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Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases uncovers a requirement for versican and glypican-6 proteolysis in cardiac development.

ABSTRACT: Background: Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. Two similar matrix-degrading metalloproteases, ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each have mild cardiac anomalies, but their combined genetic inactivation is precluded by tight linkage. We coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and mechanisms in mouse cardiac development. Methods: ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows covering myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant mouse mutants. Mass spectrometry-based N-terminomics was used to identify relevant substrates. Results: Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which we also observed in mice with ADAMTS cleavage-resistant versican. Subsequently, combined knockout impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect independently of versican proteolysis. N-terminomics of combined ADAMTS knockout and wild-type hearts identified a cleaved glypican-6 peptide only in the wild-type and showed that ADAMTS1 and ADAMTS5 each cleaved glypican-6. Paradoxically, ADAMTS1 and ADAMTS5 inactivated hearts lacked glypican-6 despite unaltered Gpc6 transcription. Gpc6-/- mice demonstrated similar rotational defects as the combined ADAMTS knockout and both had reduced Hedgehog signaling. Conclusions: ADAMTS1 and ADAMTS5 ensure proper cardiac development via cleavage of distinct proteoglycans, each with independent roles in cardiac development. Whereas versican clearance in cardiac jelly is required for proper ventricular cardiomyogenesis, glypican-6 cleavage may activate/stabilize this cell-surface proteoglycan which is required for Hedgehog signaling during outflow tract development.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Sumit Bhutada

LAB HEAD: Suneel Apte

PROVIDER: PXD045706 | Pride | 2025-05-06

REPOSITORIES: Pride

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Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases in temporally specific windows uncovers distinct roles for versican proteolysis and glypican-6 in cardiac development.

Mead Timothy J TJ Bhutada Sumit S Foulcer Simon J SJ Peruzzi Niccolò N Nelson Courtney M CM Seifert Deborah E DE Larkin Jonathan J Tran-Lundmark Karin K Filmus Jorge J Apte Suneel S SS

Matrix biology : journal of the International Society for Matrix Biology 20240513

Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncov ...[more]

PMID: 38750698

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Project description:Background: Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. Two similar matrix-degrading metalloproteases, ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each have mild cardiac anomalies, but their combined genetic inactivation is precluded by tight linkage. We coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and mechanisms in mouse cardiac development. Methods: ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows covering myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant mouse mutants. Mass spectrometry-based N-terminomics was used to identify relevant substrates. Results: Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which we also observed in mice with ADAMTS cleavage-resistant versican. Subsequently, combined knockout impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect independently of versican proteolysis. N-terminomics of combined ADAMTS knockout and wild-type hearts identified a cleaved glypican-6 peptide only in the wild-type and showed that ADAMTS1 and ADAMTS5 each cleaved glypican-6. Paradoxically, ADAMTS1 and ADAMTS5 inactivated hearts lacked glypican-6 despite unaltered Gpc6 transcription. Gpc6-/- mice demonstrated similar rotational defects as the combined ADAMTS knockout and both had reduced Hedgehog signaling. Conclusions: ADAMTS1 and ADAMTS5 ensure proper cardiac development via cleavage of distinct proteoglycans, each with independent roles in cardiac development. Whereas versican clearance in cardiac jelly is required for proper ventricular cardiomyogenesis, glypican-6 cleavage may activate/stabilize this cell-surface proteoglycan which is required for Hedgehog signaling during outflow tract development.

Project description:In mammals, the A Disintegrin-like And Metalloproteinase domain with Thrombospondin type 1 motifs (ADAMTS) family comprises 19 secreted proteases, including 6 members with the ability to cleave large aggregating proteoglycans: ADAMTS1, 4, 5, 9, 15, and 20. ADAMTS9 has an essential, non-redundant role during embryogenesis, as Adamts9 null embryos die prior to completing gastrulation and Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 proteolytic activity is required for proteostasis of versican, a widely distributed large aggregating proteoglycan/hyalectan in the provisional extracellular matrix present during early development. Despite its importance, ADAMTS9 proteoglycanase activity has not been directly compared to that of ADAMTS1, 4, and 5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, 4, and 5, ADAMTS9 cleaves versican V1 isoform at the Glu441-Ala442 site, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 variant (ADAMTS9 MDTCS) consisting of all ADAMTS ‘core domains’ that render it more comparable in size and domain structure to ADAMTS1, 4, and 5 and compared its activity against versican, aggrecan and the small leucine-rich proteoglycan biglycan. A catalytically inactive ADAMTS9 MDTCS variant (ADAMTS9 EQ) was used as the control in all experiments. We identified cleavage sites in versican (V1 and V2 isoforms) and biglycan by all 4 ADAMTS family members using a quantitative label-free proteomics strategy. Moreover, using a quantitative substrate cleavage assay, we established that ADAMTS9 MDTCS versicanase activity at the Glu441-Ala442 site is 175-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 5.5-fold higher lower than ADAMTS1. These provide systematic analysis of the proteoglycanase activity in the ADAMTS family and highlight differences and similarities in cleavage site specificities that could potentially be leveraged to design selective small molecule inhibitors for therapeutic applications.

