Progressive Left Ventricular Pressure Overload Reprograms Neonatal Left Ventricular Development via Sustained Proliferation, Metabolic Arrest, and Compensatory Angiogenesis

1. Introduction

Left ventricular pressure overload (LVPO) is a defining hemodynamic feature of various cardiovascular diseases, including hypertension and aortic valve stenosis [1,2,3]. In adult mammals, LVPO invariably triggers maladaptive remodeling characterized by cardiomyocyte (CM) hypertrophy, interstitial fibrosis, and eventual contractile dysfunction—a pathological progression extensively modeled via transverse aortic constriction (TAC) in adult rodents [4,5,6]. However, the neonatal heart exhibits a fundamentally distinct response to increased afterload [7,8,9,10]. Unlike adult CMs, neonatal CMs retain a remarkable, albeit transient, capacity for proliferation and regeneration during the first postnatal week [7,8,9,10]. Harnessing this intrinsic regenerative window holds immense therapeutic promise; yet, how LVPO intersects with and rewires this developmental program remains poorly understood.

To date, most mechanistic insights into neonatal LVPO have been derived from neonatal TAC models, which induce an acute, fixed, and often severe pressure overload [11,12,13]. While valuable, this fixed-load paradigm presents two major limitations. First, it mimics only a narrow spectrum of pediatric conditions, such as critical coarctation or interrupted aortic arch, and fails to recapitulate the progressive nature of LVPO commonly encountered in clinical practice—including postoperative aortic arch re-obstruction, evolving left ventricular outflow tract obstruction, and childhood-onset hypertension. Second, and more importantly, severe fixed overload has been shown to restrain CM proliferation and even provoke apoptosis [13], thereby obscuring the genuine adaptive capacity of the neonatal ventricle. In stark contrast, progressive LVPO, as induced by abdominal aortic banding surgery (ABS), preserves the buffering function of the elastic aorta, allowing a gradual ramp-up of afterload that more closely parallels the clinical trajectory [14,15,16]. This progressive nature may create a fundamentally different microenvironment—one that possibly sustains or even amplifies the proliferative response rather than suppressing it. Nevertheless, the precise impact of such progressive loading on the transcriptional and cellular architecture of the developing left ventricle (LV) has remained largely uncharted.

We have recently established a neonatal rat ABS model at postnatal day 1 (P1) that yields a 100% survival rate and induces a sustained, stepwise elevation of blood pressure without thoracotomy [14]. This model provides a unique experimental platform to dissect how progressive LVPO dynamically intersects with the intrinsic developmental clock of the neonatal LV.

In the present study, we address a central question: How does progressive LVPO reprogram the neonatal LV developmental trajectory during the critical window from P3 to p7? By performing comparative RNA-sequencing, we distinguish three categories of biological events: (i) developmental processes that remain unaltered despite the overload, (ii) normal LV maturational events that are extinguished under loading, and (iii) novel adaptive programs that emerge exclusively in response to progressive pressure stress. Our findings reveal that, contrary to the fixed-overload paradigm, progressive LVPO sustains and even enriches cell-cycle activity while simultaneously attenuating metabolic maturation and external-stimulus responsiveness, and it actively triggers angiogenic programs to secure oxygen and nutrient supply. This work not only elucidates the adaptive logic of the neonatal LV under clinically relevant loading conditions but also identifies potential molecular levers—such as mechanosensors and angiogenic factors—that could be therapeutically targeted to rejuvenate cardiac regeneration in adult settings. Ultimately, understanding how progressive LVPO reshapes the neonatal LV may bridge the gap between developmental biology and pediatric cardiovascular therapeutics, offering new strategies to manage congenital heart diseases and to prevent long-term maladaptive remodeling.

2. Materials and Methods

2.1. Ethics and Animal Care

All animal procedures were approved by the Animal Welfare and Human Studies Committee of Shanghai Children’s Medical Center (IRB approval No. SCMC-LAWEC-2024-1127, dated November 24, 2024) and conformed to the Guide for the Care and Use of Laboratory Animals. Sprague-Dawley rat pups of both sexes were bred in our specific pathogen-free facility under a 12-h light/dark cycle with ad libitum access to food and water.

2.2. Abdominal Aortic Banding Surgery Model and Validation

Abdominal aortic banding (ABS) or sham surgery was performed on postnatal day 1 (P1) as detailed in our recently published protocol [14]. Briefly, under a surgical microscope, the abdominal aorta was exposed through a midline laparotomy. A 12-0 suture was tied around the aorta together with a blunted 30-G padding needle, which was then gently withdrawn to create a standardized luminal narrowing. Sham-operated littermates underwent the same laparotomy and closure without ligation(Supplemental Video S1).

