Quantitative second harmonic generation microscopy for region-specific assessment of cardiac remodeling and treatment response in non-ischemic heart failure

Second harmonic generation (SHG) microscopy is a nonlinear, label-free imaging technique that enables precise visualization and quantification of fibrillar collagen microarchitecture, an essential component of cardiac extracellular matrix implicated in the progression of heart failure (HF). Despite the well-established role of myocardial fibrosis in HF pathophysiology, non-destructive and spatially resolved tools to characterize and distinguish region-specific collagen remodeling and treatment response are lacking. Addressing this unmet need, we introduce a spatially resolved, SHG-based fibrosis phenotyping framework using whole-slide SHG microscopy in a non-ischemic murine model of HF and antifibrotic treatment. SHG images were manually segmented based on anatomical definitions within the left ventricular (LV) free wall, and a comprehensive set of multiscale quantitative metrics capturing collagen orientation, frequency content, and structural complexity was extracted using intensity, tensor, Fourier-transform, and wavelet-based analyses. Across 17, 28, and 37 FOVs derived from control, HF, and treated hearts, respectively, the Wilcoxon rank sum test revealed multiple SHG-derived features that were statistically significantly different between groups (p < 0.001). Several metrics distinguished between fibrotic and non-fibrotic states, capturing hallmark features such as disorganized collagen clustering and sharply demarcated fibrotic boundaries. These findings demonstrate the potential of SHG-derived metrics as quantitative, spatial biomarkers of myocardial fibrosis capable of discriminating health, disease, and treatment states, laying the groundwork for integrating SHG imaging into preclinical and clinical pipelines for early HF detection, fibrosis monitoring, and evaluation of emerging antifibrotic therapies.