Sanofi: Information concerning the total number of voting rights and shares – February 2024

Information concerning the total number of voting rights and shares, provided pursuant to article L. 233-8 II of the Code de commerce (the French Commercial Code) and article 223-16 of the Règlement général de l’Autorité des Marchés Financiers (Regulation of the French stock market authority)

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Sanofi: Information concerning the total number of voting rights and shares - February 2024

XHANCE Approved by FDA as First and Only Medication Indicated for Treatment of Adults with Chronic Rhinosinusitis without Nasal Polyps

ReOpen was the first ever large placebo-controlled clinical trial program to demonstrate statistically significant reduction of symptoms in chronic sinusitis patients without nasal polyps

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XHANCE Approved by FDA as First and Only Medication Indicated for Treatment of Adults with Chronic Rhinosinusitis without Nasal Polyps

Rani Therapeutics to Report Fourth Quarter and Full Year 2023 Financial Results

SAN JOSE, Calif., March 15, 2024 (GLOBE NEWSWIRE) -- Rani Therapeutics Holdings, Inc. (“Rani Therapeutics” or “Rani”) (Nasdaq: RANI), a clinical-stage biotherapeutics company focused on the oral delivery of biologics and drugs, today announced that it plans to release financial results for the fourth quarter and full year ended December 31, 2023 and provide a business update on Wednesday, March 20 after the close of trading. Rani’s management team will host a conference call and webcast beginning at 4:30 p.m. ET.

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Rani Therapeutics to Report Fourth Quarter and Full Year 2023 Financial Results

NurExone’s Strategic Update: Submission of OTCQB Listing Application to Initiate US financial presence

TORONTO and HAIFA, Israel, March 15, 2024 (GLOBE NEWSWIRE) -- NurExone Biologic Inc. (TSXV: NRX) (Germany: J90) (the “Company” or “NurExone”), a pioneering biopharmaceutical company, developing regenerative medicine therapies, announces today its intention to broaden its market reach through a recently filed application for listing on the OTCQB® Venture Market (the "OTCQB") in the United States. Listing on the OTCQB is subject to approval of the OTC Markets Group.

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NurExone's Strategic Update: Submission of OTCQB Listing Application to Initiate US financial presence

Psyence Biomedical Ltd. Receives Nasdaq Notifications Regarding Market Value of Listed Securities and Market Value of Publicly Held Shares

NEW YORK, March 15, 2024 (GLOBE NEWSWIRE) -- Psyence Biomedical Ltd. (the “Company”) (Nasdaq: PBM) announced that on March 11, 2024, it received two letters from the listing qualifications department staff of The Nasdaq Stock Market (“Nasdaq”), one notifying the Company (the “MVLS Notice”) that for the last 30 consecutive business days, the Company’s Market Value of Listed Securities (“MVLS”) was below the minimum of $50 million required for continued listing on the Nasdaq Global Market pursuant to Nasdaq Listing Rule 5450(b)(2)(A) (the “Market Value Standard”), and the other notifying the Company (the “MVPHS Notice”) that for the last 30 consecutive business days, the Company’s Market Value of Publicly Held Shares (“MVPHS”) was below the minimum of $15 million required for continued listing on the Nasdaq Global Market pursuant to Nasdaq Listing Rule 5450(b)(2)(C) (the “MVPHS Standard”).

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Psyence Biomedical Ltd. Receives Nasdaq Notifications Regarding Market Value of Listed Securities and Market Value of Publicly Held Shares

Extracorporeal Shock Wave Therapy Combined With Platelet-Rich Plasma Injection to Treat the Nonunion of a Stress … – Cureus

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Extracorporeal Shock Wave Therapy Combined With Platelet-Rich Plasma Injection to Treat the Nonunion of a Stress ... - Cureus

Pediatric Hemophilic Arthropathy of the Knee: Treatment With Circular External Fixator and Intra-articular Injection of … – Cureus

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Please choose I'm not a medical professional. Allergy and Immunology Anatomy Anesthesiology Cardiac/Thoracic/Vascular Surgery Cardiology Critical Care Dentistry Dermatology Diabetes and Endocrinology Emergency Medicine Epidemiology and Public Health Family Medicine Forensic Medicine Gastroenterology General Practice Genetics Geriatrics Health Policy Hematology HIV/AIDS Hospital-based Medicine I'm not a medical professional. Infectious Disease Integrative/Complementary Medicine Internal Medicine Internal Medicine-Pediatrics Medical Education and Simulation Medical Physics Medical Student Nephrology Neurological Surgery Neurology Nuclear Medicine Nutrition Obstetrics and Gynecology Occupational Health Oncology Ophthalmology Optometry Oral Medicine Orthopaedics Osteopathic Medicine Otolaryngology Pain Management Palliative Care Pathology Pediatrics Pediatric Surgery Physical Medicine and Rehabilitation Plastic Surgery Podiatry Preventive Medicine Psychiatry Psychology Pulmonology Radiation Oncology Radiology Rheumatology Substance Use and Addiction Surgery Therapeutics Trauma Urology Miscellaneous

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Pediatric Hemophilic Arthropathy of the Knee: Treatment With Circular External Fixator and Intra-articular Injection of ... - Cureus

Vitamin A’s Puzzling Effects Unraveled: New Research Sheds Light on Stem Cell Repair Mechanisms – SciTechDaily

Hair follicle stem cells (green) mobilize and expand (white) to help repair the skins barrier by differentiating into epidermal lineages (red). Credit: Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development at The Rockefeller University

When a child falls off her bike and scrapes her knee, skin stem cells rush to the rescue, growing new epidermis to cover the wound. However, only a portion of these stem cells, which eventually repair the damage, are typically assigned the task of replenishing the epidermis that protects her body.

