Hypothesis

Targeting ZBP1-mediated PANoptosis in senescent cardiac progenitor cells (c‑Kit⁺ CPCs) will blunt the senescence-associated secretory phenotype (SASP) and interrupt the paracrine senescence loop, thereby preserving CPC regenerative capacity and limiting clonal hematopoiesis-driven inflammaging.

Mechanistic Rationale

Senescent c‑Kit⁺ CPCs accumulate mitochondrial DNA damage and oxidative stress, which can activate cytosolic DNA sensors such as ZBP1 (Z‑DNA binding protein 1) and AIM2, igniting PANoptosis—a lytic programmed cell death pathway that converges pyroptosis, apoptosis, and necroptosis [6]. PANoptosis releases damage-associated molecular patterns (DAMPs) and further fuels SASP cytokine production (IL‑6, IL‑1β, GM‑CSF) [2], creating a feed-forward loop that spreads senescence to neighboring healthy CPCs and promotes systemic inflammation via clonal hematopoiesis mutations like JAK2^V617F^. Recent work shows that ZBP1 deficiency reduces inflammasome activation and cytokine storm in aged tissues, suggesting that ZBP1 is a nodal point linking mitochondrial distress to SASP amplification.

We hypothesize that inhibiting ZBP1-driven PANoptosis in senescent CPCs will: (1) lower mitochondrial ROS‑induced inflammasome signaling, (2) decrease SASP secretion, (3) break the paracrine senescence cascade, and (4) attenuate the expansion of JAK2^V617F^-bearing hematopoietic clones that are fueled by chronic inflammatory milieu.

Testable Predictions

1. Genetic knockout or pharmacologic inhibition of ZBP1 in aged mice will reduce the proportion of p16^INK4A^⁺/SA‑β‑gal⁺ c‑Kit⁺ CPCs by >40% compared with wild‑type controls.
2. Conditioned media from ZBP1‑deficient senescent CPCs will exhibit ≥50% lower levels of IL‑6, IL‑1β, and GM‑CSF (ELISA) and will fail to induce p16^INK4A^ upregulation in naïve c‑Kit⁺ CPCs in vitro.
3. Aged ZBP1‑deficient mice subjected to myocardial infarction will show a two‑fold increase in c‑Kit⁺ CPC‑derived cardiomyocytes (lineage tracing) and improved ejection fraction (≥15% absolute increase) relative to littermate controls.
4. Peripheral blood from aged ZBP1‑deficient mice will display reduced allele frequency of JAK2^V617F^-like clonal hematopoietic mutations (droplet digital PCR) and lower plasma CRP levels.

Experimental Approach

• Model: Use c‑Kit‑CreERT2; Zbp1^fl/fl^ mice crossed with a p16‑3MR reporter to track senescent CPCs upon tamoxifen induction at 18 months of age.
• Interventions: (a) Tamoxifen‑induced ZBP1 deletion; (b) Small‑molecule ZBP1 inhibitor (e.g., necrosulfonamide analog) administered via osmotic pump.
• Readouts: Flow cytometry for p16^INK4A^, SA‑β‑gal, mtROS; SASP cytokine panel; clonogenic assay; lineage tracing with Rosa26‑tdTomato; echocardiography; ddPCR for JAK2^V617F^-equivalent mutations.
• Controls: Littermate Zbp1^+/+^ mice receiving same treatments; senolytic (D+Q) group as positive control.

If ZBP1 loss mitigates SASP and preserves CPC function, this would reposition PANoptosis inhibition as a complementary strategy to senolytics, targeting the upstream inflammasome‑SASP axis rather than merely clearing senescent cells.