Decreased soluble guanylate cyclase contributes to cardiac dysfunction induced by chronic doxorubicin treatment in mice

Aims: The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. Results: Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC alpha 1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGC alpha 1 allele [sGC alpha 1(-/-CM)]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGC alpha 1(-/-CM) than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGC alpha 1 mutant (DNsGC alpha 1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGC alpha 1(tg/+), but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGC alpha 1(tg/+) and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGC alpha 1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGC alpha 1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity

Ferulic acid-4-O-sulfate rather than ferulic acid relaxes arteries and lowers blood pressure in mice

Consumption of foods rich in ferulic acid (FA) such as wholegrain cereals, or FA precursors such as chlorogenic acids in coffee, is inversely correlated with risk of cardiovascular disease and type 2 diabetes. As a result of digestion and phase II metabolism in the gut and liver, FA is converted predominantly into ferulic acid-4-O-sulfate (FA-sul), an abundant plasma metabolite. Although FA-sul may be the main metabolite, very little has been reported regarding its bioactivities. We have therefore compared the ex vivo vasorelaxing effect of FA and FA-sul (10−7 - 3.10−5 M) on isolated mouse arteries mounted in tissue myographs. FA-sul, but not FA, elicited a concentration-dependent vasorelaxation of saphenous and femoral arteries and aortae. The FA-sul mediated vasorelaxation was blunted by 1H- [1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor. The role of sGC was confirmed in femoral arteries isolated from sGCα1(−/−) knockout mice. Furthermore, 4-aminopyridine, a specific inhibitor of voltage-dependent potassium channels, significantly decreased FA-sul mediated effects. In anesthetized mice, intravenous injection of FA-sul decreased mean arterial pressure, whereas FA had no effect, confirming the results obtained ex vivo. FA-sul is probably one of the major metabolites accounting for the blood pressure-lowering effects associated with FA consumption

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and Results We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands. Conclusions Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD. Translational Perspective Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD

The Factors Influencing the Sense of Home in Nursing Homes: A Systematic Review from the Perspective of Residents

. Purpose. To provide an overview of factors influencing the sense of home of older adults residing in the nursing home. Methods. A systematic review was conducted. Inclusion criteria were (1) original and peer-reviewed research, (2) qualitative, quantitative, or mixed methods research, (3) research about nursing home residents (or similar type of housing), and (4) research on the sense of home, meaning of home, at-homeness, or homelikeness. Results. Seventeen mainly qualitative articles were included. The sense of home of nursing home residents is influenced by 15 factors, divided into three themes: (1) psychological factors (sense of acknowledgement, preservation of one's habits and values, autonomy and control, and coping); (2) social factors (interaction and relationship with staff, residents, family and friends, and pets) and activities; and (3) the built environment (private space and (quasi-)public space, personal belongings, technology, look and feel, and the outdoors and location). Conclusions. The sense of home is influenced by numerous factors related to the psychology of the residents and the social and built environmental contexts. Further research is needed to determine if and how the identified factors are interrelated, if perspectives of various stakeholders involved differ, and how the factors can be improved in practice

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle

Deaths among wild birds during highly pathogenic avian influenza A(H5N8) virus outbreak, the Netherlands

During autumn–winter 2016–2017, highly pathogenic avian influenza A(H5N8) viruses caused mass die-offs among wild birds in the Netherlands. Among the ≈13,600 birds reported dead, most were tufted ducks (Aythya fuligula) and Eurasian wigeons (Anas penelope). Recurrence of avian influenza outbreaks might alter wild bird population dynamics

An Abundant Evolutionarily Conserved CSB-PiggyBac Fusion Protein Expressed in Cockayne Syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein