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Background: The TOPCAT trial (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) suggested clinical benefits of spironolactone treatment among patients with heart failure with preserved ejection fraction enrolled in the Americas. However, a comprehensive assessment of biologic pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction has not been performed. Methods: We conducted aptamer-based proteomic analysis utilizing 5284 modified aptamers to 4928 unique proteins on plasma samples from TOPCAT participants from the Americas (n=164 subjects with paired samples at baseline and 1 year) to identify proteins and pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction. Mean percentage change from baseline was calculated for each protein. Additionally, we conducted pathway analysis of proteins altered by spironolactone. Results: Spironolactone therapy was associated with proteome-wide significant changes in 7 proteins. Among these, CARD18 (caspase recruitment domain-containing protein 18), PKD2 (polycystin 2), and PSG2 (pregnancy-specific glycoprotein 2) were upregulated, whereas HGF (hepatic growth factor), PLTP (phospholipid transfer protein), IGF2R (insulin growth factor 2 receptor), and SWP70 (switch-associated protein 70) were downregulated. CARD18, a caspase-1 inhibitor, was the most upregulated protein by spironolactone (−0.5% with placebo versus +66.5% with spironolactone, P Conclusions: Proteomic analysis in the TOPCAT trial revealed proteins and pathways altered by spironolactone, including the caspase inhibitor CARD18 and multiple pathways that involved collagens. In addition to effects on fibrosis, our studies suggest potential antiapoptotic effects of spironolactone in heart failure with preserved ejection fraction, a hypothesis that merits further exploration.

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

The human EYA1 gene is an ortholog of the fruit fly eyes absent gene (eya), which was identified as a regulator of compound eye development. In contrast to the single eya gene found in Drosophila melanogaster, mammals possess four Eya paralogs, designated Eya1-4. The respective gene products are characterized by a C-terminal domain called the Eya domain, which is conserved in both length (271 to 274 amino acids [aa]) and sequence. The Eya domain has an intrinsic phosphatase activity (24, 39, 53) and is required for protein-protein interactions (38). Recent studies provided evidence that the phosphatase function of Eya is involved in the innate immune system and the regulation of DNA damage response (8, 35). Interestingly, all Eya interaction partners that have been identified so far, for example, the Six and Dach proteins or inhibitory G protein α (Gα) subunits, bind to the Eya domain (5, 10, 34). It has been demonstrated that the cooperative action of Eya and Six is essential for the development of several tissues and organs in a variety of species throughout evolution (reviewed in reference 6). In vertebrates, Six induces nuclear translocation of Eya and recruitment to the DNA, and Eya enhances Six-mediated activation of target gene expression (34). The N termini of Eya proteins, which are highly divergent between Eya family members, harbor a proline-serine-threonine (PST)-rich transactivation domain, which is indispensable for their function as coactivators of transcription (31, 45, 50, 58). Natural target genes of the vertebrate Eya-Six complex are, for example, Six2, Sall1, and Myogenin. Activation of Six2 and Sall1 expression has been shown to be essential for proper kidney development in the mouse, whereas activation of Myogenin is required for muscle development (3, 4, 46). Mutations in the human EYA1 gene are associated with several congenital disorders, like BOR (branchio-oto-renal) and BO (branchio-oto) syndrome, as well as ocular defects. BOR syndrome patients suffer from severe malformations of the ears, the branchial arch derivatives, and the kidneys, while in BO syndrome patients, the kidneys are not affected (11, 12, 30). The physiological importance of Eya1 was underlined by knockout studies in mice. It was shown that the phenotype of Eya1-heterozygous mice resembles the characteristic symptoms of human patients suffering from BOR syndrome with renal abnormalities, including hypoplasia and unilateral agenesis, and conductive hearing loss. In contrast, Eya1 homozygote mice die at birth, showing severe craniofacial and skeletal defects and the complete absence of the thymus, parathyroid glands, ears, and kidneys due to defective inductive tissue interactions (57, 59). Interestingly, similar to EYA1, the paralogous EYA4 gene has also been demonstrated to be associated with human disease. EYA4 is most closely related to EYA1 rather than EYA2 and EYA3. Mutations in EYA4 have been linked to cardiomyopathy and hearing loss (37, 42, 55). In contrast, there is no evidence for the existence of disease-associated mutations in EYA2 and EYA3 up to now. The identification and characterization of an Eya1 ortholog in zebrafish revealed a remarkable conservation in both the structure and expression pattern between Eya1 genes in fish and humans. In zebrafish, eya1 expression was detected in several organs during embryogenesis, such as the ears, the lateral line organ, the branchial arches, and the somites (41). As in mammals, eya1 in zebrafish is essential for the development of several tissues and organs. A quite well-studied example is the zebrafish ear, whose proper formation depends on eya1 expression, as shown by morpholino-mediated knockdown analysis (23). Since Eya1 is involved in the development of many different organs, we proposed that additional factors bind to it and, thereby, modulate its function. Using a yeast two-hybrid-based approach, we identified the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as so-far-unknown Eya1 interaction partners. We characterized the interactions and demonstrated that both Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Furthermore, we showed that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. Importantly, the morpholino-mediated knockdown of Sipl1 and Rbck1 orthologs in zebrafish led to a BOR syndrome-like phenotype, indicating the relevance of the Eya1-Sipl1/Rbck1 interaction in vivo.

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation., The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.