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Nascent polypeptide-Associated Complex and Signal Recognition Particle have cardiac-specific roles in heart development and remodeling [1]

['Analyne M. Schroeder', 'Development', 'Aging', 'Regeneration Program', 'Center For Genetic Disorders', 'Aging Research', 'Sanford Burnham Prebys Medical Discovery Institute', 'La Jolla', 'California', 'United States Of America']

Date: 2022-12 

Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve our understanding of its oligogenic underpinnings. Our studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the alpha- (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. Our data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, we demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes.

Identifying novel genes involved in cardiac development could help patients with Congenital Heart Disease through improved understanding of the developmental missteps, more precise patient diagnosis, and invention of targeted medical interventions. We identified protein biogenesis cofactors Nascent polypeptide Associated Complex (NAC) and Signal Recognition Peptide (SRP) to be involved in cardiac specific roles during development. Disruption of NAC and SRP subunits led to distinct and cell targeted disruptions in the heart, which would be unexpected if NAC and SRP merely had generic cellular function. Specifically, in flies, knockdown (KD) of the alpha- subunit of NAC, Nacα, led to an adult fly with no heart that could be rescued by co-KD with the posterior patterning Hox gene Abd-B, indicating a critical developmentally relevant genetic interaction, and not merely a generic protein biogenesis defect. This interaction was recapitulated in human Cardiac Progenitors, whereby human NACA KD redirected progenitor differentiation away from cardiomyocyte and toward fibroblast fates, which was rescued by concomitant Hox gene KD. Lastly, Nacα activity was required at specific times during development, and depending on when Nacα was knocked down, the resulting adult heart could be mildly or severely malformed. Thus, the work presents a new class of genes involved in protein biogenesis that display tissue- and temporal-specific activities that are crucial for proper heart development.

Funding: This work was funded by grants from the National Institutes of Health R01 HL054832 to RB and F32 HL131425-01 to AMS. Support also includes funding from The Department of Defense (W81XWH-21-1-0159) to AMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we provide evidence for a cardiac developmental role for NAC and SRP in Drosophila and NACA in human Multipotent Cardiac Progenitors (MCPs). In flies, cardiac KD of NACα (mentioned above) and bic throughout development led to complete loss of the heart. We demonstrate that this phenotype is dependent on the timing of NACα KD, which requires KD during both embryonic heart development and pupal cardiac remodeling for a complete loss of the adult heart. KD in embryos only led to cardiac constriction and loss of the terminal chamber, while retaining heart structures in the anterior segments. KD of Nacα only during pupal stages did not affect adult heart structure. This suggests that NACα KD primes cardiac cells already in the embryo for aberrant responses to morphogenic cues during later developmental stages. Persistent Nacα KD during pupal stages remained required for complete loss of the adult heart. Consistent with this idea, NACα KD throughout cardiac development induced ectopic expression of the posterior patterning Hox gene Abdominal-B (Abd-B) into anterior regions of the remodeling heart during pupation. Concurrent KD of NACα and Abd-B significantly rescued the heart. This interaction between Nacα and Hox gene was recapitulated in MCPs, whereby NACA KD led to deviations in progenitor cell differentiation away from cardiomyocytes and toward fibroblast cell fates, which was reversed with combined KD of NACA and Hox genes HOXC12 or HOXD12. Because NAC associates and influences the activity of SRP, we tested the effects of individual SRP subunit KD on the fly heart. Interestingly, KD of individual SRP subunits produced cardiac phenotypes, some of which were distinctly different from NACα KD. These results suggest specific roles for ubiquitously expressed protein biogenesis factors NAC and SRP in heart morphogenesis, in part through alterations in the Hox gene expression. Translational regulation adds to our growing knowledge of biological pathways that may specifically contribute to cardiac pathogenesis leading to CHD.