Model efficacy was validated using two complementary approaches: (i) abdominal ultrasound (Vevo 2100 system) at P3 and P7 to measure peak flow velocity at the banding site, and (ii) serial non-invasive tail-cuff plethysmography (IITC Life Science MRBP system) from P21 to P35 to monitor systolic, diastolic, and mean arterial pressures in the left upper limb [14,17]. These validation data, confirming a progressive pressure overload, were fully described in our prior work and served as inclusion criteria for the subsequent analyses.

2.3. Experimental Design and Tissue Collection

A total of 84 pups of both sexes were randomly assigned to four experimental groups (n = 21 per group): Sham-P3, ABS-P3, Sham-P7, and ABS-P7 (Figure 1A). From each group, 3 randomly selected animals were designated for RNA sequencing, 6 randomly selected animals for immunofluorescence staining, 6 randomly selected animals for qRT-PCR, and 6 remaining for serial non-invasive tail-cuff plethysmography. At the respective time points (P3 or P7), animals were euthanized, and hearts were rapidly excised. The left ventricular (LV) free wall was carefully dissected from the atria and right ventricle in ice-cold phosphate-buffered saline (PBS). LV tissues designated for RNA sequencing were snap-frozen in liquid nitrogen and stored at –80°C. Specimens for immunofluorescence were fixed in 4% paraformaldehyde, dehydrated in graded sucrose solutions, embedded in optimal cutting temperature (OCT) compound, and stored at –80°C until sectioning.

2.4. RNA Extraction and Library Preparation

Total RNA was extracted from LV free-wall samples using TRIzol® reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA purity and concentration were assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific), and integrity was verified on an Agilent 2100 Bioanalyzer (RIN ≥ 8.0 for all samples). Sequencing libraries were constructed from 1 μg of total RNA per sample using the NEBNext® Ultra™ RNA Library Prep Kit (New England Biolabs, USA), following the recommended protocol. Library quality was validated on the Agilent 2100 system, and paired-end 150-bp (PE150) sequencing was performed on an Illumina NovaSeq 6000 platform. Raw sequence data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE289819.

2.5. Bioinformatics Processing and Differential Expression Analysis

Raw reads were processed with Trimmomatic (v0.39) to remove adapter sequences, low-quality bases (Phred score < 20), and reads with excessive poly-N content, yielding high-quality clean reads. Clean reads were aligned to the rat reference genome (Ensembl Rnor_6.0) using STAR (v2.7.3a). Gene-level read counts were quantified with featureCounts (v1.6.3) and normalized as fragments per kilobase of transcript per million mapped reads (FPKM).

Differentially expressed genes (DEGs) were identified using the DESeq2 R package (v1.30.0). To evaluate the developmental transcriptional changes from P3 to P7 under normal and overloaded conditions, we compared Sham-P7 versus Sham-P3 (normal development) and ABS-P7 versus ABS-P3 (pressure-overload development). DEGs were defined as those with |log₂(fold change)| ≥ 0 and a P value < 0.05 [18,19,20]. This approach enables the capture of all statistically significant expression changes, including subtle ones, thereby facilitating exploratory analyses [18,19,20].

2.6. Classification of DEGs and Functional Enrichment

To discern the specific impact of progressive pressure overload on the neonatal LV developmental program, the DEG sets from the two comparisons were intersected using Venn diagram analysis and categorized into three non-overlapping groups:Shared DEGs – genes differentially expressed in both normal and overloaded development;Normal-unique DEGs – genes altered only in Sham-P7 versus Sham-P3 (i.e., developmental events that disappeared under overload);Overload-unique DEGs – genes altered only in ABS-P7 versus ABS-P3 (i.e., newly emerged events under overload).Functional enrichment analyses were performed using the clusterProfiler R package (v4.0.5), covering Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Significance was set at FDR-adjusted P < 0.05.

