IF a combinatorial repair strategy — (1) intravenous infusion of syngeneic, Y-chromosome-replete hematopoietic stem cells (Y-HSCs; Lin⁻Sca1⁺cKit⁺ cells from young male donors, ~5×10⁶ cells/mouse) preceded by non-myeloablative busulfan conditioning, combined with (2) intracoronary or intramyocardial delivery of ionizable lipid nanoparticles (LNPs) co-encapsulating synthetic mRNA encoding the Y-linked histone demethylases KDM5D and UTY (targeted to cardiac CD68⁺/F4/80⁺ macrophages via mannose-receptor-decorated LNPs, single administration) — is administered to aged male C57BL/6 mLOY bone marrow chimeras (18–22 months, generated via CRISPR-mediated Y centromere editing followed by HSC transplant, replicating the Sano et al. 2022 model described in the Evidence Set),

THEN a statistically significant reversal of established cardiac fibrosis — defined as ≥30% reduction in myocardial collagen deposition (Masson's trichrome, hydroxyproline assay), restoration of left ventricular fractional shortening toward age-matched Y-replete controls (echocardiography), and ≥50% reduction in cardiac TGF-β1 protein and downstream pSMAD2/3 signaling — will be observed at 12 weeks post-intervention, compared to mLOY chimeras receiving vehicle + mock LNPs,

BECAUSE the following sequential causal chain operates:

1. mLOY in hematopoietic stem and progenitor cells (HSPCs) generates a clonally expanded population of Y-chromosome-deficient circulating monocytes that continuously infiltrate the myocardium, differentiate into resident-like macrophages, and sustain a pro-fibrotic inflammatory niche through TGF-β hypersecretion and IL-1β/IL-6 production, as established in the Sano et al. 2022 preclinical model described in the Evidence Set and epidemiologically supported by the association of mLOY with cardiac disease outcomes (mLOY associated with elevated incident heart disease risk, HR reported in multivariable Cox regression)[https://doi.org/10.1101/2024.05.29.24308171].

2. The mechanistic driver of this pro-fibrotic phenotype is loss of Y-linked epigenetic regulators — specifically KDM5D (H3K4me3/me2 demethylase) and UTY (H3K27me2/me3 demethylase) — which normally constrain TGF-β pathway gene expression and inflammatory cytokine loci in macrophages; their absence in mLOY cells causes epigenetic drift toward a hyperactivated transcriptional state, as supported by single-cell evidence that LOY disrupts autosomal transcriptional networks and cell-fate decisions via disruption of Y-linked epigenetic regulatory machinery (LOY contributes to immune dysfunction and altered cell-fate decisions via autosomal transcriptional changes and disruption of Y-linked epigenetic regulators)[https://doi.org/10.1101/2025.06.01.25328624].

3. [SPECULATIVE] Critically, cardiac-resident macrophages in established fibrotic tissue are long-lived, partially self-renewing cells that persist independently of continuous bone marrow replenishment. Y-HSC infusion alone will replace the circ...

SENS category: GlycoSENS

Key references: • doi.org/10.1101/2024.05.29.24308171]. • doi.org/10.1101/2025.06.01.25328624]. • doi.org/10.1101/2024.05.25.595885]. • doi.org/10.1101/2024.05.25.595885] • doi.org/10.1016/j.gde.2016.12.002].