Cardiac development requires interplay between the regulation of gene expression and the assembly of functional sarcomeric proteins. We report that UNC-45b recessive loss-of-function mutations in C3H and C57BL/6 inbred mouse strains cause arrest of cardiac morphogenesis at the formation of right heart structures and failure of contractile function. Wild-type C3H and C57BL/6 embryos at the same stage, E9.5, form actively contracting right and left atria and ventricles. The known interactions of UNC-45b as a molecular chaperone are consistent with diminished accumulation of the sarcomeric myosins, but not their mRNAs, and the resulting decreased contraction of homozygous mutant embryonic hearts. The novel finding that GATA4 accumulation is similarly decreased at the protein but not mRNA levels is also consistent with the function of UNC-45b as a chaperone. The mRNAs of known downstream targets of GATA4 during secondary cardiac field development, the cardiogenic factors Hand1, Hand2 and Nkx-2.5, are also decreased, consistent with the reduced GATA4 protein accumulation. Direct binding studies show that the UNC-45b chaperone forms physical complexes with both the alpha and beta cardiac myosins and the cardiogenic transcription factor GATA4. Co-expression of UNC-45b with GATA4 led to enhanced transcription from GATA promoters in naïve cells. These novel results suggest that the heart-specific UNC-45b isoform functions as a molecular chaperone mediating contractile function of the sarcomere and gene expression in cardiac development.
Genetic studies in the mouse have resulted in important contributions to the understanding of cardiac development. These studies not only serve to understand embryonic development, but also have ramifications for cardiac remodeling in adult disease (Wessels and Sedmera, 2003). Two critical features of mammalian cardiac development are the assembly of a functional contractile system and the sequential formation of the left and right structures of the final four-chambered heart with its proper connections to the arterial and venous vasculatures. Underlying these processes is the coordinate expression of key cardiac proteins (Olson, 2004; Srivastava, 2006). The expression and function of the cardiogenic transcription factors and the contractile and calcium regulatory proteins that assemble into the sarcomeres of the differentiated cardiac myocytes have been central to molecular studies of cardiac development.
Genetic and molecular studies in the mouse have produced major breakthroughs in understanding the transcriptional control of cardiogenesis. A network of genes encoding multiple cardiogenic transcription factors are coordinately expressed as a sequence of processes leading to the fully developed functional mammalian heart: the ordered formation of the cardiac tube, the looping to form the atrial and ventricular chambers of the left heart, followed by the formation of the atrial and ventricular chambers of the right heart together with the specialized conducting system of the interventricular septum and the aortic outflow tract (Lyons et al., 1995; Liang et al., 2001; Nishida et al., 2002; Garg et al., 2003; Watt et al., 2004; Niu et al., 2008; Maitra et al., 2009). The key transcription factors essential to the function of this network include the homeodomain protein Nkx-2.5 and the cysteine zinc finger protein GATA4, representatives of larger protein families playing additional roles in general development. The genetic knockout of GATA4 produces embryonic lethality due to block of cardiogenesis during embryonic days 9–9.5 (E9–E9.5), when the right heart structures are forming (Watt et al., 2004).
Cardiac contraction is essential for viability. The major proteins of heart are the cardiac myosins, which are responsible for contraction (Jones et al., 1996; Lee et al., 2011). The myosin heavy chains (MHCs) of the cardiac myosins contain the molecular motor domains responsible for contractile function. There are two cardiac MHC isoforms, alpha-myosin heavy chain (α-MHC or Myh6) and beta-myosin heavy chain (β-MHC or Myh7). In the mouse, β-MHC is expressed first during heart development, followed by α-MHC gene expression at later stages (Kaufman and Navaratnam, 1981; Evans et al., 1988; Stewart et al., 1991; DeRuiter et al., 1992; Wei et al., 1996; Luther et al., 1997; Oana et al., 1998).
UNC-45 has been demonstrated to act as a molecular chaperone or activator protein of myosins in multiple eukaryotes (Barral et al., 2002; Lord and Pollard, 2004; Shi and Blobel, 2010). There are three distinct regions of UNC-45 proteins, an N-terminal tetratricopeptide repeat motif (TPR), a central region, and a carboxyl terminal UCS domain (Barral et al., 1998; Price et al., 2002). The TPR domain binds Heat Shock Protein 90 (Hsp90), and the UCS domain binds and functionally interacts with myosin motor domains (Barral et al., 1998). Prince and colleagues have shown that the UNC-45b mouse homolog (the human gene and protein are designated B; other vertebrates b) of the Caenorhabditis elegans myosin chaperone UNC-45 is expressed at the mRNA level in hearts at the E8 stage (Price et al., 2002). This gene is also expressed in skeletal muscle and is closely related to orthologs in vertebrate organisms from zebrafish to humans. Studies with C. elegans, cultured myocytes and purified muscle proteins have shown the significance of UNC-45b functioning in the myofibrillogenesis and maturation of full contractile functions in multiple striated muscles (Barral et al., 1998; Barral et al., 2002; Hutagalung et al., 2002; Price et al., 2002; Srikakulam and Winkelmann, 2004; Srikakulam et al., 2008).
