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Fluorescence microscopy has undergone rapid advancements, offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However, the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here, we introduce sHAPR, a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings, enabling acquisition at the maximum camera readout rate, typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems, including high-throughput flow cytometry, cardiomyocyte contraction, and neuronal calcium waves, using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities, opening up new avenues for investigating high-speed biological phenomena., (© 2024. The Author(s).)

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca 2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels., (© 2024. The Author(s).)

Efficient generation of cardiomyocytes from human-induced pluripotent stem cells (hiPSCs) is important for their application in basic and translational studies. Space microgravity can significantly change cell activities and function. Previously, we reported upregulation of genes associated with cardiac proliferation in cardiac progenitors derived from hiPSCs that were exposed to space microgravity for 3 days. Here we investigated the effect of long-term exposure of hiPSC-cardiac progenitors to space microgravity on global gene expression. Cryopreserved 3D hiPSC-cardiac progenitors were sent to the International Space Station (ISS) and cultured for 3 weeks under ISS microgravity and ISS 1 G conditions. RNA-sequencing analyses revealed upregulation of genes associated with cardiac differentiation, proliferation, and cardiac structure/function and downregulation of genes associated with extracellular matrix regulation in the ISS microgravity cultures compared with the ISS 1 G cultures. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes mapping identified the upregulation of biological processes, molecular function, cellular components, and pathways associated with cell cycle, cardiac differentiation, and cardiac function. Taking together, these results suggest that space microgravity has a beneficial effect on the differentiation and growth of cardiac progenitors., (© 2023. The Author(s).)

Background: Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation., Methods: hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli., Results: Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features-including enhanced Ca 2+ transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells., Conclusion: AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases., (© 2023. The Author(s).)

Fluorescence microscopy is one of the most indispensable and informative driving forces for biological research, but the extent of observable biological phenomena is essentially determined by the content and quality of the acquired images. To address the different noise sources that can degrade these images, we introduce an algorithm for multiscale image restoration through optimally sparse representation (MIRO). MIRO is a deterministic framework that models the acquisition process and uses pixelwise noise correction to improve image quality. Our study demonstrates that this approach yields a remarkable restoration of the fluorescence signal for a wide range of microscopy systems, regardless of the detector used (e.g., electron-multiplying charge-coupled device, scientific complementary metal-oxide semiconductor, or photomultiplier tube). MIRO improves current imaging capabilities, enabling fast, low-light optical microscopy, accurate image analysis, and robust machine intelligence when integrated with deep neural networks. This expands the range of biological knowledge that can be obtained from fluorescence microscopy.

Background: Chronic alcohol consumption in adults can induce various cardiac toxicities such as arrhythmias, cardiomyopathy, and heart failure. Prenatal alcohol exposure can increase the risk of developing congenital heart defects among offspring. Understanding the molecular mechanisms underlying long-term alcohol exposure-induced cardiotoxicity can help guide the development of therapeutic strategies., Methods: Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) were engineered into cardiac spheroids and treated with clinically relevant concentrations of ethanol (17 and 50 mM) for 5 weeks. The cells were then analyzed for changes in mitochondrial features, transcriptomic and metabolomic profiles, and integrated omics outcomes., Results: Following chronic ethanol treatment of hiPSC-CMs, a decrease in mitochondrial membrane potential and respiration and changes in expression of mitochondrial function-related genes were observed. RNA-sequencing analysis revealed changes in various metabolic processes, heart development, response to hypoxia, and extracellular matrix-related activities. Metabolomic analysis revealed dysregulation of energy metabolism and increased metabolites associated with the upregulation of inflammation. Integrated omics analysis further identified functional subclusters and revealed potentially affected pathways associated with cardiac toxicities., Conclusion: Chronic ethanol treatment of hiPSC-CMs resulted in overall decreased mitochondrial function, increased glycolysis, disrupted fatty acid oxidation, and impaired cardiac structural development., (© 2023 Research Society on Alcohol.)

In microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca 2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function., Competing Interests: Conflicts of interest J.F. and G.B. were employees of Techshot. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a cell model for drug-induced cytotoxicity in combination with traction force microscopy, functional assessments, high-throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC-CMs. Following the treatment of hiPSC-CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration-dependent increase in carfilzomib-induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC-CMs in 3-dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca 2+ transients and reduced integrin-mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC-CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses.

Background: Alcohol use in pregnancy increases the risk of abnormal cardiac development, and excessive alcohol consumption in adults can induce cardiomyopathy, contractile dysfunction, and arrhythmias. Understanding molecular mechanisms underlying alcohol-induced cardiac toxicity could provide guidance in the development of therapeutic strategies., Methods: We have performed proteomic and bioinformatic analysis to examine protein alterations globally and quantitatively in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) treated with ethanol (EtOH). Proteins in both cell lysates and extracellular culture media were systematically quantitated., Results: Treatment with EtOH caused severe detrimental effects on hiPSC-CMs as indicated by significant cell death and deranged Ca 2+ handling. Treatment of hiPSC-CMs with EtOH significantly affected proteins responsible for stress response (e.g., GPX1 and HSPs), ion channel-related proteins (e.g. ATP1A2), myofibril structure proteins (e.g., MYL2/3), and those involved in focal adhesion and extracellular matrix (e.g., ILK and PXN). Proteins involved in the TNF receptor-associated factor 2 signaling (e.g., CPNE1 and TNIK) were also affected by EtOH treatment., Conclusions: The observed changes in protein expression highlight the involvement of oxidative stress and dysregulation of Ca 2+ handling and contraction while also implicating potential novel targets in alcohol-induced cardiotoxicity. These findings facilitate further exploration of potential mechanisms, discovery of novel biomarkers, and development of targeted therapeutics against EtOH-induced cardiotoxicity., (© 2020 by the Research Society on Alcoholism.)

