Turner, G. M. | Search bioRxiv

bioRxiv2023

Effect size of delayed freezing, diurnal variation, and hindgut location on the mouse fecal microbiome relative to a standardized biological variable

BackgroundWhile murine fecal collection is central to microbiome research, there are a number of practical considerations that may vary during fecal sample collection, including time to sample storage, time of day the sample is collected, and position within the colon during terminal collections. While the need to control these factors is recognized, the relative effect on microbial community of duration at room temperature, time of day, and hindgut position, in the context of a known biological variable, is unclear. To answer these questions, and assess reproducibility of results across different microbiome compositions, parallel experiments were performed to investigate the effect of those factors on the microbiome of age- and sex-matched isogenic mice colonized with two different vendor-origin microbiomes. Results16S rRNA amplicon sequencing data from flash-frozen fecal samples showed no statistical difference in alpha or beta diversity compared to samples incubated for 1, 2, 3, 4, 6, and 9 hours at room temperature. Overall, samples collected in the AM period showed greater richness and alpha-diversity compared to samples collected in the PM period. While a significant effect of time was detected in all hindgut regions, the effect increased from cecum to distal colon. When using two vendor-origin microbiomes as a biological variable, its effect size vastly outweighed the effect size of the time samples spent at room temperature, the time of day samples were collected, and the position within the colon from which samples were collected. ConclusionsThis study has highlighted multiple scenarios encountered in microbiome research that may affect outcome measures of microbial diversity and composition. Unexpectedly, delayed time to sample cold storage up to nine hours did not affect the alpha or global beta diversity of fecal sample. We then presented evidence of location- and time-dependent effects within the hindgut on microbial richness, diversity, and composition. We finally demonstrated a relatively low effect size of these technical factors when compared to a primary experimental factor with large intergroup variability.

microbiology↗

bioRxiv2021

Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to better characterize atrial fibrillation (AF), we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined are poor representations of human AF, and raise a cautionary note about the use of any murine model to study AF.

physiology↗