MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes.

Cry toxins are primarily a family of insecticidal toxins produced by the bacterium Bacillus thuringiensis (Bt). However, some Cry toxins, called parasporins (PSs), are non-insecticidal and have been shown to differentially kill human cancer cells. Based on amino acid homology, there are currently six different classes of parasporins (PS1-6). It is not known what role parasporins play in nature, nor if certain PSs are associated with Bt found in particular environments. Herein, we present ten parasporin-containing isolates of Bt from the Caribbean island of Trinidad. Genes coding for PS1 and PS6 were found in isolates associated mainly with artificial aquatic environments (e.g., barrels with rain water), while Bt possessing two novel PS5-like genes (ps5-1 and ps5-2), were isolated from manure collected directly from the rectum of cattle. The amino acid sequences inferred from the two PS5-like genes were 51 % homologous to each other, while being only 41 or 45 % similar to PS5Aa1/Cry64Aa, the only reported member of the parasporin five class. The low level of amino acid homology between the two PS5-like genes and PS5Aa1 indicate that the two PS5-like genes may represent a new class of parasporins, or greatly expand the level of diversity within the current parasporin 5 class.

Perceived stress and hair cortisol concentration in a study of Mexican and Icelandic women.

Hair cortisol concentration (HCC) represent a potential biomarker of chronic psychological stress. Previous studies exploring the association between perceived stress and HCC have been limited to relatively small and selected populations. We collected hair samples from 881 women from the Mexican Teachers' Cohort (MTC) and 398 women from the Icelandic SAGA pilot-cohort following identical protocols. HCC was quantified using liquid chromatography coupled with tandem mass spectrometry. The self-reported Perceived Stress Scale (PSS, 10 and 4 item, range 0-40 and 0-16) was used to assess psychological stress. We conducted multivariable linear regression analyses to assess the association between perceived stress and log-transformed HCC in the combined sample and in each cohort separately. MTC participants had slightly higher HCC and PSS scores than SAGA participants (median HCC 6.0pg/mg vs. 4.7pg/mg and mean PSS-10 score 12.4 vs. 11.7, respectively). After adjusting for sociodemographic factors and health behaviors, we observed a 1.4% (95% CI 0.6, 2.1) increase in HCC for each unit increase in the PSS-10 score in the combined sample. Furthermore, PSS-10 quintiles were associated with a 24.3% (95% CI 8.4, 42.6, mean logHCC 1.8 vs 1.6) increase in HCC when comparing the highest to the lowest quintile, after multivariable adjustment. Similar results were obtained when we analyzed each cohort separately and when using the PSS-4. Despite relatively small absolute differences, an association between perceived stress and HCC was found in a sample of women from two diverse geographical and cultural backgrounds supporting the hypothesis that HCC is a viable biomarker in studies of chronic psychological stress.