conditions 
. For instance, heart regions adjacent to a myocardial infarction show a significant increase of Ki-67 proliferation index, while no changes were noted distant from the lesion. Additionally, mitotic activity in cardiomyocytes is increased nearly 10-fold increase in end-stage ischemic heart disease and in idiopathic dilated cardiomyopathy 
. Therefore, our data, in addition to other studies, strongly suggests that the reactivation of cell cycle in adult cardiomyocytes plays an important role in the development of cardiomyopathy and heart failure. This said, further study into the relationship to cell cycle re-entry as an adaptive or maladaptive process is clearly necessary.
Another important finding of our study is that Myc induces the morphological and functional alterations of mitochondria in MHC-Myc mice. Since PGC-1α related pathways can be directly regulated by Myc 
, we suspect that Myc-mediated PGC-1α regulation, which is tightly related with mitochondrial biogenesis and function, is a key pathway for the development of hypertrophic cardiomyopathy and heart failure in MHC-Myc mice 
. In support of this, cardiac-specific overexpression of PGC-1α, driven by the α-myosin heavy chain promoter, leads to marked mitochondrial proliferation, severe cardiomyopathy and death 
. Further, of more physiologic relevance, is a model of inducible PGC-1α overexpression in the adult mouse, which leads to a more subtle increase in mitochondrial number and the development of a reversible cardiomyopathy, with both systolic and diastolic functional impairment 
. The potential mechanism(s) of mitochondrial-mediated cardiac dysfunction by PGC-1α overexpression likely involves defects in mitochondrial oxidative metabolism or the increased production of reactive oxygen species 
. Consistent with this notion, the increase of PGC-1α in our MHC-Myc mice was concomitant with an increase in mitochondrial number, a decrease in mitochondria size, and a reduction in the activity of mitochondrial oxidative metabolism. While the mechanistic basis for PGC-1α-mediated cardiomyopathy is unclear, it is likely that dysregulated mitochondrial metabolism and/or morphological alteration play an important role in the development of cardiomyopathy and heart failure. Interestingly, a recent study demonstrated an increased level of PGC-1α, but not activity, in failing human hearts suggesting that, despite the increase of PGC-1α, downstream pathways were disturbed by other mechanisms to induce mitochondrial pathologies 
. Our observations in MHC-MYC mouse are consistent with these data from human patients as we also observed an increase of level of PGC-1α accompanied by functional deficits in mitochondrial energy metabolism.
Notably, it is well established that defects in electron transport chain complexes I 
, complex III 
and IV 
lead to dilated cardiomyopathy in human patients. Moreover, the enzyme activity of electron transport chain complexes is decreased in hearts explanted from patients with end-stage heart failure 
and, importantly, end-stage heart failure also results in re-expression of Myc as well as decreases in complexes I, III, and IV, and mutations in mtDNA 
. Therefore, the functional defect in mitochondrial energy metabolism in MHC-Myc mice is likely a key mechanism for the development of cardiac phenotypes such as hypertrophy and heart failure. Regarding this, the smaller size of mitochondria may also explain the defect in mitochondrial function coupled with an increase of mitochondrial number. In fact, our biochemical analyses show that citrate synthase, amount and activity, is decreased in MHC-Myc mice. Since citrate synthase is a mitochondrial matrix marker enzyme, reflecting relative mitochondrial abundance and purity, these results suggest that Myc may increase the number of immature mitochondria. The decrease in cytochrome content and multiple defects in the electron transport chain further supports the notion that mitochondrial development may be incomplete in Myc-ON mice 
. Collectively, these findings could explain increased mitochondrial number coupled with decreased activities of citrate synthase, complex I, and complex III.
In conclusion, our findings using a novel transgenic mouse with cardiomyocyte-specific expression of Myc (MHC-Myc mice) clearly demonstrate that increased expression of Myc can induce hypertrophic cardiomyopathy and heart failure in vivo. Mechanistically, dysregulation of cell cycle and concomitant mitochondrial alteration are likely key pathogenic mechanisms for subsequent hypertrophy-induced heart failure. As such, therapeutics targeted toward Myc, cell cycle re-activation, and/or mitochondrial dysfunction may provide opportunities for the treatment and management of cardiomyopathies.