Proposed pathway stimulated by feeding in the Burmese python. Feeding provides the stimulus for increased energy demand. Increased intracellular AMP stimulates transcription of nuclear transcription factors that promote transcription of both structural mitochondrial protein and other transcription factors that act on mitochondrial DNA. Transcription of mRNAs responsible for mitochondrial biogenesis is increased, as are transcripts that are translated into components of the machinery responsible for mitochondrial fission. As the stimulus decreases, the active mitochondrial pool is decreased by reduced transcription of the mRNAs that contribute to mitochondrial biogenesis and fusion is promoted to decrease the number of mitochondria.
The postprandial metabolic rate of the Burmese python increases a remarkable 44-fold compared to fasting. The mechanisms mediating the rapid production of ATP required for increased cardiac output have not been fully described and in fact, are contradictory in the literature. Increased oxidative capacity was previously observed in the ventricle of the Burmese python, as evidence by a lack of lipid accumulation, increased cytochrome oxidase 2 expression, and increased expression of superoxide dismutase 2, which mitigates the effects of reactive oxygen species production by mitochondria. This dramatic increase in cardiac metabolism likely exceeds the capacity of existing mitochondria and requires an increase in either mitochondrial size or number.
In human hearts, when AMP accumulates from high levels of metabolic activity, AMPK is activated. Indeed, both we and others report an increase in AMPK signaling in the postprandial python ventricle. Interestingly, we also observed decreased p70 S6 kinase phosphorylation, which further increases AMPK signaling in other tissues including muscle by increasing AMP:ATP ratios. AMPK phosphorylates PGC1α, which promotes expression of genes that regulate mitochondrial biogenesis such as ESRRα. Coactivation of ESRRα causes ESRRα to autodimerize and initiate transcription of genes that regulate processes ranging from energy homeostasis to ATP generation. Increases in both ESRRα and NRF2 expression at 1dpf and 3dpf are consistent with increased energy usage of the ventricle and suggest that the heart is responding in a physiological manner to increased energy demand. Unexpectedly, expression of PGC1α was inversely proportional to heart growth, with its lowest expression at peak hypertrophy. Although PGC1α is a known activator of ESRRα and NRFs, it is possible that the true peak expression of PGC1α was not captured during the time points used in this study. Peak expression of PGC1α may have occurred within hours of feeding. An early increase could have been followed by a decrease to negatively regulate mitochondrial biogenesis; indeed, mitochondrial mutations induced by PGC1α constitutive activity lead to cardiomyopathy. Additionally, previous studies and our antibody array data revealed increased activation of AMPK that activates PGC1α beginning at 1dpf 5. Therefore, we hypothesize that a rapid increase in PGC1α activity is required for activation of ESRRα and other genes responsible for mitochondrial remodeling.
Transcription factor MEF2A regulates genes that specify cardiac muscle cell differentiation, manage the build up of ROS, and are crucial to mitochondrial localization. The early 4-fold decrease in expression at 1dpf was unexpected, although MEF2A activity may be regulated by histone deacetylases at the onset of hypertrophic stimuli. The 2-fold increase in expression of MEF2A at 3dpf corresponds with an observed 1.26-fold increase in p38α activity. p38α is part of the MAP kinase signaling pathway, which is known to phosphorylate MEF2A to promote activation during cardiac muscle growth and differentiation. Additionally, EM imaging shows mitochondrial alignment at this time, indicative of activated mitochondrial localization genes that lie downstream of MEF2A. These data support the conclusion that growth factor signaling may cause activated MAP kinase signaling cascades and eventually downstream gene expression through post-translational modifications.
NRF2 mediates repair of the damaging effects of ROS and regulates metabolic responses by changing mitochondrial bioenergetics. The 2.08-fold increase in expression of NRF2 would cause upregulation of genes involved in mitochondrial gene transcription such as mitochondrial transcription factor A (TFAM) and TFB2M to maintain the stoichiometry of structural and functional elements of existing and/or new mitochondria in the energetically demanding ventricle. These factors, along with POLRMT and TFB1M, are responsible for initiating transcription of mammalian mitochondrial DNA. Indeed, expression of POLRMT, TFB1M, and TFB2M increased at 1dpf. The observed expression patterns of each of these mitochondrial transcription factors should result in increased mitochondrial DNA transcription and thus increased concentration of the 13 necessary subunits of the respiratory pathway associated with the inner mitochondrial membrane. Indeed, increased expression of CS, essential for the first step of the citric acid cycle, TOMM22, an organizer of the TOM complex, and VDAC1, an important aquaporin was observed. In fact, increased CS expression has been observed with mitochondrial biogenesis following exercise training in mammals and is considered predictive of mitochondrial number. The upregulation of FIS1 further supports increased mitochondrial biogenesis because its expression has been shown to correlate with the number of mitochondria in human cells. Its role in recruiting mitochondrial fission proteins to the outer membrane of the mitochondria and its measured increase in expression signifies that the mitochondria should be increasing in number if all required proteins of the fission complex are functioning within the cell.
Unexpectedly, MTERFD2, an important regulator of mitochondrial ribosomal synthesis, continued to increase through all time points. There are two potential interpretations for this pattern of gene expression. The first is that MTERFD2 expression is relatively low in the fasted python heart and must increase dramatically in order to meet the increased demands of energy production during cardiac hypertrophy. The sustained increase in expression of this gene may represent the role of MTERFD2 in the translation of regulatory components important to mitochondrial function or number. The second involves the largely unexplored function of MTERFD2 as a mitochondrial transcription terminator. MTERFD2 is structurally similar to other MTERF isoforms; it contains the same nucleic acid-binding protein domain. Thus, it is possible that MTERFD2 begins to regulate the transcription of mitochondrial genes involved in biogenesis at 3dpf as regression begins and continues to down-regulate these genes at 10dpf.
Despite a need to understand and address mitochondrial dysfunction that occurs with CVD, there exist no approved drugs to increase energy production in the failing heart. One approach to treating this issue is to improve energy production, however, the mechanisms by which energy production is sustained in physiological cardiac hypertrophy are not well understood due to conflict reports and incomplete studies in various mammalian models. This study is the first to our knowledge to comprehensively examine the patterns of intracellular signaling and gene expression regulating mitochondrial function in the Burmese python during physiological cardiac hypertrophy. Our data provide a greater understanding of the significance of mitochondrial biogenesis in physiologic hypertrophy, which could lead to the development of novel therapeutics capable of reverting pathological hypertrophy to physiological hypertrophy and potentially reducing the progression of CVD.
Heat map showing expression of genes encoded in the nucleus that regulate transcription of nuclear genes (a), mitochondrial genes (b), genes encoding structural proteins (c), and genes encoding functional proteins (d) in the mitochondria. Expression was measured 28 days after feeding (fasted), 1 day after feeding (1dpf), 3 days after feeding (3dpf), and 10 days after feeding (10dpf). Values are reported relative to expression in the fasted state, which is shown as zero fold change (black boxes). n = 2 pooled python ventricles and three technical replicates (reference and gene of interest) per time point, 8 pythons total.
For complete research details, see Killian, et al., 2015, a research article published by the students in The Python Project: