Multimodality Molecular Profiling Nominates Targetable Mechanisms in Progressive RV Dysfunction

This study utilizes multi-omics profiling of a pig model to characterize the cellular and molecular landscape of progressive right ventricular dysfunction, identifying specific mechanisms such as impaired mitochondrial proteostasis, defective macrophage efferocytosis, and activated ribotoxic stress responses as potential therapeutic targets.

The Gist

Imagine your heart as a two-pump system. The left pump pushes blood to your whole body, and the right pump pushes blood to your lungs. In many heart diseases, the right pump gets clogged or blocked (like a clogged pipe), forcing it to work much harder. This is called Right Ventricular Dysfunction (RVD).

Think of the right ventricle like a marathon runner. At first, when the road gets steep (the blockage), the runner speeds up and muscles get bigger to keep going. This is the "mild" stage. But if the road stays steep too long, the runner eventually collapses. This is the "severe" stage.

Until now, doctors knew the runner collapsed, but they didn't fully understand why the muscles failed at the cellular level, making it hard to fix the problem with medicine.

This study is like a team of high-tech detectives who took a "snapshot" of the runner's muscles at different stages of the race (Control, Mild, and Severe) using advanced microscopes and computers. They looked at the DNA, the proteins, and the chemical signals inside the cells to find out exactly what went wrong.

Here is what they discovered, explained simply:

1. The "Garbage Truck" Broke Down

Inside the heart, there are special cells called macrophages. Think of them as the heart's sanitation workers or garbage trucks. Their job is to clean up dead or dying cells so the heart can stay healthy.

• What happened: In the severe stage, these garbage trucks showed up in huge numbers, but they were broken. They couldn't pick up the trash (dead cells).
• The result: The heart became filled with garbage and inflammation, making it harder for the heart to beat.
• The Fix: The researchers found that a "Do Not Eat Me" sign (a protein called CD47) was stuck on the dying cells, telling the garbage trucks to back off. They suggest using existing drugs to remove that sign so the trucks can start cleaning again.

2. The Power Plant Lost Its Spark

The heart cells have tiny power plants inside them called mitochondria. These generate the energy the heart needs to pump.

• What happened: In the severe stage, the power plants started to get damaged and filled with broken parts. Normally, the cell has a "repair crew" (called the mitochondrial unfolded protein response) that fixes these broken parts.
• The result: In severe RVD, the repair crew stopped working. The power plants got clogged with junk, and the heart ran out of energy.
• The Fix: The researchers found a common allergy/vertigo drug called Meclizine that might wake up this repair crew, helping the power plants get back to work.

3. The Construction Crew Got Confused

When the heart is under stress, it tries to build bigger muscles (hypertrophy) to handle the load. This requires a lot of construction equipment (ribosomes).

• What happened: In the severe stage, the heart built too many construction machines, but they were defective. Instead of building muscle, these broken machines started screaming "Alert!" (a stress response called the ribotoxic stress response).
• The result: This alarm signal told the cells to commit suicide (a process called pyroptosis), causing more heart cells to die.
• The Fix: They found a way to silence this alarm using a drug called Disulfiram (usually used for alcoholism) or a new experimental drug that stops the alarm from ringing.

4. The Blood Vessels Got Messy

The heart needs a network of tiny blood vessels to get oxygen.

• What happened: In severe RVD, the heart tried to grow more blood vessels, but they were messy and disconnected. The "support beams" (pericytes) that hold the vessels together were missing.
• The result: Even though there were more vessels, they didn't work well, leaving the heart muscle starving for oxygen (hypoxia).

The Big Picture: Why This Matters

This study is a game-changer because it doesn't just say "the heart is failing." It gives a specific instruction manual on how to fix it.

The researchers found three main "levers" they can pull to potentially save the heart:

1. Wake up the repair crew (using Meclizine) to fix the power plants.
2. Turn off the suicide alarm (using Disulfiram or ZAK inhibitors) to stop cells from dying.
3. Unstick the garbage trucks (using anti-CD47 antibodies) to clean up the inflammation.

The Takeaway:
For years, we've tried to treat heart failure by just making the heart pump harder. This study suggests we should instead fix the internal machinery—cleaning the trash, repairing the power plants, and silencing the false alarms. Since many of the drugs they identified are already approved for other conditions (like allergies or alcoholism), we might be able to repurpose them to treat heart failure much faster than waiting for brand-new drugs to be invented.

Technical Summary

1. Problem Statement

Right ventricular dysfunction (RVD) is a critical predictor of mortality in various cardiovascular diseases (CVDs), including pulmonary arterial hypertension, valvular cardiomyopathy, and congenital heart disease. Despite its prognostic importance, there are currently no effective therapies that directly augment RV function.

• Knowledge Gap: It remains unclear whether the severity of RVD corresponds to distinct, progressive cellular and molecular alterations. Most existing studies focus on rodent models or specific disease states (like PAH) without an integrated, multi-omic view across the spectrum of dysfunction (from compensated/mild to decompensated/severe).
• Objective: To define the cellular landscape and molecular mediators of progressive RVD using a large animal model and to nominate druggable pathways for therapeutic intervention.

2. Methodology

The study utilized a Pulmonary Artery Banded (PAB) pig model to induce pressure overload, creating a spectrum of RV dysfunction.

• Animal Cohort: 16 Yorkshire male pigs (29–73 days old) stratified into three groups based on Cardiac MRI-derived RV Ejection Fraction (RVEF):
  • Control: RVEF ~58% (n=5)
  • Mild RVD: RVEF 35–43% (n=5)
  • Severe RVD: RVEF <30% (n=6)
• Multi-Omic Profiling:
  • Single-Nucleus RNA Sequencing (snRNAseq): Performed on 4 pigs per group to map cell-type specific transcriptomic changes. Identified 7 major cell types (cardiomyocytes, macrophages, fibroblasts, endothelial cells, pericytes, neuronal cells, lymphocytes).
  • Proteomics: TMT 16-plex proteomics was conducted on mitochondrial-enriched and cytoplasmic fractions from all 16 pigs to assess protein abundance and pathway activity.
  • Phosphoproteomics: Mass spectrometry analysis of phosphorylated peptides to map kinase and phosphatase activity (kinome and phosphatome).
• Validation:
  • Histology/Immunohistochemistry: WGA staining for cardiomyocyte hypertrophy, Isolectin-B4 for capillary density, and Galectin-3 for macrophages.
  • Confocal Microscopy: Quantification of macrophage abundance and pericyte-endothelial colocalization.
• Data Integration: Computational integration of transcriptomic, proteomic, and phosphoproteomic data using tools like Seurat, CellChat, KEGG, and Kinase Enrichment Analysis (KEA).

3. Key Results

A. Physiological and Structural Progression

• RVD progression was characterized by a stepwise decline in RVEF, RV-pulmonary artery coupling, and an increase in RV dilation, mass, and tricuspid regurgitation.
• Crucial Finding: Cardiomyocyte hypertrophy (cross-sectional area) was comparable between mild and severe RVD, suggesting that functional decline is driven by molecular/cellular dysfunction rather than just structural hypertrophy.

B. Cellular Landscape Remodeling (snRNAseq)

• Cellular Composition: Severe RVD was marked by a significant loss of cardiomyocytes (dropping from ~74% to ~49%) and a compensatory expansion of non-cardiomyocyte populations, particularly macrophages (resident and recruited), lymphocytes, endothelial cells, and pericytes.
• Macrophage Dysfunction:
  • Both resident and recruited macrophages accumulated in severe RVD.
  • Impaired Efferocytosis: Pathway analysis revealed downregulation of efferocytosis, phagosome, and lysosomal pathways in severe RVD macrophages.
  • CD47 Upregulation: Fibroblasts and cardiomyocytes in severe RVD upregulated CD47 ("don't eat me" signal), which inhibits macrophage engulfment of dying cells, creating a pro-inflammatory environment.
• Microvascular Compromise: Despite increased endothelial cell density, severe RVD showed reduced colocalization between pericytes and endothelial cells and upregulated cardiomyocyte HIF1A, indicating functional microvascular failure and hypoxia.
• Fibroblast Activation: While total fibrosis did not significantly increase, fibroblasts in severe RVD shifted to an activated phenotype (high FN1, POSTN, FAP).

C. Metabolic and Proteostatic Derangements

• Metabolic Collapse: Progressive RVD correlated with the suppression of oxidative phosphorylation, the TCA cycle, and fatty acid oxidation at both transcript and protein levels, most pronounced in severe RVD.
• Mitochondrial Proteostasis Failure:
  • Severe RVD showed a specific deficit in mitochondrial proteostasis, including downregulation of mitochondrial ribosomes, import proteins, chaperones, and proteases.
  • MitoUPR Deficit: The Mitochondrial Unfolded Protein Response (mitoUPR) was impaired, specifically the downregulation of LONP1 (a critical mitochondrial protease).
• Cytoplasmic Proteostasis Paradox: Unlike mitochondria, cytoplasmic ribosomes and ubiquitin-proteasome components were upregulated in severe RVD. However, this coincided with the activation of the Ribotoxic Stress Response (RSR).
  • RSR Activation: Phosphorylation of ZAKα (a stress kinase) was elevated only in severe RVD, suggesting ribosomal stress leading to pyroptosis (inflammatory cell death).

D. Signaling Kinome and Phosphatome

• Distinct kinase activation profiles were observed between mild and severe RVD.
• Severe RVD uniquely activated kinases including TTN, FYN, GSK3β, ABL1, LATS2, STK11, and AKT1, suggesting a shift toward inflammatory and stress-signaling pathways.

4. Key Contributions & Proposed Therapeutic Targets

The study integrates multi-omic data to nominate three specific, druggable pathways for reversing severe RVD:

1. Restoring Mitochondrial Proteostasis (mitoUPR):
   • Mechanism: Impaired mitoUPR leads to metabolic failure and ROS accumulation.
   • Therapeutic Candidate: Meclizine (an antihistamine) is known to activate mitoUPR and could be repurposed to enhance mitochondrial protein quality control.
2. Enhancing Macrophage Efferocytosis:
   • Mechanism: Failure to clear dying cells drives inflammation and remodeling via CD47 signaling.
   • Therapeutic Candidates: Disulfiram (FDA-approved for alcoholism) and Protocatechuic acid are known to enhance efferocytosis. Anti-CD47 antibodies are also suggested.
3. Inhibiting the Ribotoxic Stress Response (RSR):
   • Mechanism: Ribosomal stress activates ZAKα, triggering NLRP1 inflammasome-mediated pyroptosis.
   • Therapeutic Candidates: ZAK-N-1 (a small-molecule ZAKα inhibitor) and Disulfiram (which suppresses pyroptosis).

5. Significance

• Mechanistic Insight: This is the first study to provide a comprehensive, multi-omic map of RVD progression in a large animal model, distinguishing between compensated (mild) and decompensated (severe) states.
• Paradigm Shift: It challenges the notion that RV failure is solely a problem of hypertrophy or fibrosis, highlighting metabolic collapse, mitochondrial proteostasis failure, and impaired immune clearance as primary drivers.
• Translational Potential: By identifying existing FDA-approved drugs (Meclizine, Disulfiram) and investigational agents that target these specific molecular defects, the study provides a clear roadmap for repurposing drugs to treat severe RV failure, a condition currently lacking effective pharmacotherapy.
• Model Validity: The use of pigs provides a physiologically relevant model for human cardiac biology, bridging the gap between rodent studies and clinical application.

Limitations

• The study used only young, castrated male pigs, limiting insights into sex and age differences.
• The study is descriptive; future interventional studies are required to confirm that targeting these pathways improves RV function.
• Fibrosis was not significantly elevated, which may be specific to the age/sex of the model, though fibroblast activation was noted.