Formation of a TBX20-CASZ1 protein complex is protective against dilated cardiomyopathy and critical for cardiac homeostasis

By the age of 40, one in five adults without symptoms of cardiovascular disease are at risk for developing congestive heart failure. Within this population, dilated cardiomyopathy (DCM) remains one of the leading causes of disease and death, with nearly half of cases genetically determined. Though genetic and high throughput sequencing-based approaches have identified sporadic and inherited mutations in a multitude of genes implicated in cardiomyopathy, how combinations of asymptomatic mutations lead to cardiac failure remains a mystery. Since a number of studies have implicated mutations of the transcription factor TBX20 in congenital heart diseases, we investigated the underlying mechanisms, using an unbiased systems-based screen to identify novel, cardiac-specific binding partners. We demonstrated that TBX20 physically and genetically interacts with the essential transcription factor CASZ1. This interaction is required for survival, as mice heterozygous for both Tbx20 and Casz1 die post-natally as a result of DCM. A Tbx20 mutation associated with human familial DCM sterically interferes with the TBX20-CASZ1 interaction and provides a physical basis for how this human mutation disrupts normal cardiac function. Finally, we employed quantitative proteomic analyses to define the molecular pathways mis-regulated upon disruption of this novel complex. Collectively, our proteomic, biochemical, genetic, and structural studies suggest that the physical interaction between TBX20 and CASZ1 is required for cardiac homeostasis, and further, that reduction or loss of this critical interaction leads to DCM. This work provides strong evidence that DCM can be inherited through a digenic mechanism.

Introduction Heart failure is a major cause of morbidity in the United States with more than 5 million people in the US living with this disease [1]. A major risk factor for developing heart failure is dilated cardiomyopathy (DCM). Clinically recognized as systolic dysfunction accompanied by dilation of one or both ventricles, DCM is a predominating cardiomyopathy and the most common disease requiring heart transplantation in the US [2,3]; however, nearly half of DCM cases are of unknown etiology [4].
In efforts to understand the etiology of idiopathic DCM, mutations in over 50 genes including components of the contractile apparatus and cell cytoskeleton, as well as in factors involved in excitation-conduction coupling, have been identified as causative in DCM [5,6]. However, few studies have explored the potential for aberrant transcriptional regulation of these factors to contribute to disease pathogenesis. In exception to this, recent studies have identified mutations in the T-box transcription factor TBX20 associated with DCM [7][8][9].
While TBX20 is an essential transcription factor for heart development and its disease relevance is well established, many fundamental questions remain about the mechanism of TBX20 function. Principle among these is how TBX20 mutations associated with DCM circumvent the essential embryonic cardiac requirement for TBX20.
To elucidate the mechanisms by which mutations in TBX20 lead to human adult pathological states, we identified endogenous TBX20 cardiac protein-protein interactions by coupling a tagged endogenous allele of Tbx20 with unbiased proteomic analysis. Results from these studies revealed TBX20 interacts with the essential cardiac transcription factor Castor (CASZ1), a gene that was also recently linked to DCM [26]. We confirmed that TBX20 and CASZ1 interact biochemically and genetically, and we go on to show that while mice singularly haploinsufficient for Tbx20 or Casz1 are asymptomatic, mice heterozygous for both Tbx20 and Casz1 die, beginning at 4 to 8 weeks post birth, and exhibit cardiomyocyte hypertrophy, interstitial fibrosis, and severe DCM. Interestingly, the human mutant TBX20 F256I bypasses the early essential requirement for TBX20 but leads to DCM. We report here that TBX20 F256I disrupts the TBX20-CASZ1 interaction, ascribing clinical relevance to this protein complex. Further, by using quantitative proteomics we have identified the molecular pathways altered in TBX20-CASZ1-mediated DCM. Together, these results identify a novel interaction between TBX20 and CASZ1 that is essential for maintaining cardiac homeostasis. These findings imply that DCM can be inherited through a digenic mechanism.

