Fig 2: CFs proliferation on myocardial slices. Representative images of (A–D) VIM+ cell (green) proliferation on the myocardial slice with time. (E) We observed a significant increase in the percentage of the slice surface covered by VIM+ cells at Day 3, with a further significant increase seen at Days 7 and 10. (F) By Day 10 the cells completely covered the surface of the slices and started to express aSMA (red). Three images per slice were acquired and the data obtained were averaged before statistical analysis. Six slices were used for this experiment.

Fig 3: CFs proliferation and contractile capacity of culture human myocardial slices. Human myocardial slices cultured in vitro progressively lose their contractility (A). Number of slices/number of experiments. As observed in canine myocardial slices, we noticed a significant increase in the percentage of the slice surface covered by VIM+ cells (B). Representative images of VIM+ cells proliferating on the human slice surface at Days 0, 1, 3, and 7 (Green, C–F). Three images were acquired from each slice and the data obtained were averaged before statistical analysis. The slices were prepared from at least three human samples (K). The Table summarizes the groups of end-stage heart patient biopsies used in the study. (G) DCM, dilated cardiomyopathy; DCM with LVAD, dilated cardiomyopathy with left ventricular assist device; ICM, ischaemic cardiomyopathy; ICM with LVAD: ischaemic cardiomyopathy with left ventricular assist device.

Fig 4: Cellular composition of dog myocardial slices. (A,B) transverse and longitudinal section of a myocardial slice; cardiomyocytes, endothelial cells and fibroblasts were stained for CAV3 (green), ISOB4 (white), and VIM (red). (C,D) Higher magnification of CFs (red) and their location in proximity to capillaries (white). (E) The cardiac cellular composition is made up by 48% endothelial cells, 14.2% stromal cells, 37.9% myocytes. 44 images generated from 10 myocardial slices were processed and a total of 3259 cells were counted.

Fig 5: Isolated CFs in vitro. Freshly isolated dog CFs (VIM+/aSMA-), were seeded on bottom glass dishes coated with fibronectin. (A–E) Within 3 days, CFs started to express aSMA. By Day 7 30% of the cells were aSMA+ and the number increased to 50% by Day 10 (F). The experiment was repeated three times and three images were acquired from each dish and the data obtained were averaged before statistical analysis. VIM+ cells that proliferated out of the myocardial slices and onto the Transwells membrane expressed aSMA at Day 7. (G). VIM+ cells, trypsinized from Day 7 slice surface and cultured on glass, expressed aSMA after 24 h (H). The cells were isolated after collagenase digestion, labelled with Qtracker 585 (white) and seeded onto dog slices (I, J). Labelled CFs were then cultured for 7 days and observed to be aSMA-. (K) CF aSMA expression was assessed in different conditions. CF proliferating on the slice surface were aSMA(-) after 7 days of in vitro culture. CFs migrating onto the culture plastic support or isolated with enzymatic digestion and cultured on glass were aSMA(+). CFs proliferating on myocardial slices for 7 days [aSMA(-)] were trypsinized and seeded onto glass dishes and on myocardial slices. These cells were respectively aSMA(+) and aSMA(-) suggesting a crucial role in the slice as a substrate to preserve a more physiological phenotype. Each experiment was repeated at least three times.