Fig 2: GCa Vericiguat exerts anti-inflammatory properties in human cardiac cells and skeletal muscle cells exposed to subclinical anthracycline levels. Intracellular NLRP-3 levels (fold of control) (A,C) and MyD-88 (fold of control) (B,D) human cardiomyocytes (AC-16 cells) (A,B) and primary skeletal muscle cells (HSkMC cells) (C,D) untreated (control) or treated for 24 h with DOXO 1 µM or pretreated for 1 h with sGCa Vericiguat (0.1, 1 or 10 µM) and then incubated with DOXO for 24 h. After treatments, cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 20 mM NaF, 3 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail). Lysates were centrifuged at 2500 rpm, and supernatants were collected and analyzed for NLRP-3 and Myd-88 cellular levels through the human MyD88 and NLRP-3 ELISA Kit. Error bars depict means ± SD (n = 3). Statistical analysis was performed using paired t-test *** p < 0.001.** p < 0.01.* p < 0.05. ns: No Significance.

Fig 3: Proposed molecular mechanism by which inclisiran protects cardiomyocytes against doxorubicin and trastuzumab-induced injury. This schematic illustrates the sequential cardiotoxic effects of anthracyclines and trastuzumab and the protective mechanism of inclisiran via RNA interference. At the top left, doxorubicin and trastuzumab bind to their respective targets on the cardiomyocyte membrane. Doxorubicin induces mitochondrial dysfunction and increases reactive oxygen species (ROS) production (red dots), while trastuzumab contributes to oxidative stress and immune-related damage. Inclisiran (green icon) is an siRNA therapeutic targeting PCSK9 mRNA. Upon cellular uptake via receptor-mediated endocytosis, inclisiran enters the cytoplasm and engages the RNA-induced silencing complex (RISC). Within RISC, inclisiran guides the cleavage of PCSK9 mRNA (blue-red duplex), resulting in gene silencing and subsequent reduction of PCSK9 protein levels. This inhibition leads to downstream suppression of pro-inflammatory signaling pathways, including the NLRP3 inflammasome (green specks) and MyD88-dependent cytokine release, reducing mitochondrial damage and oxidative stress. Green arrows indicate decreased expression or activity of oxidative and inflammatory mediators. The reduction in ROS levels and lipid peroxidation (gray clusters) ultimately prevents the initiation of ferroptosis (indicated at bottom right). The diagram also highlights the prevention of cytoskeletal degradation and improved cellular integrity through reduced inflammatory burden. This multifaceted protective mechanism underlines the therapeutic potential of inclisiran in preventing cancer therapy-related cardiac dysfunction (CTRCD).

Fig 4: Inclisiran reduces intracellular NLRP-3, MyD88 and pro-inflammatory cytokines in cardiomyocytes during anthracycline and trastuzumab therapy in sequential therapy regimen. (A) Intracellular NLRP-3 (fold of control) in hiPSC-CMS cells as measured using selective ELISA method. (B) Myocardial MyD-88 levels (fold of control) in HIPSC-CMS cells as measured using selective ELISA method. (C) Cytokines and growth factors expression (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IFN-γ, TNF-α, G-CSF, GM-CSF) in pg/mL in HIPSC-CMS cells as measured using selective ELISA method. In all experiments, cardiomyocytes were untreated (control) or treated with PCSK9i inclisiran (PSCK9i group) or with anthracyclines and HER-2 mAbs (trastuzumab) in sequential therapy regimen alone (DOXO-Anti HER-2 mAb group) or combined with PCSK9i inclisiran (PCSK9i + DOXO-Anti HER-2 mAb group). (n = 3 independent cell culture experiments.) Data are presented as mean ± SD.

Fig 5: PI3Kα inhibition and estrogen receptor blockade activate inflammasome-related signaling in cardiomyocytes, attenuated by dapagliflozin. Human iPSC-derived cardiomyocytes were treated for 24 h with alpelisib (100 nM), fulvestrant (100 nM), or their combination, in the absence or presence of dapagliflozin (1 µM) under hyperglycemic conditions. Intracellular levels of (A) NLRP3 and (B) MyD88 were quantified in cardiomyocyte lysates and normalized to total protein content. Drug treatments were associated with increased expression of inflammasome-related signaling components, while dapagliflozin reduced these responses. Data are presented as mean ± SEM with individual data points representing independent biological replicates (n = 6). Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple-comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001.