2.7. Immunofluorescence Staining

Frozen OCT-embedded tissues were sectioned at 8 μm thickness. Sections were air-dried, washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 5% goat serum for 1 h at room temperature. Primary antibodies—rabbit anti-Ki67 (1:200, ab15580, Abcam) and mouse anti-cardiac troponin T (cTnT, 1:400, ab8295, Abcam)—were applied overnight at 4°C. After PBS washes, sections were incubated with appropriate Alexa Fluor® 488- or 555-conjugated secondary antibodies (1:500, Thermo Fisher) for 1 h at room temperature in the dark, followed by DAPI counterstaining. Fluorescence images were captured on a Leica SP8 confocal microscope. For each animal, at least 5 random fields (×400 magnification) were analyzed using ImageJ software (NIH, USA). The percentage of Ki67-positive cardiomyocytes among cTnT-positive cells was calculated.

2.8. Quantitative Real-Time PCR Validation

To validate the RNA-seq findings, total RNA was extracted from LV samples (n = 6 per group) and reverse-transcribed into cDNA using the PrimeScript™ RT Kit (Takara, Japan). Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler® 480 II system (Roche) using SYBR® Premix Ex Taq™ (Takara). The primer sequences for mechanosensory genes (Piezo2, Sik1, Impact,Tnfrsf1a,Tlr4,Slc39a4, Nppa, and Srebf2) and angiogenesis-related genes (Adam8, Dll1, Ptgs2,Aplnr,F3,FIt1,Tgfbr2, and Ntrk2) are listed in Supplemental Table S1, with Gapdh as the internal control. Each reaction was run in triplicate, and relative expression was calculated using the 2^-ΔΔCt method.

2.9. Statistical Analysis

All continuous data are expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using two-tailed Student’s t-test; multiple group comparisons were assessed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) post-hoc test. Data normality was confirmed by the Shapiro-Wilk test and homogeneity of variances by Levene’s test; when these assumptions were violated, the Mann-Whitney U test was applied. All statistical analyses were conducted using SAS software (v12.0, SAS Institute Inc.), and a two-sided P < 0.05 was considered statistically significant.

3. Results

3.1. Establishment of a Progressive LVPO Model in Neonatal Rats

To investigate how progressive pressure overload remodels left ventricular (LV) development during the neonatal period, we performed ABS on P1 rats. This established a model of LVPO [14]. At P3 and P7, we conducted RNA-seq, hematoxylin-eosin (HE) staining, and abdominal ultrasound examinations (Figure 1A). Because neonatal rats are too small for upper-limb blood pressure measurement, we randomly selected 6 rats per group and measured their blood pressure from P21 to P35 (Figure 1A). As shown in Figure 1B, a thread-fixed aorta was visible in the abdomen at P7, and HE staining revealed marked narrowing and thickening of the arterial wall at the banding site (Figure 1C–D). These findings confirmed successful ABS. The procedure yielded a nearly 100% success rate with no mortality, consistent with our previous report [14].

3.2. Validation of the Progressive Neonatal LVPO Model

To verify that ABS effectively induces a progressive LVPO, we first performed abdominal ultrasound. At both P3 and P7, increased blood flow velocity and turbulent flow were observed at the banding site, indicating hemodynamic alterations distal to the aortic constriction (Figure 2A–C). Furthermore, continuous monitoring of upper-limb blood pressure revealed sustained elevations in systolic, diastolic, and mean arterial pressures (Supplemental Figure S1A–C), confirming that ABS induces a progressive LVPO. These results are consistent with our previous observations [14].

3.3. Progressive LVPO Alters the Neonatal LV Developmental Trajector

To explore how progressive LVPO modifies LV development during the neonatal period (from P3 to P7), we performed RNA-sequencing on LV free walls collected at P3 and P7. In normal neonatal LV development, we identified 2,927 differentially expressed genes (DEGs), of which 1,697 were downregulated and 1,230 upregulated. Under LVPO, 3,132 DEGs were detected, including 1,742 downregulated and 1,390 upregulated genes (Supplemental Figure S2A–B), suggesting only modest changes in total DEG counts. The heatmap of DEGs showed good intra-group similarity and inter-group divergence (Figure 3A). Principal component analysis (PCA), however, revealed little difference between ABS and sham groups at P3, but a marked separation at P7, indicating that progressive LVPO substantially shifts the developmental trajectory of the neonatal LV (Figure 3B). The Venn diagram showed 1,550 DEGs shared between LVPO-exposed and normal development; 1,582 DEGs were uniquely expressed in the ABS group, while 1,377 were unique to Sham group (Figure 3C). Collectively, these results indicate that progressive LVPO notably alters neonatal LV development.

3.4. Neonatal LV Developmental Events Unaltered by Progressive LVPO

To understand how LVPO affects neonatal LV development, we first examined the developmental events that remain unchanged. Functional enrichment analysis of the 1,550 shared DEGs was performed. Gene Ontology (GO) analysis revealed that these genes were primarily enriched in cardiac regeneration, cell proliferation, and cardiomyocyte morphogenesis, suggesting that these essential postnatal cardiac developmental processes are preserved (Figure 4A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis yielded consistent results, showing enrichment in cell cycle and DNA replication (Figure 4B). To validate the transcriptomic findings, we performed immunofluorescence staining for Ki67, a marker of proliferation. Under progressive LVPO, the number of proliferating (Ki67-positive) CMs increased in the neonatal LV (Figure 4C–D). In contrast, during normal development, proliferating CMs decreased (Supplemental Figure S3A–B). Thus, although both conditions showed enrichment for cardiac regeneration and CM proliferation, the actual cellular responses were opposite.

3.5. Neonatal LV Developmental Events Lost Under LVPO

To understand how LVPO affects neonatal LV development, we first examined the developmental We next analyzed the 1,377 DEGs uniquely expressed during normal development to identify events that disappear under progressive LVPO. GO enrichment indicated that these genes were mainly associated with response to external stimuli, metabolism, and oxidative phosphorylation (Figure 5A), suggesting that maturation of the neonatal LV may be compromised. Given that cardiac regeneration and CM maturation are opposing processes [21,22], this finding is consistent with the enhanced CM proliferation observed under LVPO (Figure 4C–D). KEGG pathway analysis similarly highlighted oxidative phosphorylation and metabolic pathways (Figure 5B). These results also imply a blunted response to external stimuli in pressure-overloaded LVs. To corroborate this, we performed heatmap and qRT-PCR analyses for eight DEGs within the “response to external stimulus” term. We observed significant upregulation of mechanosensors such as Piezo2, Sik1, and IMPACT (Figure 5C–D), validating the enrichment results.

3.6. Neonatal LV Developmental Events Newly Emergent Under LVPO

To understand how LVPO affects neonatal LV development, we first examined the developmental Finally, we performed enrichment analysis on the 1,582 DEGs uniquely expressed under LVPO to identify newly emergent developmental events. GO analysis showed predominant enrichment in angiogenesis, cell migration, and cell cycle (Figure 6A), indicating that the neonatal LV may generate new vessels to augment blood and oxygen supply in response to progressive LVPO. KEGG analysis corroborated these findings, with enrichment in cell cycle, DNA replication, and the VEGF signaling pathway (Figure 6B). To verify these results, we examined eight DEGs within the “blood vessel morphogenesis” term using heatmap and qRT-PCR. We found significant upregulation of angiogenesis-related genes, including Adam8, Dll1, and Ptgs2 (Figure 6C–D), confirming the enrichment analysis.

4. Discussion

The transition of the mammalian heart from the neonatal regenerative state to the adult non-regenerative state represents a critical window for therapeutic intervention [23,24]. While fixed pressure overload (e.g., TAC) is known to either suppress proliferation or induce apoptosis in neonatal CMs [13], the impact of progressive LVPO on the endogenous developmental trajectory of the neonatal LV has remained obscure. By leveraging a clinically relevant P1 neonatal rat model of progressive LVPO (as recently established and verified in our prior work [14]), the present study provides the transcriptomic and cellular dissection of how a gradually LVPO dynamically rewires LV development during the critical postnatal window (P3–P7). Our results reveal a paradigmatic shift: rather than simply superimposing pathological stress onto normal development, progressive LVPO actively reprograms the balance between proliferation, maturation, and angiogenesis, creating a unique adaptive state that fundamentally alters the cardiac maturation clock.

One of the most striking findings is the functional dichotomy observed among the 1,550 shared DEGs. Although both normal development and progressive LVPO enriched for "cardiac regeneration" and "cell proliferation" (Figure 4A–B), the cellular phenotypes were diametrically opposite: normal development saw a progressive decline in Ki67⁺ CMs, whereas progressive LVPO resulted in a substantial increase (Figure 4C–D). This suggests that the shared gene signature does not represent a fixed proliferative trajectory but rather a plastic transcriptional repertoire that responds to mechanical cues.

