High levels of the cardiomyocyte-specific kinase Tnni3k impair zebrafish heart regeneration by driving chronic myocardial inflammation.

BACKGROUND: Zebrafish regenerate their hearts after injury, and defining the barriers that block this capacity in mammals may reveal targets for heart failure treatment. Elevated levels of the cardiomyocyte-specific kinase TNNI3K are associated with human cardiomyopathy, and its overexpression drives adverse remodeling in mice. Recent work has linked elevated TNNI3K to cardiomyocyte polyploidization and loss of regenerative competence, but whether this cell-cycle effect accounts for the pathology seen in patients has not been established. Because TNNI3K is restricted to cardiomyocytes, whether it also acts non-cell-autonomously is unknown. METHODS: We generated an allelic series of zebrafish lines comprising cardiomyocyte-specific Tnni3k overexpression, an expression-matched kinase-dead variant, a full-locus deletion, and a line overexpressing mouse Tnni3k. Regeneration, cardiomyocyte proliferation, and ploidy were assessed in uninjured hearts and after cryoinjury. We combined RNA-sequencing, macrophage depletion before or after injury, tamoxifen-inducible Cre-lox switches, and cardiomyocyte-restricted CRISPR mutagenesis. Hearts from mice overexpressing human TNNI3K were analyzed in parallel. RESULTS: Elevated Tnni3k impaired heart regeneration in a kinase-dependent manner. Cardiomyocyte proliferation, redifferentiation, and productive cell division were preserved, and polyploidization remained well below the threshold compatible with robust regeneration, arguing against a cell-cycle defect. Instead, Tnni3k overexpression established an inflammatory and fibrotic state in the uninjured myocardium, marked by interferon, NF-κB, and antigen-presentation programs and by leukocyte and macrophage accumulation. This state amplified after injury, and these animals retained substantially more scar at 60 days post-injury. Depleting macrophages before injury, but not after, prevented the excess fibrosis. Switching Tnni3k off after injury attenuated both inflammation and fibrosis. Cardiomyocyte-specific deletion of pkmb , a glycolytic gene strongly repressed in Tnni3k-overexpressing animals and previously associated with inflammation, reproduced both phenotypes. Mice overexpressing TNNI3K showed comparable fibrosis and macrophage accumulation. CONCLUSIONS: Elevated Tnni3k impairs cardiac regeneration by sustaining a chronic inflammatory and fibrotic state rather than by driving polyploidization. Because this state requires continued kinase activity and remains reversible after injury, TNNI3K inhibition may warrant exploration as a strategy to interrupt the inflammation-fibrosis loop in inflammation-driven cardiac disease. WHAT IS NEW?: Elevated Tnni3k (troponin I-interacting kinase) impairs heart regeneration in zebrafish without reducing cardiomyocyte proliferation or productive cell division, and with only marginal polyploidization, arguing against the prevailing ploidy-based model.Cardiomyocyte-specific Tnni3k overexpression establishes an inflammatory and fibrotic state in the uninjured heart that is amplified after injury and fails to resolve; depleting macrophages before injury, but not after, prevents the excess fibrosis. Switching Tnni3k overexpression off after injury attenuates inflammation and fibrosis. Cardiomyocyte-specific mutagenesis identifies the downregulation of pyruvate kinase ( pkmb ) as a candidate downstream effector linking Tnni3k to innate immune activation. WHAT ARE THE CLINICAL IMPLICATIONS?: Chronic myocardial inflammation can act as a barrier to cardiac repair independently of cardiomyocyte cell-cycle activity, suggesting that strategies aimed solely at stimulating cardiomyocyte proliferation may be insufficient in hearts carrying a pre-existing inflammatory substrate.Because elevated Tnni3k primes the myocardium before injury, patients with increased TNNI3K expression or gain-of-function variants might enter an ischemic event with an inflammatory substrate already in place.Since the phenotype remains reversible after injury and selective TNNI3K inhibitors already exist, post-injury TNNI3K inhibition could potentially be explored to limit fibrosis in cardiac disease driven by persistent inflammation.