Others are former hair follicle stem cells, which usually promote hair growth but respond to the more urgent needs of the moment, morphing into epidermal stem cells to bolster local ranks and repair efforts. To do that, these hair follicle stem cells first enter a pliable state in which they temporarily express the transcription factors of both types of stem cells, hair, and epidermis.

Now, new research demonstrates that once stem cells have entered this state, known as lineage plasticity, they cannot function effectively in either role until they choose a definitive fate. In a screen to identify key regulators of this process, retinoic acid, the biologically active form of Vitamin A, surfaced as a surprising rheostat. The findings shed light on lineage plasticity, with potential clinical implications.

Our goal was to understand this state well enough to learn how to dial it up or down, says Rockefellers Elaine Fuchs. We now have a better understanding of skin and hair disorders, as well as a path toward preventing lineage plasticity from contributing to tumor growth.

Lineage plasticity has been observed in multiple tissues as a natural response to wounding and an unnatural feature of cancer. But minor skin injuries are the best place to study the phenomenon, because the skins outer layers are subject to perpetual abuse. And when the scratches or abrasions damage the epidermis, hair follicle stem cells are the first responders.

Fuchs and colleagues began to look more closely at lineage plasticity because it, can act as a double-edged sword, explains Matthew Tierney, lead author on the paper and an NIH K99 pathway to independence postdoctoral awardee in the Fuchs lab. The process is necessary to redirect stem cells to parts of the tissue most in need but, if left unchecked, it can leave those same tissues vulnerable to chronic states of repair and even some types of cancer.

To better understand how the body regulates this process, Fuchs and her team screened small molecules for their ability to resolve lineage plasticity in cultured mouse hair follicle stem cells, under conditions that mimicked a wound state. They were surprised to find that retinoic acid, a biologically active form of vitamin A, was essential for these stem cells to exit lineage plasticity and then be coaxed to differentiate into hair cells or epidermal cells in vitro.

Through our studies, first in vitro and then in vivo, we discovered a previously unknown function for vitamin A, a molecule that has long been known to have potent but often puzzling effects on skin and many other organs, Fuchs says. The team found that genetic, dietary, and topical interventions that boosted or removed retinoic acid from mice all confirmed its role in balancing how stem cells respond to skin injuries and hair regrowth. Interestingly, retinoids did not operate on their own: their interplay with signaling molecules such as BMP and WNT influenced whether the stem cells should maintain quiescence or actively engage in regrowing hair.

The nuance did not stop there. Fuchs and colleagues also demonstrated that retinoic acid levels must fall for hair follicle stem cells to participate in wound repairif levels are too high, they fail to enter lineage plasticity and cant repair woundsbut if the levels are too low, the stem cells focus too heavily on wound repair, to the expense of hair regeneration.

This may be why vitamin As effects on tissue biology have been so elusive, Fuchs says.

One result of retinol biology remaining obscure for so long is that retinoid and vitamin A applications have long produced confusing results. Topical retinoids are known to stimulate hair growth in wounds, but excessive retinoids have been shown to prevent hair cycling and cause alopecia; both positive and negative effects of retinoids on epidermal repair have been documented through various studies. The present study brings greater clarity by casting retinoids in a more central roleat the helm of regulating both hair follicle and epidermal stem cells.

By defining the minimal requirements needed to form mature hair cell types from stem cells outside the body, this work has the potential to transform the way we approach the study of hair biology, Tierney says.

How retinoids impact other tissues remains to be seen. When you eat a carrot, vitamin A gets stored in the liver as retinol where it is sent to various tissues, Fuchs says. Many tissues that receive retinol and convert it to retinoic acid need wound repair and use lineage plasticity, so it will be interesting to see how broad the implications of our findings in skin will be.

The Fuchs lab is also interested in how retinoids impact lineage plasticity in cancer, particularly squamous and basal cell carcinoma. Cancer stem cells never make the right choicethey are always doing something off-beat, Fuchs says. As we were studying this state in many types of stem cells, we began to realize that, when lineage plasticity goes unchecked, its a key contributor to cancer.

Basal cell carcinomas have relatively little lineage plasticity and are far less aggressive than squamous cell carcinomas. If future studies demonstrate that suppressing lineage plasticity is key to controlling tumor growth and improving outcomes, retinoids may have a key role to play in treating these cancers.

Its possible that suppressing lineage plasticity can improve prognoses, Fuchs says. This hasnt been on the radar until now. Its an exciting front to now investigate.

