The complex architecture of cardiac extracellular matrix (ECM) provides a unique 3D environment fundamental for cardiac development, homeostasis, and functionality. The derangement of ECM integrity occurring during the onset and progression of cardiac diseases drives a modification in the nanotopography and mechano-physical properties of the ECM itself, thus impairing cardiac cell contractility and proper organ function. Furthermore, our immunohistochemistry and gene expression analysis, on infarcted mouse hearts and human cardiac biopsies, confirms that the dramatic structural changes triggered by myocardial infarction and tissue remodelling, leading to heart failure, causes aswitch in cardiac cell mechanosome.Cardiac decellularized ECM (dECM) obtained from physiological and pathological specimens feature the three dimensional cues, mechanical properties, chemical complexity and the native organization of heart tissue in healthy and diseased condition. For this reason, they are here proposed as an in vitromodel to investigate the mechanisms of cell-ECM interaction, especially consequent to cardiac pathologies.In this study, we show that second harmonic generation imaging, scanning electron microscopy and atomic force microscopy can be implemented to obtain high-resolution 3D maps of cardiac dECM structure and mechanical properties, as an essential asset to the conventional immunostaining and protein analyses. Furthermore, recellularization of physiological and pathological myocardial scaffolds resulted in distinct cell-dECM interactions, thus bringing further hints to disclose a possible window of action for heart cell therapies. Altogether, our findings show that cardiac dECMs are powerful and reliable toolboxes to monitor cardiac nanostructural changes and to investigate cardiac system mechanobiology.