Fig 2: Quantification of nuclear and intercalated disc p‐MLKL levels. (A) The number of phosphorylated MLKL (p‐MLKL)‐positive cardiomyocyte nuclei and the number of total cardiomyocyte nuclei in myocardial sections were counted, and nuclear p‐MLKL level was expressed as the percentage of the number of p‐MLKL‐positive cardiomyocyte nuclei to the number of total cardiomyocyte nuclei. Using the median value, that is, 25%, patients were classified into a high nuclear p‐MLKL level group (H‐nucMLKL) and a low nuclear p‐MLKL level group (L‐nucMLKL). (B) p‐MLKL expression levels in the intercalated discs of cardiomyocytes were analysed in a field (200 × 200 μm) in which cardiomyocytes were sectioned longitudinally. Intercalated disc areas stained with anti‐p‐MLKL antibodies were selected and quantified using BZ‐X analyzer software, and the percentage of the positive area to the total area of cardiomyocytes was calculated. Using the median value, that is, 0.38%, patients were classified into a high intercalated disc p‐MLKL level group (H‐idMLKL) and a low intercalated disc p‐MLKL level group (L‐idMLKL).

Fig 3: Adverse event‐free survival curves for different levels of p‐MLKL in the myocardium. Kaplan–Meier survival curves for groups divided by the median value of total p‐MLKL level, nuclear p‐MLKL, or intercalated disc p‐MLKL. (A) A high p‐MLKL level group (H‐MLKL) and a low p‐MLKL level group (L‐MLKL). (B) A high nuclear p‐MLKL (p‐nucMLKL) level group (H‐nucMLKL) and a low nuclear p‐MLKL level group (L‐nucMLKL). (C) A high intercalated disc p‐MLKL (p‐idMLKL) level group (H‐idMLKL) and a low intercalated disc p‐MLKL level group (L‐idMLKL). (D) Receiver operating characteristic curves of brain‐type natriuretic peptide (BNP) and p‐MLKL levels for predicting adverse events. Area under the curve (AUC) value for each variable is as follows: BNP, 0.823 [95% confidence interval (CI), 0.688–0.958]; p‐nucMLKL, 0.826 (95% CI, 0.657–0.995); and p‐idMLKL, 0.547 (95% CI, 0.300–0.795).