Bingyan J. Wang

BACKGROUND: Unlike conventional microsecond pulsed electrical fields that primarily target the cell membranes, nanosecond pulses are thought to primarily electroporate intracellular organelles. We conducted a comprehensive preclinical assessment of catheter-based endocardial nanosecond pulsed field ablation in swine. METHODS: A novel endocardial nanosecond pulsed field ablation system was evaluated in a total of 25 swine. Using either a low-dose (5-second duration) or high-dose (15-second duration) strategy, thoracic veins and discrete atrial and ventricular sites were ablated. Predetermined survival periods were P =0.007). The esophagus revealed nontransmural adventitial surface lesions in 5 of 5 (100%) animals euthanized early (2 days) post-ablation. In the 10 animals euthanized later (14–28 days), all animals demonstrated significant esophageal healing—8 with complete resolution, and 2 with only trace fibrosis. CONCLUSIONS: A novel, endocardial nanosecond pulsed field ablation system provides acute and durable venous isolation and linear lesions. Transient phrenic injury and nontransmural esophageal lesions can occur with worst-case assessments suggesting limits to pulsed field ablation tissue selectivity and the need for dedicated assessments during clinical studies.

Background: Pulsed field ablation (PFA) has recently been shown to penetrate ischemic scar, but details on its efficacy, risk of arrhythmias, and imaging insights are lacking. In a porcine model of myocardial scar, we studied the ability of ventricular PFA to penetrate scarred tissue, induce ventricular arrhythmias, and assess the influence of QRS gating during pulse delivery. Methods: Of a total of 6 swine, 5 underwent coronary occlusion and 1 underwent radiofrequency ablation to create infarct scar and iatrogenic scar models, respectively. Two additional swine served as healthy controls. An 8 Fr focal PFA catheter was used to deliver bipolar, biphasic PFA (2.0 kV) lesions guided by electroanatomical mapping, fluoroscopy, and intracardiac echocardiography over both scarred and healthy myocardium. Swine underwent magnetic resonance imaging 2–7 days post-PFA. Results: PFA successfully penetrated scar without significant difference in lesion depth between lesion at the infarct border (5.9±1.0 mm, n=41) and healthy myocardium (5.7±1.3 mm, n=26; P =0.53). PFA penetration of both infarct and iatrogenic radiofrequency abalation scar was observed in all examined sections. Sustained ventricular arrhythmias requiring defibrillation occurred in 4 of 187 (2.1%) ungated applications, whereas no ventricular arrhythmias occurred during gated PFA applications (0 of 64 [0%]). Dark-blood late-gadolinium–enhanced sequences allowed for improved endocardial border detection as well as lesion boundaries compared with conventional bright-blood late-gadolinium–enhanced sequences. Conclusions: PFA penetrates infarct and iatrogenic scar successfully to create deep lesions. Gated delivery eliminates the occurrence of ventricular arrhythmias observed with ungated porcine PFA. Optimized magnetic resonance imaging sequences can be helpful in detecting lesion boundaries.

Background: Our understanding of catheter-based pulsed field ablation (PFA) of the ventricular myocardium is limited. We conducted a series of exploratory evaluations of ventricular PFA in swine ventricles. Methods: A focal lattice-tip catheter was used to deliver proprietary biphasic monopolar PFA applications to swine ventricles under general anesthesia, with guidance from electroanatomical mapping, fluoroscopy, and intracardiac echocardiography. We conducted experiments to assess the impact of (1) delivery repetition (2×, 3×, or 4×) at each location, (2) epicardial PFA delivery, and (3) confluent areas of shallow healed endocardial scar created by prior PFA (4 weeks earlier) on subsequent endocardial PFA. Additional assessments included PFA optimized for the ventricle, lesion visualization by intracardiac echocardiography imaging, and immunohistochemical insights. Results: Experiment no. 1: lesions (n=49) were larger with delivery repetition of either 4× or 3× versus 2×: length 17.6±3.9 or 14.2±2.0 versus 12.7±2.0 mm ( P P =0.22), width 13.4±1.8 or 10.6±1.3 versus 10.5±1.1 mm ( P P =1.00), and depth 6.1±2.1 or 5.1±1.3 versus 4.2±1.0 mm ( P P =0.21). Experiment no. 2: epicardial lesions (n=18) were reliably created and comparable to endocardial lesions: length 24.6±9.7 mm (n=5), width 15.6±4.6 mm, and depth 4.5±3.7 mm. Experiment no. 3: PFA (n=16) was able to penetrate to a depth of 4.8 (interquartile range, 4.5–5.4) mm in healthy myocardium versus 5.6 (interquartile range, 3.6–6.6) mm in adjacent healed endocardial scar ( P =0.79), suggesting that superficial scar does not significantly impair PFA. Finally, we demonstrate, PFA optimized for the ventricle yielded adequate lesion dimensions, can result in myocardial activation, can be visualized by intracardiac echocardiography, and have unique immunohistochemical characteristics. Conclusions: This in vivo evaluation offers insights into the behavior of endocardial or epicardial PFA delivered using the lattice-tip catheter to normal or scarred porcine ventricular myocardium, thereby setting the stage for future clinical studies.