Defining the endogenous cardiac TBX20 interactome
To identify endogenous protein interactions that regulate TBX20 function, we introduced the Avitag, in-frame, to the carboxy terminus of mouse Tbx20 through homologous recombination in mouse embryonic stem cells (ESCs) (Tbx20 Avi ) (S1A and S1B Fig). Since the Avi-tag can be biotinylated through recognition of the Avi-tag sequence by the E. coli biotin ligase BirA [27, 28], we generated a lentivirus expressing BirA and transduced it into mouse Tbx20 Avi/+ ESCs. After hygromycin selection, Tbx20 Avi/+ ESCs that stably expressed BirA (S1C Fig) were differentiated into induced cardiomyocytes (iCM) using a serum-free differentiation method that routinely generates cultures containing >60% cardiomyocytes (Fig 1A) [29].
Expression analysis at each day of differentiation confirmed the Tbx20 Avi :BirA ESCs recapitulated the wild-type cardiomyocyte differentiation program (Fig 1B). We further showed by Myosin Heavy Chain (MHC) expression and time lapse imaging that the Tbx20 Avi :BirA ESCs differentiated into beating neonatal cardiomyocytes ( Fig 1C, S1 Movie). Our analysis further verifies that Tbx20 Avi expression recapitulates endogenous Tbx20 expression with highest levels in immature cardiomyocytes at day 4 and differentiated cardiomyocytes at day 7 ( Fig 1B).
Published data has demonstrated a requirement for TBX20 in adult mice, with loss of TBX20 leading to abrupt cardiac failure [25]. Recently, mutations in TBX20 in humans were associated with DCM [7][8][9]. To delineate the mechanisms of how TBX20 DCM-associated mutations circumvent the essential requirements for TBX20 in cardiac development, we isolated and characterized the endogenous TBX20 cardiac protein interactome under physiological conditions from Tbx20 Avi ; BirA iCMs at day 7 of differentiation. As a control for non-specific interactions, identical affinity isolations were performed from BirA-negative iCMs. Proteins coisolated from TBX20 Avi affinity purifications (APs) were analyzed by an SDS-PAGE tandem mass spectrometry-based proteomics approach, as in [30]. TBX20 was detected in the AP from BirA-expressing iCMs, with 19 unique tryptic peptides covering 54% of the TBX20 sequence (out of a theoretical maximum coverage of~75%) (Fig 1D and 1E; S2A and S2B Fig; S1 Table).
Identification of candidate high-confidence TBX20 interactions that have the potential to regulate cardiac functions was achieved using a multi-step bioinformatics approach based on the number of identified spectra per protein. First, interacting proteins identified by less than 10 spectra did not meet the identification requirement and were excluded from further analysis. Further, proteins identified in the BirA-expressing isolations were required to have at least a 4-fold increase in identified spectra over isolations from control iCMs. Due to the ascribed function of TBX20 as a critical cardiac transcription factor, we specifically focused on proteins with a nuclear or unknown subcellular localization. Finally, these interaction candidates were ranked by their AP enrichment (AP abundance versus whole cell abundance, S1 Table), which we have previously used to highlight the most prominent associations suitable for functional validation [31,32]. Interestingly, the top 50 most enriched proteins in the TBX20 AP were predominately (32/50) annotated to Chromatin and Transcription gene ontologies (Fig 2A; S1 Table). Functional annotation of these proteins in the STRING database [33] revealed an interconnected network containing components of chromatin remodeling and RNA polymerase transcriptional complexes (including four components of the INO80 complex-Ino80, Actr5, Actr5, Nfrkb, and five components of the RNA Pol II mediator complex-Med13, Med14, Med17, Med19, Med27) (Fig 2A). These data suggest that TBX20 predominantly acts to regulate transcription in neonatal cardiomyocytes, likely via interactions with the INO80 and RNA Pol II mediator complexes.

TBX20 complexes with CASZ1
In addition to identifying components of broadly expressed multiprotein chromatin machines, our analysis revealed the association of TBX20 with the essential cardiac transcription factor CASZ1 in BirA-expressing iCMs (25 unique peptides and 21% sequence coverage. As previously reported [34,35], CASZ1 protein runs as three bands on at approximately 191kD, presumably due to post translational processing) ( Fig 2B, S2C Fig). Surprisingly, this was the only developmentally-regulated cardiac transcription factor we found to interact with TBX20 in Day 7 cardiomyocytes. The low estimated cellular abundance of CASZ1 and the relatively high AP enrichment ratio (S1 Table) highlighted CASZ1 as a potential in vivo TBX20 interacting protein. We further confirmed the TBX20-CASZ1 interaction through reciprocal immunoisolation of endogenous CASZ1 in cardiac nuclei from adult mouse hearts (Fig 2C) thus, verifying our ESC differentiation-based approach can successfully identify bona fide TBX20 interaction partners under physiological conditions. Since expression analysis and genetic fate mapping studies have shown CASZ1 is expressed only in cardiomyocytes and no other cardiac cell types [36], and since we were unable to identify this interaction at Day 4 of iCM differentiation, these studies imply the interaction between TBX20 and CASZ1 is temporally regulated and cardiomyocyte-specific.
Phylogenic analysis shows that TBX20 and CASZ1 are highly conserved across vertebrate orthologs [34,35], suggesting that the TBX20-CASZ1 interaction may also be evolutionarily conserved. To confirm the interaction and to determine whether it is evolutionarily conserved, we injected X. laevis embryos with the Xenopus orthologous mRNAs of TBX20 and CASZ1. In parallel, we co-expressed murine versions of tagged TBX20 and CASZ1 proteins in HEK293 cells. Immunoaffinity purification of TBX20 protein complexes from both of these sources, followed by immunoblotting confirms the formation of a TBX20-CASZ1 interaction in human cells and in X. laevis embryos (S3A and S3B Fig). Taken together, our findings are supportive of an evolutionarily conserved role for the formation of a TBX20-CASZ1 protein complex in differentiated cardiomyocytes.

Combined haploinsufficiency of Tbx20 and Casz1 results in dilated cardiomyopathy
To determine the biological relevance of the TBX20-CASZ1 interaction, we tested for genetic interaction between Tbx20 and Casz1 by generating mice with cardiac-specific heterozygous loss of Tbx20 and Casz1 (Tbx20 flox/+ ; Casz1 flox/+ ; Nkx2.5 Cre ) [21,36]. Compound heterozygous mice, hereafter referred to as Tbx20 flox/+ ; Casz1 flox/+ , were born and appeared normal. However, beginning at 4 weeks of age we observed an increased incidence of death among Tbx20 flox/+ ; Casz1 flox/+ mice (2.7%) compared to the single heterozygotes (0%) ( Table 1). This effect was amplified at later timepoints, with survival rates of 90.5% and 62.5% at 8 and 16 weeks of age, respectively, compared to a 100% survival rate in the single heterozygotes. Furthermore, we did not observe any overt phenotypes in Tbx20 flox/+ ; Nkx2.5 Cre or Casz1 flox/+ ; Nkx2.5 Cre mice. Since we were able to demonstrate that loss of Casz1 does not affect Tbx20 expression in adult heart tissue and that loss of Tbx20 does not affect Casz1 expression (S4A and S4B Fig), these studies are supportive of a genetic requirement for a functional interaction between TBX20 and CASZ1. The TBX20-CASZ1 interaction is essential for normal cardiac function To determine the cause of the reduced survival rate we observe in Tbx20 flox/+ ; Casz1 flox/+ mice, we performed detailed physiological analysis, using echocardiography, of single and compound heterozygous mice. These studies revealed that cardiac function is significantly compromised in Tbx20; Casz1 compound heterozygotes compared to single heterozygotes. Compound heterozygotes exhibit significantly decreased ejection fraction and fractional shortening, increased left ventricular blood volume, and increased left ventricular diameter ( Fig  3A-3D; Tables 2 and 3; S2 Table; S2-S6 Movies). Further, Tbx20 flox/+ ; Casz1 flox/+ heterozygous mice display dilated ventricles with a striking decrease in ventricular wall thickness compared to single heterozygotes (Fig 4A and 4B). These findings were observed in both male and female mice (S3 Table). Thus, Tbx20 flox/+ ; Casz1 flox/+ mice display defining anatomical features of DCM that progress to cardiac failure. Collectively, these findings suggest that the genetic interaction between Tbx20 and Casz1 is essential for normal cardiac homeostasis, and perturbation of this interaction leads to DCM. Tbx20; Casz1 compound heterozygous hearts undergo cardiac remodeling One of the defining clinical features of severe DCM is an accumulation of myocardial collagen leading to interstitial fibrosis, a contributing and compounding factor in cardiac dysfunction [37][38][39]. To confirm that the severe cardiac dysfunction we observe in Tbx20; Casz1 compound heterozygotes is associated with advanced DCM, we examined collagen fibers and found robust collagen deposition in the interstitium of Tbx20 flox/+ ; Casz1 flox/+ hearts ( Fig 4B  and 4C). Despite the interstitial fibrosis and severely impaired systolic function, the fact that over half of these mice survive to adulthood with some degree of cardiac function led us to hypothesize that Tbx20; Casz1 compound heterozygous cardiomyocytes undergo compensatory pathological hypertrophy. To test this hypothesis, we measured cardiomyocyte cross-sectional areas and found that MF20-positive cells in compound heterozygotes were indeed increased in size relative to controls (Fig 4D and 4E). This data suggests that disrupting the TBX20-CASZ1 interaction leads to severe DCM and cardiac fibrosis. In response to this heightened cardiac stress, Tbx20; Casz1 compound heterozygote hearts appear to undergo pathological hypertrophy as an adaptive response.
A human DCM-associated TBX20 mutation reduces the TBX20-CASZ1 interaction Our data implies the TBX20-CASZ1 interaction is essential for normal cardiac homeostasis. To define the region of TBX20 that mediates interaction with CASZ1, we conducted immunoisolations with wild-type and deletion mutants in which either the T-Box or the C-terminus of TBX20 has been removed ( Fig 5A). Immunopurifications of CASZ1 in the presence of wildtype TBX20, TBX20 ΔT-box , or TBX20 ΔC , show that the T-box domain is required for interaction with CASZ1, but that the C-terminus is dispensable ( Fig 5A). In reciprocal studies, we find the four most amino-terminal zinc finger domains of CASZ1 are necessary for interaction with TBX20 ( Fig 5B). The CASZ1-interacting region of TBX20, as well as the TBX20-interacting region of CASZ1, are highly conserved across species implying functional relevance to these regions ( Fig 5C).
Recently, human TBX20 mutations have been identified that are associated with DCM; however, only one of these mutations, TBX20 F256I , co-segregates in a dominant manner with complete penetrance in a family with DCM [9]. Moreover, DCM was found in all affected     family members reported as healthy during health assessments performed when they were juveniles. The functional relevance of the F256I mutation is further underscored by the finding that the amino acid disrupted by this mutation is 100% conserved across all TBX20 orthologs and by the observation that no F256I mutations were identified in 600 control samples [9]. Interestingly, the F256I mutation associated with DCM lies within the TBX20 T-box domain, the region we found essential for interaction with CASZ1 (Figs 5A and 6A). To test if TBX20 F256I perturbs the TBX20-CASZ1 interaction, we performed immunoaffinity purifications of CASZ1 in the presence of wild-type TBX20 or TBX20 F256I . Interestingly, the F256I mutation significantly reduces the interaction with CASZ1 ( Fig 6A). These data imply that the DCM mutation F256I may contribute to the development of cardiac disease by disrupting a critical physical interaction between TBX20 and CASZ1.
