Inhibition of Gasdermin D by Disulfiram Attenuates Cardiac Inflammation and Fibrosis following Ischaemia Reperfusion Injury

This study demonstrates that repurposing the FDA-approved drug Disulfiram to inhibit Gasdermin D significantly attenuates cardiac inflammation and fibrosis while improving cardiac function following ischemia-reperfusion injury in mice.

The Gist

The Big Picture: A Heart Attack is Like a House Fire

Imagine your heart is a house. When you have a heart attack (myocardial infarction), it's like a fire breaks out in one room because the power lines (blood vessels) got cut off.

When the fire department (doctors) rushes in to turn the power back on (reperfusion), they save the house from burning down completely. But here's the problem: The act of turning the power back on creates a massive amount of smoke and sparks. This "smoke" is inflammation.

In the days and weeks after the fire, this smoke doesn't just clear away. Instead, it causes the walls of the house to get thick, stiff, and scarred (fibrosis). The house becomes a rigid, broken shell that can't pump water (blood) effectively anymore. This leads to Heart Failure.

The Villain: Gasdermin D (The "Explosive" Switch)

Inside the cells of your heart and your immune system, there is a protein called Gasdermin D (or GSDMD for short). Think of GSDMD as a dangerous "self-destruct" switch.

When the heart is injured, this switch gets flipped. It punches holes in the cell walls (like blowing a hole in a balloon).

1. The Cell Dies: The cell bursts open (a process called pyroptosis).
2. The Smoke Spreads: When the cell bursts, it dumps a bucket of toxic, inflammatory chemicals (like IL-1β) into the surrounding tissue.
3. The Chain Reaction: This attracts more "firefighters" (immune cells) who get confused and start attacking healthy tissue, making the scarring and stiffness worse.

The Hero: Disulfiram (The "Fire Extinguisher")

The researchers wanted to know: Can we stop this self-destruct switch?

They looked at an old, FDA-approved drug called Disulfiram. You might know it as a medicine used to treat alcoholism (it makes you feel sick if you drink). But recently, scientists discovered it has a superpower: it jams the GSDMD switch.

Think of Disulfiram as a heavy-duty padlock that you put on the self-destruct switch. Even if the fire alarm goes off, the switch can't flip, the holes don't get punched in the cells, and the toxic smoke stays contained.

What the Scientists Did (The Experiment)

The team tested this idea on mice that had a simulated heart attack.

1. The Setup: They blocked a heart artery in mice for an hour (simulating a heart attack) and then opened it up (reperfusion).
2. The Treatment: Immediately after opening the artery, they gave the mice Disulfiram.
3. The Comparison: Some mice got a high dose, some a low dose, and some got a fake treatment (placebo).

The Results: A Stronger, Less Scarred Heart

The results were very promising, especially when looking at the heart one week after the "fire":

• The Heart Pumped Better: Mice treated with Disulfiram had much better heart function. Their hearts were still flexible and could pump blood efficiently, unlike the placebo group whose hearts were stiff and weak.
• Less Scarring: The hearts of the treated mice had much less "scar tissue" (fibrosis). The walls remained flexible rather than turning into a brick wall.
• Fewer "Firefighters": The treated hearts had fewer angry immune cells invading the tissue. Because the self-destruct switch was jammed, the cells didn't burst, so they didn't send out the "come help me" signals that attract the inflammatory crowd.
• The Mechanism: In the lab (using human and mouse cells), they confirmed that Disulfiram stopped the cells from bursting and stopped them from releasing the toxic inflammatory chemicals.

The Catch: Timing and Dosage

The study found that Disulfiram worked best early on (7 days after the injury). At this stage, the inflammation is raging, and the drug acts like a powerful fire extinguisher.

By 28 days, the heart had already started to remodel. While the drug still reduced the genetic markers of inflammation and scarring, it didn't fully restore the heart's pumping power to normal levels. This suggests that timing is everything. You have to catch the fire while it's still burning to save the house.