Using Drosophila melanogaster and zebrafish (Etheridge et al., 2002; Etard et al., 2007; Wohlgemuth et al., 2007; Anderson et al., 2008; Lee et al., 2011) as a genetically tractable invertebrate and vertebrate, respectively, genetic studies have shown the significance of UNC-45b in later phases of cardiac and skeletal muscle development. Specific mutations and mRNA knockdowns have demonstrated the functional significance of the UNC-45b ortholog during cardiac development in both species, particularly in the organization of muscle sarcomeres. These studies confirm the earlier work in C. elegans (Epstein and Thomson, 1974), in which unc-45 mutations produce temperature-sensitive loss-of-function and embryonic lethal phenotypes related to the differentiation of body wall muscle cells (Venolia and Waterston, 1990; Barral et al., 1998; Hoppe et al., 2004; Kachur et al., 2004; Landsverk et al., 2007; Kachur et al., 2008), the assembly and contractile properties of their myosins (Barral et al., 2002; Kachur and Pilgrim, 2008), and the role of actively functioning muscle cells in overall embryonic morphogenesis.
Studies of the regulation of gene-expression programs in heart development are likely to suggest new therapeutic targets for cardiovascular disease (Epstein, 2010). UNC-45B, GATA4, and cardiac MHC have been implicated in human disease. The altered regulated turnover of UNC-45B protein has been implicated in the pathogenesis of inclusion body myositis (Janiesch et al., 2007). Partial loss-of-function mutations affecting GATA4 protein are associated with clinically significant, congenital atrioventricular septal defects in multiple affected families (Garg et al., 2003). In the mouse, GATA4 has also been shown to be essential for early heart formation and development (Kuo et al., 1997; Molkentin et al., 1997; Watt et al., 2004; Zeisberg et al., 2005).
In this study, we describe three independently derived lines of mouse UNC-45b loss-of-function mutants in both the C3H and C57BL/6 inbred mouse strains that demonstrate the essential function of UNC-45b in cardiac myosin heavy chain accumulation and function and the formation of right heart structures concomitant with the onset of GATA4 function in cardiogenesis. We studied the three independent lines to rule out secondary mutations as well as the UNC-45b mutations leading to the dual phenotype. Immunochemical experiments showed that the accumulation of both cardiac myosins and GATA4 protein were reduced in the UNC-45b mutants and that both cardiac myosin and GATA4 protein could interact with UNC-45b. Our experiments show that UNC-45b is necessary for the proper accumulation and function of both sarcomeric myosins and the GATA4 transcription factor during embryonic cardiogenesis in a mammalian species.
Generation of UNC-45b mutant lines
To determine the biological significance of UNC-45b expression, mutant UNC-45b mouse lines were generated in both the C3H and C57BL/6 inbred strains of mice. To avoid an unrelated mutation in each genetic background, we generated UNC-45b mutant mouse lines by two different methods, in two different mouse strains, insertion of a gene trap cassette and chemical modification to produce nucleotide substitution mutations (Miller and Nadon, 2000; Partridge and Gems, 2007; Toivonen et al., 2007). An UNC-45b mutant line was generated in the C57BL/6 strain by insertion of a gene trap cassette between exon 2 and exon 3 of the UNC-45b gene. The insertion created a frame-shift leading to generation of multiple stop-codons downstream (Austin et al., 2004). The gene trap cassette in one chromosome and the wild-type UNC-45b gene in the other chromosome were detected by RT-PCR (Fig. 1A). To avoid possibly confounding effects associated with either mouse strain or mutagenic method, we identified two independent base substitution mutations in the UNC-45b gene in C3H, creating distinct, independently derived mutant UNC-45b lines. The mutant lines were caused by replacing Y735 with a stop-codon in the UCS domain, confirmed by RT-PCR (Fig. 1B), and substituting L486 to R in the central region, tested by RT-PCR followed by restriction endonuclease digestion (Fig. 1C).
Heterozygous mutants were backcrossed to wild-type females to generate G1 mutants. To further minimize the possibility of presence of secondary mutations, heterozygous males were backcrossed with wild-type females until G6. G6 animals were used for all final analyses (Augustin et al., 2005; Keays et al., 2006). The heterozygotes showed normal behavior, morphology, growth, and fertility. Therefore, the phenotypes of all three mutations appeared fully recessive, consistent with loss of function in UNC-45b. All three homozygous mutants (85/85 embryos) were lethal between E9.5 and E10.5 and then resorbed. An additional test of specificity was the construction of heteroallelic mutants (UNC-45bL486R/Y735stop) that showed both phenotypes, again reducing the possibility of non-UNC-45b mutations having significant effects in the mutants.
Anti-UNC-45B antibody reacts with the three identified UNC-45b regions
In order to characterize UNC-45b protein in the mutant mice, we tested the reactivity of an affinity-purified anti-UNC-45B antibody. We expressed the full-length UNC-45b, TPR domain, central region and UCS domain protein fragments in Escherischia coli (Fig. 2A). The expression of recombinant UNC-45b and its regional fragments were detected by Ponceau S staining and confirmed by immunoblotting with the antibody (Fig. 2B,C). Reaction was detected with all three identified regions, spanning the amino and carboxyl terminals. This antibody, therefore, was suitable for the identification of any stable UNC-45b protein truncates that might be produced in the stop-codon and gene trap mutants, or alternatively, to verify the null state of these mutants at the protein level.
