Decellularized Extracellular Matrix Powder Accelerates Metabolic Maturation at Early Stages of Cardiac Differentiation in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45-500 µm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (~70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Ion Channels and Transporters in Muscle Cell Differentiation

Investigations on ion channels in muscle tissues have mainly focused on physiological muscle function and related disorders, but emerging evidence supports a critical role of ion channels and transporters in developmental processes, such as controlling the myogenic commitment of stem cells. In this review, we provide an overview of ion channels and transporters that influence skeletal muscle myoblast differentiation, cardiac differentiation from pluripotent stem cells, as well as vascular smooth muscle cell differentiation. We highlight examples of model organisms or patients with mutations in ion channels. Furthermore, a potential underlying molecular mechanism involving hyperpolarization of the resting membrane potential and a series of calcium signaling is discussed.

Single-cell RNA sequencing reveals that BMPR2 mutation regulates right ventricular function via ID genes

Mutations in bone morphogenetic protein type II receptor (BMPR2) have been found in patients with congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH). Our study aimed to clarify whether deficient BMPR2 signalling acts through downstream effectors, inhibitors of DNA-binding proteins (IDs), during heart development to contribute to the progress of PAH in CHD patients. To confirm that IDs are downstream effectors of BMPR2 signalling in cardiac mesoderm progenitors (CMPs) and contribute to PAH, we generated Cardiomyocytes (CMs)-specific Id 1/3 knockout mice (Ids cDKO), and 12/25 developed mild PAH with altered haemodynamic indices and pulmonary vascular remodelling. Moreover, we generated ID1 and ID3 double-knockout (IDs KO) human embryonic stem cells that recapitulated the BMPR2 signalling deficiency of CHD-PAH iPSCs. CMs differentiated from induced pluripotent stem cells (iPSCs) derived from CHD-PAH patients with BMPR mutations exhibited dysfunctional cardiac differentiation and reduced Ca2+ transients, as evidenced by confocal microscopy experiments. Smad1/5 phosphorylation and ID1 and ID3 expression were reduced in CHD-PAH iPSCs and in Bmpr2+/– rat right ventricles. Moreover, Ultrasound revealed that 33% of Ids cDKO mice had detectable defects in their ventricular septum and pulmonary regurgitation. CMs isolated from the mouse right ventricles also showed reduced Ca2+ transients and shortened sarcomeres. Single-cell RNA(scRNA)-seq analysis revealed impaired differentiation of CMPs and downregulated USP9X expression in IDs KO cells compared with wild-type (WT) cells. We found that BMPR2 signals through IDs and USP9X to regulate cardiac differentiation, and the loss of ID1 and ID3 expression contributes to CM dysfunction in CHD-PAH patients with BMPR2 mutations.

ASB2 is a novel E3 ligase of SMAD9 required for cardiogenesis

Cardiogenesis requires the orchestrated spatiotemporal tuning of BMP signalling upon the balance between induction and counter-acting suppression of the differentiation of the cardiac tissue. SMADs are key intracellular transducers and the selective degradation of SMADs by the ubiquitin–proteasome system is pivotal in the spatiotemporal tuning of BMP signalling. However, among three SMADs for BMP signalling, SMAD1/5/9, only the specific E3 ligase of SMAD9 remains poorly investigated. Here, we report for the first time that SMAD9, but not the other SMADs, is ubiquitylated by the E3 ligase ASB2 and targeted for proteasomal degradation. ASB2, as well as Smad9, is conserved among vertebrates. ASB2 expression was specific to the cardiac region from the very early stage of cardiac differentiation in embryogenesis of mouse. Knockdown of Asb2 in zebrafish resulted in a thinned ventricular wall and dilated ventricle, which were rescued by simultaneous knockdown of Smad9. Abundant Smad9 protein leads to dysregulated cardiac differentiation through a mechanism involving Tbx2, and the BMP signal conducted by Smad9 was downregulated under quantitative suppression of Smad9 by Asb2. Our findings demonstrate that ASB2 is the E3 ligase of SMAD9 and plays a pivotal role in cardiogenesis through regulating BMP signalling.

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner

Background: Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RNA-binding motif protein 24 (RBM24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim isto study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. Methods: We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. Results: Although RBM24 -/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (e.g. ACTN2, TTN, and MYH10) were misspliced. Consequently, MYH6 cannot replace non-muscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the actin-binding domain (ABD; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24 -/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. Conclusions: RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

Nitro-Oleic Acid Inhibits Stemness Maintenance and Enhances Neural Differentiation of Mouse Embryonic Stem Cells via STAT3 Signaling

Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and β-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.