Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques.

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

Studies of a mosaic patient with DBA and chimeric mice reveal erythroid cell-extrinsic contributions to erythropoiesis.

We follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.

Assessment of arrhythmia mechanism and burden of the infarcted ventricles following remuscularization with pluripotent stem cell-derived cardiomyocyte patches using patient-derived models.

AIMS: Direct remuscularization with pluripotent stem cell-derived cardiomyocytes (PSC-CMs) seeks to address the onset of heart failure post-myocardial infarction (MI) by treating the persistent muscle deficiency that underlies it. However, direct remuscularization with PSC-CMs could potentially be arrhythmogenic. We investigated two possible mechanisms of arrhythmogenesis-focal vs. re-entrant-arising from direct remuscularization with PSC-CM patches in two personalized, human ventricular computer models of post-MI. Moreover, we developed a principled approach for evaluating arrhythmogenicity of direct remuscularization that factors in the VT propensity of the patient-specific post-MI fibrotic substrate and use it to investigate different conditions of patch remuscularization. METHODS AND RESULTS: Two personalized, human ventricular models of post-MI (P1 and P2) were constructed from late gadolinium enhanced (LGE)-magnetic resonance images (MRIs). In each model, remuscularization with PSC-CM patches was simulated under different treatment conditions that included patch engraftment, patch myofibril orientation, remuscularization site, patch size (thickness and diameter), and patch maturation. To determine arrhythmogenicity of treatment conditions, VT burden of heart models was quantified prior to and after simulated remuscularization and compared. VT burden was quantified based on inducibility (i.e. weighted sum of pacing sites that induced) and severity (i.e. the number of distinct VT morphologies induced). Prior to remuscularization, VT burden was significant in P1 (0.275) and not in P2 (0.0, not VT inducible). We highlight that re-entrant VT mechanisms would dominate over focal mechanisms; spontaneous beats emerging from PSC-CM grafts were always a fraction of resting sinus rate. Moreover, incomplete patch engraftment can be particularly arrhythmogenic, giving rise to particularly aberrant electrical activation and conduction slowing across the PSC-CM patches along with elevated VT burden when compared with complete engraftment. Under conditions of complete patch engraftment, remuscularization was almost always arrhythmogenic in P2 but certain treatment conditions could be anti-arrhythmogenic in P1. Moreover, the remuscularization site was the most important factor affecting VT burden in both P1 and P2. Complete maturation of PSC-CM patches, both ionically and electrotonically, at the appropriate site could completely alleviate VT burden. CONCLUSION: We identified that re-entrant VT would be the primary VT mechanism in patch remuscularization. To evaluate the arrhythmogenicity of remuscularization, we developed a principled approach that factors in the propensity of the patient-specific fibrotic substrate for VT. We showed that arrhythmogenicity is sensitive to the patient-specific fibrotic substrate and remuscularization site. We demonstrate that targeted remuscularization can be safe in the appropriate individual and holds the potential to non-destructively eliminate VT post-MI in addition to addressing muscle deficiency underlying heart failure progression.

Computational modeling of aberrant electrical activity following remuscularization with intramyocardially injected pluripotent stem cell-derived cardiomyocytes.

Acute engraftment arrhythmias (EAs) remain a serious complication of remuscularization therapy. Preliminary evidence suggests that a focal source underlies these EAs stemming from the automaticity of immature pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in nascent myocardial grafts. How these EAs arise though during early engraftment remains unclear. In a series of in silico experiments, we probed the origin of EAs-exploring aspects of altered impulse formation and altered impulse propagation within nascent PSC-CM grafts and at the host-graft interface. To account for poor gap junctional coupling during early PSC-CM engraftment, the voltage dependence of gap junctions and the possibility of ephaptic coupling were incorporated. Inspired by cardiac development, we also studied the contributions of another feature of immature PSC-CMs, circumferential sodium channel (NaCh) distribution in PSC-CMs. Ectopic propagations emerged from nascent grafts of immature PSC-CMs at a rate of <96 bpm. Source-sink effects dictated this rate and contributed to intermittent capture between host and graft. Moreover, ectopic beats emerged from dynamically changing sites along the host-graft interface. The latter arose in part because circumferential NaCh distribution in PSC-CMs contributed to preferential conduction slowing and block of electrical impulses from host to graft myocardium. We conclude that additional mechanisms, in addition to focal ones, contribute to EAs and recognize that their relative contributions are dynamic across the engraftment process.

Mechanisms of Sinoatrial Node Dysfunction in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: The ability to increase heart rate during exercise and other stressors is a key homeostatic feature of the sinoatrial node (SAN). When the physiological heart rate response is blunted, chronotropic incompetence limits exercise capacity, a common problem in patients with heart failure with preserved ejection fraction (HFpEF). Despite its clinical relevance, the mechanisms of chronotropic incompetence remain unknown. METHODS: Dahl salt-sensitive rats fed a high-salt diet and C57Bl6 mice fed a high-fat diet and an inhibitor of constitutive nitric oxide synthase (Nω-nitro-L-arginine methyl ester [L-NAME]; 2-hit) were used as models of HFpEF. Myocardial infarction was created to induce HF with reduced ejection fraction. Rats and mice fed with a normal diet or those that had a sham surgery served as respective controls. A comprehensive characterization of SAN function and chronotropic response was conducted by in vivo, ex vivo, and single-cell electrophysiologic studies. RNA sequencing of SAN was performed to identify transcriptomic changes. Computational modeling of biophysically-detailed human HFpEF SAN was created. RESULTS: Rats with phenotypically-verified HFpEF exhibited limited chronotropic response associated with intrinsic SAN dysfunction, including impaired ß-adrenergic responsiveness and an alternating leading pacemaker within the SAN. Prolonged SAN recovery time and reduced SAN sensitivity to isoproterenol were confirmed in the 2-hit mouse model. Adenosine challenge unmasked conduction blocks within the SAN, which were associated with structural remodeling. Chronotropic incompetence and SAN dysfunction were also found in rats with HF with reduced ejection fraction. Single-cell studies and transcriptomic profiling revealed HFpEF-related alterations in both the "membrane clock" (ion channels) and the "Ca2+ clock" (spontaneous Ca2+ release events). The physiologic impairments were reproduced in silico by empirically-constrained quantitative modeling of human SAN function. CONCLUSIONS: Chronotropic incompetence and SAN dysfunction were seen in both models of HF. We identified that intrinsic abnormalities of SAN structure and function underlie the chronotropic response in HFpEF.

