Date Published: February 3, 2017
Publisher: Public Library of Science
Author(s): Chris McDermott-Roe, Marion Leleu, Glenn C. Rowe, Oleg Palygin, John D. Bukowy, Judy Kuo, Monika Rech, Steffie Hermans-Beijnsberger, Sebastian Schaefer, Eleonora Adami, Esther E. Creemers, Matthias Heinig, Blanche Schroen, Zoltan Arany, Enrico Petretto, Aron M. Geurts, Manuel Portolés.
Mitochondrial dysfunction contributes to myriad monogenic and complex pathologies. To understand the underlying mechanisms, it is essential to define the full complement of proteins that modulate mitochondrial function. To identify such proteins, we performed a meta-analysis of publicly available gene expression data. Gene co-expression analysis of a large and heterogeneous compendium of microarray data nominated a sub-population of transcripts that whilst highly correlated with known mitochondrial protein-encoding transcripts (MPETs), are not themselves recognized as generating proteins either localized to the mitochondrion or pertinent to functions therein. To focus the analysis on a medically-important condition with a strong yet incompletely understood mitochondrial component, candidates were cross-referenced with an MPET-enriched module independently generated via genome-wide co-expression network analysis of a human heart failure gene expression dataset. The strongest uncharacterized candidate in the analysis was Leucine Rich Repeat Containing 2 (LRRC2). LRRC2 was found to be localized to the mitochondria in human cells and transcriptionally-regulated by the mitochondrial master regulator Pgc-1α. We report that Lrrc2 transcript abundance correlates with that of β-MHC, a canonical marker of cardiac hypertrophy in humans and experimentally demonstrated an elevation in Lrrc2 transcript in in vitro and in vivo rodent models of cardiac hypertrophy as well as in patients with dilated cardiomyopathy. RNAi-mediated Lrrc2 knockdown in a rat-derived cardiomyocyte cell line resulted in enhanced expression of canonical hypertrophic biomarkers as well as increased mitochondrial mass in the context of increased Pgc-1α expression. In conclusion, our meta-analysis represents a simple yet powerful springboard for the nomination of putative mitochondrially-pertinent proteins relevant to cardiac function and enabled the identification of LRRC2 as a novel mitochondrially-relevant protein and regulator of the hypertrophic response.
Mitochondria are highly abundant organelles, found in almost every eukaryotic cell, and are best known for production of adenosine triphosphate via oxidative phosphorylation. However, the functional remit of the mitochondrion extends far beyond this canonical role as the ‘powerhouse of the cell’ . Mitochondria regulate multiple essential facets of cellular metabolism and physiology including synthesis, breakdown and interconversion of amino acids, maintenance and modulation of ion gradients, and regulation of pyrimidine and heme biosynthesis . Given the diverse and essential nature of such processes, it follows that perturbations therein result in varied and often severe phenotypic manifestations. Mitochondrial diseases (MD) aggregately represent the most prevalent cause of inborn errors of metabolism and encompass a clinically heterogenous group of multisystemic disorders—with symptoms including myopathy, encephalopathy, lactic acidosis, neuropathy, liver failure, ataxia, deafness and optic atrophy—that result from defective mitochondrial function . Mitochondrial dysfunction is also a feature of many common diseases including Alzheimer’s , Parkinson’s , hypertrophic cardiomyopathy , and cancer . Elucidating how mitochondrial dysfunction contributes to disease states, either as the principal cause of monogenic disorders or as a compounding feature in complex disease, will undoubtedly augment diagnostic success and therapeutic prospects. The stochastic nature of mitochondrial disease (e.g., multiple modes of inheritance, variable age of onset and wide symptom spectrum  and challenges associated with reverse-genetics approaches means functional genomics, proteomics and forward genetics-based strategies have played an important role in the discovery of disease-associated genes. Capturing the full complement of mitochondrial proteins has been the focus of much effort for the last two decades  and mass spectrometry-based methods have been especially useful in the shaping understanding of the mitochondrial proteome [10, 11]. MitoCarta is a collection of approximately 1,100 proteins with experimental evidence of mitochondrial localization and is the most expansive self-contained survey to date . As well as highlighting the functional heterogeneity of the organelle, MitoCarta has enabled the identification of multiple disease-causing genes [12–16]. In addition to the proteins reported in the MitoCarta database and companion studies [17, 18], it is estimated that a substantial number, perhaps several hundred, remain unassigned and efforts to fill this gap continue.