Long noncoding RNAs (lncRNAs) are crucial in many cellular processes, yet relatively few have been shown to regulate human cardiomyocyte differentiation. Here, we demonstrate an essential role of GATA6 antisense RNA 1 (GATA6-AS1) in cardiomyocyte differentiation from human pluripotent stem cells (hPSCs). GATA6-AS1 is adjacent to cardiac transcription factor GATA6. We found that GATA6-AS1 was nuclear-localized and transiently upregulated along with GATA6 during the early stage of cardiomyocyte differentiation. The knockdown of GATA6-AS1 did not affect undifferentiated cell pluripotency but inhibited cardiomyocyte differentiation, as indicated by no or few beating cardiomyocytes and reduced expression of cardiomyocyte-specific proteins. Upon cardiac induction, the knockdown of GATA6-AS1 decreased GATA6 expression, altered Wnt-signaling gene expression, and reduced mesoderm development. Further characterization of the intergenic region between genomic regions of GATA6-AS1 and GATA6 indicated that the expression of GATA6-AS1 and GATA6 were regulated by a bidirectional promoter within the intergenic region. Consistently, GATA6-AS1 and GATA6 were co-expressed in several human tissues including the heart, similar to the mirror expression pattern of GATA6-AS1 and GATA6 during cardiomyocyte differentiation. Overall, these findings reveal a previously unrecognized and functional role of lncRNA GATA6-AS1 in controlling human cardiomyocyte differentiation., (© 2020 Federation of American Societies for Experimental Biology.)

Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease._Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/- vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs._Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b(+) Gr-1(+) MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b(+) Gr-1(+) MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment. Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Myeloid derived suppressor cells (MDSCs) from tumor-bearing mice are important negative regulators of anti-cancer immune responses, but the role for immature myeloid cells (IMCs) in non-tumor-bearing mice in the regulation of immune responses are poorly described. We studied the immune-suppressive activity of IMCs from the bone marrow (BM) of C57Bl/6 mice and the mechanism(s) by which they inhibit T-cell activation and proliferation. IMCs, isolated from BM by high-speed FACS, inhibited mitogen-induced proliferation of CD4(+) and CD8(+) T-cells in vitro. Cell-to-cell contact of T-cells with viable IMCs was required for suppression. Neither neutralizing antibodies to TGFβ1, nor genetic disruption of indolamine 2,3-dioxygenase, abrogated IMC-mediated suppressive activity. In contrast, suppression of T-cell proliferation was absent in cultures containing IMCs from interferon-γ (IFN-γ) receptor KO mice or T-cells from IFN-γ KO mice (on the C57Bl/6 background). The addition of NO inhibitors to co-cultures of T-cells and IMC significantly reduced the suppressive activity of IMCs. IFN-γ signaling between T-cells and IMCs induced paracrine Nitric Oxide (NO) release in culture, and the degree of inhibition of T-cell proliferation was proportional to NO levels. The suppressive activity of IMCs from the bone marrow of tumor-free mice was comparable with MDSCs from BALB/c bearing mice 4T1 mammary tumors. These results indicate that IMCs have a role in regulating T-cell activation and proliferation in the BM microenvironment.

Myeloid Derived Suppressor Cells (MDSCs) are a mixed group of bone marrow-derived myeloid cells containing macrophages, granulocytes, immature DCs and early myeloid precursors that have immune suppressive activity. MDSCs infiltrate the BM, spleen and peripheral blood of tumors-bearing experimental animals and are found in the blood of cancer patients as a result of tumor-induced alterations in myelopoiesis. Evidence from murine model systems indicated that myeloid-derived cells with suppressor activity also accumulate in non-tumor bearing hosts in response to infection, chemotherapy, stress, and immune senescence. MDSCs are considered key negative regulators of immune responses. Their association with tumor-associated immune defects make MDSCs an attractive target for therapeutic intervention in cancer.

Galectin-1 (Gal-1) and galectin-3 (Gal-3) molecules are involved in many vital, biological, and pathological processes. In this study the expression pattern of these two molecules was investigated in leukemic cells from 85 Iranian chronic lymphocytic leukemia (CLL) patients, classified into indolent, progressive, mutated, and unmutated subtypes by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry methods. Our results showed significant downregulation of Gal-3, but not Gal-1, in CLL patients compared with normal subjects (p < .001). Higher representation of Gal-3 mRNA was observed in indolent patients compared with the progressive group (p < .05). Our findings imply a regulatory role for Gal-3 gene in initiation and/or progression of CLL.