Reference: Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices by Matthew T. Tierney, Lisa Polak, Yihao Yang, Merve Deniz Abdusselamoglu, Inwha Baek, Katherine S. Stewart and Elaine Fuchs, 8 March 2024, Science. DOI: 10.1126/science.adi7342

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Vitamin A's Puzzling Effects Unraveled: New Research Sheds Light on Stem Cell Repair Mechanisms - SciTechDaily

Synthetic Circuits Reveal the Key to Rewinding the Cellular Clock – The Scientist

Most people wonder how their lives would change if they could turn back time and remake past decisions. While a seemingly impossible feat, stem cell biologists Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University first turned back the cellular clock in 2006.1 By overexpressing four transcription factors in fully differentiated fibroblasts, Takahashi and Yamanaka reprogrammed the cells to a pluripotent state and called them induced pluripotent stem cells (iPSC).

Although researchers employ iPSC in the laboratory and the clinic, scientists struggle to efficiently produce large quantities of iPSC.2 Reprogramming is still very inefficient, said Thorsten Schlaeger, a stem cell biologist at the Boston Childrens Hospital. It is still not fully understood why 98 or 99.9 percent of the cells do not end up reprogramming into iPSC. In a recently published Science Advances paper, Schlaeger and his team developed a system for tracking the fate of cells with different transcription factor expression dynamics during reprogramming.3 These findings will enable researchers in the field to improve iPSC yield.

Scientists suspected that the heterogeneity in reprogramming outcomes results from variations in the levels and durations of transcription factors expression. Consequently, several research groups have attempted to correlate the success of reprogramming to the levels of the octamer-binding transcription factor 4 (OCT4), which is one of Takahashi and Yamanakas transcription factors that is essential for the reprogramming process. However, these studies used population-based measurements and failed to consider the contribution of endogenous OCT4 to reprogramming.

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Compelled to overcome these limitations, Schlaegers coauthor Domitilla Del Vecchio, a discipline-straddling mechanical engineer at the Massachusetts Institute of Technology, developed an innovative OCT4 expression system. The idea really was to try to use a more sophisticated way of overexpressing transcription factors to reprogram stem cells, Del Vecchio recalled.

Del Vecchios team designed a synthetic gene circuit to ectopically overexpress a fluorescently-tagged version of the OCT4 transcription factor, while simultaneously blocking the expression of the endogenous OCT4 through microRNA. This allowed the researchers to control the total OCT4 protein levels within the cell and quantify them by measuring the fluorescence. Additionally, the ectopic OCT4 gene was controlled by an inducible and noisy promoter, which meant that the system generated variability in the expression of the OCT4 conjugate and a broad range of trajectories to assess, such as cells that maintained high OCT4 expression throughout reprogramming or those whose expression decreased over time.

Thorsten Schlaeger and Domitilla Del Vecchio developed a synthetic gene circuit-based system that allowed them to control and monitor the total OCT4 protein levels within fibroblasts during reprogramming.

Thorsten Schlaeger and Domitilla Del Vecchio

To determine which OCT4 trajectories successfully reprogrammed human dermal fibroblasts into iPSC, Del Vecchio, Schlaeger, and their team transduced the cells with lentiviral vectors encoding their OCT4 trajectory generator and followed the levels of fluorescently-tagged OCT4 proteins within the cells over time through imaging. The researchers then fixed the resulting cell colonies and immunostained them for two pluripotent stem cell surface markers.

They observed that the colonies fell into three categories: type I colonies were positive for only one of the surface markers; type II colonies showed the exact opposite staining pattern from type I; and type III colonies were positive for both markers. They considered cells within type III colonies as iPSC and categorized the cells within type I and II colonies as incompletely reprogrammed. Despite these differences, cells in all three colony types stably expressed supraphysiological levels of OCT4 during reprogramming, indicating that successfully reprogramming human dermal fibroblasts into iPSC requires consistently high levels of the OCT4 transcription factor. But this parameter alone is not sufficient to promote iPSC generation.

The paper is innovative in a technical sense. It is consistent with work that has been done showing that elevated levels of OCT4 are important for the reprogramming process, said Dean Tantin, a geneticist at the University of Utah, who was not involved in the study.

Although Tantin thought Del Vecchios system presented a clever way to directly examine total OCT4 protein levels within live cells, he suggested that examining protein levels alone may not convey the whole story. The level of a protein based on a fluorescent marker is not the same as the activity of a proteinits ability to bind DNA [or] its ability to regulate transcription once it binds," he noted. "So, I think where the field needs to go now is [to find out] how OCT4 activity is really dynamically regulated during [reprogramming]. he noted. Building on this idea, Tantin and his team recently determined that OCT4 activity during reprogramming and differentiation is redox-regulated, and he suspects that regulation of other reprogramming components will be of interest in the years to come.4

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Del Vecchio hopes that her work will inspire other researchers to think beyond the standard methods they employ to dissect molecular pathways. This study is showing how more sophisticated genetic engineering tools can be used in the context of a highly complex biological process and help you get information that will be difficult to get otherwise, Del Vecchio said.

Schlaeger wants to leverage the knowledge gained in this study to develop off-the-shelf iPSC-based therapies, such as CAR T cells, and he believes that precision engineering will be the key to safely bringing these products to the clinic. With the cells, we want to get to this precise control and that can only be done with complex genetic switches and circuits, Schlaeger said.