UNC-45b localizes in the heart and somites of E9.5 embryos
UNC-45b mRNA has been reported to be highly expressed only in heart and skeletal muscle of adult mice and in the developing hearts of E8.5 C57BL/6 mouse embryos (Price et al., 2002). Its expression at the protein level in mice has not been reported previously. Fig. 3A shows the expression of UNC-45b protein in an embryonic heart of a 9.5-day wild-type C3H mouse embryo. Only the myocardium and lateral structures within somites showed reaction with the affinity-purified antibody. Sarcomeric myosins and GATA4 protein showed localizations to the heart similar to UNC-45b. Human UNC-45B has been predicted to have two distinct isoforms on the basis of alternatively spliced mRNA sequences (GenBank ID: 146862). Fig. 3B shows that wild-type adult mouse C3H cardiac and skeletal muscle each uniquely expressed immunoreactive protein bands by SDS-PAGE whose molecular weights were consistent with the two human splice variants of UNC-45b predicted to contain 931 and 850 amino acids, respectively. No reaction with any UNC-45a and UNC-45A isoforms (Guo et al., 2011) was detected with this antibody in non-muscle cells and tissues of mice and humans. In Fig. 3C, we studied the stop codon and missense mutant heterozygotes and wild-type C3H mice by western blot to confirm that no truncated proteins were generated in the UNC-45b loss-of-function mutants. The reduced protein levels of UNC-45b in the stop-codon heterozygotes compared to wild-type C3H hearts were consistent with the undetectable UNC-45b levels of the homozygous mutant hearts (see below).
Decreased rates of contraction in mutant embryonic hearts
The major function of cardiac myosins is to produce cardiac contraction in order to circulate the blood. The rates of contraction of isolated E9.5 embryonic hearts from wild type and all three mutants were measured. The C3H missense mutant hearts (UNC-45bL486R/L486R) beat at one-half the rate of wild-type hearts consistent with a partial loss of UNC-45b function. The stop-codon (UNC-45bY735stop/Y735stop) and gene trap (UNC-45bgt/gt) nonsense mutant hearts consistently showed no detectable beating, consistent with more severe loss of function in UNC-45b (Table 1). Heteroallelic mice (UNC-45bL486R/Y735stop) showed contraction rates intermediate between the two homozygous C3H mutants. These results suggest that the reduced or absent function of UNC-45b might lead to decreased or absent accumulation or function of its myosin client proteins in a quantitative manner. It should be noted that the cardiac conduction system does not form until E13–E16, significantly later than the affected E9.5 embryos, and therefore, is unlikely to be related to their contractile behavior (Virágh and Challice, 1977; Christoffels and Moorman, 2009).
|Mutant line||Heart rate (beats /minute)|
|Mutant line||Heart rate (beats /minute)|
ND, not detectable.
Defective cardiac looping in mutant embryonic hearts
Because all three UNC-45b mutant embryos were arrested at E9.5 similar to that in GATA4 knockouts (Kuo et al., 1997; Molkentin et al., 1997; Watt et al., 2004; Zeisberg et al., 2005), we studied the gross cardiac morphology of wild-type and mutant embryos at E9.5. In wild-type C3H and C57BL/6 mouse embryos, three of the chambers of the heart (right ventricle, left atrium, left ventricle) and the one outflow tract are formed at E9.5. In comparison, all 85 hearts examined from C3H and C57BL/6 homozygous mutant embryos at this stage showed only one atrium and one ventricle with a single outflow tract, indicating failure of normal looping (Fig. 4). At the earlier E8.5 stage, development of the mutant and wild-type homozygotes was morphologically indistinguishable from one another in 52/52 embryos (supplementary material Fig. S1). Therefore, the UNC-45b mutant phenotype appeared between E8.5 and E9.5. In GATA4 knockout mice, the defective cardiac looping leads to a very similar timing of embryonic lethality and structural defects (Kuo et al., 1997; Molkentin et al., 1997; Watt et al., 2004; Zeisberg et al., 2005). The defects in UNC-45b mutants suggested that the three loss-of-function UNC-45b mutations were similarly affecting GATA4 activity or expression.
Reduced levels of sarcomeric myosin and GATA4 in mutant embryonic hearts
UNC-45 proteins and their fungal UCS-domain homologues have been implicated in the functional maturation of target myosins in several organisms including C. elegans, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, zebrafish and cultured mouse myocytes (Hutagalung et al., 2002; Price et al., 2002; Toi et al., 2003; Wesche et al., 2003; Lord and Pollard, 2004; Kachur and Pilgrim, 2008; Kim et al., 2008; Lee et al., 2011). We used immunohistochemistry to determine the localization of UNC-45b, myosin, GATA4 and UNC-45a in wild-type embryonic heart tissues. The results showed that UNC-45b, myosin and UNC-45a were localized in the cytoplasm, and that GATA4 was detected in both the cytoplasm where it is synthesized and presumably folds and nucleus where it ultimately functions (Fig. 5).
