Companion Animals in Zoonoses Research – Ethical Considerations

Non-human animals are commonly classified according to their "role", such as "livestock", "wild" or "companion" animals. But what if those classifications overlap? This article presents a report of the retreat week "ZooCan – Zoonoses of companion animals as case study for animal ethics" at the University of Veterinary Medicine Hannover, Germany, in November 2022. The workshop included participants from different European countries with interdisciplinary backgrounds (animal law, bioethics, epidemiology, philosophy, biology and veterinary medicine). We address ethically relevant issues that emerge when companion animals are used as research animals, particularly in zoonoses research. The outcomes of the multi-disciplinary approach are used to i) define criteria to classify "companion" and "research" animals, ii) provide guidance to overcome the challenges with classificational overlaps, iii) give insights into cutting-edge zoonoses research with an example of SARS-CoV-2 in cats, and iv) discuss animal ethics approaches with regard to classifications

Module M1 of Zebrafish Neuroglobin Acts as a Structural and Functional Protein Building Block for a Cell-Membrane-Penetrating Activity

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro- fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration

Loss of single cluster does not change critical heart function parameters.

<p>Deletion of a single miR-1/133a cluster did not impair embryonic survival, as indicated by the Mendelian distribution of genotypes after mating of heterozygous parents. The distribution was determined at the time of weaning (A).We did not observe morphological changes during heart development at E15.5, especially no ventricular septum defects are observed (B). At E15.5 the thickness of the left ventricular wall (LV) was not changed, similarly the thickness of the interventricular septum (IS) was not significantly reduced (C). Weight gain (D) and survival (E) of single cluster mutant animals was comparable to WT litter mate animals. Heart functional parameters were determined using MRI (F); ejection fraction (EF), stroke volume (SV), end-diastolic volume (EDV), end-systolic volume (ESV), left ventricular (LV) mass was analyzed and no significant differences in heart function were detected (F). The scale bar in B corresponds to 200 µm.</p

MiRNA-1/133a Clusters Regulate Adrenergic Control of Cardiac Repolarization

<div><p>The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the <i>in vivo</i> effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity <i>in vivo</i> abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.</p></div

miR-1/133a controls impact of β-adrenergic regulation on L-type calcium-channel.

<p>The increased slope of ΔQT/ΔRR indicates LQT at low heart rates in miR-1-1/133a-2 and miR-1-2/133a-1 mutant mice. The LQT was rescued in vivo by inhibition of β-adrenergic signaling using Propranolol or by inhibition of L-type calcium channel using Verapamil, respectively (A). This result confirms the in vitro measurements proving that the miR-1/133a clusters modulate β-adrenergic signaling mediated regulation of L-type calcium channel activity and that loss of this modulation causes LQTS after deletion of single miR-1/133a clusters. Thus the miR-1/133a clusters are essential for repression of the smooth muscle gene program in post-natal heart and for maintenance of repolarization properties that are essential for normal function of the heart in its physiological context (B).</p

Loss of miR-1/133a impairs cardiac repolarization.

<p>Analysis of surface ECGs from immobilized animals revealed an increased QT duration (A). The increased QT duration was obvious especially at low heart rates (longer RR interval) induced by anesthesia with Isoflurane (B). We did not observe changes in PR or QRS interval length, nor arrhythmia or changes in the morphology of the ECG traces. The increased QT interval length is based on a longer ST duration. As shown in (C) the slope of a linear fit (ΔQT/ΔRR) is greater in miR-1/133a single cluster compared to the WT animals.</p

β-adrenergic signaling is intact in after loss of miR-1/133a.

<p>The concentration of cAMP was not changed in the adult heart of miR-1/133a single cluster mutant mice (A). Adrenergic signaling of cardiomyocytes isolated form adult hearts of WT and single miR-1/133a cluster mutant mice was investigated by stimulation with 1 µM Isoproterenol (ISO; B–G). Adrenergic signaling affects multiple components involved in cardiomyocyte calcium handling and contraction. Phosphorylation of several targets of the adrenergic signaling cascade was analyzed to detect modulation of the adrenergic signaling in cardiomyocytes isolated from mutant and WT animals. Proteins of cardiomyocytes isolated from of ≥5 animals was used for statistical evaluation (C–G), representative blots are shown (B).</p