Nacα has been demonstrated to be critical for development of several tissues using various model organisms. In mouse, the Nacα subunit can function as a transcriptional coactivator regulating bone development [ 23 – 25 ] and hematopoiesis in zebrafish [ 26 ]. In vertebrates, a skeletal muscle- and heart-specific variant of Nacα has been associated with myofibril organization in zebrafish [ 27 ] and muscle and bone differentiation in mouse [ 28 – 32 ]. Recent studies in the fly showed that Nacα knockdown (KD) specifically in the heart led to a ‘no adult heart’ phenotype [ 10 ], suggesting that Nacα could play a role in sarcomeric biogenesis, but its exact role in cardiac development had been unclear.

In Drosophila, NAC plays an important role in translational regulation critical for embryonic development [ 19 , 20 ]. Fly homologs of NAC subunits, Nacα and bicaudal (bic), were shown to repress protein translation of the posterior patterning gene oskar (osk) in anterior regions of the embryo, which was despite association of osk mRNA with polysomes, usually indicative of active translation. Restricting OSK protein translation and accumulation to the posterior pole of the embryo is critically required for patterning the posterior body plan [ 21 ]. Depletion of either Nacα or bic, expands OSK protein localization anteriorly resulting in a bicaudal phenotype, where the embryo develops with mirror-image duplication of the posterior axis [ 19 , 22 ]. These studies suggest that within the developing embryo, NAC appears to regulate the expression of select proteins for proper spatial distribution. NAC could have a similar role in the timing and spatial targeting of translation within specific tissues.

Previous data suggest that the human gene Nascent polypeptide Associated Complex-alpha (NACA) is a candidate CHD gene that could provide novel insights into biological pathways in cardiac morphogenesis and pathogenesis. Using a GWAS approach, NACA was located within a genomic locus associated with increased myocardial mass [ 10 ], while Whole Exome Sequencing in families with Tetralogy of Fallot identified a single nucleotide polymorphism within NACA [ 11 ]. NACA is a highly conserved alpha subunit of a heterodimeric complex called Nascent polypeptide Associated Complex (NAC). Along with its heterodimeric partner, NAC-beta (NACβ/BTF3), NAC is one of several chaperones found near the ribosome exit tunnel that bind to select emerging nascent polypeptides [ 12 ]. NAC-ribosome complexes facilitate transport of nascent polypeptides to the mitochondria as has been demonstrated in yeast [ 13 – 15 ]. At the ribosome exit site, NAC gates the activity of other nascent polypeptide chaperones. For example, NAC enhances the fidelity of Signal Recognition Particle (SRP) binding to only those nascent polypeptides destined for import to the Endoplasmic Reticulum (ER) [ 16 – 18 ]. Depletion of NAC leads to promiscuous binding of SRP onto nascent polypeptides, drawing mistargeted ribosome-nascent polypeptide complexes to the ER for aberrant insertion into the membrane or secretion. NACA’s function as part of NAC therefore regulates the localization and posttranslational quality control of proteins in the cell.

Congenital Heart Disease (CHD) is characterized by structural malformations of the heart present at birth caused by deviations from the normal course of cardiogenesis [ 1 ]. Genetics is a critical driver of CHD [ 2 , 3 ]. Chromosomal anomalies as well as variants in genes involved in heart development have been identified in CHD patients [ 4 ]. These genetic features and disease presentations are heritable and cluster in families [ 5 , 6 ]. Identifying the genes associated with disease helps piece together genetic networks that could uncover mechanisms underlying pathogenesis. Approximately 400 genes have been implicated in CHD [ 2 ], some that cluster within defined pathways, which permits a genetic diagnosis for approximately 20% of CHD patients. However, this leaves the vast majority of CHD cases with unknown genetic origins [ 3 ]. Therefore, expansion of the genetic data pertinent to CHD, such as functional analysis of genes with variants of uncertain significance (VUS), would advance our understanding of the disease and may offer, in the future, a diagnosis and targeted treatment for CHD patients. A better understanding of additional genetic risk factors and patient-specific combinations of such factors is aided by identification of candidate disease genes through patient-specific genomics, such as whole genome sequencing (WGS), followed by their evaluation in cardiac developmental platforms and assays from various genetic model systems [ 7 , 8 ]. These validation efforts can accelerate candidate gene identification and focus on new potentially pathogenic genes, including genes located within larger genomic anomalies such as de novo Copy Number Variants [ 9 ].

Results

Nacα is required in the embryo to pre-program cardioblasts for appropriate cardiac remodeling Prior to pupal cardiac remodeling, a larval heart was still present despite Nacα KD using the strong cardiac driver Hand4.2-GAL4 (Figs 1E and 3E). However, these larval heart tubes were thinner and the cytoskeletal structures less prominent than controls (Figs 3E and S1B), reminiscent to the constricted adult heart phenotypes with the weaker driver tinHE-Gal4 (Figs 2 and S7). These observations suggest that Nacα may have earlier developmental functions in addition to a role in metamorphosis. We therefore wanted to temporally dissect Nacα’s function in the heart by knocking down its expression during different developmental stages. We generated a Hand4.2-GAL4 driver line that included two copies of a temperature-sensitive allele of GAL80 driven by a ubiquitous promoter (tubulin-GAL80ts), which we termed HTT [9]. At the permissive temperature (18°C), the GAL80 transcriptional repressor prevents GAL4 activation of UAS sites thereby inhibiting transcription of downstream constructs [40]. This temperature-sensitive form of GAL80 protein is unstable at higher temperatures (28–29°C), thus permitting GAL4 activity at higher ambient temperatures. Maintaining HTT flies crossed to Nacα-RNAi or controls at 18°C throughout development resulted in normal heart structure (Fig 4A) and produced no differences in diastolic diameter, systolic diameter or fractional shortening compared to controls (Fig 4I), effectively demonstrating GAL80’s ability to suppress Nacα-RNAi transcription at 18°C temperatures. Constant exposure to 28°C throughout development phenocopies the absence of the heart in adults produced by Hand4.2-GAL4 driver (Fig 4B). KD of Nacα in adults only for one week by exposure to high temperatures led to largely normal heart structure and function compared to controls (Fig 4C and 4I), suggesting that Nacα is primarily required developmentally for establishing a normal heart in adults, rather than maintaining its function or structure with age. Remarkably, although lifelong cardiac Nacα KD using Hand4.2-GAL4 led to histolysis of the heart during metamorphosis, KD of Nacα during pupation only (~3 days at 28°C) did not produce gross heart defects (Fig 4D). The diastolic and systolic diameters were slightly increased, which caused some reduction in fractional shortening (Fig 4I). Even when we induced Nacα KD earlier, starting at mid-larval stages through metamorphosis until eclosion (~5 days at 28°C), a period with substantial developmental growth requiring high levels of protein translation, heart structure was unaffected (Fig 4E). This lack of phenotype is remarkable, as disruption of NAC is associated with proteostasis and ER stress, which often leads to cell death [41–43]. These results suggest that KD of Nacα in the developing heart, even for relatively long durations does not unequivocally lead to cell death. When Nacα was knocked down during embryonic stages only (egg-lay up to 24 hours) and subsequently reared at 18°C until dissection to prevent Nacα KD at later stages, most adult hearts remained intact, but considerably constricted with smaller diastolic and systolic diameters (Fig 4F and 4I). Interestingly, the posterior terminal heart chamber was absent in most cases (Fig 4F), which suggests that the no-heart phenotype observed with continuous KD likely arises from developmental defects already in the embryo. Extending exposure to higher temperature during embryonic stages until 48 hours after egg lay, produced similar phenotypes compared to 24hr exposure, with the presence of a heart tube but without a terminal chamber (Fig 4G). Remarkably, when flies were exposed to higher temperatures during both embryonic stages (24 hours) and pupal stages (3 days; for a total of 4 days), with a return to 18°C during larval development, an almost complete ‘no-heart’ phenotype was reproduced in most cases (Fig 4H). This suggests that Nacα KD only during combined embryonic and pupal stages produces a nearly complete loss of heart structures, but at either stage alone was insufficient for such a severe phenotype. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Temporal regulation of Nacα-RNAi expression in the heart. Using a temperature inducible driver specifically in the heart (HTT, Hand4.2-GAL4, tubulin-GAL80ts; tubulin-GAL80ts), Nacα-RNAi was expressed during specific stages of development by controlling ambient temperature to determine its contribution to cardiogenesis. Controls lacking RNAi are kept in similar temperature conditions to account for any developmental effects of temperature on the heart. A-H. Phalloidin staining to visualize cytoskeletal structural effects of Nacα knockdown (KD). A, As a test of GAL80 control of transcription, flies held at 18°C throughout development did not produce changes to heart structure indicating an inhibition of Nacα-RNAi transcription. B, Exposing flies to high temperatures (28°C) throughout development produced a no heart phenotype similar to the effects of driving Nacα-RNAi using Hand4.2-GAL4 alone, suggesting an induction of Nacα-RNAi transcription and subsequent Nacα KD with exposure to higher temperatures. * indicates absence of the heart. C, Exposing flies to high temperature during adulthood only for 1 week, D, pupae to eclosion, or E, mid-larvae to eclosion did not produce gross structural defects in the heart. F, Exposing embryos to high temperatures starting at egg-lay up until 24 hours resulted in the absence of the terminal chamber, indicated by white bar. Only thin alary muscles were present. G, Extending the high temperature exposure to 48 hours led to similar loss of the posterior heart, indicated by white bar. H, Only when the hearts were exposed to higher temperatures during embryonic stage (24 hours) and pupal stage until eclosion (~3 days), were we able to recapitulate the no heart phenotype produced by exposing the heart to constant high temperatures. I, Functional analysis of the adult heart following Nacα KD at various developmental stages. Maintaining flies at 18°C throughout development or exposure of adult flies to high temperature for 1 week led to no changes in diastolic diameter, systolic diameter, or fractional shortening. High temperature exposure from pupae to eclosion or from mid-larvae to eclosion led to subtle changes in diameters and fractional shortening. Exposure of embryos to high temperatures for 24hr led to constricted diastolic and systolic diameters. * p<0.05, ** p<0.01, *** p<0.001. https://doi.org/10.1371/journal.pgen.1010448.g004 We measured Nacα mRNA expression in adult hearts of HTT flies subject to pupae only Nacα KD (S8 Fig). We detected significantly reduced Nacα expression shortly after eclosion, suggesting that despite reduced Nacα levels, the heart remodels into a largely normal heart (Fig 4D). We also measured Nacα mRNA levels in early pupal hearts, that were subject to Nacα KD only in the embryo (24 hours) before being returned to 18°C (S8 Fig). Interestingly, we also found reduced Nacα mRNA levels in early pupae at the onset of metamorphosis, suggesting that embryonic KD of Nacα reprograms cells leading to longer term effects such as reduced Nacα expression later in development even without RNAi induction. This reduced Nacα expression early in pupae leads to changes in cardiac remodeling and constricted adult hearts (Fig 4F and 4I). However, this is still insufficient to completely histolyze the heart, unless subject to additional KD during pupal stages. Some extra-cardiac tissues are also collected during heart dissection in pupae and adult, such as few fat and muscle cells where Hand4.2-Gal4 is not expressed, thus the level of Nacα KD is likely an underestimation. These results suggest that Nacα’s role in driving heart morphogenesis has a temporal component, perhaps regulating several different processes during cardiac development. Furthermore, the observation that Nacα KD during embryonic as well as pupal stages is needed to induce histolysis of nearly the entire heart tube during pupal cardiac remodeling suggests that Nacα plays an essential role in embryonic heart development by programing cardiac cell fate. This embryonic requirement seems to be partially compensated for by sufficient Nacα function during heart remodeling. However, additional reduction of pupal Nacα exacerbates the changes in embryonic cardioblast programming, together leading to a failure of the larval heart to respond to remodeling cues during metamorphosis, thus causing histolysis.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010448

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