The TBX20 F256I mutation acts to sterically inhibit the TBX20-CASZ1 interaction To gain a structural understanding of how the F256I mutation disrupts the TBX20-CASZ1 interaction, we conducted molecular modeling of the wild-type and TBX20 F256I T-box domain (Fig 5B and 5C). The predicted structures were based on the range of fluctuations in the structure that occur over a period of 100 ns (S4 Movie). Three regions are highlighted which show conformational changes induced by the mutation (Fig 6B). Our models find F256 is not predicted to contact DNA but the conversion of phenylalanine to isoleucine at position 256 leads to steric clashes with the conserved T-box residues E258 and T259 (Fig 6C). The critical functional nature of this region of TBX20 is underscored by the complete conservation of amino acids at residues F256, E258, and T259 across 250 members of the T-box gene family (S5A and S5B Fig). Taken together, these findings imply that F256I leads to a conformational change across the surface predicted to interact with CASZ1, and that disruption of this interaction leads to alteration in DNA binding.
To determine the transcriptional consequences of TBX20 F256I on the TBX20:CASZ1 interaction, we conducted transcriptional assays with TBX20, CASZ1 and TBX20 F256I alone and in combination. Results demonstrate TBX20 synergistically acts with CASZ1 and that TBX20 F256I significantly diminishes transcriptional activation by TBX20:CASZ1 (S6 Fig). These data together with our structural studies provide a mechanistic basis for how F256I disrupts TBX20: CASZ1 function.
Tbx20; Casz1 compound heterozygous hearts have an altered DCMassociated proteome To identify the molecular pathways altered in DCM haploinsufficient mutant (Tbx20 flox/+ ; Casz1 flox/+ ; Nkx2-5 Cre ) mouse hearts, we used quantitative multiplexed mass spectrometry to identify proteins with altered abundances relative to control hearts. Proteins were extracted from nuclear-enriched mouse cardiac fractions of mutant and control (Nkx2-5 Cre ) mice in duplicate and digested in-solution with trypsin. Peptides from each sample were labeled with different isobaric tandem mass tagging (TMT) reagents, pooled, fractionated, and analyzed by reverse phase nanoliquid chromatography coupled to a high resolution quadrupole Orbitrap tandem mass spectrometer. Using this strategy, 3164 proteins were identified and quantified based on their respective sequenced peptides and TMT reporter ions, respectively (S4 Table).
To define the TMT ratio threshold for differential relative abundance, protein abundance values were compared between biological duplicates (S7 Fig). For both the control and mutant replicates, the correlation of abundances was high (R 2 = 0.99) and the majority of proteins had low dispersion from a 1:1 linear curve (S7A and S7B Fig), indicating low biological and technical variation. Curve-fit analysis of TMT abundance ratio histograms for the control and mutant biological duplicates showed that, on average, 90% of the ratios varied less than ±30% (S7 Fig). Based on this result, a relative abundance ratio of at least ±1.3-fold between mutant and control mice in both replicates were used to identify a protein as differential. From the total number of quantified proteins, 175 met this criterion, of which 86 and 89 were up and down-regulated, respectively (Fig 7C; S4 Table). Further verifying the role of the TBX20; CASZ1 interaction, 165 of the 175 of the proteins identified by this approach were encoded by a gene previously demonstrated to be a TBX20 target [40](S5 Table).
To generate an initial picture of potentially dysregulated pathways, the known functional connectivity among differential proteins can be determined using databases of annotated pathways and protein-protein interaction. Towards this goal, known functional associations among the 175 differential proteins were scored based on the STRING bioinformatics database [41], and the relational networks visualized in Cytoscape [42] (Fig 7D; S8 Fig). A high degree of interconnectivity was observed among the differential proteins as 127 of the 175 annotated proteins had at least one other connection and each protein on average was connected to 4.6 neighbors. Network clustering was performed to identify subsets of highly connected proteins, which likely share similar functions. Overall, there are 10 functional clusters containing at least 3 proteins, indicated by the color-coding in Fig 6C. To identify the most significant biological processes and pathways that are perturbed in Tbx20; Casz1 hypomorphic DCM hearts, we performed comparative Gene Ontology over-representation analysis of the differentially regulated proteins using ClueGO [43] (Fig 7E). Consistent with studies demonstrating an association between DCM and inflammation [44,45], we found components of the pro-inflammatory response (i.e. complement activation) significantly up-regulated.
In addition to inflammation-associated proteins, our data further revealed a dysregulation of mitochondrial proteins known to be associated with impaired cardiomyocyte contractile function in DCM [46]. In line with these findings and the observation that reduced contractile force is linked to altered glycogen metabolism and cardiomyopathy [47][48][49], we found an over-representation of proteins associated with the glycogen metabolic pathway. We note that these were exclusively down-regulated proteins, represented by glycogen synthase (Gys1), glycogen phosphorylases (Pygm/Pygb), and phosphorylase kinase gamma 1 (Phkg1). Interestingly, proteins involved in glycogen regulation and in myosin-dependent muscle contractility were part of the same functional cluster (Fig 7D, yellow nodes); however, their individual abundances were down-and up-regulated, respectively (Fig 7D, circle vs. square nodes). Taken together, these data confirm at the protein level the DCM pathology in Tbx20; Casz1 hypomorphic DCM mice.