Why This Matters

This is a story of drug repurposing. Disulfiram is already a safe, approved drug that humans have used for decades. If we can prove it works for heart attacks, we don't need to spend 10 years inventing a new drug from scratch. We could potentially start using this "padlock" for the self-destruct switch in heart attack patients very quickly.

In summary:
Heart attacks cause a chain reaction where cells blow themselves up, spreading inflammation and scarring the heart. This study shows that an old drug, Disulfiram, can jam the mechanism that causes these cells to blow up. By stopping the explosion, it reduces the damage, keeps the heart flexible, and helps it keep pumping strong. It's a simple, clever way to stop the heart from hurting itself after a heart attack.

Technical Summary

1. Problem Statement

Acute Myocardial Infarction (AMI) remains a leading cause of heart failure (HF) and mortality. Despite advances in revascularization (e.g., PCI), approximately 20–30% of survivors develop HF within a year, driven largely by adverse cardiac remodeling characterized by persistent inflammation and fibrosis.

• Pathophysiology: Post-AMI, the innate immune system is activated, leading to neutrophil and macrophage infiltration. This triggers the NLRP3 inflammasome, which activates Gasdermin D (GSDMD). GSDMD forms pores in the cell membrane, causing pyroptosis (inflammatory cell death) and the release of pro-inflammatory cytokines, specifically IL-1β and IL-18.
• Therapeutic Gap: While IL-1β inhibition (e.g., CANTOS trial) showed promise, it carries infection risks. There is a need for targeted therapies that inhibit the upstream pyroptotic machinery (GSDMD) to limit cytokine release and tissue damage without completely abolishing host defense.
• Hypothesis: The FDA-approved drug Disulfiram (DSF), recently identified as a GSDMD pore-formation inhibitor, can be repurposed to attenuate cardiac inflammation, fibrosis, and dysfunction following Ischemia-Reperfusion (I/R) injury.

2. Methodology

The study utilized a multi-modal approach combining in vitro mechanistic studies and in vivo preclinical models.

A. In Vitro Models:

• Cell Lines: Mouse Bone Marrow-Derived Macrophages (BMDMs) and human PMA-differentiated THP-1 monocytes.
• Stimulation: Cells were primed with Lipopolysaccharide (LPS) and activated with ATP or Nigericin to induce NLRP3 inflammasome activation.
• Intervention: Treatment with Disulfiram (DSF) at doses ranging from 0.1 to 50 µM.
• Assays:
  • Cytokine Secretion: ELISA for IL-1β and IL-6.
  • Gene Expression: RT-qPCR for inflammatory markers (Il-1b, Il-6, Nlrp3, Gsdmd, Tnf-a, Mcp-1).
  • Cell Viability/Pyroptosis: Lactate Dehydrogenase (LDH) release and Propidium Iodide (PI) staining.
  • Protein Analysis: Immunoblotting for Caspase-1, GSDMD (full-length and cleaved N-terminal fragment), and IL-1β.

B. In Vivo Model:

• Subjects: C57BL/6 male mice (12 weeks old).
• Procedure: Surgical induction of cardiac I/R injury via 60-minute ligation of the Left Anterior Descending (LAD) coronary artery, followed by reperfusion.
• Treatment Groups:
  • Sham (surgery without ligation).
  • I/R + Vehicle (Sesame oil).
  • I/R + DSF (25 mg/kg or 50 mg/kg).
  • Administration: DSF was administered via intraperitoneal (IP) injection immediately at reperfusion and daily thereafter.
• Endpoints: Assessments were conducted at 7 days (early remodeling) and 28 days (late remodeling).
• Measurements:
  • Cardiac Function: Echocardiography (Ejection Fraction, Fractional Shortening, Volumes).
  • Histology: Picrosirius Red and Masson's Trichrome staining for fibrosis; CD68 immunohistochemistry for macrophage infiltration.
  • Flow Cytometry: Analysis of immune cell populations (neutrophils, Ly6C-high/low monocytes, macrophages) in blood, spleen, bone marrow, and heart tissue.
  • Molecular Analysis: RT-qPCR and Western Blotting for inflammatory, fibrotic, and cell death markers in cardiac tissue.