The disappearance of normal maturational events under LVPO has profound implications. The GO and KEGG enrichment of the 1,377 lost DEGs highlighted deficits in oxidative phosphorylation and metabolic reprogramming, which are hallmarks of normal postnatal heart maturation. This finding echoes the "regenerative-metabolic switch" hypothesis [21,25], wherein the neonatal heart's regenerative capacity is intrinsically linked to its reliance on glycolysis [21,25]. By blunting the normal shift toward mitochondrial oxidative metabolism, progressive LVPO forces the LV to remain in an "immature" metabolic niche. Notably, the attenuated response to external stimuli suggests that the overloaded LV downregulates its sensitivity to systemic signals—potentially a protective mechanism to isolate the developing myocardium from inflammatory or neurohormonal overactivation, thereby preventing fibrosis and maladaptive remodeling, which is consistently observed in adult overload models but absent in this neonatal ABS model.

A third layer of adaptation is the de novo emergence of pro-angiogenic programs exclusively in the overloaded LV (1,582 DEGs, Figure 6A–B). The enrichment of VEGF signaling, alongside upregulated Adam8, Dll1, and Ptgs2 (Figure 6C–D), indicates that increased wall stress rapidly precipitates a vasculogenic response. In adult hearts, angiogenesis usually lags behind myocyte hypertrophy, leading to relative hypoxia and fibrotic substitution. In contrast, the neonatal LV appears to couple myocyte proliferation with active vascular morphogenesis. This coordinated response is likely critical for sustaining the increased metabolic demand of expanded cell populations. Without this concurrent angiogenesis, the proliferative surge observed in Figure 4 would likely be futile or even harmful due to ischemia. This integrated proliferative-angiogenic response distinguishes progressive LVPO from fixed severe overload, where rapid and severe pressure elevation often leads to capillary rarefaction and necrosis [4,5].

Our previous method paper [14] demonstrated that ABS induces a gradual increase in BP, contrasting sharply with the acute afterload of TAC [4,5]. The present data provide the biological rationale for this technical distinction. In the fixed TAC model, the sudden stress causes a massive surge of DEGs early on, overwhelming the adaptive capacity and leading to apoptosis. However, the "gradual ramping" in ABS allows the neonatal LV to phase its responses: initial metabolic retention (P3), followed by robust proliferation and angiogenesis (P7). This stepwise adaptation likely prevents the unfolded protein response and ER stress observed in acute models. Therefore, progressive LVPO does not merely "damage" the developing heart; it co-opts the developmental machinery to build a larger, more vascularized, yet functionally preserved myocardium. This has critical implications for pediatric cardiology: children with postoperative residual stenosis or evolving hypertension often present with preserved systolic function despite hypertrophy. Our model provides a mechanistic explanation—the preservation of proliferation and angiogenesis outweighs the fibrotic and apoptotic pathways.

This study underscores that the neonatal heart possesses an inherent "adaptive rheostat" sensitive to the rate of load increase. The identification of Piezo2/Sik1/IMPACT as potential mechano-metabolic sensors opens new avenues for therapeutic intervention. If we can pharmacologically "trick" the adult heart into mimicking this metabolic stunting and angiogenic response, it may be possible to rekindle proliferation without eliciting fibrosis. Furthermore, the 1,582 uniquely expressed genes in the overloaded heart represent a reservoir of novel targets—particularly those regulating angiogenesis (e.g., Dll1 and Ptgs2)—that could be harnessed to improve coronary perfusion in ischemic heart diseases.

Several limitations should be acknowledged. First, our transcriptomic analysis was limited to the P3-P7 window; whether the observed "maturational delay" persists into adulthood or eventually catches up requires long-term follow-up. Second, while we identified robust gene expression changes, causal relationships (e.g., whether Piezo2 directly mediates the metabolic shift) need to be validated via cardiac-specific knockout or knockdown experiments. Third, we primarily focused on CMs; the contribution of non-myocytes (fibroblasts, endothelial cells) to the observed transcriptomic changes, particularly the angiogenic signature, warrants further single-cell resolution investigation.

5. Conclusions

In summary, this study demonstrates that progressive LVPO fundamentally alters the neonatal LV developmental trajectory by orchestrating a three-pronged adaptation: (i) sustaining CM proliferation through metabolic maturation arrest mediated by mechanosensors; (ii) extinguishing normal maturational pathways to maintain cellular plasticity; and (iii) actively inducing compensatory angiogenesis to support the expanding myocardium. These findings redefine our understanding of cardiac adaptation to afterload and establish the neonatal ABS model as an indispensable tool for uncovering the molecular logic of cardiac regeneration, with profound implications for treating both pediatric congenital heart diseases and acquired heart failure in adults.