Loss-of-function UNC-45 mutants in C. elegans nematodes show decreased accumulation of the two myosin heavy chains in striated body wall muscle (Barral et al., 1998; Landsverk et al., 2007), suggesting that the decreased protein levels of the chaperone lead to enhanced degradation of its client proteins. Ubiquitination and proteasomal degradation were then shown to mediate muscle myosin accumulation in C. elegans. Accordingly, we studied the protein levels of UNC-45b, sarcomeric myosin heavy chains and GATA4 in both C3H and C57BL/6 lines by immunohistochemistry (Fig. 6). Anti-UNC-45B antibody showed robust reaction with cardiac sections of hearts of wild-type E9.5 embryos from both the C3H (UNC-45bC3H/C3H) and C57BL/6 (UNC-45bC57/C57) lines. Although isolated mutant hearts, particularly in the severe loss-of-function mutants, showed significant friability upon isolation in comparison to the wild type, the mutants exhibited structured hearts in situ (Fig. 4). The homozygous UNC-45b missense (UNC-45bL486/L486) mutant hearts showed decreased immunohistochemical reactions with anti-UNC-45B antibody, whereas the homozygous UNC-45b stop-codon (UNC-45bY735stop/Y735stop) and gene trap (UNC-45bgt/gt) nonsense mutant hearts showed no detectable immunohistochemical reaction with anti-UNC-45B.
Immunohistochemistry with the MF20 monoclonal antibody to vertebrate sarcomeric myosins showed parallel results to the decreased levels of UNC-45b in the mutants. Immunohistochemical reaction with E9.5 embryonic hearts was robust in both wild-type C3H (UNC-45bC3H/C3H) and C57BL/6 (UNC-45bC57/C57) lines. We observed a decreased level of sarcomeric myosins in the homozygous UNC-45b missense (UNC-45bL486/L486) mutant hearts, and no detectable immunohistochemical reactions with the homozygous UNC-45b stop-codon (UNC-45bY735stop/Y735stop) and gene trap (UNC-45bgt/gt) mutant hearts. These results were consistent with their decreased cardiac contractions.
The finding that the different mutants showed similar blocks in cardiac morphogenesis but distinct levels of myosin and contraction rates suggested that a second, non-myosin target of UNC-45b such as GATA4 might be affected. The functional interaction of UNC-45b with GATA4 would require a threshold level of UNC-45b protein or activity instead of the distinctly linear effects of UNC-45b levels on myosin to explain the similarity of the developmental blocks. The GATA4 cardiogenic transcription factor has been found necessary for the proper formation of right heart structures, concomitant with the morphogenetic defects of the UNC-45b mutants. GATA4 protein was robustly expressed in cardiac muscle of E9.5 embryos in both the wild-type C3H (UNC-45bC3H/C3H) and C57BL/6 (UNC-45bC57/C57) strains. GATA4 protein appeared reduced in the homozygous UNC-45b missense (UNC-45bL486/L486) mutant and was not detectable in both the homozygous UNC-45b stop-codon (UNC-45bY735stop/Y735stop) and gene trap (UNC-45bgt/gt) mutant hearts. The reduced protein accumulation of GATA4 and the cardiac myosins is similar to the myosin-related phenotype observed with reduced levels of active UNC-45 chaperone seen previously in C. elegans UNC-45 mutants (Barral et al., 1998; Hoppe et al., 2004; Landsverk et al., 2007).
UNC-45a expression could not compensate for UNC-45b loss of function during heart development
Immunohistochemistry of the wild-type and mutant hearts with monoclonal antibody to UNC-45A, a molecular chaperone for non-muscle myosin II involved in cell proliferation and migration (Price et al., 2002; Bazzaro et al., 2007; Guo et al., 2011), showed no differences in the UNC-45a protein accumulation of wild-type and the three mutant hearts (Fig. 7). These results indicate that expression of UNC-45a did not compensate for the loss of function in UNC-45b. The absence of mutant effects on UNC-45a levels also serves as a control for the specificity of the decreased accumulation of UNC-45b and its target proteins.
Myocardial proliferation was unaffected in UNC-45b mutant hearts
In order to determine whether the UNC-45b mutations affected cardiac development by reducing myocardial cell proliferation, we counted the number of nuclei per area in 15 locations each of the wild-type and homozygous UNC-45b mutant myocardiums. Because the development of right atrium and ventricle was blocked in the UNC-45b mutants, we could only examine the primary heart fields of left atriums and ventricles (supplementary material Fig. S2). The average nuclear numbers of a given area were the same in both the wild type and the mutants, indicating that general cardiomyocyte proliferation was not influenced by the reduced UNC-45b protein levels.
Cardiac myosin heavy chain and GATA4 mRNA expression in mutant embryonic hearts are unaffected, but GATA4 downstream target mRNAs are decreased
Because UNC-45b is expected as a molecular chaperone to facilitate the folding of its target proteins but not affect the expression of their genes at the mRNA level, we quantified the mRNAs of the UNC-45b and its target proteins in isolated wild-type and UNC-45b mutant embryonic hearts to test this prediction. Quantitative real-time PCR of mRNA isolated from embryonic hearts was performed (Fig. 8). The mRNA contents were measured on a fixed amount of total RNA, 0.5 µg, extracted from isolated hearts. The results showed barely detectable levels of UNC-45b mRNA in the severe UNC-45b loss-of-function mutants (UNC-45bY735stop/Y735stop and UNC-45bgt/gt), consistent with the known effects of nonsense mediated mRNA decay (Maquat, 2004) and their putative status as null mutants at the protein level. Importantly, the UNC-45b mRNA was unaffected in the UNC-45b missense mutant (UNC-45bL486R/L486R). Because the cardiac myosin heavy chains and GATA4 are putative target proteins of UNC-45b, we also tested whether the decreased function of UNC-45b chaperone reduced either the expression cardiac myosin heavy chain (HC) or GATA4 mRNAs. Our results showed that unlike UNC-45b mRNA, which was significantly reduced in the severe UNC-45b mutants, the mRNAs of Myh6 (alpha cardiac myosin HC), Myh7 (beta cardiac myosin HC) and GATA4 did not change in the mutant E9.5 embryonic hearts in comparison with wild type, consistent with the effects of UNC-45b as a chaperone. We then examined whether the expression of genes normally activated by GATA4 during secondary cardiac field development was decreased when GATA4 protein was reduced. The results showed that the mRNAs of three GATA4 targets, Nkx-5, Hand1 and Hand2, were decreased in the mutant hearts, consistent with the observed blocks of right heart development.