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Synthetic Circuits Reveal the Key to Rewinding the Cellular Clock - The Scientist

PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived … – Nature.com

Generation of homozygous PLEKHM2-KO hiPSCs and differentiating into cardiomyocytes

The generation of homozygous PLEKHM2-KO hiPSCs was carried out using the CRISPR-Cas9 system, and the guide RNA (gRNA) was designed and synthesized to target exon 2 of PLEKHM2 (Fig. 1A). Subsequent screening confirmed the successful knockout of PLEKHM2 gene, which revealed a one-nucleotide deletion in one allele and a one-nucleotide insertion in another allele (Fig. 1B and Supplementary Fig. 1B). The cell line under investigation was found to express the human pluripotency markers SSEA4 and OCT4 (Fig. 1C) and tested negative for mycoplasma contamination (Supplementary Fig. 1C). Western blot (WB) analyses revealed the absence of PLEKHM2 protein expression in PLEKHM2-KO hiPSC-CMs at day 20 (Fig. 1D). Wild-type (WT) and PLEKHM2-KO hiPSCs were differentiated into cardiomyocytes using the small molecule-based method (Fig. 1E). The efficacy of hiPSC-CMs differentiation was evaluated using flow cytometry, which indicated that PLEKHM2-KO and WT hiPSC-CMs exhibited similar proportions of cardiac Troponin T (cTnT)-positive cells (around 93%) at day 20 post differentiation (Fig. 1F, G). These results indicate that the PLEKHM2-KO hiPSC-CMs were successfully constructed.

A The PLEKHM2 gene structure and the location of the guide RNA (gRNA) used for epigenome editing with CRISPR/Cas9. B Sequencing analysis confirmed a homozygous PLEKHM2-KO hiPSC line with a 1-nucleotide deletion in one allele and a 1-nucleotide insertion in the other allele. C Pluripotent stem cell markers SSEA4 and OCT4 were detected by immunofluorescence staining in PLEKHM2-KO colonies. Scale bar, 20 m. D Western blot analysis of PLEKHM2 in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at day 20. E Protocol of small molecule-based methods to induce cardiac differentiation. F, G Flow cytometry analysis for cTnT from representative WT and PLEKHM2-KO differentiation at day 20. The results are presented as meansSD of 3 independent experiments. N.S. not significant.

We next investigate the dynamic changes in myocardial contractility and calcium transients of PLEKHM2-KO hiPSC-CMs. The HCell series single myocardial cell function detection system was utilized to measure myocardial contractility [10] (Supplementary Fig. 2A), and the green fluorescent calcium-modulated protein 6 fast type (GCaMP6f) calcium imaging system was employed to track myocardial calcium transients [11] (Supplementary Fig. 2B, C).

At the early stage of myocardial differentiation, specifically on 20 day, no significant alterations in myocardial contractility were observed between the WT hiPSC-CMs and the PLEKHM2-KO hiPSC-CMs. But at day 30, PLEKHM2-KO hiPSC-CMs exhibited a minor reduction in systolic displacement, as well as systolic and diastolic velocities compared to WT hiPSC-CMs, but no change in contractile force. And at day 40, the systolic displacement, contractile force, as well as systolic and diastolic velocities were significantly reduced in PLEKHM2-KO hiPSC-CMs compared to the WT hiPSC-CMs (Fig. 2AE, and Supplementary Fig. 2D, E), showing that the PLEKHM2-KO hiPSC-CMs developed systolic dysfunction phenotype. Calcium transient is a principal mechanism responsible for myocardial contraction, wherein the magnitude of contraction force is contingent upon variations in calcium ion concentration within the cell [12]. Hence, we next evaluated the alterations in calcium transients in the myocardium. In accordance with the trend observed in myocardial contractility, no significant variation was detected in calcium transient of PLEKHM2-KO hiPSC-CMs during the early phase post myocardial differentiation. However, a decline in calcium transient amplitude was observed at day 30, alongside a decrease in upstroke and recovery velocity of calcium transients in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs, and further exacerbated by 40 day (Fig. 2FJ, and Supplementary Fig. 2F). Interestingly, we also found that compared to WT hiPSC-CMs, the baseline values of calcium transients in PLEKHM2-KO hiPSC-CMs showed a significantly increased at day 40, indicating abnormal calcium handling in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 2G). These results suggest that abnormal calcium handling is a potential cause of the impaired myocardial contractility in PLEKHM2-deficient cardiomyopathy.

A Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40. BE Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group). F Representative line scan images of calcium transients in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40. GJ Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group). K Quantitative PCR analysis of heart failure and calcium handling -related genes in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 40. Data are shown as meanSD of 3 independent experiments. L Representative immunofluorescence staining and transmission electron microscope (TEM) of sarcomeric. M Quantification of complete organization, intermediate disorganization, and complete organization in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at days 40 based to immunofluorescence staining (more than 120 cells per group). Scale bar, 10 m. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

Subsequently, we evaluated the expression of key genes involved in heart failure and calcium handling. We found a significant increase in the expression of both NPPB and the MYH7/MYH6 ratio in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs, whereas ryanodine receptor 2 (RYR2) expression significantly decreased at day 40 (Fig. 2K). Moreover, we observed significantly disordered sarcomeres in PLEKHM2-KO hiPSC-CMs at day 40 (Fig. 2L, M). Moreover, transmission electron microscopy (TEM) showed significant abnormalities in the myofilaments of PLEKHM2-KO hiPSC CMs, including disordered myofilament arrangement and blurred Z-disc morphology (Fig. 2L). Overall, these findings corroborate the strong link between PLEKHM2 deficiency and DCM, which manifests as reduced contractility and impaired calcium handling, along with sarcomeric disorganization and dysregulated expression of heart failure markers.

