New research highlights postnatal regeneration limits and pathological remodeling of the cardiac conduction system after myocardial infarction, uncovering mechanisms behind clinical arrhythmogenesis

In a recent study published in the Nature Cardiovascular Research, a group of researchers investigated how myocardial infarction (MI) disrupts the cardiac conduction system (CCS) and explored mechanisms of conduction system regeneration versus pathological remodeling to prevent arrhythmias (irregularities in the heart's rhythm).

The CCS generates and propagates electrical impulses critical for the heart's rhythmic contractions. Following MI, arrhythmias such as ventricular tachycardia, ventricular fibrillation, atrioventricular block, and bundle branch block significantly increase mortality.

While ischemic necrosis (tissue death due to loss of blood supply) and scarring are major contributors, the specific role of the CCS, particularly the His–Purkinje network and bundle branches, in arrhythmogenesis remains unclear.

The extent of cell loss, remodeling, and repair in the ventricular conduction system after MI is poorly understood. Further research is needed to clarify these mechanisms and develop targeted interventions for post-MI conduction system dysfunction and associated arrhythmias.

The Connexin 40 -enhanced green fluorescent protein (Cx40eGFP), Cx40- Cre recombinase fused to a tamoxifen-inducible estrogen receptor (Cx40-CreERT2), NK2 Homeobox 5 gene with a Cre recombinase knock-in (NK2 Homeobox 5-Cre (Nkx2-5Cre/+)), and Rosa26-membrane-targeted tomato/membrane-targeted green fluorescent protein (Rosa26mTmG) mouse strains were used to study CCS dynamics.

Female CD1 mice were bred with Cx40eGFP/eGFP males for neonatal imaging and single-cell RNA sequencing (scRNA-seq) studies, while CD1 females crossed with C57BL/6 males were used for electrocardiogram (ECG) recordings. All animal experiments adhered to United Kingdom (UK) regulations under the Animals (Scientific Procedures) Act 1986.

Neonatal MI was induced by ligating the left anterior descending (LAD) coronary artery. Neonatal mice were anesthetized, underwent thoracotomy, and the LAD was tied to mimic MI or left untied for sham surgeries. Post-operative care included warming, respiratory monitoring, and returning pups to their mothers. Tamoxifen-induced Cre recombinase (CreERT2) lineage tracing was performed at postnatal day 1 (P1), with injury responses analyzed by day 4 (P4).

High-resolution imaging of the ventricular conduction system (VCS) was performed using the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis (CUBIC) method. Purkinje fiber disorganization was observed post-MI, particularly in non-regenerative stages.

Ribonucleic Acid fluorescence in situ hybridization (RNA-FISH) and scRNA-seq revealed molecular changes within CCS cells, differentiating regenerative and non-regenerative responses. Computational modeling of human Purkinje networks simulated post-MI conduction delays and electrocardiographic abnormalities.

The VCS originates from working cardiomyocytes during embryogenesis and undergoes specialization around embryonic day 16.5 in mice. This specialization involves downregulating the sarcomeric apparatus and expression of unique ion channels and connexin isoforms.

VCS growth and remodeling were analyzed using a CUBIC protocol combined with immunostaining in Gja5 (Cx40)-eGFP reporter mice. Broad CX40 expression, initially observed in trabecular myocardium at embryonic day 16.5, became restricted to the mature VCS by P1.

Postnatally, significant VCS growth occurred, with a twofold increase in network volume and filament length by P10, primarily through the extension of existing Purkinje fibers rather than new branch formation. Growth was more pronounced in the right ventricle compared to the left, reflecting asymmetrical maturation.

Following MI, induced by LAD coronary artery ligation, the His–Purkinje network exhibited structural disruptions. At three days post-MI, both regenerative P1 and non-regenerative P7 hearts showed gaps in the network and altered fiber morphology. P1 hearts demonstrated increased CX40 expression in distal Purkinje fibers, while P7 hearts showed reduced fiber formation and disorganization. This disruption was critical as fiber bundling ensures electrical insulation and efficient conduction.

scRNA-seq revealed transcriptional heterogeneity in the VCS after MI, identifying cardiomyocyte-like and fibroblast-like subpopulations. Regenerative P1 hearts maintained a cardiomyocyte-like VCS population with enriched conduction-related genes, including Nkx2-5 and Slc8a1, critical for calcium signaling. In contrast, P7 hearts showed a shift toward fibroblast-like populations and downregulation of conduction-related genes, suggesting a loss of electrical identity.

At 21 days post-MI, regenerative P1 hearts restored VCS architecture and electrical function, as confirmed by ECG recordings showing no conduction delays. Non-regenerative P7 hearts exhibited persistent CX40 downregulation and gaps in the network, resulting in prolonged PR intervals and first-degree atrioventricular block, reflecting conduction delays.

In silico modeling of human hearts with reduced His–Purkinje conduction replicated observed mouse findings, linking VCS disruptions to clinical ECG signatures of conduction blocks and arrhythmias.

To summarize, this study investigated the repair and remodeling of the His–Purkinje network in neonatal mouse hearts post-MI. While regenerative P1 hearts restored their VCS via surviving CX40-positive cells, non-regenerative P7 hearts exhibited sustained abnormalities, including disrupted fiber bundling, altered cell composition, CX40 downregulation, and changes in ion channel expression.

These anomalies corresponded to conduction delays and increased dyssynchrony arrhythmias, as observed in human heart models. The findings highlight the molecular pathways driving VCS regeneration and remodeling, offering insights into arrhythmia prevention and potential therapeutic targets.