Interactions between UNC-45b and its client proteins
In order to further characterize the interactions of GATA4 and the cardiac myosins with UNC-45b, we studied the formation and functional significance of their complexes. Our previous work shows that recombinant C. elegans UNC-45 binds directly to sarcomeric myosin and shows chaperone-like functional and physical interactions (Barral et al., 1998; Barral et al., 2002; Hoppe et al., 2004; Landsverk et al., 2007). To confirm the binding between UNC-45b and sarcomeric myosin in the mouse, the anti-UNC-45B antibody was used to pull down sarcomeric myosin from homogenates of adult mouse heart (Fig. 9A). As suggested by previous studies with the ubiquitously expressed homolog UNC-45A, its binding to the progesterone and glucocorticoid receptors (Chadli et al., 2006) and its effects on the retinoic acid receptor alpha isoform (Epping et al., 2009), we first demonstrated that UNC-45b participated in a complex with GATA4. Plasmids of UNC-45b–FLAG and GATA4–Myc were co-transfected into HeLa S3 cells. When UNC-45b–FLAG was pulled down by anti-FLAG tag antibody, GATA4–Myc was present as detected by anti-Myc antibody, and vice versa (Fig. 9B). In contrast, UNC-45b failed to pull down the glucocorticoid receptor in similar experiments (data not shown). To show direct binding between UNC-45b and GATA4, UNC-45b was expressed and purified from E. coli, whereas GATA4 was expressed and purified from HEK293T cells. Experiments with the purified recombinant proteins showed that UNC-45b bound GATA4 directly (Fig. 9C).
We then tested whether UNC-45b facilitated GATA4-mediated activation of GATA promoter elements for transcription (Thuerauf et al., 1994). Upregulation of a GATA4-sensitive BNP promoter-luciferase reporter was enhanced significantly by co-transfection with GATA4 and UNC-45b expression plasmids in HeLa S3 cells, compared to transfections with GATA4 or UNC-45b alone (Fig. 9D). These results demonstrated the interaction of UNC-45b with GATA4 is functionally significant.
In this study, we demonstrated that UNC-45b is essential for cardiac development in mouse embryos. Mutation of UNC-45b led to embryonic lethality caused by blocking of cardiac morphogenesis and decreased or absent cardiac contraction. We first used genetic experiments to establish two distinct phenotypes produced by mutation of the UNC-45b chaperone in mouse embryonic cardiogenesis. Here, one target was the cardiac sarcomeric myosins, which are necessary for contraction and sarcomere organization, consistent with previous studies in C. elegans, Drosophila and zebrafish (Beall et al., 1989; Venolia and Waterston, 1990; Barral et al., 2002; Etheridge et al., 2002). The mutant mouse hearts showed reduced or absent contractions compared to wild type. These effects are unlikely to be the result of defects in the heart conduction system which does not completely form and function until E16, significantly later than the reduced contraction rates observed at E9.5 (Virágh and Challice, 1977; Christoffels and Moorman, 2009). The UNC-45b mutants also showed blocks in right heart formation similar to those of GATA4 mutants. Here, the putative target, not shown by previous studies, was the cardiogenic transcription factor GATA4 which is necessary for the formation of right heart structures (Kuo et al., 1997; Molkentin et al., 1997; Liang et al., 2001; Garg et al., 2003; Watt et al., 2004; Zeisberg et al., 2005; Maitra et al., 2009).
The genetic evidence also showed that the UNC-45b molecular chaperone was necessary for the normal accumulation of the cardiac myosins and GATA4, consistent with its function as a chaperone (Landsverk et al., 2007). The three UNC-45b loss-of-function mutations led to decreased accumulation of sarcomeric myosin and GATA4 proteins as assayed by immunohistochemistry of mutant embryos. In contrast, quantitative measurement of the mRNAs for cardiac alpha and beta myosin heavy chains and GATA4 by the real-time PCR method did not detect any significant differences between the wild type and the UNC-45b mutants. However, in all of the UNC-45b loss-of-function mutants, the Nkx-2.5, Hand1, and Hand2 cardiogenic transcription factor mRNAs, products of known target genes of GATA4 during secondary cardiac field development, were decreased when GATA4 protein was reduced (Firulli et al., 1998; Riley et al., 1998; Tanaka et al., 1999; Yamagishi et al., 2001; McFadden et al., 2005; Zeisberg et al., 2005).