To assess for potentially pathogenic effects of PLEKHM2-deficient cardiomyopathy, we performed quantitative transcriptome profiling by RNA-seq (Supplementary Fig. 3AC). We identified 8725 differentially expressed genes in PLEKHM2-KO hiPSC-CMs versus WT hiPSC-CMs at day 40, including 4426 upregulated and 4299 downregulated genes (Fig. 3A). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that these dysregulated genes were enriched in pathways mainly involved in regulating autophagy, lysosome, cardiomyopathy, apoptosis and metabolism (Fig. 3B). Of particular a significant finding was that the dysregulated genes were enriched in autophagy with the highest enrichment score in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs (Fig. 3B). The molecular-level analysis of Gene Ontology (GO) enrichment demonstrated a marked dysregulation in gene expression related to mitochondria, apoptosis, and autophagy in PLEKHM2-KO hiPSC-CMs (Fig. 3C, D). Notably, mitochondria-related pathways show the most significant differences between PLEKHM2-KO and WT hiPSC-CMs (Fig. 3C). Overall, these results indicate that PLEKHM2 deficiency leads to widespread dysregulation of signaling pathways in cardiomyocytes. Subsequently, we conducted quantitative PCR to validate the expression of genes associated with substantial dysregulation of mitochondria, apoptosis, and autophagy in RNA seq. Our results indicate a significant downregulation of BNIP3, DNM1L, OPA1, and MFN1 in addition to an upregulation of TSPO expression in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs at day 40 (Fig. 3E). It is widely acknowledged that BNIP3 and TSPO participate in various physiological processes such as mitophagy, apoptosis, oxidative stress, and the oxidative respiratory chain [13, 14]. While DNM1L, OPA1, and MFN1 play crucial roles in maintaining and regulating mitochondrial morphology and stability [15, 16]. The observed dysregulation in these genes highlight a disruption of mitochondrial homeostasis in PLEKHM2-deficient cardiomyopathy.

A Volcano plot shows 8725 genes with altered expression in PLEKHM2-KO hiPSC-CMs compared with WT. Blue and red dots indicate genes with increased and decreased expression, respectively, based on a P value<0.05 and a log2 fold change >1 (n=3 for each group). B Enrichment analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) databases revealed that pathways related to lysosomal function, autophagy, cardiomyopathy, apoptosis and metabolism were disrupted in PLEKHM2-KO hiPSC-CMs. ARVC, arrhythmogenic right ventricular cardiomyopathy. C Gene Ontology (GO) enrichment analysis showed significant changes in gene expression associated with mitochondrial function, apoptosis and autophagy in PLEKHM2-KO hiPSC-CMs. The color scale indicates the P values of the top 15 altered pathways in GO molecular function and the bubble size reflects the number of genes involved in each pathway. D Heatmap of differentially expressed genes involved in mitochondrial function, apoptosis and autophagy. E Quantitative PCR confirmed the altered expression of a representative subset of genes identified by RNA sequencing in PLEKHM2-KO hiPSC-CMs. Data are shown as meanSD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

RNA-seq analysis revealed notable anomalies in autophagy and mitochondrial-related pathways, as indicated by significant findings in the KEGG and GO enrichment analysis. Following this discovery, we proceeded to investigate alterations in mitochondrion and autophagic processes. To further investigate the impacts of PLEKHM2 deficiency on the mitochondrion, we next assessed mitochondrial morphology and content using Mitotracker at day 40. Compared to the typical linear arrangement of mitochondria along sarcomeres in WT hiPSC-CMs, the mitochondria within PLEKHM2-KO hiPSC-CMs display distinctive fragmented and punctate patterns, along with irregular distribution throughout the cytoplasm (Fig. 4A, and Supplementary Fig. 4A, B). TEM revealed matrix swelling, empty spaces, and loose, disordered, and wider cristae in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 4A, C). This suggest that the PLEKHM2 deficiency significantly affects the localization and tissue structure of mitochondria in cardiomyocytes. Mitochondrial morphology disruption usually trigger mitophagy, which targets damaged or dysfunctional mitochondria for degradation and clearance from the cell, and the number of mitochondria within the cell usually decrease due to their removal [17]. However, further analysis using flow cytometry revealed an increasing mitochondrial content within the PLEKHM2-KO hiPSC-CMs, compared to the WT hiPSC-CMs (Fig. 4B, C). These results suggested that mitochondrial morphological abnormalities and impaired mitochondrial clearance occur in PLEKHM2-KO hiPSC-CMs.