3. Key Contributions

• Mechanistic Validation: Confirmed that DSF directly inhibits GSDMD pore formation and subsequent IL-1β secretion in both murine and human macrophages, independent of Caspase-1 levels.
• Repurposing Strategy: Demonstrated the efficacy of an existing FDA-approved drug (DSF) in a clinically relevant I/R injury model (mimicking PCI/CABG scenarios), rather than permanent ligation models used in previous studies.
• Dual Action: Uncovered that DSF not only blocks pyroptosis but also modulates the systemic immune response, shifting bone marrow monocyte populations from pro-inflammatory (Ly6C-high) to anti-inflammatory (Ly6C-low) phenotypes.
• Temporal Analysis: Provided a comprehensive timeline of therapeutic effects, showing early functional improvements (7 days) and sustained structural benefits (28 days), despite functional metrics plateauing at the later time point.

4. Key Results

A. In Vitro Findings:

• Cytokine Suppression: DSF dose-dependently reduced IL-1β secretion by up to 95% and IL-6 by ~91% in WT BMDMs. This effect was absent in Gsdmd^-/- cells, confirming the mechanism relies on GSDMD inhibition.
• Cell Viability: DSF significantly reduced LDH release and membrane permeability (pyroptosis) in a dose-dependent manner.
• Protein Mechanism: DSF treatment reduced the cleavage of GSDMD into its pore-forming N-terminal fragment (GSDMD-NT) and reduced pro-IL-1β levels, without altering total Caspase-1 levels.

B. In Vivo Findings (7 Days Post-I/R):

• Cardiac Function: DSF (50 mg/kg) significantly improved Ejection Fraction (EF) and reduced ventricular dilation (diastolic/systolic volume) compared to vehicle-treated I/R mice.
• Fibrosis: DSF treatment significantly reduced free wall scar size and total fibrosis area.
• Immune Modulation:
  • Heart: Significant reduction (~57%) in CD68⁺ macrophage infiltration.
  • Bone Marrow: A phenotypic shift occurred: a 96% reduction in pro-inflammatory Ly6C-high monocytes and a 7-fold increase in anti-inflammatory Ly6C-low monocytes.
  • Spleen: ~51% reduction in total monocytes.
• Gene Expression: Downregulation of pro-inflammatory genes (Il-1b, Il-6, Tnf-a, Nlrp3) and pro-fibrotic genes (Ctgf, Fibronectin, Tgf-b).

C. In Vivo Findings (28 Days Post-I/R):

• Structural Preservation: DSF continued to significantly reduce fibrosis and scar size.
• Functional Outcome: While fibrosis was reduced, improvements in Ejection Fraction were not statistically significant at 28 days compared to the vehicle group. The authors suggest this may be due to the preservation of contractility preventing the heart from overworking (pressure overload), resulting in "preserved" but not "improved" EF in the context of severe injury.
• Molecular Persistence: DSF maintained suppression of Nlrp3, Gsdmd, Caspase-3, and Nox4 (ROS-producing enzyme) gene expression.

5. Significance and Conclusion

• Therapeutic Potential: This study provides strong preclinical evidence that repurposing Disulfiram can mitigate post-MI cardiac remodeling by targeting the GSDMD-mediated pyroptotic pathway.
• Clinical Relevance: By inhibiting GSDMD, DSF limits the release of IL-1β and subsequent IL-6 production, breaking the cycle of inflammation-driven fibrosis and cardiomyocyte death.
• Safety Profile: As an FDA-approved drug with a known safety profile, DSF offers a rapid pathway to clinical translation for post-AMI therapy, potentially reducing the burden of heart failure.
• Future Directions: The study highlights the need for dose optimization (50 mg/kg was more effective at 7 days) and further investigation into long-term functional outcomes and pressure-volume dynamics to fully understand the preservation of cardiac function.

In summary, the paper establishes Disulfiram as a potent cardioprotective agent that attenuates I/R injury by inhibiting GSDMD pore formation, thereby reducing pyroptosis, systemic inflammation, and cardiac fibrosis.