Differential regulation of cardiomyocyte inflammasome signaling and contractile function by immune check point inhibitors and IFNγ-induced GBP5

Cardiovascular disease and cancer are the two leading causes of death worldwide, together accounting for more than 18 million deaths each year through distinct but increasingly overlapping disease mechanisms. Besides their desired effects, anti-cancer drugs exhibit adverse effects that depends on their mechanism of action, often due to unintended interactions within healthy tissues, and in severe cases this results in fatal cardiac dysfunction. Chemotherapeutic agents are grouped by their mechanism of action. Doxorubicin, an anthracycline antibiotic, inhibits topoisomerase II to block DNA replication, whereas Imatinib, a tyrosine kinase inhibitor, specifically targets aberrantly active kinases driving chronic myeloid leukemia.The early 2000s introduced immune checkpoint inhibitors (ICIs) as a paradigm shift in oncology, offering potentially improved safety profiles compared to traditional cytotoxic therapies. ICIs, primarily targeting the PD-1/PD-L1 and CTLA-4 receptors, antagonize physiological brakes on T-cell activation. These checkpoints, expressed on T-cells and antigen-presenting cells, deliver inhibitory signals upon ligand binding to regulate immune activity and prevent self-reactive immune responses. Monoclonal blockade unleashes T-cell effector functions, enhancing antitumor immunity and enabling tumor clearance without direct cytotoxicity. Despite these advantages, similar to chemotherapeutic drugs, ICIs can also provoke serious immune-related adverse events, including myocarditis, with an incidence of approximately 1.5% where the patient a mortality rate is up to 50%. Recently, Finke et al. recently identified upregulation of guanylate-binding proteins 5 and 6 in the cardiac tissue of patients with myocarditis followed by ICIs treatment. This thesis investigated the cardiomyocyte specific and immune-independent role of GBP5 as a potential mediator of cardiac inflammation in the context of anti-cancer drugs (ICIs,Doxorubicin and Imatinib) and characterized the IFNγ-induced GBP5 role in cardiomyocyte inflammasome and contractile function. The immune checkpoint inhibitors Nivolumab and Ipilimumab, alone or in combination, failed to induce expression of GBP5 and other inflammasome markers in cardiomyocytes (neonatal rat ventricular cardiomyocytes and hiPSC-derived cardiomyocytes), cardiac fibroblasts, and macrophages. However, combined ICI treatment increased Ccl2 and Stat1 expression in cardiomyocytes, suggesting a modest immune activation without full inflammasome engagement. Functional assays in hiPSCderived cardiomyocytes showed preserved mitochondrial respiration and contractility under monotherapy but significant impairment with combination treatment, recapitulating the higher clinical risk of combined ICI regimen. Of note, Doxorubicin induced Gbp5 mRNA expression during both acute and chronic treatment without upregulating STAT1 protein levels in cardiomyocytes. In contrast, Imatinib triggered rapid pSTAT1/STAT1 activation without inducing Gbp5 mRNA or protein levels, indicating drug-specific modulation of the STAT1-GBP5 axis. Both Doxorubicin and Imatinib altered cardiomyocyte contraction and mitochondrial respiration in neonatal rat ventricular cardiomyocytes. This study shows that hiPSC-derived cardiomyocytes exhibit robust potential to upregulate inflammasome and inflammation signaling in response to type I and type II interferon response. Interestingly, hiPSC-derived cardiomyocytes subjected to IFNγ showed increased inflammasome signalling with elevated AIM2/NLRC4/CASP1 levels. Gain- and loss-of-function experiments in hiPSC-derived cardiomyocytes revealed a dual regulatory role of GBP5. Cardiomyocytes with GBP5 overexpression in the presence of IFNγ attenuated the gene expression of AIM2, NLRC4, CASP1, IL1B, CCL2, CCL5, STAT1, TGFb, and IL10, consistent with a moderating or anti-inflammatory effect on IFNγ-driven responses in hiPSC-CM. Surprisingly, the canonical NLRP3 inflammasome remained unaltered with GBP5 modulation. In contrast, GBP5 knockdown in hiPSC-derived cardiomyocytes disrupted inflammatory homeostasis, increasing gene expression of STAT1, CCL2, CCL5, NLRP3, IL18, and IL1b, while decreasing TGFb, IL10, NLRC4, and AIM2, suggesting impaired anti-inflammatory regulation and altered inflammasome-associated sensing. Overall, this study redefines GBP5 as a context-dependent regulator of cardiac inflammation, integrating its role in the AIM2-NLRC4-CASP1 axis and highlighting how GBP5 expression differentially shape inflammatory gene network. This research work demonstrates that ICI treatment neither induces GBP5 expression nor activates the NLRP3 inflammasome in cardiomyocytes, although it impairs cardiac contractility and mitochondrial respiration. These observations identify an immune cell-independent role of GBP5 in cardiomyocytes. These findings suggest an important role of interferon signalling wherein IFNγ induced GBP5 can exert either protective or detrimental effects in inflammation-related cardiac failure, depending on the cellular and contextual environment and highlight GBP5 as a potential mediator in inflammation-associated cardiomyopathy.