A Mitotracker staining revealed that PLEKHM2-KO altered the mitochondrial structure from the filamentous form aligned with the sarcomere in WT hiPSC-CMs to a punctate and fragmented morphology at day 40. Scale bar, 10 m. B, C Quantification of Mitotracker green intensity obtained by flow cytometry demonstrates a significantly increased fluorescence intensity in PLEKHM2-KO hiPSC-CMs at day 40 as compared with WT hiPSC-CMs (n=4). DF Autophagic flux was assessed in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs using mRFP-EGFP-LC3 adenovirus and subjected them to starvation medium for 0, 1, 2, and 4h at day 40. Representative images and quantification of GFP+, RFP+, and GFP, RFP+ puncta are shown in (D)(F). 12 cells per cell line per condition were analyzed. Scale bars, 10 m. G, H Representative western blot and quantification of P62 expression in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at day 40 (n=3). CQ: chloroquine. Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

The damaged mitochondria undergo degradation and digestion with the participation of lysosomes [4], while impaired autophagy or lysosomal acidification disorders usually impair mitophagy, resulting in delayed clearance of damaged mitochondria [18]. Consequently, we proceeded to investigate alterations in lysosomal localization and autophagy in the PLEKHM2-KO hiPSC-CMs. In this study, we utilized LAMP1 as a marker to identify lysosomal localization. Our results indicate that lysosomes in PLEKHM2-KO hiPSC-CMs exhibit significant clustering around the nucleus, whereas the lysosomes in the WT hiPSC-CMs demonstrate scattered distribution throughout the cytoplasm (Supplementary Fig. 4C, D), which is consistent with previous study [2]. To investigate the effects of PLEKHM2 deficiency on autophagy, we next monitored alterations in autophagic flux at 0, 1, 2, and 4hours post-starvation. Day 3 after Ad-mRFP-EGFP-LC3 infection, we observed a significant accumulation of autophagosomes (GFP+/RFP+ puncta) with increasing starvation duration in both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs. Notably, at the 2-hour after starvation, the number and proportion of autophagosome-lysosome fusion (GFP-/RFP+ puncta) in PLEKHM2-KO hiPSC-CMs was significantly lower than that of the WT hiPSC-CMs, indicating that the autophagic degradation of PLEKHM2-KO hiPSC-CMs was impaired (Fig. 4DF). In this study, we observed that in the late phase of autophagy (4hour after staving), most autophagosomes in the WT group accumulated around the nucleus and fused with lysosomes to form autolysosomes [19]. However, in the PLEKHM2-KO hiPSC-CMs, a substantial number of autophagosomes remained scattered within the cytoplasm and were not yet concentrated around the nucleus (Supplementary Fig. 4E), suggesting that PLEKHM2 deficiency affected the aggregation of autophagsomes to the perinuclear and fusion with lysosomes. In the subsequent WB results, we also observed that PLEKHM2 deficiency led to accumulation of p62 (Fig. 4G, H, and Supplementary Fig. 4F, G). In summary, these findings suggested that PLEKHM2 deficiency lead to abnormal lysosomal localization and blocking of autophagic flux, resulting in impaired autophagy and damaged mitochondrial accumulation.

Mitophagy is a fundamental cellular self-cleaning mechanism that plays a critical role in maintaining mitochondrialfunction and preventing the accumulation of reactive oxygen species (ROS) by selectively removing damaged mitochondria [20, 21]. m is a crucial indicator of mitochondrial health and function. To investigate the impact of PLEKHM2 deficiency on mitochondrial function, the carbocyanine compound JC-1, a fluorescent voltage-sensitive dye that possesses membrane-permeant fluorescent lipophilic cationic properties, was utilized to assess m and mitochondrial health. Our results revealed that JC-1 in PLEKHM2-KO hiPSC-CMs, exhibited a robust red fluorescence and weak green fluorescence similar to the WT hiPSC-CMs at day 20. However, over time, the red fluorescence of JC-1 in the PLEKHM2-KO hiPSC-CMs decreased gradually, while the green fluorescence increased (Fig. 5A). Notably, at day 30 and 40, the ratio of aggregate to monomeric JC-1 fluorescence in the PLEKHM2-KO hiPSC-CMs significantly reduced compared to that of the WT hiPSC-CMs (Fig. 5B). Futhrtmore, the destabilization in m lead to the release of cytC from mitochondria, which activated the caspase-3 in PLEKHM2-KO hiPSC-CMs at 40 day (Supplementary Fig. 5AC). These results suggest that PLEKHM2 deficiency leads to extensive depolarization of m and impaired mitochondrial function.

