Human induced pluripotent stem cell-derived closed-loop cardiac tissue for drug assessment.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) exhibit functional immaturity, potentially impacting their suitability for assessing drug proarrhythmic potential. We previously devised a traveling wave (TW) system to promote maturation in 3D cardiac tissue. To align with current drug assessment paradigms (CiPA and JiCSA), necessitating a 2D monolayer cardiac tissue, we integrated the TW system with a multi-electrode array. This gave rise to a hiPSC-derived closed-loop cardiac tissue (iCT), enabling spontaneous TW initiation and swift pacing of cardiomyocytes from various cell lines. The TW-paced cardiomyocytes demonstrated heightened sarcomeric and functional maturation, exhibiting enhanced response to isoproterenol. Moreover, these cells showcased diminished sensitivity to verapamil and maintained low arrhythmia rates with ranolazine-two drugs associated with a low risk of torsades de pointes (TdP). Notably, the TW group displayed increased arrhythmia rates with high and intermediate risk TdP drugs (quinidine and pimozide), underscoring the potential utility of this system in drug assessment applications.

SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes.

Although an increased risk of myocarditis has been observed after vaccination with mRNA encoding severe acute respiratory syndrome coronavirus 2 spike protein, its underlying mechanism has not been elucidated. This study investigated the direct effects of spike receptor-binding domain (S-RBD) on human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs). Immunostaining experiments using ACE2 wild-type (WT) and knockout (KO) iPSC-CMs treated with purified S-RBD demonstrated that S-RBD was bound to ACE2 and internalized into the subcellular space in the iPSC-CMs, depending on ACE2. Immunostaining combined with live cell imaging using a recombinant S-RBD fused to the superfolder GFP (S-RBD-sfGFP) demonstrated that S-RBD was bound to the cell membrane, co-localized with RAB5A, and then delivered from the endosomes to the lysosomes in iPSC-CMs. Quantitative PCR array analysis followed by single cell RNA sequence analysis clarified that S-RBD-sfGFP treatment significantly upregulated the NF-kß pathway-related gene (CXCL1) in the differentiated non-cardiomyocytes, while upregulated interferon (IFN)-responsive genes (IFI6, ISG15, and IFITM3) in the matured cardiomyocytes. S-RBD-sfGFP treatment promoted protein ISGylation, an ISG15-mediated post-translational modification in ACE2-WT-iPSC-CMs, which was suppressed in ACE2-KO-iPSC-CMs. Our experimental study demonstrates that S-RBD is internalized through the endolysosomal pathway, which upregulates IFN-responsive genes and promotes ISGylation in the iPSC-CMs.

Tenascin-C as a potential marker for immunohistopathology of doxorubicin-induced cardiomyopathy.

Aims: Doxorubicin is used in classical chemotherapy for several cancer types. Doxorubicin-induced cardiomyopathy (DOX-CM) is a critical issue among cancer patients. However, differentiating the diagnosis of DOX-CM from that of other cardiomyopathies is difficult. Therefore, in this study, we aimed to determine novel histopathological characteristics to diagnose DOX-CM. Methods and results: Twelve consecutive patients with DOX-CM who underwent cardiac histopathological examination in two medical centres were included. Twelve patients with dilated cardiomyopathy, who were matched with DOX-CM patients in terms of age, sex, and left ventricular ejection fraction, formed the control group. Another control group comprised five consecutive patients with cancer therapy-related cardiac dysfunction induced by tyrosine kinase inhibitors or vascular endothelial growth factor inhibitors were the controls. The positive area of tenascin-C, number of infiltrating macrophages, and presence of p62- and ubiquitin-positive cardiomyocytes were evaluated. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used for in vitro investigation. The myocardium exhibited significantly greater tenascin-C-positive area and macrophage number in the DOX-CM group than in the control groups (P < 0.01). The tenascin-C-positive area correlated with the number of both CD68- and CD163-positive cells (r = 0.748 and r = 0.656, respectively). Immunostaining for p62 was positive in 10 (83%) patients with DOX-CM. Furthermore, western blotting analysis revealed significant increase in tenascin-C levels in hiPSC-CMs upon doxorubicin treatment (P < 0.05). Conclusion: The combined histopathological assessment for tenascin-C, macrophages, and p62/ubiquitin may serve as a novel tool for the diagnosis of DOX-CM. Doxorubicin may directly affect the expression of tenascin-C in the myocardium.

Case report: Usefulness of angioscopy in determining antiplatelet drug reduction after carotid artery stenting.

We report a case in which neointima was confirmed by angioscopy and antiplatelet drug administration was reduced 2 months after carotid artery stenting (CAS). A patient in their 80s was scheduled to undergo resection for renal cancer; however, he also had right cervical internal carotid artery stenosis. Because this was a risk for general anesthesia, CAS was performed after first starting dual antiplatelet therapy. Urologically, early reduction of antiplatelet drugs was necessary for a nephrectomy. Although no obvious neointima could be identified on ultrasound 2 months after CAS, thin neointima was observed using angioscopy. Based on the above results, we reduced the antiplatelet drug administration, and then the nephrectomy was performed. Ultimately, no cerebral infarction occurred in the perioperative or postoperative periods. Angioscopy allows for visual confirmation of thin neointima. If sufficient neointima can be confirmed, antiplatelet drug reduction can be performed more safely and reliably.

