Advancing Cardiovascular Drug Screening Using Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a promising tool for studying cardiac physiology and drug responses. However, their use is largely limited by an immature phenotype and lack of high-throughput analytical methodology. In this study, we developed a high-throughput testing platform utilizing hPSC-CMs to assess the cardiotoxicity and effectiveness of drugs. Following an optimized differentiation and maturation protocol, hPSC-CMs exhibited mature CM morphology, phenotype, and functionality, making them suitable for drug testing applications. We monitored intracellular calcium dynamics using calcium imaging techniques to measure spontaneous calcium oscillations in hPSC-CMs in the presence or absence of test compounds. For the cardiotoxicity test, hPSC-CMs were treated with various compounds, and calcium flux was measured to evaluate their effects on calcium dynamics. We found that cardiotoxic drugs withdrawn due to adverse drug reactions, including encainide, mibefradil, and cetirizine, exhibited toxicity in hPSC-CMs but not in HEK293-hERG cells. Additionally, in the effectiveness test, hPSC-CMs were exposed to ATX-II, a sodium current inducer for mimicking long QT syndrome type 3, followed by exposure to test compounds. The observed changes in calcium dynamics following drug exposure demonstrated the utility of hPSC-CMs as a versatile model system for assessing both cardiotoxicity and drug efficacy. Overall, our findings highlight the potential of hPSC-CMs in advancing drug discovery and development, which offer a physiologically relevant platform for the preclinical screening of novel therapeutics.

Yeast Small Ubiquitin-Like Modifier (SUMO) Protease Ulp2 is Involved in RNA Splicing.

In eukaryotes, RNA splicing, an essential biological process, is crucial for precise gene expression. Inaccurate RNA splicing can cause aberrant mRNA production, disrupting protein synthesis. To regulate splicing efficiency, some splicing factors are reported to undergo Ubiquitin-like Modifier (SUMO)ylation. Our data indicate that in Saccharomyces cerevisiae, the SUMO protease, Ulp2, is involved in splicing. In the ulp2Δ mutant, some ribosomal protein (RP) transcripts exhibited a significant increase in the levels of intron-containing pre-mRNA because of improper splicing. Moreover, we confirmed Ulp2 protein binding to the intronic regions of RP genes. These findings highlight a critical Ulp2 role in RP transcript splicing.

Tho2-mediated escort of Nrd1 regulates the expression of aging-related genes.

The relationship between aging and RNA biogenesis and trafficking is attracting growing interest, yet the precise mechanisms are unknown. The THO complex is crucial for mRNA cotranscriptional maturation and export. Herein, we report that the THO complex is closely linked to the regulation of lifespan. Deficiencies in Hpr1 and Tho2, components of the THO complex, reduced replicative lifespan (RLS) and are linked to a novel Sir2-independent RLS control pathway. Although transcript sequestration in hpr1Δ or tho2Δ mutants was countered by exosome component Rrp6, loss of this failed to mitigate RLS defects in hpr1Δ. However, RLS impairment in hpr1Δ or tho2Δ was counteracted by the additional expression of Nrd1-specific mutants that interacted with Rrp6. This effect relied on the interaction of Nrd1, a transcriptional regulator of aging-related genes, including ribosome biogenesis or RNA metabolism genes, with RNA polymerase II. Nrd1 overexpression reduced RLS in a Tho2-dependent pathway. Intriguingly, Tho2 deletion mirrored Nrd1 overexpression effects by inducing arbitrary Nrd1 chromatin binding. Furthermore, our genome-wide ChIP-seq analysis revealed an increase in the recruitment of Nrd1 to translation-associated genes, known to be related to aging, upon Tho2 loss. Taken together, these findings underscore the importance of Tho2-mediated Nrd1 escorting in the regulation of lifespan pathway through transcriptional regulation of aging-related genes.

Increased ER stress by depletion of PDIA6 impairs primary ciliogenesis and enhances sensitivity to ferroptosis in kidney cells.

Primary cilia are crucial for cellular balance, serving as sensors for external conditions. Nephronophthisis and related ciliopathies, which are hereditary and degenerative, stem from genetic mutations in cilia-related genes. However, the precise mechanisms of these conditions are still not fully understood. Our research demonstrates that downregulating PDIA6, leading to cilia removal, makes cells more sensitive to ferroptotic death caused by endoplasmic reticulum (ER) stress. The reduction of PDIA6 intensifies the ER stress response, while also impairing the regulation of primary cilia in various cell types. PDIA6 loss worsens ER stress, hastening ferroptotic death in proximal tubule epithelial cells, HK2 cells. Counteracting this ER stress can mitigate PDIA6 depletion effects, restoring both the number and length of cilia. Moreover, preventing ferroptosis corrects the disrupted primary ciliogenesis due to PDIA6 depletion in HK2 cells. Our findings emphasize the role of PDIA6 in primary ciliogenesis, and suggest its absence enhances ER stress and ferroptosis. These insights offer new therapeutic avenues for treating nephronophthisis and similar ciliopathies.