Neonatal Apical Resection Preserves the Proliferative Capacity of Cardiomyocytes Throughout the Left Ventricle

I. Academic Background: The Cardiac Mystery Facing Regenerative Medicine

Cardiovascular diseases, especially myocardial infarction (MI), are among the leading causes of death and disability worldwide due to heart damage. However, the adult mammalian heart has long been regarded as having essentially lost its endogenous regenerative ability, with the vast majority of mature cardiomyocytes entering permanent cell cycle arrest. Once injured, these cells irreversibly transform into scar tissue, resulting in heart failure and death. In contrast, lower vertebrates such as fish and salamanders possess strong myocardial regenerative capabilities, while in mammals, only neonatal individuals (within a few days after birth) have been reported to possess a brief window of myocardial regeneration. This limitation has greatly impeded progress in cardiac regenerative medicine and makes diseases such as heart failure difficult to fundamentally cure.

For early-stage regenerative capacity in mammals, previous research has confirmed that neonatal mice and large mammals such as pigs can trigger a limited regenerative response of cardiomyocytes upon injury on postnatal day 1 (P1), but this ability rapidly disappears within several days. Meanwhile, both clinical and laboratory research have focused primarily on the proliferative response of cardiomyocytes in the injury “border zone” (BZ). However, whether cardiomyocytes in the remotely located “remote zone” (RZ) can be activated, whether they possess proliferative potential, and whether injury-induced regenerative mechanisms can cover the entire ventricle remain to be systematically and quantitatively addressed. In addition, systematic studies are extremely scarce regarding the role of regulatory molecules—particularly heat shock proteins (HSPs)—in injury-induced regeneration of cardiomyocytes. Therefore, comprehensively elucidating the mechanisms of neonatal cardiac regeneration in large mammals, and seeking interventions that combine surgical and molecular strategies to promote regeneration, are pressing scientific issues in cardiac regeneration and translational medicine.

II. Source of the Paper and Team Introduction

This paper, entitled “newborn apical resection preserves the proliferative capacity of cardiomyocytes located throughout the left ventricle,” is an original research article published in Stem Cells (vol. 43, issue 5, 2025; advance access publication April 17, 2025). The major authors include Kaili Hao, Thanh Nguyen, Yuji Nakada, Gregory Walcott, Yuhua Wei, Yalin Wu, Daniel J Garry, Peng Yao, and Jianyi Zhang, mainly from the University of Alabama at Birmingham, University of Minnesota, and University of Rochester. Peng Yao and Jianyi Zhang are corresponding authors. This team has long focused on the mechanistic analysis of large mammalian cardiac regeneration, single-cell/single-nucleus transcriptomics, artificial intelligence data mining, and related fields, and has profound expertise in cardiac regeneration and translational medicine.

III. Research Workflow and Innovative Methods

1. Overall Experimental Design and Animal Model

This research centers on neonatal pigs (piglets) as experimental subjects, comparing cardiac regenerative capacity in two main models:

• On postnatal day 1 (P1), apical resection (AR, denoted as ARP1) was performed, followed by myocardial infarction (MI) induced by left anterior descending coronary artery ligation at day 28 (P28) and healing assessed at P56.
• In the control group, MI was induced only at P28, without early resection.

As previously reported, the MI-only group developed severe cardiac scarring, while the ARP1+MI group exhibited almost no visible scarring, with regeneration primarily dependent on proliferation of the cardiomyocytes themselves. The paper further hypothesizes whether ARP1 can preserve the proliferative capacity of cardiomyocytes across all regions of the left ventricle, rather than only in the vicinity of the resection.

2. Sampling and Immunofluorescence Analysis

At various time points (P2, P4, P8, P15, P28) in neonatal pig hearts, regions following apical resection (~5 mm above apex) and MI (~2 cm from apex) were partitioned, and samples collected from the border zone (BZ, adjacent to resection), remote zone (RZ, distant from resection), and healthy controls. Each group included samples from 3 pigs. Key detection parameters included:

• Cardiomyocyte cell cycle activity markers—phosphorylated histone 3 (PH3) and symmetric Aurora B kinase (SAUB) expression.
• Immunofluorescence labeling of CTnT (cardiac troponin T), α-Actin (α-actin) to identify cardiomyocytes.
• Tissue sections imaged at multiple points (n≥20 per group), positive cell ratios quantified.

