**Background:** Cardiovascular diseases (CVD) often originate from adverse conditions during early development, including growth retardation, inflammation, and malnutrition. While intrauterine triggers are well-studied, early postnatal triggers remain less understood. Lactose intolerance commonly accompanies gastroenteritis in infants and can cause inflammation, oxidative stress, and metabolic disturbances. This study tested whether neonatal lactose intolerance (NLI) exerts long-term detrimental effects on cardiac structure, ploidy, and transcriptome in rats.
**Methods:** Eight-day-old rat pups were fed lactose-containing water daily until weaning at day 21, overloading intestinal lactase capacity. Control pups received distilled water. At 140 days of age (4 months post-treatment), body weight was measured. Left ventricle (LV) cardiomyocytes were isolated by enzymatic perfusion. Ploidy was assessed by cytophotometry of DAPI-stained nuclei (≥500 cells/animal, 24 animals/group). DNA instability was quantified as percentage of cells with chromatin bridges or aneuploid nuclei. Protein content per cell and per genome was measured by naphthol yellow staining and image analysis (≥300 cells/animal). LV apical mRNA was sequenced (Illumina HiSeq2500; 3 biological samples/group). Differentially expressed genes showing qualitative (on/off) switching were identified using two-dimensional histogram peak analysis. Stringent filtering via protein–protein interaction networks (String server, high confidence) and Glay clustering in Cytoscape identified tight functional gene clusters, which were analyzed by Metascape for pathway enrichment and MCODE complex detection. qRT-PCR validated expression of Tgfb2, Egr1, and Ccna2 (5 animals/group).
**Key Results:** Experimental animals weighed 199 ± 7.3 g vs. 248 ± 9.2 g in controls (Mann–Whitney, p < 0.01), a ~25% reduction. Cardiomyocyte ploidy increased ~20%: 4.7 ± 0.27 c in experiment vs. 3.7 ± 0.23 c in controls (p < 0.005), driven by increased tetraploid (2cx2, 4c, 4cx2) and octoploid (8c) cells. Chromatin bridges were present in 1.8 ± 0.4% of experimental cardiomyocytes vs. 0% in controls (p < 0.001). Aneuploid cells were 5.1 ± 1.6% vs. 0.94 ± 0.05% (p < 0.004). Protein content per cardiomyocyte decreased ~1.28-fold (p < 0.03); protein per genome decreased ~1.7-fold (p < 0.01). RNA-seq identified 13,742 genes; 1,322 switched-on and 1,028 switched-off genes were selected. After interactome filtering, 496 upregulated and 355 downregulated hub genes formed four upregulated clusters (DNA repair, immunity/inflammation, fibrosis, transcription/ribosome biogenesis) and two downregulated clusters (calcium/thyroid hormone/circadian signaling; glutathione/detoxification). The DNA repair cluster (55 genes) was enriched in mismatch repair, base excision repair, double-strand break repair, and telomere maintenance (including Terf1, Terf2, ATM). The immunity cluster (146 genes) showed enrichment in JAK-STAT signaling, interferon/cytokine production, and innate immunity (including Stat5A, c-Kit, Irf1, Irf7, Irf9). The fibrosis cluster (29 genes) included TGF beta signaling (Tgfb1, Tgfb2, Tgfb3, Nbl1, Bambi) and collagen biosynthesis (Col3A, Col1a2, Col16a1, Col1a1, Col6a6). The transcription cluster (164 genes) was enriched in gene expression, mRNA processing, rRNA metabolism, and ribosome biogenesis, plus "negative regulation of chromosome organization." Downregulated clusters included muscle contraction, thyroid hormone synthesis, circadian entrainment, and calcium signaling (47 genes; including Cacna1f, Cacna1d, Adcy1, Gng3, Plcb1, Rgs19), and glutathione metabolism/detoxification (including Gstm4, Gstm1, Gpx1, Gsta1, Gpx7, Gss, Gpx). The Myh6/Myh7 ratio shifted from 3.19 in controls to 2.01 in experiment. qRT-PCR confirmed upregulation of Tgfb2 and downregulation of Egr1 and Ccna2, consistent with RNA-seq. Protein interaction networks showed coherent expression changes among direct regulators and interactants of these genes.
**Clinical Implications:** This study provides the first evidence that neonatal lactose intolerance can serve as a trigger for developmental programming of adult cardiovascular disease. The identified mechanisms—cardiomyocyte hyperpolyploidy, DNA instability, inflammation, fibrosis, fetal gene reactivation, and impaired thyroid hormone/calcium/glutathione signaling—are all well-established features of human heart disease. Because cardiomyocyte polyploidization is irreversible, these effects may have lifelong consequences for cardiac function. The findings suggest that preventing or mitigating NLI in early life could reduce long-term cardiovascular risk. Limitations include the use of a rodent model (differing from humans in cardiac maturation trajectory) and focus on left ventricle only. Future work should examine other cardiac chambers, larger animal models, and the effects of varying lactose concentrations on iPSC-derived cardiomyocyte maturation.