A, B Representative immunofluorescence staining and quantification of JC-1 revealed that mitochondrial monomers (green fluorescence) increased and the mitochondrial aggregates (red fluorescence) decreased gradually in PLEKHM2-KO hiPSC-CMs compared to WT at day 20, 30, and 40 (more than 120 cells per group). Scale bar, 10 m. C Heatmap of differentially expressed genes involved in oxidative stress in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs. D GSEA analysis revealed dysregulation of the respose to oxidative stress signaling pathway in PLEKHM2-KO hiPSC-CMs. E, F Representative flow cytometry analysis and quantification of cell reactive oxygen species (ROS) intensity demonstrated a continuous increased fluorescence intensity in PLEKHM2-KO hiPSC-CMs at days 20, 30, and 40 as compared with WT hiPSC-CMs. G, H Oxygen consumption rate (OCR) of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs at 40 day was measured using a seahorse analyzer. Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Numerous studies have shown that ROS plays a crucial role in inducing widespread m depolarization by directly triggering mPTP opening. And mPTP opening can further enhance ROS production by impairing m, ultimately triggering a vicious cycle of m depolarization and ROS production [22,23,24]. RNA-seq suggested significant changes in the expression gene profile associated with oxidative stress in PLEKHM2-KO hiPSC-CMs compared to WT hiPSC-CMs (Fig. 5C, D). Thus, to investigate whether the PLEKHM2 deficiency leads to an increase in ROS levels, we assessed the dynamic changes in ROS levels in PLEKHM2-KO hiPSC-CMs. We found a continuous increase in ROS levels in PLEKHM2-KO hiPSC-CMs than WT hiPSC-CMs (Fig. 5E, F), which indicated that PLEKHM2 deficiency causes progressive oxidative stress in hiPSC-CMs. To further investigate the effect of PLEKHM2 deficiency on mitochondrial OXPHOS activity, the oxygen consumption rates (OCR) of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs was analyzed at day 40 (Fig. 5G). These kinetic results revealed that PLEKHM2 deficiency significantly impaired ATP production, basal respiration and spare capacity (Fig. 5H). These results suggest that PLEKHM2 deficiency causes extensive mitochondrial dysfunction.

Previous studies have shown a strong link between oxidative stress and cardiomyopathy. To investigate whether ROS plays an important role in the pathogenesis of PLEKHM2-deficient cardiomyopathy, we administered oxidative stress activator, lipopolysaccharides (LPS) to WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs, and observed the effects on myocardial mitochondrial function, calcium handling, and contractility at day 40. After LPS administration, both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs exhibited significantly higher levels of ROS than untreated CMs (Fig. 6A, B). Next, the JC-1 was utilized to assess the effect of LPS treatment on mitochondrial function. Our results showed that LPS treatment induced the same mitochondrial dysfunction phenotype in WT hiPSC-CMs as in PLEKHM2-KO hiPSC-CMs. Moreover, LPS treatment exacerbated the m destabilization in PLEKHM2-KO hiPSC-CMs compared to untreated hiPSC-CMs (Fig. 6C, D). To investigate whether oxidative stress accelerates the progression of PLEKHM2-deficient cardiomyopathy, we evaluated the effects of LPS administration on the calcium transient and myocardial contractility of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs. We found that LPS treatment decreased calcium transients (Fig. 6EI, and Supplementary Fig. 6) in both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs compared to untreated hiPSC-CMs. And LPS treatment significantly decreased myocardial contractility (Fig. 6JN) in WT hiPSC-CMs compared to untreated hiPSC-CMs. These results suggested that oxidative stress may play a significant role in mitochondrial dysfunction, abnormal calcium handling and impaired myocardial contractility in the development of PLEKHM2-deficient cardiomyopathy.

A, B Representative flow cytometry analysis and quantification of cellular ROS levels showed that both WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs had significantly increased ROS production compared to untreated controls after LPS exposure (n=4 independent experiments). C, D Representative immunofluorescence staining and quantification of JC-1 revealed that LPS treatment impaired mitochondrial membrane potential of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs, as indicated by increased green fluorescence (monomeric form) and decreased red fluorescence (aggregated form) of JC-1 (more than 120 cells per group). Scale bar, 10 m. E Representative line scan images of calcium transients in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40. FI Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs (n=12 cells per group) with or without LPS treatment at day 40. J Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40. KN Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without LPS treatment at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Reduced glutathione (GSH) is an important antioxidant helps to prevent and reduce oxidative stress by neutralizing free radicals, widely used in the treatment of various types of oxidative stress-related diseases, including neurodegenerative diseases, cardiovascular diseases, and diabetes [25]. Hence, we treated PLEKHM2-KO hiPSC-CMs with GSH at day 30 to observe whether it could rescue the disease phenotype caused by PLEKHM2 deficiency. We found that PLEKHM2-KO hiPSC-CMs treated with GSH exhibited a considerable reduction in ROS levels (Fig. 7A, B) and significantly elevation of m level compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 7C, and Supplementary Fig. 7A). This indicates that inhibiting ROS helps improve mitochondrial function by preventing oxidative stress-induced damage to nearby mitochondria. Next, we observed the effects of GSH on the calcium transient and myocardial contractility of PLEKHM2-KO hiPSC-CMs. After GSH treatment, the diastolic calcium concentration and recovery velocity of PLEKHM2-KO hiPSC-CMs were comparable to that of WT hiPSC-CMs (Fig. 7D-H, and Supplementary Fig. 7B). Similarly, after GSH treatment, the PLEKHM2-KO hiPSC-CMs also showed significant improvements in contractile force (Fig. 7IM). These results further suggested the critical role of oxidative stress in mediating the disease phenotype of PLEKHM2-deficient cardiomyopathy.