Modeling Reduced Contractility and Stiffness Using iPSC-Derived Cardiomyocytes Generated From Female Becker Muscular Dystrophy Carrier.

Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT-DMD, Δ45-48-DMD, or Δ45-48-DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48-DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier.

Peripartum Cardiomyopathy with the Cardiac Function Restored by Cabergoline.

Peripartum cardiomyopathy (PPCM) is a left ventricular systolic dysfunction associated with heart failure (HF) in late-term pregnancy or peripartum. A 29-year-old pregnant woman with no history of cardiac disease noted lower extremity edema around 34 weeks' gestation with significant weight gain. She delivered twins via caesarean section, and the edema regressed postpartum. On postpartum day 4, however, she experienced difficulty breathing at night and was diagnosed with HF owing to PPCM. HF treatment along with cabergoline was initiated. With low prolactin blood levels, her symptoms and cardiac function improved over time. This case demonstrated the usefulness of anti-prolactin therapy with cabergoline in PPCM.

Human-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for TNNT2 Δ160E-Induced Cardiomyopathy.

BACKGROUND: The Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development. METHODS: We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]). RESULTS: Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs. CONCLUSIONS: Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.

Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes.

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.

Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent stem cell-derived cardiomyocytes.

Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.

Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.

Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

Presence of increased inflammatory infiltrates accompanied by activated dendritic cells in the left atrium in rheumatic heart disease.

AIMS: Left atrial (LA) structural remodelling develops in rheumatic heart disease (RHD) according to the disease severity of the mitral valve and the presence of atrial fibrillation. Sustained active inflammation has been previously reported in the LA of patients with RHD, suggesting a direct role of cell-mediated immunity in the pathogenesis of LA remodelling. Dendritic cells (DCs) have a major antigen-presenting role, and are known as crucial modulators of innate and adaptive immunity. We investigated whether DCs are involved in the pathogenesis of LA remodelling in RHD. METHODS AND RESULTS: Immunohistochemical analyses were performed using antibodies to CD11c, CD209 and CD80 as markers of myeloid DCs, migratory-active DCs, mature DCs and infiltrated inflammatory cells including T lymphocytes (CD3) and M1 (CD68; pro-inflammatory profile) and M2 (CD163; pro-resolution profile) macrophages. Furthermore, tenascin-C, an extracellular matrix (ECM) protein that appears during ECM remodelling and inflammatory response, was examined. Infiltrated myeloid DCs, migratory-active DCs, mature DCs and other inflammatory infiltrates including T lymphocytes and M1 and M2 macrophages, were significantly higher in the RHD group than the non-RHD group. The positive area fraction for tenascin-C was significantly higher in the RHD group than in the non-RHD group. CONCLUSION: Our histological findings suggest that inflammation may persist long after a bout of rheumatic fever, ultimately leading to ECM remodelling. We identified and quantitatively assessed several subsets of DCs and other immunocompetent cells, and our results indicated that activation of DCs has some role in persistence of LA inflammation in patients with chronic RHD.

Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes.

Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in non-dividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2. Imaging-based sequential evaluation of endogenously tagged protein revealed that HDR occurs in cardiomyocytes, independently of DNA synthesis. We sought to repair a pathological mutation in Tnnt2 in cardiomyocytes of cardiomyopathy model mice. An sgRNA that avoided the mutated exon minimized deleterious effects on Tnnt2 expression, and AAV-mediated HDR achieved precise genome correction at a frequency of ~12.5%. Thus, targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.

Congenitally Corrected Transposition of the Great Arteries and Situs Inversus in an Octogenarian With Systemic Right Ventricular Failure.

Systemic right ventricular (RV) failure in patients with congenitally corrected transposition of the great arteries (ccTGA), a major cause of mortality in the long-term follow-up, is usually induced by concomitant severe morphologically tricuspid regurgitation (TR) with/without Ebstein's anomaly or progressive conduction tissue disturbances. However, whether or not myocardial fibrosis is a common cause of systemic RV failure in patients with ccTGA remains unclear. Here, we describe an 82-year-old man who had been diagnosed previously as having uncomplicated ccTGA and situs inversus and recently developed systemic RV failure, which was neither associated with severe TR nor advanced conduction tissue abnormalities. Cardiovascular magnetic resonance (CMR) with delayed-enhancement imaging clearly detected extensive myocardial scars (presumably fibrosis) in the RV wall as well as prominent dilatation, hypertrophy, and systolic dysfunction of the systemic RV. These findings suggest that myocardial fibrosis can cause systemic RV failure in elderly patients with uncomplicated ccTGA despite the absence of severe TR or advanced conduction tissue abnormalities and that CMR may be a useful examination to accurately detect systemic RV failure associated with myocardial fibrosis and to subsequently clarify the prognosis in these patients.