The premise of co-expression analysis is that transcripts that correlate with a particular query transcript have an increased likelihood of being functionally-related to that particular query transcript. Certain gene classes are particularly well suited to co-expression-based analyses. For example, many heat shock and chaperone protein-encoding genes exhibit coordinated changes in expression following modulations in their upstream regulator HSF1 . Similarly, many nuclear genes that give rise to mitochondrial proteins are expressed in a coordinated fashion due to a relatively finite collection of upstream regulators. By way of example, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) regulates the expression of 70% of the one hundred or so electron transport chain sub-unit (and ATP synthase) genes as well as all eight enzymes in the Citric Acid cycle . Such synchronicity affords significant predictive power and has been exploited to identify multiple mitochondrial protein-encoding genes [10, 12]. In this study, we used a predictive methodology based on gene co-expression to nominate proteins relevant to mitochondrial function in the context of the metabolic and signaling derangement associated with pathological cardiac hypertrophy. We first conducted a lenient and inclusive correlation analysis to nominate novel mitochondrial protein-encoding transcripts (MPETs). This was accomplished by probing almost 10,000 publicly available datasets with the CORD database  for transcripts whose expression signatures resemble those of bona fide MPETs. In the second phase of the study, a less agnostic approach was adopted in that we asked if the phase one candidates could be rediscovered in a disease-relevant setting. To accomplish this, we created a gene network from a human heart gene expression dataset  via WGCNA and identified a module highly enriched in MPETs. Leucine Rich Repeat Containing 2 (LRRC2) was among this recurrent population and attracted interest on account of it correlating with transcripts associated with cardiac remodeling as well as a general paucity of knowledge regarding its function. We found LRRC2 to be at least partially localized to the mitochondrion and regulated by the mitochondrial master regulator PGC-1α. As well as being elevated in modeled hypertrophic settings, we also observed paralleled expression between LRRC2 and MYH7, an established biomarker of heart failure, in the human heart . Moreover, Lrrc2 suppression in a cardiomyocyte cell line invoked transcriptional changes typically associated with pathological hypertrophy and a concomitant elevation in mitochondrial mass in the context of increased Pgc-1α expression. These data implicate a role for LRRC2 in the control of processes that dictate cardiomyocyte hypertrophy and mitochondrial abundance. How, mechanistically, LRRC2 loss-of-function invokes an increase in mitochondrial biogenesis is uncertain and requires further examination. In light of the association between compensated cardiac hypertrophy and Pgc-1α-driven mitochondrial biogenesis, it’s tempting to speculate that Lrrc2-mediated regulation of Pgc-1α could influence the hypertrophic response. Defining how LRRC2 fits into this model warrants additional study but one possibility, suggested by the regulatory interplay observed (i.e., that Pgc-1α overexpression and suppression leads to increased and decreased Lrrc2 expression, respectively and that Lrrc2 loss-of function elevates Pgc-1α expression) is that a protein-protein interaction between Lrrc2 and Pgc-1α may dampen the capacity of the latter to auto-regulate. Indeed, when Pgc-1α was over-expressed in H9c2 cells in combination with Lrrc2, the increase in Pgc-1α transcript abundance was far less than that observed when it was over-expressed alone. Enhanced presence of such repressive signals may contribute to the gradual reduction in mitochondrial function observed in heart failure and type 2 diabetes [48–50], and hence represents a rational target for therapeutic intervention.