Importance: SARS-CoV-2 infection directly causes severe acute respiratory illness, leading to systemic tissue hypoxia and ischemia including the heart. Myocardial cytopathy associated with hypoxic response has been largely overlooked in COVID-19 patients. Additionally, histology analysis and cardiac function of COVID-19 cases are often reported separately, rendering an incomplete understanding of COVID-19 cardiac symptoms. Objective: To examine the relationship between myocardial cellular responses to hypoxic stress versus cardiac functional alterations within the same COVID-19 patients. Design, Setting, and Participants: Cellular hypoxia Inducible Factor 1 alpha (HIF1α) expression was analyzed by immunohistochemistry using post-mortem COVID-19 heart and lung tissues with known cardiac echocardiography records from a total of 8 patients. Clinical echocardiography data were obtained from Mount Sinai Heart between March to December, 2020. All gender and age groups were considered as long as cardiac involvement meets the preserved (EF > 50%) or moderate to severe (EF < 45%) criteria with confirmed SARS-CoV-2 infection. Cell-type specific subcellular localization of HIF1α expression and nuclear stability was examined by immunohistochemistry and transmission electronic microscopy (TEM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to quantify apoptosis. Main Outcomes and Measures: No planned outcomes of this study as this is a retrospective analysis based on post-mortem specimens exclusively. Results: Cardiac HIF1α expression was found to be significantly higher in patients with preserved EF levels than it was in the low EF group. In the preserved EF group, HIF1α is protective against apoptosis predominantly in endothelial cells and cardiac fibroblasts. In the low EF group, HIF1α protects cardiomyocyte nuclear integrity as evident by its nuclear accumulation with nuclear envelope preservation. Conclusions and Relevance: This study establishes a direct link of cardiac cellular responses to hypoxic stress with matching functional and histological data, serving as one of the first studies to bridge previous stand-alone clinical data and cellular data. The protective role of HIF1α in hearts may help predict cardiac involvement in not only COVID-19 patients, but also decipher the underlying mechanisms in other forms of viral cardiomyopathy. KEY POINTS: Question: Are hypoxic signaling pathways associated with cardiac functional alterations in COVID-19 patients? Findings: Cardiac HIF1α expression of COVID-19 patients with EF>50% or EF+ cells were found in patients with higher EF. HIF1α ⁺ endothelial cells are resistant to apoptosis, and HIF1α ⁺ cardiomyocytes are able to retain nuclear envelope under hypoxic stress. Meaning: HIF1α is cardioprotective in hearts of COVID-19 patients.

Aims: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFβ) signalling pathway where inhibiting TGFβ signalling maintains telomere length. The relationship between Pim1 and TGFβ has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFβ signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. Methods and results: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFβ signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFβ signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFβ pathway genes. Conclusion: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFβ pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein–protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such ‘next generation’ implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit⁺cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c-Kit⁺ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra-embryonic tissue formation.

Background: Cellular therapy to treat heart failure is an ongoing focus of intense research and development, but progress has been frustratingly slow due to limitations of current approaches. Engineered augmentation of established cellular effectors overcomes impediments, enhancing reparative activity with improved outcomes relative to conventional techniques. Such ‘next generation’ implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, as previously reported by our group, prompted design of a three-dimensional (3D) structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell losses upon delivery. Methods: Three distinct populations of human cardiac interstitial cells including mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and c-Kit ⁺ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. Biological consequences of CardioCluster formation were assessed by multiple assays including single cells RNA-Seq transcriptional profiling. Protective effects of CardioClusters in vitro were measured using cell culture models for oxidative stress and myocardial ischemia in combination with freshly isolated neonatal rat ventricular myocytes. Long-term impact of adoptively transferred CardioClusters upon myocardial structure and function in a xenogenic model of acute infarction using NOD ^scid mice was assessed over a longitudinal time course of 20-weeks. Results: CardioCluster design enables control over composite cell types, cell ratios, size, and preservation of structural integrity during delivery. Profound changes for biological properties of CardioClusters relative to constituent parental cell populations include enhanced expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines. The CardioCluster 3D microenvironment maximizes cellular interaction while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster delivery improves cell retention following intramyocardial injection with preservation of long-term cardiac function relative to monolayer-cultured cells when tested in an experimental murine infarction model followed for up to 20 weeks post-challenge. CardioCluster-treated hearts show increases in capillary density, preservation of cardiomyocyte size, and reduced scar size indicative of blunting pathologic infarction injury. Conclusions: CardioClusters are a novel ‘next generation’ development and delivery approach for cellular therapeutics that potentiate beneficial activity and enhance protective effects of human cardiac interstitial cell mixed populations. CardioClusters utilization in this preclinical setting establishes fundamental methodologic and biologic insights, laying the framework for optimization of CardioCluster design to provide greater efficacy in cell-based therapeutic interventions intended to mitigate cardiomyopathic damage.

Cardiac interstitial cells (CIC) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c-Kit ⁺ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast-like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast-like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra-embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra-embryonic tissue formation.