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

Tissue resident (TR) immune cells play important roles in facilitating tissue homeostasis, coordinating immune responses against infections and tumors, and maintaining immunological memory. While studies have shown these cells are distinct phenotypically and functionally from cells found in the peripheral blood (PB), the clonal relationship between these populations across tissues has not been comprehensively studied in primates or humans. We utilized autologous transplantation of rhesus macaque hematopoietic stem and progenitor cells containing high diversity barcodes to track the clonal distribution of T, B, myeloid and natural killer (NK) cell populations across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract, in comparison with PB longitudinally post-transplantation, in particular we focused on NK cells which do not contain endogenous clonal markers and have not been previously studied in this context. T cells demonstrated tissue-specific clonal expansions as expected, both overlapping and distinct from blood T cells. In contrast, B and myeloid cells showed a much more homogeneous clonal pattern across various tissues and the blood. The clonal distribution of TR NK was more heterogenous between individual animals. In some animals, as we have previously reported, we observed large PB clonal expansions in mature CD56-CD16+ NK cells. Notably, we found a separate set of highly expanded PB clones in CD16-CD56- (DN) NK subset that were also contributing to TR NK cells in all tissues examined, both in TR CD56-CD16+ and DN populations but absent in CD56+16- TR NK across all tissues analyzed. Additionally, we observed sets of TR NK clones specific to individual tissues such as lung or GI tract and sets of TR NK clones shared across liver and spleen, distinct from other tissues. Combined with prior functional data that suggests NK memory is restricted to liver or other TR NK cells, these clonally expanded TR NK cells may be of interest for future investigation into NK cell tissue immunological memory, with implications for development of NK based immunotherapies and an understanding of NK memory.

Accurate Conduction Velocity Maps and Their Association With Scar Distribution on Magnetic Resonance Imaging in Patients With Postinfarction Ventricular Tachycardias.

BACKGROUND: Characterizing myocardial conduction velocity (CV) in patients with ischemic cardiomyopathy (ICM) and ventricular tachycardia (VT) is important for understanding the patient-specific proarrhythmic substrate of VTs and therapeutic planning. The objective of this study is to accurately assess the relation between CV and myocardial fibrosis density on late gadolinium-enhanced cardiac magnetic resonance imaging (LGE-CMR) in patients with ICM. METHODS: We enrolled 6 patients with ICM undergoing VT ablation and 5 with structurally normal left ventricles (controls) undergoing premature ventricular contraction or VT ablation. All patients underwent LGE-CMR and electroanatomic mapping (EAM) in sinus rhythm (2960 electroanatomic mapping points analyzed). We estimated CV from electroanatomic mapping local activation time using the triangulation method that provides an accurate estimate of CV as it accounts for the direction of wavefront propagation. We evaluated the association between LGE-CMR intensity and CV with multilevel linear mixed models. RESULTS: Median CV in patients with ICM and controls was 0.41 m/s and 0.65 m/s, respectively. In patients with ICM, CV in areas with no visible fibrosis was 0.81 m/s (95% CI, 0.59-1.12 m/s). For each 25% increase in normalized LGE intensity, CV decreased by 1.34-fold (95% CI, 1.25-1.43). Dense scar areas have, on average, 1.97- to 2.66-fold slower CV compared with areas without dense scar. Ablation lesions that terminated VTs were localized in areas of slow conduction on CV maps. CONCLUSIONS: CV is inversely associated with LGE-CMR fibrosis density in patients with ICM. Noninvasive derivation of CV maps from LGE-CMR is feasible. Integration of noninvasive CV maps with electroanatomic mapping during substrate mapping has the potential to improve procedural planning and outcomes. Visual Overview: A visual overview is available for this article.

Understanding AF Mechanisms Through Computational Modelling and Simulations.

AF is a progressive disease of the atria, involving complex mechanisms related to its initiation, maintenance and progression. Computational modelling provides a framework for integration of experimental and clinical findings, and has emerged as an essential part of mechanistic research in AF. The authors summarise recent advancements in development of multi-scale AF models and focus on the mechanistic links between alternations in atrial structure and electrophysiology with AF. Key AF mechanisms that have been explored using atrial modelling are pulmonary vein ectopy; atrial fibrosis and fibrosis distribution; atrial wall thickness heterogeneity; atrial adipose tissue infiltration; development of repolarisation alternans; cardiac ion channel mutations; and atrial stretch with mechano-electrical feedback. They review modelling approaches that capture variability at the cohort level and provide cohort-specific mechanistic insights. The authors conclude with a summary of future perspectives, as envisioned for the contributions of atrial modelling in the mechanistic understanding of AF.