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.

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.

Efficacy of Intravascular Ultrasound-Based 3D Wiring Using the Tip Detection Method for CTO Intervention.

To perform intravascular ultrasound (IVUS)-based real-time 3-dimensional wiring in chronic total occlusion percutaneous coronary intervention, we devised a tip detection method and developed AnteOwl WR (AO)-IVUS, which is an upgraded version of Navifocus WR (Navi)-IVUS with an added pull back transducer system. We compared the procedural outcomes of AO-IVUS-based 3-dimensional wiring using the tip detection method (n = 30) and Navi-IVUS-based conventional wiring (n = 17) in chronic total occlusion percutaneous coronary intervention. The success rate of IVUS-guided wiring was markedly improved in the AO-IVUS group compared with the Navi-IVUS group (93% vs 59% of cases, respectively; P = 0.007). In cases of successful IVUS-guided wiring, the IVUS-guided wiring time was markedly improved in the AO-IVUS group compared with the Navi-IVUS group (9 ± 8 minutes vs 24 ± 26 minutes, respectively; P = 0.001). There were 2 successful cases of tip detection-antegrade dissection and re-entry in the AO-IVUS group.

Guide extension proximal locking method: standardization of maximum backup force in percutaneous coronary intervention.

We developed the smallest diameter guide-extension catheter (GUIDE PLUS® 5Fr) to enable a new technique, the guide-extension proximal locking method (GP-Lock), and assessed its efficacy in the present experimental and clinical study. Sufficient guide catheter backup is sometimes crucial for PCI. We developed the KIWAMI-Lock direct anchoring method to obtain the strongest backup force by locking a Kiwami® 4Fr child catheter (Terumo Corp. Tokyo, Japan) directly to the coronary artery by ballooning from outside the child catheter. However, this method is complicated due to the requirement for a child catheter. We compared the backup power of the GP-Lock method and other conventional methods in an experimental study and compared the procedural outcomes of 17 cases treated using the initial GP-Lock method with 17 cases using the recent KIWAMI-Lock method before GP-Lock. The GP-Lock method had the highest backup force among the methods examined (GP-Lock: 293.7 ± 10.2 g force (gf), KIWAMI-Lock: 270.4 ± 12.9 gf, side branch balloon anchoring technique: 182.7 ± 8.1 gf, respectively, P < 0.0001). The preparation time was significantly shorter for the GP-Lock group than the KIWAMI-Lock group (5.0 [4.0, 5.0] min vs. 11.0 [8.0, 13.0] min, respectively, P < 0.001). The GP-Lock method makes it possible to easily obtain the strongest backup force, which can overcome situations where devices cannot pass through, especially in complex PCI procedures.

End-stage Hypertrophic Cardiomyopathy with Advanced Heart Failure in Patients Carrying MYH7 R453 Variants: A Case Series.

The MYH7 R453 variant has been identified in inherited hypertrophic cardiomyopathy (HCM) and is associated with sudden death and a poor prognosis. The detailed clinical course of HCM with the MYH7 R453 variant, from a preserved to a reduced left ventricular ejection fraction, has not been reported. We identified the MYH7 R453C and R453H variants in three patients who progressively developed advanced heart failure requiring circulatory support and summarized the clinical course and echocardiographic parameters of these patients over the years. Because of the rapid disease progression, we consider genetic screening for patients with HCM imperative for future prognosis stratification.

Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption.

Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.

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.

Polycythemia Vera Associated with Pulmonary Hypertension and Diffuse Large B-Cell Lymphoma: A Case Report.

BACKGROUND Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), are associated with pulmonary hypertension (PH) and malignant lymphomas. Although the underlying mechanisms have not been completely clarified, it has been suggested that the Janus kinase 2 (JAK2) mutation, which is frequently identified in PV, can be involved in the development and/or progression of these distinct diseases in patients with MPNs. However, no reports have described the coexistence of PH and malignant lymphoma in patients with MPNs. CASE REPORT A 79-year-old man being treated for PV for 27 years and PH for 5 years was hospitalized due to severe dyspnea at rest. His soluble interleukin-2 receptor levels gradually increased and the chest computed tomography showed remarkable progression of the lung lesions and an enlargement of the mediastinal and axillary lymph nodes. A lymph node biopsy was performed and the patient was diagnosed with diffuse large B-cell lymphoma (DLBCL). Owing to his poor condition, chemotherapy was not initiated, and he died on the 89th day of hospitalization. The pathological autopsy revealed the destruction of alveolar structures with neoplastic space-occupying lesions of DLBCL. Multifactorial features of PH associated with MPNs, including the intimal thickening of pulmonary arteries accompanied by megakaryocytes and obstructed pulmonary arteries with organized thrombi in the lung tissue specimens, were observed. We found a JAK2 mutation based on a genetic analysis of the patient's bone marrow. CONCLUSIONS We present the rare case of a patient who had PV with a JAK2 mutation, which coexisted with PH and DLBCL, and he developed severe refractory respiratory failure.

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.

Guide catheter extension lock enables the strongest backup force during the antegrade approach in percutaneous coronary intervention.

Sufficient guide catheter backup is crucial for successful percutaneous coronary intervention (PCI). We have developed a new technique for locking a small child catheter to the vessel wall by balloon dilatation outside the child catheter at the proximal site of the main branch. We call this the "guide catheter extension lock" ("Kiwami® lock", Kiwami® child catheter, Terumo Corp. Tokyo, Japan). The guide catheter extension lock backup force was evaluated in an experimental model, and we report a case of chronic total occlusion treated with PCI using this technique. The guide catheter backup force was measured using an artificial cardiac vessel model with a right coronary artery and side branch. We evaluated the backup forces of the guide catheter extension lock and other techniques using an 8Fr Judkins right guide catheter (child catheter: 45.0 ± 4.6 g force [gf]; a 7Fr delivery catheter (Guide-Liner®, Vascular Solutions, Minneapolis MN, USA): 67.4 ± 14.4 gf; an anchoring balloon technique at the side branch: 98.3 ± 11.9 gf; and the guide catheter extension lock: 112.1 ± 13.0 gf). The guide catheter extension lock technique provided the strongest backup force. Our clinical experience shows that this technique is effective during PCI.