There is very limited regenerative potential in cardiomyocytes of adult human hearts, as cytokinesis is practically absent after the age of 20. Previous studies have identified cyclin A2 (CCNA2) as a key regulator of the cardiomyocyte cell cycle, capable of inducing mitosis and regeneration in animal models. This study assessed the ability of CCNA2 gene therapy, which is a cardiac Troponin T (cTnT)-driven adenoviral [Ad5-cTnT-hCCNA2], to induce complete cytokinesis in adult human cardiomyocytes and investigated its underlying transcriptional pathways through an integrative transcriptomic analysis.
Isolation and culturing of adult human cardiomyocytes were done on 21-, 41-, and 55-year-old donors based on modified porcine cardiomyocyte culture protocols. Cells were transduced with either the cTnT-hCCNA2 adenovirus or a control cTnT-eGFP (MOI 100) to determine cytokinesis. The integrity of sarcomeres and cell division were visualized through live-cell epifluorescence time-lapse microscopy using eGFP (green) and α-actinin (red) labeling. Cytokinesis events were measured in 42 regions of interest, and the formation of daughter cells was confirmed by DAPI staining and the nuclear GFP gene expression.
Calcium flux (Ca²⁺) was measured in transduced cardiomyocytes embedded in a 3D agarose matrix under pacing at 0.5 Hz and 1 Hz, using Fluo3 dye to measure functional differentiation. Bulk RNA sequencing, ultra-deep fetal versus adult heart RNA-seq, and single-nucleus RNA-seq (snRNA-seq) of CCNA2-transgenic (CCNA2-Tg) and non-transgenic (nTg) mice were subjected to transcriptional reprogramming. Transcriptomic data on transverse aortic banding (TAB)-induced models of hypertrophy were also compared. Pathway activation was analyzed using pathway enrichment and gene set enrichment analysis (GSEA).
Cardiomyocytes transduced with cTnT-hCCNA2 demonstrated robust CCNA2 expression and cytokinesis, particularly in cells from 41- and 55-year-old donors, without disrupting sarcomere architecture. Daughter cells exhibited mononuclear morphology, retained contractile characteristics, and developed active Ca²⁺ transients that were similar to the native cells. CCNA2 reactivated fetal-like gene expression programs, such as those controlling calcium homeostasis and sarcomere formation (e.g., TNNC1, TTN, NEB, CALM2, CALM1, SLC8A1, ATP2A2, RYR2). Calcium-handling (Ryr2, Atp2a2, Slc8a1) and reprogramming and dedifferentiation (Itpr1, Orai2, Stim2) signatures were down- and up-regulated, respectively, in CCNA2-Tg mice and matched fetal transcriptional reactivation.
Analysis of snRNA-seq confirmed increased expression of genes associated with cell cycle progression, proliferation, and mesenchymal plasticity, with enrichment of NOTCH, TGF-b, Wnt, MAPK, PDGF, and Jak-STAT signaling pathways, and suppression of oxidative phosphorylation and mitochondrial metabolism, reflecting a metabolic shift toward a proliferative, fetal-like phenotype. In previous large-animal models, CCNA2-Tg cardiomyocytes exhibited regenerative rather than hypertrophic remodeling, with reduced fibrosis and preserved cell size.
This study provides the first direct evidence that cardiomyocyte-specific expression of the CCNA2 can trigger full cytokinesis in adult human cardiomyocytes until 55 years of age, effectively overcoming the conventional cell-cycle block of postnatal cells. The cTnT-based Ad5 vector demonstrated cardiac tissue specificity and safety, minimizing off-target proliferation and oncogenic risks. Transcriptomic analyses suggest that CCNA2 reactivates developmental and regenerative gene programs, enabling cell division without causing additional structural damage.
Despite technical challenges such as variability in human cell cultures and the immunogenicity of adenoviral vectors, these findings position CCNA2 as a promising next-generation therapeutic agent for cardiac regeneration. Future research should focus on developing efficient delivery systems and mapping the molecular landscape of proliferative CCNA2-positive cardiomyocytes to advance precision heart repair strategies.
Reference: Bouhamida E, Vadakke-Madathil S, Mathiyalagan P, et al. Cyclin A2 induces cytokinesis in human adult cardiomyocytes and drives reprogramming in mice. NPJ Regenerative Medicine. 2025;10:47. doi:10.1038/s41536-025-00438-7