These results were compatible with the proposed chaperone action of UNC-45 in C. elegans muscle where loss-of-function mutants led to decreased myosin heavy chain accumulation as the result of enhanced degradation by the ubiquitin-proteasome system in the presence of normal myosin mRNA levels (Landsverk et al., 2007). In the mutant hearts studied, both cardiac sarcomeric myosin heavy chains and GATA4 were likely, therefore, to have been rapidly degraded in the absence or reduced accumulation of the UNC-45 chaperone. The limited amounts of material in E9.5 embryonic hearts prevents direct analysis of the degradation of UNC-45b and related proteins as was done with C. elegans and human cancer cells (Landsverk et al., 2007; Guo et al., 2011). The participation of UNC-45b in intermolecular complexes with either cardiac myosin or GATA4 was shown by pull-down experiments with cell lysates. The direct binding between UNC-45b and GATA4 was further confirmed by binding experiments with purified components as has been shown previously for myosin (Barral et al., 2002; Srikakulam and Winkelmann, 2004; Srikakulam et al., 2008). The luciferase reporter assay indicated that GATA4-mediated transcriptional activation was enhanced by UNC-45b. These results are consistent with UNC-45b being an essential chaperone for the physical binding, functional activation and in vivo accumulation of the cardiac sarcomeric myosins and the cardiogenic GATA4 transcription factor.
To reduce the possibility of the complex mutant phenotypes being caused by a second mutation, the experiments reported here were performed on the progeny of heterozygous mutant mice that underwent six backcrosses, which would make the possibility of mutations in the Gata4 locus on chromosome 17 co-segregating with the two UNC-45b mutations in the C3H strain on chromosome 11 of the order of 1 in 106 (Augustin et al., 2005; Keays et al., 2006). The two mutations in the inbred C3H strain arose from distinct pools of sperm mutagenized with ethylnitrosourea, indicating their independent origin. Most critically, the dual effects on contraction and right heart formation were observed in heteroallelic constructs where one chromosome 11 contained the missense mutation, and the complementary chromosome contained the stop codon mutation. Independent mutations would have had to occur twice in each of two genes that would also be closely linked, another very unlikely possibility. The independent, severe loss-of-function gene trap mutation in the C57BL/6 inbred strain again strongly confirmed the dual functions of UNC-45b.
Our genetic results, which demonstrate the dual roles of UNC-45b in affecting sarcomeric myosin and the GATA4 transcription factor during mouse cardiogenesis, are reminiscent of studies reporting the interactions of UNC-45A (A is for humans; a for other vertebrates) with the progesterone and glucocorticoid receptors (Chadli et al., 2006), and the retinoic acid receptor pathway (Epping et al., 2009). Both the hormone receptors and the retinoic acid receptor, like GATA4, perform significant roles in development and homeostasis.
GATA4 and the receptors share homology in their DNA binding domain containing cysteine zinc fingers (Arceci et al., 1993; Lowry and Atchley, 2000). The sequences of the domains fall into two groups, the progesterone, glucocorticoid, and alpha retinoic acid receptor receptors show about 75% similarity and interact with UNC-45A, whereas GATA4 shows only about 33% similarity with them, consistent with its distinct specificity in UNC-45b interaction (Fig. 10).
That UNC-45 chaperones appear to function in dual roles essential for the maturation of myosins of the cytoskeleton and sarcomere and for transcription factors en route to the nucleus represents an important addition to the increasing number of proteins implicated in nuclear–cytoplasmic transactions. Examples of such nuclear–cytoplasmic signaling pathways include the role of beta-catenin in cadherin-mediated adhesion and the Wnt pathway (Kikuchi, 2000; Lilien and Balsamo, 2005), LIM proteins and their regulation by specific E3–ubiquitin ligases that perform dual roles in differentiation of multiple cell types (Arber and Caroni, 1996; Ostendorff et al., 2002), and UNC-97 and UNC-98 that link myosin assembly membrane adhesion sites and the nucleus (Mercer et al., 2003; Miller et al., 2006; Qadota et al., 2007).
Our studies reported here supported the hypothesis that UNC-45b and very likely its human UNC-45B ortholog, perform as regulatory chaperones affecting distinct pathways in cardiac development: sarcomere assembly and contraction and morphogenesis of the right heart. These putative roles raise the broader question as to whether other UCS-domain family members are also involved in nuclear-cytoplasmic transactions.
Materials and Methods
Screening of mutants in C3H mice
Mutant mice were generated at Ingenium Pharmaceuticals, Martinsried, Germany using N-ethyl-N-nitrosourea (ENU) (Augustin et al., 2005; Keays et al., 2006). Two mutations were identified by PCR screening in exons coding for sequences in the central and carboxyl-terminal UCS regions. The transversion of T to G leads to leucine substitution for arginine in the UNC-45bL486R mutant whereas T to A leads to tyrosine replaced by a stop-codon in the UNC-45bY735 mutant mouse chromosomes. UNC-45bL486R/C3H and UNC-45bY735/C3H males were outcrossed to wild-type C3H females to generate G1 mutants. In order to significantly reduce the presence of secondary mutations, heterozygous males of _ENREF_48both strains were outcrossed with wild-type C3H females through G6. G6 animals were used for all final analyses (Miller and Nadon, 2000; Partridge and Gems, 2007). As a further test of specificity to the UNC-45b gene, UNC-45bL486R/C3H and UNC-45bY735/C3H were crossed to create heteroallelic (UNC-45bL486R/Y735) mice. Care and handling of all mice was carried out as per the institutional animal care guidelines approved by Institutional Animal Care and Use Committee (IACUC) of the University of Texas Medical Branch.