A, B Representative flow cytometry analysis and quantification of cellular ROS levels showed that underwent GSH treatment PLEKHM2-KO hiPSC-CMs exhibited a considerable reduction in ROS levels comparable to WT hiPSC-CMs (n=4 independent experiments). C Quantification of JC-1 revealed that GSH treatment increased significantly the m level in PLEKHM2-KO hiPSC-CMs (more than 120 cells per group). D Representative line scan images of calcium transients of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40. EH Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40 (n=12 cells per group). I Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40. JM Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without GSH treatment at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S., not significant.

Previous studies have shown that PLEKHM2 deficiency lead to abnormal lysosomal localization and impaired autophagic flux [2], causing damaged mitochondrial accumulation and ROS production. mTORC1 signaling pathway is the main negative regulator of autophagy, inhibiting autophagy by phosphorylating and inactivating key autophagy proteins such as ULK1 and ATG13 [26]. Hence, we investigated whether inhibition of mTORC1 by RAPA could boost autophagy and rescue the mitochondrial dysfunction and ROS generation in PLEKHM2-KO hiPSC-CMs. We found that p-mTOR levels were significantly higher in PLEKHM2-KO hiPSC-CMs than WT hiPSC-CMs (Supplementary Fig. 8A, B). Administration of RAPA significantly reduced the p-mTOR levels in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 8A, B). We further observed the effect of RAPA on autophagic flux in PLEKHM2-KO hiPSC-CMs. RAPA increased the number of autophagosomes (GFP+/RFP+ puncta) and autophagolysosome (GFP-/RFP+ puncta) in PLEKHM2-KO hiPSC-CMs, indicating that RAPA induced autophagy (Supplementary Fig. 8C). However, the number and proportion of GFP-/RFP+ puncta in PLEKHM2-KO hiPSC-CMs was still significantly lower than that WT hiPSC-CMs (Supplementary Fig. 8C), indicating that rapamycin cannot completely improve obstruction of autophagic flux caused by PLEKHM2 deficiency. We then evaluated the effects of RAPA treatment on mitochondrial function and myocardial contractility in PLEKHM2-KO hiPSC-CMs at day 40. We found that RAPA treatment partially improved m level and reduced ROS generation in PLEKHM2-KO hiPSC-CMs (Supplementary Fig. 8EG). Next, we observed the effects of RAPA treatment on myocardial contraction and calcium transient in PLEKHM2-KO hiPSC-CMs. We found that RAPA treatment enhanced the calcium transient amplitude (Supplementary Fig. 8HL) of PLEKHM2-KO hiPSC-CMs, but the myocardial contractility (Supplementary Fig. 8MR) was still significantly lower than those in WT hiPSC-CMs. These results indicated that administering RAPA cannot completely correct impaired autophagy caused by PLEKHM2 deficiency, but partially improves the disease phenotype of PLEKHM2-deficient cardiomyopathy.

We next investigated whether PLEKHM2-WT overexpression could restore autophagic flux in PLEKHM2-KO hiPSC-CMs and rescued the disease phenotype of PLEKHM2-deficient cardiomyopathy. We found that PLEKHM2-WT overexpression corrected the abnormal lysosomal localization (Supplementary Fig. 9) and increased the number and proportion of GFP-/RFP+ puncta in PLEKHM2-KO hiPSC-CMs, compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 8A, B). This indicates that PLEKHM2-WT overexpression improve the autophagic degradation in PLEKHM2-KO hiPSC-CMs. We further observed the effects of PLEKHM2-WT overexpression on the mitochondrial function of PLEKHM2-KO hiPSC-CMs. PLEKHM2-KO hiPSC-CMs treated with PLEKHM2-WT overexpression exhibited a significant increase in the m level and decrease in ROS levels compared to untreated PLEKHM2-KO hiPSC-CMs (Fig. 8CF). Subsequently, we evaluated the effects of PLEKHM2-WT overexpression on the calcium transient and myocardial contractility of PLEKHM2-KO hiPSC-CMs. PLEKHM2-WT overexpression significantly enhanced calcium transient amplitude (Fig. 8GK) and myocardial contractility (Fig. 8LP) compared to untreated PLEKHM2-KO hiPSC-CMs. This further demonstrates that PLEKHM2 plays a crucial role in regulating autophagy and clearing damaged mitochondria.

A, B Autophagic flux was assessed in hiPSC-CMs using mRFP-EGFP-LC3 adenovirus. Representative images and quantification of GFP and RFP+ puncta are shown in (A) and (B). C, D Representative immunofluorescence staining and and quantitative analysis of JC-1 revealed that PLEKHM2-WT overexpression ameliorated m of PLEKHM2-KO hiPSCs-CMs (more than 70 cells per group). Scale bars, 10 m. E, F Representative flow cytometry analysis and quantification of cell reactive oxygen species (ROS) intensity demonstrated PLEKHM2-WT overexpression reduced ROS levels of PLEKHM2-KO hiPSCs-CMs. G Representative line scan images of calcium transients of WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40. HK Quantification of amplitude, diastolic Ca2+ concentration, upstroke and recovery velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40 (n=12 cells per group). L Representative line scan images of myocardial contractility in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40. MP Quantification of displacement, force, contraction and relaxation velocity in WT hiPSC-CMs and PLEKHM2-KO hiPSC-CMs with or without PLEKHM2-WT overexpression at day 40 (n=12 cells per group). Data are shown as meanSD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; N.S. not significant.

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