In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Surgical explantation of failed transcatheter heart valves: indications and results.

Given the recent surge in transcatheter heart valve replacement (THVR), cardiac surgeons will surely face the challenge of eventual explantation. The aim of this study was to determine indications for reoperation, while exploring pertinent technical aspects and survival after THV explantation in a cohort originally deemed high risk or even inoperable. Between February 2008 and March 2019, 31 patients with failed transcatheter aortic valve replacement (TAVR) underwent surgical explantations at our facility. Data were prospectively collected for retrospective analysis of procedural indications, technical issues, and postoperative survival. The major reason for TAVR removal was bioprosthetic valve failure (BVF) due to infective endocarditis (IE: 16/31 [51.6%]), non-structural (NSVD: 14/31 [45.2%]) and structural (SVD: 1/31 [3.2%]) valve deterioration accounting for the rest. Mean age at THV explantation was 76.3 ± 8.3 years, and median time from TAVR to explantation was 153 days (0 days-56.6 months). Median ICU and hospital stay were 6 days (1-44 days) and 23 days (8-62 days), respectively. Thirty-day and 1-year survival rates were 74.2% and 67.2%, respectively. Median follow-up interval after explantation was 364 days (3 days-80 months). Mean cardiopulmonary bypass time was 124.6 ± 46.8 min, and mean aortic cross-clamp time was 84.3 ± 32.9 min. There was no need for unplanned aortic root repair owing to tissue damage during dissection of the TAVR from surrounding tissue. The most common reason for THV explantation was (a) BVF for IE and (b) BVF secondary to NSVD. Although 30-day and 1-year mortality rates in this multimorbid cohort were predictably high, no procedural mortalities occurred.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Congenital heart disease risk loci identified by genome-wide association study in European patients.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

In vitro comparison of everting vs. non-everting suture techniques for the implantation of a supra-annular biological heart valve.

BACKGROUND: The aim of this study was to evaluate the hemodynamic effect of different suturing techniques for aortic valve replacement (AVR) in vitro. Whether or not the applied suturing technique impacts the outflow tract diameter by narrowing the annulus diameter was examined. METHODS: The commonly applied non-everting pledget forced suture technique (NE, n=13) was compared with an everting pledget forced suture (ET, n=13) for AVR using the 25 mm St. Jude Trifecta aortic valve. Hemodynamic parameters were obtained in a pulsatile flow simulator. A high speed camera captured the visual aspects of the suturing technique. RESULTS: Despite some kind of left ventricular outflow narrowing due to protruding pledgets using the NE suture technique, mean pressure gradients of both techniques were nearly similar (NE 5.88±2.7 mmHg, ET 5.23±1.31 mmHg, P=0.44). Closing volume (NE 3.16±0.48 mL; ET 3.51±0.68 mL; P=0.14) and the leakage volume (NE: 8.09±2.53 mL; ET: 8.35±3.65 mL; P=0.83) also showed no differences. CONCLUSIONS: AVR using either suturing techniques leads to a similar hemodynamic performance in vitro. The impact of the suturing technique may be higher in a smaller annulus. Therefore, further studies using smaller prostheses are necessary.

Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3ß (GSK-3ß) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3ß inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3ß inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

Levitating Cells to Sort the Fit and the Fat.

Density is a core material property and varies between different cell types, mainly based on differences in their lipid content. Sorting based on density enables various biomedical applications such as multi-omics in precision medicine and regenerative repair in medicine. However, a significant challenge is sorting cells of the same type based on density differences. Here, a new method for real-time monitoring and sorting of single cells based on their inherent levitation profiles driven by their lipid content is reported. As a model system, human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with neutral lipid storage disease (NLSD) due to loss of function of adipose triglyceride lipase (ATGL) resulting in abnormal lipid storage in cardiac muscle are used. This levitation-based strategy detects subpopulations within ATGL-deficient hiPSC-CMs with heterogenous lipid content, equilibrating at different levitation heights due to small density differences. In addition, sorting of these differentially levitating subpopulations are monitored in real time. Using this approach, sorted healthy and diseased hiPSC-CMs maintain viability and function. Pixel-tracking technologies show differences in contraction between NLSD and healthy hiPSC-CMs. Overall, this is a unique approach to separate diseased cell populations based on their intracellular lipid content that cannot be achieved using traditional flow cytometry techniques.

Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage.

Myosin binding protein H-like (MYBPHL) is a protein associated with myofilament structures in atrial tissue. The protein exists in two isoforms that share an identical amino acid sequence except for a deletion of 23 amino acids in isoform 2. In this study, MYBPHL was found to be expressed preferentially in atrial tissue. The expression of isoform 2 was almost exclusively restricted to the atria and barely detectable in the ventricle, arteria mammaria interna, and skeletal muscle. After atrial damage induced by cryo- or radiofrequency ablation, MYBPHL was rapidly and specifically released into the peripheral circulation in a time-dependent manner. The plasma MYBPHL concentration remained substantially elevated up to 24 hours after the arrival of patients at the intensive care unit. In addition, the recorded MYBPHL values were strongly correlated with those of the established biomarker CK-MB. In contrast, an increase in MYBPHL levels was not evident in patients undergoing aortic valve replacement or transcatheter aortic valve implantation. In these patients, the values remained virtually constant and never exceeded the concentration in the plasma of healthy controls. Our findings suggest that MYBPHL can be used as a precise and reliable biomarker to specifically predict atrial myocardial damage.

Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues.

PURPOSE OF REVIEW: 3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues. RECENT FINDINGS: Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.

Consecutive operative procedures in patients with Marfan syndrome up to 28 years after initial aortic root surgery.

OBJECTIVES: Most patients (75%) with Marfan syndrome present with aortic root dilatation that may require surgical intervention. However, associated cardiovascular disorders are not limited to the aortic root. These patients frequently require consecutive operations on the remaining thoracic aorta or the heart valves. Our intent was to characterize the spectrum of such procedures. METHODS: Data from all patients with Marfan syndrome undergoing aortic root surgery at our centre between 1988 and 2016 were analysed retrospectively. RESULTS: Overall, 73 patients (26 women) were selected for the study. The median age at 1st operation was 30 years (3-68 years). Indications for aortic root surgery were aneurysm (78%) and dissection (22%). Initially, 33 Bentall procedures and 40 valve-sparing root replacement procedures were performed, with a 97% rate of follow-up completion. The median follow up was 8 years (0-28 years). Survival at 1, 10 and 15 years was 100%, 85% and 82%, respectively. During follow-up monitoring, 48 subsequent procedures were performed in 33 patients (aorta, 23; aortic valve, 11; mitral valve, 7 and combined procedures, 7). The 30-day mortality rate after subsequent procedures was 4.2%. Freedom from subsequent operation and death (combined end-point) after 5, 10 and 15 years was 70%, 53% and 34%, respectively. CONCLUSIONS: After the initial surgery, subsequent procedures required in the setting of Marfan syndrome most often involve the remaining native aorta, followed by the aortic and mitral valves. The continued need for additional operative procedures remains high, even decades after the initial surgeries are undertaken. Therefore, long-term patient monitoring at specialized centres is imperative.

Cardiac fibroblasts: more than mechanical support.

Fibroblasts are cells with a structural function, synthesizing components of the extracellular matrix. They are accordingly associated with various forms of connective tissue. During cardiac development fibroblasts originate from different sources. Most derive from the epicardium, some derive from the endocardium, and a small population derives from the neural crest. Cardiac fibroblasts have important functions during development, homeostasis, and disease. However, since fibroblasts are a very heterogeneous cell population no truly specific markers exist. Therefore, studying them in detail is difficult. Nevertheless, several lineage tracing models have been widely used. In this review, we describe the developmental origins of cardiac fibroblasts, comment on fibroblast markers and related lineage tracing approaches, and discuss the cardiac cell composition, which has recently been revised, especially in terms of non-myocyte cells.

Impact of previous cardiac surgery in patients undergoing transcatheter aortic valve implantation: a systematic review.

INTRODUCTION: Redo surgical aortic valve replacement after prior cardiac surgery is usually related to a higher risk of mortality and morbidity. Transcatheter aortic valve implantation (TAVI) became an alternative therapy for those patients in the past couple of years. EVIDENCE ACQUISITION: We aimed in this study to analyze the outcomes of patients undergoing TAVI after a prior cardiac surgery especially those who underwent coronary artery bypass grafting (CABG) and to see if TAVI offers any advantages for those patients than conventional surgical aortic valve replacement. EVIDENCE SYNTHESIS: We searched for relevant articles in Medline and abstracted clinical information based on pre-defined criteria and endpoints. Data of nine studies including the baseline characteristics, implantation data, postoperative outcomes and major adverse cardiac complications, which were published between 2011 and 2015 were collected and evaluated. From all reviewed studies, 769 patients had a prior cardiac surgery and underwent TAVI for symptomatic severe aortic stenosis. Of these, 738 patients (96%) had prior CABG. Patients' age ranged from 78±3 to 82±5.8 years. The STS and EuroSCORE ranged from 4.5±3% to 14.7±12.3% and 25.6±16.2% to 37±18%, respectively. In all reviewed studies the 30-day mortality was about 5.6% and was not significantly higher compared to patients with no history of prior cardiac surgery. The total incidence of stroke was about 3.6%, myocardial infarction was 1.7%, acute kidney injury was 13.8% and permanent pacemaker implantation was about 14.2%. CONCLUSIONS: However, patients presented with severe aortic valve disease after a previous cardiac surgery exhibited a higher preoperative STS and EuroSCORE than those without previous cardiac surgery. The 30-day mortality was not significantly higher in comparison to those patients without history of prior cardiac surgery. According to that, transcatheter aortic valve implantation should be considered as an attractive alternative for those patients.