Cardiomyocyte Apoptosis in Dilated Cardiomyopathy: Limitations of TUNEL-Based Apoptotic Index from Endomyocardial Biopsies and Implications for Prognosis in the Era of Multimodal Assessment

Background: Dilated cardiomyopathy (DCM) is characterized by progressive left ventricular dilation and systolic dysfunction, often leading to advanced heart failure. Cardiomyocyte loss is a key pathological feature, and apoptosis has long been implicated as a contributing mechanism. Early studies primarily relied on the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in endomyocardial biopsies to quantify the apoptotic index. However, accumulating evidence has challenged the specificity of TUNEL staining, suggesting that positive signals may reflect DNA repair, reversible injury, or other non-apoptotic processes rather than true programmed cell death. Objective: This study reevaluates the prognostic significance of TUNEL-derived apoptotic index in DCM patients, while exploring the broader context of cardiomyocyte apoptosis and related cell death pathways (e.g., regulated necrosis, pyroptosis, and autophagy) in disease progression and clinical outcomes. Methods: We analyzed endomyocardial biopsy samples from 68 patients with idiopathic or non-ischemic DCM (mean age 48 ± 11 years, left ventricular ejection fraction [LVEF] 28 ± 9%). Apoptosis was assessed using in situ TUNEL staining, supplemented by electron microscopy, immunohistochemical markers (caspase-3 activation, Bax/Bcl-2 ratio), and molecular assays for DNA laddering. Follow-up data (median 4.2 years) included all-cause mortality, heart failure hospitalization, and changes in LVEF. Multivariate Cox regression models adjusted for age, NYHA class, LVEF, and fibrosis extent were used to evaluate prognostic associations. Results: TUNEL-positive cardiomyocytes were detected in 24% of biopsies, with a mean apoptotic index of 0.52 ± 1.18% (range 0–14.2%). Consistent with prior observations, the TUNEL index showed no significant correlation with baseline functional parameters (LVEF, LV end-diastolic diameter) or follow-up outcomes, including mortality (7 deaths), hospitalization for worsening heart failure (22 events), or reverse remodeling. Electron microscopy confirmed that many TUNEL-positive cells exhibited features of viable cardiomyocytes undergoing DNA repair rather than classic apoptosis. However, when integrated with caspase-3 activation and Bcl-2 downregulation, a composite apoptosis score was modestly associated with adverse prognosis (HR 1.68, 95% CI 1.12–2.51, p=0.014). Recent evidence also highlights that pyroptosis may predominate over apoptosis in some DCM subsets, particularly in inflammatory or viral-persistent cases. Conclusions: The apoptotic index derived solely from TUNEL assays in endomyocardial biopsies does not reliably predict prognosis in DCM, likely due to assay limitations and the multifactorial nature of cardiomyocyte death. True apoptotic signaling, when confirmed by complementary molecular and ultrastructural methods, may offer incremental prognostic value. Future research should prioritize multimodal approaches, including caspase inhibitors, anti-pyroptotic therapies, and advanced imaging, to better stratify risk and guide targeted interventions in DCM patients. Keywords: Dilated cardiomyopathy, apoptosis, TUNEL assay, endomyocardial biopsy, prognosis, cardiomyocyte death, pyroptosis, heart failure.