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MYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using isogenic human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, functional, and energetic changes caused by the MYBPC3D389V variant, which is associated with increased fractional shortening and highly prevalent in South Asian descendants. Recombinant C0-C2, N’ region of cMyBP-C (wild-type and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (??m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after the treatment of the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3D389V hCOs. Lastly, various vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by the administration of a myosin inhibitor.

The MAPT gene encodes Tau protein, a member of the large family of microtubule-associated proteins. Tau forms large insoluble aggregates that are toxic to neurons in several neurological disorders, and neurofibrillary Tau tangles represent a key pathological hallmark of Alzheimer’s disease (AD) and other tauopathies. Lowering Tau expression levels constitutes a potential treatment for AD but the mechanisms that regulate Tau expression at the transcriptional or translational level are not well understood. Natural antisense transcripts (NATs) are a particular class of long non-coding RNAs (lncRNAs) that can regulate expression of their overlapping protein-coding genes at the epigenetic, transcriptional, or translational level. We and others identified a long non-coding RNA associated with the MAPT gene, named MAPT antisense 1 (MAPT-AS1). We confirmed that MAPT-AS1 is expressed in neurons in human post mortem brain tissue. To study the role of MAPT-AS1 on MAPT expression regulation, we modulated the expression of this lncRNA in human neuroblastoma cell lines and in human induced pluripotent stem cell (iPSC) derived neurons. In contrast to previous reports, we observed no changes on MAPT mRNA or Tau protein levels upon modulation of MAPT-AS1 levels in these cellular models. Our data suggest that MAPT-AS1 does not regulate Tau expression levels in human neurons in vitro. Thus, MAPT-AS1 does not represent a valuable therapeutic target to lower Tau expression in patients affected by tauopathies including AD.

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans. Graphical abstractImage 1 Highlights•REST and RCOR genes display cell-type specific expression patterns in neural cells.•RCOR3 (encodes CoREST3) is upregulated during neuronal and astrocyte differentiation.•RCOR2 (encodes CoREST2) is downregulated during differentiation of astrocytes.•Evidence of potential cell-type specific functions of the CoREST family.

Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 106 cells/kg) and high-dose (10 × 106 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload.

SummaryMutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2–4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD. Graphical abstract Highlights•Myogenic differentiation of DMD hiPSCs diverges at the somite stage•Cell junction formation is dysregulated in DMD somite cells•Somite cells from DMD hiPSCs have impaired epithelialization properties•Migration velocity of DMD-mutant somite progenitors is upregulated Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Developmental biology.

AbstractAn intronic G4C2 repeat expansion in the C9ORF72 gene is the major known cause for Amyotrophic Lateral Sclerosis (ALS), with current evidence for both, loss of function and pathological gain of function disease mechanisms. We screened 96 200 small molecules in C9ORF72 patient iPS neurons for modulation of nuclear G4C2 RNA foci and identified 82 validated hits, including the Brd4 inhibitor JQ1 as well as novel analogs of Spliceostatin-A, a known modulator of SF3B1, the branch point binding protein of the U2-snRNP. Spliceosome modulation by these SF3B1 targeted compounds recruits SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. This leads to increased repeat-associated non-canonical (RAN) translation and ultimately, enhanced cell toxicity. Our data (i) provide a new pharmacological entry point with novel as well as known, publicly available tool compounds for dissection of C9ORF72 pathobiology in C9ORF72 ALS models, (ii) allowing to differentially modulate RNA foci versus RAN translation, and (iii) suggest that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery. Graphical Abstract Graphical Abstract