Transcriptomic remodeling in gene-negative hypertrophic cardiomyopathy revealed by single-nucleus RNA sequencing

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by unexplained left ventricular hypertrophy. While sarcomeric variants explain many cases, gene-negative HCM remains poorly understood. To define its molecular basis, we performed single-nucleus RNA sequencing on septal myectomy samples and non-diseased controls and integrated the transcriptomic data with patient clinical and imaging profiles. We hypothesized that gene-negative HCM has distinct transcriptional programs and altered extracellular matrix (ECM) signaling microenvironment that are linked to disease severity. We identified 25 cardiac cell populations, including distinct cardiomyocyte (CM) states. Pseudotime analysis revealed two inferred trajectories emerging from a shared origin. One trajectory showed progressive remodeling culminating in an HCM-specific state (CM7). Along this path, gene expression changes reflected coordinated increases in growth, cytoskeletal signaling, and energy metabolism, supported by dynamic shifts in transcriptional regulons. CM7 abundance correlated with left ventricular wall thickness and cardiomyocyte hypertrophy, consistent with a terminal remodeling state. In contrast, the second trajectory terminated in CM5, which showed fewer trajectory-associated changes and instead represented a distinct functional state. CM5 was characterized by reduced expression of genes involved in action potential, calcium handling, and contractility, alongside increased mitochondrial programs. This state was associated with diastolic dysfunction, suggesting impaired excitation-contraction coupling. At the tissue level, gene-negative HCM showed an overall reduction in intercellular signaling, but selective enrichment of β1-integrin-mediated ECM pathways. Fibroblasts were the main signaling hubs, and ITGA9 emerged as the dominant α-subunit within this network. ECM-derived signals preferentially targeted cardiomyocyte states positioned later along the inferred remodeling-associated trajectory, where ITGA9 expression was increased. At the patient level, higher ITGA9 expression correlated with left atrial enlargement, linking this pathway to structural remodeling. Together, these findings suggest that gene-negative HCM is characterized three linked features: distinct cardiomyocyte states, divergence between a CM7 remodeling-associated continuum and a CM5 functional/excitation-contraction abnormality, and an altered ECM signaling environment enriched for ITGA9-β1 interactions. This integrated cardiomyocyte-microenvironment model supports gene-negative HCM as a biologically distinct remodeling phenotype rather than simply a residual category defined by the absence of identifiable sarcomere mutations.