TBX20; CASZ1-linked DCM is associated with dysregulation of cell-cell adhesion proteins
In addition to proteins previously reported to be associated with DCM, our analysis identified a distinct set of cell-cell adhesion proteins in Tbx20; Casz1 compound heterozygous hearts that were significantly overrepresented compared to whole genome annotation (Fig 7E; S8 Fig, yellow). These observations highlight the significant changes that are likely occurring in the extracellular and intracellular spaces and raise a key question-what are the signaling mediators that link these processes? To identify potential key mediators of TBX20-CASZ1-driven DCM, we constructed a gene-linked GO network for the Cellular Component ontology (S8 and S9 Figs). This network highlighted two interesting candidates, bone morphogenic protein 10 (Bmp10) and thrombospondin 1 (Thbs1), the former being a TGF-beta receptor ligand and the latter having roles in cell-cell adhesion as well as ER stress response [50]. Overall, this systems-level proteome view of DCM provides potential downstream targets and pathways that may be influenced as a result of Tbx20 and Casz1 haploinsufficiency and suggests a role for cell-cell adhesion in mediating DCM.

Discussion
One in five adults free of cardiovascular disease by the age of 40 are at risk of developing congestive heart failure over their lifetime [51,52]. Within this population DCM remains one of the leading causes of disease and death with nearly half of DCM cases genetically determined [53][54][55][56][57]. To date, most DCM mutations have been identified in genes coding for components of the contractile apparatus or the cell cytoskeleton or factors involved in excitationconduction coupling. Though these studies have provided insight into the pathology of DCM, the transcriptional regulation of DCM is poorly understood. Recently, studies have identified mutations in the T-box transcription factor TBX20 in DCM patients [7][8][9]. However, these studies have not explained how DCM-associated mutations bypass early essential requirements for TBX20. Here, we demonstrate TBX20 and CASZ1 physically and genetically interact in the adult heart and establish that this interaction is essential for cardiac homeostasis. We further find that disruption of the TBX20-CASZ1 interaction in mice and humans leads to cardiomyopathy. Mice singly heterozygous for alleles of Tbx20 or Casz1 are asymptomatic, while Tbx20; Casz1 compound heterozygotes die post-natally, exhibiting systolic dysfunction, as well as ventricular dilation and interstitial fibrosis. These cardiac defects, in the absence of coronary artery disease or substantially abnormal load, are defining features of DCM as seen in human patients [6,58,59].