3. Single-Nucleus RNA Sequencing and AI Analysis

The team employed 10X Genomics single-nucleus RNA sequencing (snRNA-seq) to obtain transcriptional data from cardiomyocyte nuclei in various heart regions and timepoints. For high-dimensional data, they innovatively used a “cell-cycle-specific autoencoder” for 10-dimensional reduction and clustering, combined with UMAP (Uniform Manifold Approximation and Projection) for cluster visualization, and applied the DBSCAN (Density-Based Spatial Clustering of Applications with Noise) algorithm for automated cell subtype identification.

Analysis covered embryonic, healthy controls at each period (P1, P2, P28), and different regions of ARP1-treatment groups. The AI tools were independently developed and optimized by the team, with previous literature publications.

4. Molecular Pathway and Mechanism Exploration

Using Ingenuity Pathway Analysis, the team analyzed enriched genes in activated cell subpopulations. Focus was placed on heat shock protein family members (HSPA5, HSP90B1, HSP90AB1) and downstream hypoxia-inducible factor HIF1 signaling. In the human AC16 cardiomyocyte cell line, overexpression/knockdown of these HSPs was achieved via lentiviral delivery, and Western blot assessed downstream effectors including PH3, PRX-V (peroxiredoxin V, an antioxidant), P53, and its phosphorylation status, revealing regulatory mechanisms.

5. Dual Validation: Tissue- and Molecular-Level

Given the potential bias in traditional immunohistochemistry, the team used:

• Small-area, high-signal targeted imaging, and
• Randomized, blinded, large-area sampling (spanning 3mm, covering ≥10% of tissue), applying Z-stack multilayer scanning and matrix segmentation quantification for PH3, HSPA5, HSP90B1, etc., processed with in-house MATLAB software for heatmap and violin plot visualization, ensuring thorough and credible quantification.

Furthermore, qRT-PCR was used to molecularly validate some key markers at the tissue level. The overall experimental design emphasized multi-level, mutually validating data.

IV. Main Results

1. Significant Upregulation of Proliferation Markers across the Entire Left Ventricle

PH3 and SAUB expression increased persistently from P2–P8 in the ARP1 model in both the resection border (BZ) and remote zone (RZ); while levels declined slightly at P14 and P28, they remained higher than controls, with extremely significant statistical differences. The proportion of PH3/SAUB-positive cells between the two regions showed no significant difference at any timepoint, overturning the traditional concept of localized regeneration. Results were consistent upon repeated validation with nuclear marker NKX2-5. Analysis of cell area and density also confirmed similar size and quantity of cells in both regions.

2. snRNA-seq Reveals Widespread Proliferative Cardiomyocyte Subpopulations

snRNA-seq AI analysis clearly identified four major cardiomyocyte subpopulations:

• CM1: Prominently enriched at the embryonic stage and at P8 and P15 in the ARP1 model, with similar ratios in BZ and RZ. This subpopulation highly co-expressed key cell cycle/division genes (Aurora B, MKI67, INCENP, CDCA8, BIRC5), designated as actively proliferative cardiomyocytes.
• CM2: More common in embryonic and early stages, associated with genes involved in intercellular junctions and ECM (extracellular matrix), mainly contributing to structural remodeling and signal conduction.
• CM3: Enriched at P8 in ARP1, involving centromere/chromatin regulatory processes, as a subpopulation related to cell cycle initiation.
• CM4: Represents the majority of terminally differentiated, non-dividing cardiomyocytes.

Analysis of temporal and subpopulation ratio trends showed that in ARP1 groups, CM1 and CM3 proliferative subpopulations appeared synchronously in both BZ and RZ, indicating that the effect of apical resection extends beyond the local site to activate regenerative capacity throughout the left ventricle.