Screening of mutants in C57BL/6 mice
Mutant mice were generated by the gene trap method at Lexicon Genetics Incorporated, TX, USA (Austin et al., 2004). The gene trap cassette carries two LTR sites, and a trapping cassette sequence in the middle of LTR sites. Insertion of the cassette into an intronic sequence results in the fusion of the N-terminus of the trapped gene of UNC-45b between exons 2 and 3.
Genotyping mutant mice
A genotyping assay was used to identify the wild type and mutations in both C3H and C57BL/6 mouse lines. Genomic DNA was isolated from mouse tails using standard protocol. Individual tails (0.5 cm) were lysed by incubating overnight at 56°C in 200 µl lysis buffer with 20 mg/ml proteinase K. The mixture was centrifuged at 14,000 rpm for 10 min, and then 100 µl of supernatant was carefully transferred in a new tube. DNA was precipitated by adding equal volumes of isopropanol, mixed by inverting tubes several times and stored at –20°C overnight. DNA was pelleted by centrifugation at 14,000 rpm for 5 min. The pellet was washed with 70% ethanol and air dried at room temperature. The purified DNA was suspended in 100 µl of RNase, DNase, protease-free water that was used for all aqueous studies including PCR and restriction digestions.
Collection of embryos and genotyping
Heterozygous male and female mutants were crossed to produce embryos. Females were examined for vaginal plug each morning. Noon of the day when the vaginal plug appeared was designated as E0.5. Pregnant mothers were sacrificed, and their uteruses dissected to harvest E8.5 and E9.5 embryos. These embryos were fixed in 4% paraformaldehyde. Embryonic tissues were taken during embryo collection for DNA isolation and genotyping. DNA isolation and the genotyping procedure for embryos were similar to that with adult mice. For production of homozygous embryos, males and females heterozygous for the same mutation were crossed.
Embryonic morphology and histology
Embryos were dissected from pregnant females and fixed in 4% paraformaldehyde (at 4°C) overnight. Embryonic and cardiac morphologies were analyzed using a Wild M5 dissecting microscope (Wild Heerburgg, Gais, Switzerland), and whole heart images were captured with a Nikon E990 camera. For histological analyses, wild-type UNC-45bC3H/C3H and homozygous UNC-45bL486R/L486R and UNC-45bY735/Y735 mutants in C3H mice, and wild-type UNC-45bC57/C57and homozygous UNC-45bgt/gt mutants in C57BL/6 mice were embedded in OCT and frozen (Sakura Finetek, Torrance, CA) for cryosectioning. Serial sagittal and transverse sections were obtained at 10 µm thickness. Sections were stained with Hematoxylin and Eosin. Images were taken using an Axioplan-2 microscope with an Axicam MRc5 camera (Carl Zeiss, Jena Germany).
RNA was extracted from dissected embryonic hearts using TriReagent (Sigma) according to the manufacturer's instructions, and 0.5 µg RNA was reverse transcribed using Superscript III (Invitrogen) strictly according to the company's directions. Real-time PCR reactions were performed in triplicate using 1 µl of the resulting cDNA per 20 µl iQ SYBR Green Supermix (Bio-Rad). Seven embryonic hearts were pooled for each analysis. The housekeeping gene GAPDH was used as control. The primers used for mouse UNC45b were 5′-ATGGCAGAGGCTGAAGCG-3′ (forward) and 5′-GCTGCTCTTGGATGCTGGTG-3′ (reverse). The primers used for mouse GATA4 were 5′-GGTGTGTAGCAGGCAGAAAG-3′ (forward) and 5′-CCTCTAGGCTCTGGTTTGCT-3′ (reverse). The primers used for mouse Nkx-2.5 were 5′-GACTTGAACACCGTGCAG-3′ (forward) and 5′-GACAGGGCATAGTGGGAG-3′ (reverse). The primers used for mouse Hand1 were 5′-TAAACCAGTGAGACCAGGC-3′ (forward) and 5′-GCTGCGTCCTTTCCTCTC-3′ (reverse). Primers used for mouse Hand2 were 5′-CCACCAGATACATCGCCTACC-3′ (forward) and 5′-CTTGTCGTTGCTGCTCACTGT-3′ (reverse). The primers for mouse GAPDH, Myh6, and Myh7 were purchased from SABiosciences. PCR was performed on the MyiQ system (Bio-Rad) according to the manufacturer's protocol. The mRNA levels were analyzed by the ΔΔCt method (Livak and Schmittgen, 2001). The data are reported as mean ± standard deviation as indicated.
Sections from wild-type and homozygous mutants in both C3H and C57BL/6 mouse backgrounds were used for immunohistochemical analyses. The ZymedHistoMouse-SP system (Invitrogen) containing biotinylated secondary antibodies to mouse, rabbit and guinea pig IgGs was used for immunostaining strictly according to manufacturer's protocol. Affinity purified rabbit polyclonal anti-UNC-45B against full-length recombinant human UNC-45B was generated by Sarah Spinette, Rhode Island College, Providence, RI. Mouse monoclonal IgG, a gift from Ahmed Chadli, Georgia Health Sciences University, against recombinant UNC-45A was produced using purified UNC-45A protein and was used for detection of UNC-45a (Chadli et al., 2006). Note that the mouse and human UNC-45A(a) and UNC-45B(b) proteins are greater than 95% identical in sequence (Price et al., 2002). Anti-sarcomeric myosin monoclonal antibody (MF20) was kindly provided by Donald Fischman, Weill Cornell Medical College, New York (Shimizu et al., 1985). Commercially available affinity purified rabbit polyclonal anti-GATA4 antibody (Santa Cruz Biotechnology) was used.