Project description:We identify a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motif), ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days of gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was established in bt/bt:Adamts9+/- mice, which have increased white spotting relative to bt mice, as previously reported, and a fully penetrant cleft palate. Palatal closure was delayed, although eventually completed, in both bt/+;Adamts9+/- and bt/bt mice, demonstrating a cooperative role of these related genes. Adamts9 and Adamts20 are both expressed in palatal mesenchyme, with Adamts9 expressed exclusively in microvascular endothelial cells. Palatal shelves from bt/bt:Adamts9+/- mice fused in culture, suggesting an intact TGF signaling pathway in palatal epithelium, and indicating a temporally specific delay in palatal shelf elevation and growth toward the midline. Palatal shelf mesenchymal cells showed a statistically significant decrease of cell proliferation at E13.5 and E14.5, as well as decreased processing of versican, an ADAMTS substrate, at these stages. Vcan haploinsufficiency led to a greater penetrance of cleft palate in bt mice, and impaired proliferation was also seen in palatal mesenchymal cells of these mice, suggesting a role for ADAMTS-mediated versican proteolysis in palatal closure. In a parallel with recent work identifying a role for a bioactive ADAMTS-generated versican fragment in regulating apoptosis during interdigital web regression, we propose that versican proteolysis may influence palatal mesenchymal cell proliferation. Palatal shelves were dissected from four E13.75 Adamts9+/-:bt/bt embyos (correspond to the 4 samples: Palate_Adamts9+/-:bt/bt_Rep1, Palate_Adamts9+/-:bt/bt_Rep2, Palate_Adamts9+/-:bt/bt_Rep3 and Palate_Adamts9+/-:bt/bt_Rep4) and age-matched 3 wild-type C57Bl/6 embryos (correspond to the 3 samples: Palate_WT_Rep1, Palate_WT_Rep2, and Palate_WT_Rep3) that were used as the controls

Project description:BACKGROUND: A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for treating BAV patients. Over the past decade, the genes involved in aortic valve development and BAV formation has been increasingly recognized. On the other hand, ADAMTS gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. METHODS: Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were then performed to evaluate the expression pattern in the aortic valve Accordingly, related genetic mouse models (both knock-out and knock-in) were generated using the CRISPR/Cas9 method to further study the roles of ADAMTS family genes. The lineage tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Both inducible pluripotent stem cells (iPSC) derived from BAV patients and genetic mouse tissue were used to study the molecular mechanism of the gene. Immunohistochemistry using different antibodies was performed to describe the phenotype of cardiac valve anomalies, especially in the ECM components. RESULTS: ADAMTS genes targeted phenotype screening in zebrafish and targeted DNA sequencing on a cohort of BAV patients identified ADAMTS16 as a BAV-causing gene and found the ADAMTS16 p.H357Q variant in an inherited BAV family. In addition, both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited an R-NC-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Conditional Adamts16 mutant mouse models further demonstrated that Adamts16 deficiency mainly in endothelial lineage cells recapitulated the BAV phenotype. This was confirmed in lineage tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using iPSC-induced endothelial cells (iPSC-ECs) and genetic mouse embryonic heart tissue unveiled enhanced FAK signaling, which was accompanied by elevated fibronectin levels. Both in vitro iPSC-ECs culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. CONCLUSIONS: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation. We then performed gene expression profiling analysis using data obtained from RNA-seq of 15 different tissues of different genetic mice.

			Action	DRS
	TS1_5_Heart_TAILS1.msf	Msf
	TS1_5_Heart_TAILS2.msf	Msf
	TS1_5_Heart_preTAILS1.msf	Msf
	TS1_5_Heart_preTAILS2.msf	Msf
	TS1_5_TAILS1.raw	Raw