CASZ1 and DCM
CASZ1 is a large para-zinc finger protein of unique structure and to date, there have been limited studies on the mechanisms of how CASZ1 regulates transcription [60][61][62]. These types of studies have been compromised by the lack of high-affinity high-specificity mammalian CASZ1 antibodies, precluding approaches such as ChIP-seq. It further remains unclear if CASZ1, as a para-zinc finger protein, directly binds DNA or is recruited via other transcription factors. Our structural studies favor a model by which the TBX20-CASZ1 interaction is required for DNA binding. This model predicts that the respective region of TBX20 that binds CASZ1 is near to or contributes to the DNA binding interface and has the potential to impact CASZ1 binding.
CASZ1 was first ascribed a role in vertebrate cardiovascular development in Xenopus [34, 62,63]. Subsequent genetic studies in mammals uncovered that like TBX20, CASZ1 functions in the embryonic heart to control cardiomyocyte proliferation, with loss of CASZ1 leading to cardiac death by E12.5 [36,64]. Our finding that Tbx20; Casz1 compound heterozygous mice die post-natally implies that CASZ1 has a second and later role in cardiac homeostasis. This

TBX20-CASZ1 protein pathways and DCM
In these studies, the Nkx2.5-Cre driver was used to generate mice null for Casz1 and Tbx20. In all cases the Nkx2.5-Cre driver alone was used, with wild-type mice as a negative control in our physiological studies and in our quantitative proteomic studies. In contrast to previous reports [65], we could detect no significant changes in any cardiac function in Nkx2.5-Cre mice relative to wild-type mice. These finding may be due to genetic background, the sex on which the Cre driver was delivered to the offspring, or environmental variability as reported for other lines [66,67]. Regardless of the reason for the variability, we did find the Nkx2.5-Cre driver had a high recombination efficiency reducing the levels of TBX20 and CASZ1 by half (S4 Table). Since, reducing CASZ1 expression by half had no detectable alteration in Tbx20 expression and vice versa, our data suggests a biochemical and genetic interaction between TBX20 and CASZ1. Our data further indicates that disruption of this complex leads to DCM in mice and humans.
A previously published model of DCM, the phospholamban R9C transgenic mouse [68], has also been studied by proteomic analysis [69,70]. This model exhibits impaired calcium regulation in cardiomyocytes, accompanied by decreased cardiac contractility and premature mortality [68]. The GO-associated proteome changes that we found in the haploinsufficient mice share similarities with the Phospholamban R9C mice. Specifically, both mouse models show up-regulation of actin-myosin cytoskeletal networks and down-regulation of mitochondria-associated proteins involved in fatty acid oxidation. Interestingly, proteomic analyses performed on ventricular tissues from human patients with inflammatory DCM had similar findings [71]. Yet some functional protein classes in our Tbx20-CASZ1 haploinsufficient DCM mice were distinct, including an up-regulation of the complement system and greater coverage of down-regulated proteins in glycogen metabolic processes. While we found the Tbx20-CASZ1 haploinsufficient mice have evidence of differential regulation in calcium-binding proteins, not surprisingly, the Phospholamban R9C mice have more pervasive effects on calcium-dependent signaling, such as involving ER stress responses, though it is possible that these distinctions may be due to differences in the progression of the fibrosis associated with DCM.
One of the hallmarks of DCM is altered cardiomyocyte force transduction that is frequently associated with alteration in the composition or functions of intercalated discs-a cardiac-specific structure at the contact site between cardiomyocytes [72,73]. Here, we observed a significant mis-regulation of proteins involved in cell-cell adhesion in heart tissue from Tbx20; Casz1 heterozygous mice. Moreover, these include three proteins which are encoded by genes that when mutated are causative to DCM-TTN, DES, and PDLIM3. Thus, our findings imply that the TBX20-CASZ1 complex acts, at least in part, to control the electrical and mechanical integration of neighboring cardiomyocytes.