Recombinant protein expression and pull-downs
GATA4 cDNA was cloned into pCMV-3Tag-4 (Stratagene) as a HindIII–Xhol fragment. UNC-45b cDNA was cloned into pCMV-3Tag-8 (Stratagene) as a HindIII–Xhol fragment. His-UNC-45b, and its regional fragments His-TPR, His-Central, and His-UCS were cloned into pPROEX-HTb (Invitrogen) as NarI–HindIII fragments. For UNC-45b–GATA4 binding studies, HeLa S3 cells were transiently transfected with UNC-45b–FLAG or GATA4–Myc or co-transfected with the UNC-45b–FLAG and GATA4–Myc by Fugene HD (Roche). Cells were collected after 48 h in 1 ml lysis buffer [phosphate-buffered saline (PBS), 4 mM EDTA, pH 7.4, 1% Triton X-100, protease inhibitor cocktail (Roche) and 1 mM PMSF], and incubated with mouse anti-FLAG M2 agarose beads (Sigma) or c-Myc monoclonal Ab-agarose beads (Clontech) in 500 µl incubation buffer (PBS, 5 mM EDTA, pH 7.4, 0.5% Triton X-100, protease inhibitor cocktail and 1 mM PMSF) at 4°C for 8 h. The samples were washed twice with 500 µl washing buffer (PBS, 5 mM EDTA, pH 7.4, 0.1% Triton X-100, protease inhibitor cocktail and 1 mM PMSF). Western blotting were performed using primary polyclonal anti-c-Myc antibody produced in rabbit (Sigma) and secondary monoclonal goat anti-rabbit IgG-horseradish peroxidase (Southern Biotech) or monoclonal anti-FLAG M2 horseradish peroxidase antibody produced in mouse (Sigma).
Recombinant His–UNC-45b protein was expressed in BL21-CodonPlus-RIL (Stratagene), and purified by Ni-affinity and gel filtration chromatography. The recombinant GATA4–Myc was expressed in HEK293T cells, and purified by the anti-DDK epitope (DYKDDDDK) column. In the UNC-45b pull-down experiments with purified recombinant His–UNC-45b, and GATA4–Myc, their proteins and His-tag beads were incubated in 2 ml binding buffer (PBS, 10 mM imidazole, 1 mM EDTA, pH 7.4, 1% Triton X-100, protease inhibitor cocktail, 5 µM ATP and 1 mM PMSF) at 4°C for 16 h. Elution was achieved by 1 ml of 1 M imidazole. In the in vitro GATA4 pull-down experiments, purified His–UNC-45b, GATA4–Myc and c-Myc monoclonal Ab-agarose beads (Clontech) were incubated in 2 ml incubation buffer (PBS, 5 mM EDTA, pH 7.4, 0.5% Triton X-100, protease inhibitor cocktail and 1 mM PMSF) at 4°C for 8 h. The complex was eluted by 1 ml of the 5 mg/ml Myc peptide solution. SDS-PAGE was used for detection.
Reporter construction and luciferase assay
BNP (−116 to +80)-luciferase recombinant plasmid was kindly provided by Chris Glembotski, San Diego State University, San Diego, CA. In the plasmid, luciferase gene expression was driven by the GATA4 responding element BNP (−116 to +80) (Thuerauf et al., 1994). Cells were co-transfected with a luciferase reporter and other plasmids. At 24–48 h after transfection, the activity of reporters was evaluated with luciferase assay system (Promega). The levels of luciferase activity were normalized to pBNP(−116 to +80)-Luc luciferase activity.
For western blotting, proteins were separated on 1.5 mm thick 10% SDS-PAGE mini gels and then were transferred to Immobilon-NC filters in 20% methanol running buffer at 80 volts for 2 h at 4°C. Coomassie Blue staining of the gels confirmed transfer. Blot were blocked with 20 ml 1% nonfat dry milk in 150 mM NaCl, 50 mM Tris pH 7.6, 0.05% Tween 20, and reacted with the different antibodies (Bader et al., 1982; Barral et al., 2002).
Counting of cardiac cells
The E9.5 embryonic hearts in wild-type and mutant mice were sectioned as above. Clear monolayer regions of the myocardiums were used to perform cell counting. Image-pro plus (MediaCybernetics) was used to measure 15 different locations within each heart examined.
The two-tailed Student's t-test was used to evaluate the statistical significance of the results at the 95% confidence level. A P-value less than 0.05 was considered to indicate statistical significance.
We thank Jose Barral, Darren Boehning and Maki Wakamiya for their invaluable suggestions during the course of this work, and Ahmad Chadli, Donald Fischman and Chris Glembotski for their generous gifts of reagents.
The work was supported by the Muscular Dystrophy Association [grant number 3993 to H.F.E.]; the National Institutes of Health [grant number R01 AR05005-A1 to H.F.E.]; and the Cecil H. and Ida M. Green Endowment at the University of Texas Medical Branch. Deposited in PMC for release after 12 months.