3. Heat Shock Proteins and HIF1α Pathway as Key Molecular Axes

Pathway enrichment and immunohistochemistry quantitation showed that HSPA5, HSP90B1, HSP90AB1 were strongly upregulated from P2–P8 in ARP1 in both BZ and RZ, with no significant difference between them. AI analysis showed that the proportion of cells with high HIF1 signaling activity/HSPA5 expression peaked at P4 in ARP1, synchronously activating downstream proliferation and antioxidant defense pathways.

4. Mechanistic Validation: HSPA5/HSP90B1 Promote Proliferation of Human Cardiomyocytes

Experiments in AC16 human cardiomyocyte cultures confirmed that:

• Overexpression of HSPA5 significantly increased expression of PH3 (cell cycle marker) and PRX-V (antioxidant), and reduced P53 and its phosphorylation (promoting proliferation);
• Knockdown of HSPA5 had the opposite effect: PH3 and PRX-V decreased, P53 increased.
• HSP90B1 likewise promoted upregulation of HSPA5 and cell proliferation and downregulated the P53 pathway.

This demonstrates that heat shock proteins efficiently maintain cardiomyocyte cell cycling and antioxidant status, promoting regeneration through mechanisms somewhat similar to those in tumor cells (such as P53 inhibition).

V. Research Conclusions and Value

1. Major Conclusions

Neonatal pig apical resection (ARP1) can significantly and durably preserve the regenerative potential of cardiomyocytes across all regions of the left ventricle, promoting widespread cardiomyocyte cell cycle activity and overturning the previous view that cardiac regeneration is limited to the injury site. This mechanism is likely closely related to upregulation of HSPA5/HSP90 class heat shock proteins, activation of HIF1 signaling, downregulation of P53, and enhanced antioxidant defense.

2. Scientific and Applied Value

This study, for the first time, systematically demonstrates in a large mammal that: (1) surgical intervention (apical resection) can permanently “reprogram” neonatal cardiomyocytes for ventricle-wide regenerative capacity; (2) HSPA5 and related heat shock proteins and HIF1 signaling are pivotal molecular regulators of cardiac regeneration; (3) a new therapeutic paradigm is proposed based on systemic intervention (surgical + molecular) to achieve widespread regeneration in mammalian hearts. This provides both theoretical foundation and translational potential for the regenerative treatment of heart failure and for molecular drug development.

3. Research Highlights and Innovations

• Pan-ventricular Regeneration Evidence: Experimentally demonstrated for the first time that apical resection can enable long-term retention of proliferative potential in all regions of the left ventricle’s cardiomyocytes;
• AI-Powered Big Data Analysis: Used cell-cycle-specific autoencoder combined with UMAP/DBSCAN for unbiased clustering, uncovering more refined cell lineages and functional subpopulations;
• Multi-layered Validation of Molecular Mechanisms: Integrated animal tissue, cell line experiments, and mechanistic molecular studies to comprehensively elucidate the multi-axis role of HSPA5/HSP90B1 in activating cardiac regeneration;
• Innovative Sampling and Quantitative Analysis: Combined large-area blinded sampling with small-area targeted imaging, used multimodal MATLAB quantitative algorithms to enhance the credibility of conclusions.

4. Other Valuable Information

The paper also discusses the potential role of heat shock protein HSPA5 as a non-classical RNA-binding protein in multiple cell types (including cardiomyocytes), suggesting that it is not just a protein chaperone but may regulate translation and protein folding via RNA actions, providing direction for future research into fundamental mechanisms.

VI. Summary and Outlook

Integrating surgical intervention, molecular immunology, single-cell genomics, artificial intelligence, and molecular functional mechanisms, this study is the first to comprehensively reveal that neonatal apical resection in large mammals can globally activate the regenerative potential of cardiomyocytes throughout the left ventricle, and to resolve its regulation network centered on heat shock proteins. These results set a paradigm for a new “pan-ventricular, multi-target, systemic promotion” approach in cardiac regenerative medicine, and provide a solid foundation for the development of novel regenerative therapeutic strategies. In the future, research focusing on the RNA-binding activity of heat shock proteins, tissue-specific targeting, and the clinical translation of combined surgical and molecular interventions will become important trends in the field of cardiac regeneration.