Inducible Depletion of Cardiac Thymosin β4: Development and Shortcomings of a Popular Technique in Cardiac Research

In reference to recent studies highlighting the importance of Thymosin beta 4 (Tβ4) during embryonic development and its therapeutic potential, an inducible cardiac-specific knockdown of Tβ4 was established to investigate the outcomes of loss of function in adult mammalian hearts. Tβ4shRNAflox/MerCreMer mice were generated with the aim of depleting cardiac Tβ4 expression by RNA interference in a tamoxifen-dependent fashion. In vivo, in vitro and ex vivo approaches showed that tamoxifen treatment of Tβ4shRNAflox/MerCreMer mice does not result in significant down-regulation of Tβ4 mRNA or protein in the heart. Interestingly, higher levels of Tβ4 were identified in cardiac fibroblasts compared to cardiomyocytes. The above analyses suggest that an inducible knockout of Tβ4 would be a more direct, more reliable and more effective means to study loss of function in the adult mammalian heart. In the course of the study, an adverse cardiac phenotype was observed in tamoxifen-treated MerCreMer-positive animals, consisting of perivascular and interstitial fibrosis, decreased cardiac function, a marked inflammatory response and increased expression of factors involved in cardiac remodelling and hypertrophy. This was not related to MerCreMer gene copy number (homo/heterozygosity) but found to be associated with tamoxifen dose, mode of delivery and genetic background. A thorough analysis of related literature revealed that a number of recent publications have failed to include tamoxifen-treated αMHC/MerCreMer controls and some of the presented data may be affected by Cre toxicity. In conclusion, αMHC/MerCreMer mice should be included as controls in the analysis of future studies or alternative inducible systems considered. Recent analyses revealed that IGF-1Ea overexpression in the heart improves the outcome of myocardial infarction in mice. This process has been described on the molecular level but a comprehensive analysis of the subcellular structure of αMHC/IGF-1Ea hearts had not previously been performed. A detailed transmission electron microscope analysis of αMHC/IGF-1Ea hearts revealed previously not described large electron-dense structures inside αMHC/IGF-1Ea cardiomyocytes. The structures resembled large autophagolysosomes although markers of autophagy were neither found to be associated with the structures nor upregulated in the hearts of αMHC/IGF-1Ea mice. Intriguingly lysosomes were identified in the proximity of the structures potentially implying a role in turnover of intracellular material. These results provide further insights into the diverse roles of IGF-1Ea in the heart

Ablation of SGK1 Impairs Endothelial Cell Migration and Tube Formation Leading to Decreased Neo-Angiogenesis Following Myocardial Infarction

Serum and glucocorticoid inducible kinase 1 (SGK1) plays a pivotal role in early angiogenesis during embryonic development. In this study, we sought to define the SGK1 downstream signalling pathways in the adult heart and to elucidate their role in cardiac neo-angiogenesis and wound healing after myocardial ischemia. To this end, we employed a viable SGK1 knockout mouse model generated in a 129/SvJ background. Ablation of SGK1 in these mice caused a significant decrease in phosphorylation of SGK1 target protein NDRG1, which correlated with alterations in NF-κB signalling and expression of its downstream target protein, VEGF-A. Disruption of these signalling pathways was accompanied by smaller heart and body size. Moreover, the lack of SGK1 led to defective endothelial cell (ECs) migration and tube formation in vitro, and increased scarring with decreased angiogenesis in vivo after myocardial infarct. This study underscores the importance of SGK1 signalling in cardiac neo-angiogenesis and wound healing after an ischemic insult in vivo

Main Routes of Entry and Genomic Diversity of SARS-CoV-2, Uganda.

We established rapid local viral sequencing to document the genomic diversity of severe acute respiratory syndrome coronavirus 2 entering Uganda. Virus lineages closely followed the travel origins of infected persons. Our sequence data provide an important baseline for tracking any further transmission of the virus throughout the country and region

Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver

Tamoxifen-inducible Cre-mediated manipulation of animal genomes has achieved wide acceptance over the last decade, with numerous important studies heavily relying on this technique. Recently, a number of groups have reported transient complications of using this protocol in the heart. In the present study we observed a previously unreported focal fibrosis and depressed left-ventricular function in tamoxifen-treated αMHC-MerCreMer-positive animals in a Tβ4shRNAflox × αMHC-MerCreMer cross at 6–7 weeks following standard tamoxifen treatment, regardless of the presence of the floxed transgene. The phenotype was reproduced by treating mice from the original αMHC-MerCreMer strain with tamoxifen. In the acute phase after tamoxifen treatment, cell infiltration into the myocardium was accompanied by increased expression of pro-inflammatory cytokines (IL-1β, IL-6, TNFα, IFNγ, Ccl2) and markers of hypertrophy (ANF, BNP, Col3a1). These observations highlight the requirement for including tamoxifen-treated MerCreMer littermate controls to avert misinterpretation of conditional mutant phenotypes. A survey of the field as well as the protocols presented here suggests that controlling the parameters of tamoxifen delivery is important in avoiding the chronic MerCreMer-mediated cardiac phenotype reported here

Modulation of NF-κB pathway and VEGF-A expression by SGK1.

<p>Western blot analysis of IκBα (A), NF-κB₂/p100 (B) and VEGF-A (C) in WT and SGK1-/- hearts. GAPDH and α-Tubulin were used as loading controls. Quantification of band intensities for each represented blot was performed using ImageJ. The number of hearts analysed was ≥4 in each group and statistical analysis is performed by Student t-test. ** Indicates p<0.01, *** p<0.001. The data are reported as Mean ± SE.</p

Lack of SGK1 correlated with reduced cardiomyocyte size.

<p>(A) Cross sectional area in heart sections from WT and SGK1-/- mice. Area was measured in ≥10 distinct microscope field for each slide. Four hearts were analysed for each slide in both WT and SGK1-/- mice. Scale bar represents 50 µm. (B) Confocal images of isolated adult cardiomyocytes form WT and SGK1-/- mice The data represent the average of three independent experiments and are reported as Mean ± SE. Cell volume was measured using a custom-written macro in ImageJ. Scale bars represent 5µm. Statistical analysis has been performed as described in Materials and Methods using Student t-test; *** indicates p<0.001. </p