TBX20, CASZ1, and digenic inheritance of DCM
The observation that the TBX20 F256I mutation leads to a decreased association with CASZ1, along with the finding that patients heterozygous for a predicted TBX20 null mutation (TBX20 Q195X ) [23] also display DCM suggest that the TBX20 F256I mutation may be acting in a haploinsufficient fashion. However, only two of the individuals within a single pedigree with the TBX20 Q195X mutation display DCM while other individuals display a wide range of cardiac abnormalities [23]. Moreover, we have screened the Exome Aggregation Consortium (ExAc) reference set and have identified four variants in TBX20 in individuals that are asymptomatic.
All variants lead to a premature stop codon in one of the TBX20 alleles and all would be predicted to be functionally null (introduction of stop codons into exons 2, 4, 7, and 8) [74]. Together, these findings imply that the function of the TBX20-CASZ1 complex in DCM is not dose dependent. Alternatively, individuals harboring the TBX20 F256I mutation have a genetically sensitized background leading to a varying degree of penetrance that is determined by modifying genes that may be carried within the CASZ1 pathway.
In cardiovascular disease, genetic mutations often result in varying degrees of penetrance, and in extreme examples, the presence of a disease-causing mutation can be asymptomatic [75][76][77][78][79][80]. These phenomena have often been explained by the action of genetic modifiers in which one gene mutation is causative to CHD and a second mutation modifies the effect of the first. However, more recent studies suggest an alternate or additional mechanism by which complete penetrance is achieved in human disease states by genetic variation at one or more loci [81]. In digenic inheritance, two genetic mutations are required for the clinical phenotype with either mutations alone being asymptomatic. Our findings provide an example of digenic inheritance in DCM and suggest that mutations in TBX20 or CASZ1 could lead to susceptibility to DCM but in many cases are not in themselves causative. We would envision these findings are not restricted to TBX20 and CASZ1 but rather are applicable to other genes and other forms of congenital heart disease (CHD) and DCM, and predict that genome sequencing of familial CHD will ultimately reveal a spectrum of additional CHD susceptibility alleles.

Generation of Tbx20 Avi allele
The Tbx20 Avi allele was created by introducing the biotin acceptor peptide (Avi) targeting cassette, similar to our previous study [82], in-frame to the terminal exon of Tbx20 in collaboration with the UNC Animal Models Core and the UNC BAC Core (Chapel Hill).

Generation of Tbx20 Avi ; BirA cell line
The Tbx20 Avi ; BirA cell line was generated by targeting a sequence containing the Avitag followed by a loxP-flanked neo cassette into the stop codon of exon 8 of a Tbx20a genomic fragment derived from a 129 Sv genomic BAC library. The targeting construct was linearized and electroporated into mouse embryonic stem cells (ESCs) of E14TG2a.4 origin. Targeted ESCs were placed under 250 μg/mL G418 selection for 7-10 days and G418-resistant ESC clones (n = 384) were screened for homologous recombination by Southern blot analysis. Three ESC clones were correctly targeted, and one of these clones was subsequently used to derive the Tbx20 Avi/+ ; BirA cell line. Briefly, Tbx20 Avi/+ ESCs were grown to approximately 40% confluence and transduced with 5 MOI Lenti-BirA for 8 hrs. Twenty-four hours following transduction, cells were placed under 200 μg/mL hygromycin selection for 4-5 days. Hygro-resistant Tbx20 Avi/+ cells were subsequently used for cardiomyocyte differentiations.

ESC differentiation
Tbx20 Avi/+ ; BirA ESCs were maintained on gelatin-coated dishes in a feeder-free culture system and differentiated [29] in serum-free (SF) media according to the Keller protocol. Briefly, ESCs were trypsinized and cultured at 75,000 cells/mL on uncoated petri dishes in SF medium without additional growth factors for 48 hrs. Two-day-old aggregated embryoid bodies (EBs) were dissociated and the cells reaggregated for 48 hr in SF medium containing 5 ng/mL human Activin A, 0.1 ng/mL human BMP4, and 5 ng/mL human VEGF (all growth factors purchased from R&D Systems). Four-day-old EBs were dissociated and 2 x 10 6 cells were seeded into individual gelatin-coated wells of a 6-well dish in StemPro-34 SF medium (Invitrogen) supplemented with 2 mM L-glutamine, 1 mM ascorbic acid, 5 ng/mL human VEGF, 20 ng/mL human bFGF, and 50 ng/mL human FGF10 (R&D Systems). Cardiomyocyte monolayers were maintained in this media for 4-5 additional days with cells typically beginning to beat 2 days after seeding onto gelatin (total of 7-8 days of differentiation).

Immunofluorescence
For immunofluorescence of cardiomyocytes, four-day-old ES cell-derived EBs were dissociated and seeded into 8-well chamber slides precoated with 0.1% gelatin. Induced cardiomyocytes were fixed on day 7 of differentiation in 4% paraformaldehyde for 20 min at room temperature, washed (3 x 1X PBS), permeabilized in 0.1% Triton X-100 in 1X PBS for 10 min, and blocked (10% fetal bovine serum [FBS], 0.1% Tween 20 in 1X PBS) for 30 min. Anti-myosin heavy chain (Abcam) was applied overnight, followed by PBS washes (3 x 1X PBS), and incubation with goat anti-mouse Alexa 546 (Invitrogen) for 1 hr. Cells were incubated in DAPI (200 ng/mL in ethanol) for 30 min and visualized by confocal microscopy on a Zeiss 710.

Proteomic analysis of Tbx20 affinity purifications
Protein preparations, conjugation of magnetic beads and immunoaffinity purification and mass spectrometry were conducted as previously reported [82]. All results are from a minimum of two independent biological replicates. Briefly, immunoisolated proteins were resolved (~4 cm) by SDS-PAGE, and visualized by Coomassie blue. Each lane was subjected to in-gel digestion with trypsin and analyzed by nanoliquid chromatography coupled to tandem mass spectrometry as previously reported [83]. Tandem mass spectra were extracted by Proteome Discoverer (ThermoFisher Scientific, ver 1.4), and searched with the SEQUEST algorithm against a theoretical tryptic peptide database generated from the forward or reverse entries of the mouse UniProt-SwissProt protein sequence database (2013/08) and common contaminants (total of 43, 007 sequences). SEQUEST search results were analyzed by Scaffold (version 4.6.1, Proteome Software Inc) using the LFDR scoring scheme to calculate peptide and protein probabilities. Peptide and protein probabilities thresholds were selected to achieve 1% FDR at the peptide level based on LFDR modeling and at the protein level, based on the number of proteins identified as hits to the reverse database. The spectral counts assigned to proteins that satisfy these criteria and had a minimum of two unique peptides were exported to Excel for data processing.

Interaction bioinformatics analysis
Proteins identified by LC-tandem MS were filtered to exclude non-specific associations. Proteins were retained as specific interaction candidates if the proteins were assigned (1) at least ten spectral counts in the Tbx20 Avi ;BirA condition, and (2) were uniquely identified or had at least a 4-fold spectral count enrichment in the Tbx20 Avi ; BirA condition versus the control. Next, the subset of candidates assigned a nuclear or unknown UniProt subcellular localization were retained for calculation of enrichment index values, as previously described [31]. Briefly, the relative protein abundance within the affinity purification was calculated using the NSAF approach [84], then normalized by each protein's respective cellular abundance estimated in the PAX database [85] (Mouse-whole organism, SC GPM 2014). Interaction candidates were ranked by their enrichment index and the top 50 proteins were analyzed by STRING [86] for interaction network analysis. Interactions with a combined STRING score of > 0.4 (medium confidence) were retained, exported, and visualized in Cytoscape (ver. 3.3). Proteins within the network were assigned into broad protein functional classes based on annotations in the UniProtKB database.

Mice
Tbx20 flox/+ mice were generously provided by Sylvia Evans (UCSD) [21]. The Casz1 flox/+ mouse has been previously reported [36]. Histological sectioning and immunohistochemistry were done as reported except as noted [36]. All mice are on a mixed B6/129/SvEv/CD-1 background and all mouse experiments were performed according to the Animal Care Committee at the University of North Carolina, Chapel Hill.

Echocardiography
Cardiac function was assessed in conscious 8-11 week-old Casz1 flox/+ ; Nkx2.5 Cre , Tbx20 flox/+ ; Nkx2.5 Cre , and Tbx20 flox/+ ; Casz1 flox/+ ; Nkx2.5 Cre mice (5-10 mice per genotype) by thoracic echocardiography using VisualSonics Vevo 770 ultrasound system (Visual Sonics, Inc.). All imaging was done by trained technicians blinded to the genotypes of the animals. Briefly, a topical hair removal agent was used on the chest and abdomen of mice. The mice placed on a warmed table in the supine position for imaging. A 30 MHz pediatric probe used to capture 2-dimensional guided M-mode views of the long and short axes at the level of the papillary muscle. VisualSonics Analytic software was used to determine mean ventricular wall and interventricular septum thickness, as well as the left ventricle diameter from at least 3 consecutive cardiac cycles. Means were used to calculate ejection fraction and fractional shortening.

Statistics
All statistical analysis performed using SAS JMP 10. Statistical significance between individual groups was calculated using Student's T-test, while significance between more than 2 groups was calculated using ANOVA.