NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro

Abstract

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Figure 1

Cardiac fibroblasts induce the expression of VCS markers in adjacent cardiomyocytes. (A) Representative image of the cardiomyocyte-fibroblast delimited co-culture. Cell nuclei (DAPI), cardiomyocytes (cardiac Troponin I positive cells, Red), cardiac fibroblasts (cardiac Troponin I negative cells), and Nav1.5 (Green). The dotted line (b) delimits the cardiomyocyte-enriched area on the left side from the beginning of the contact zone. Samples of the cardiomyocyte-enriched area and the contact zone are magnified at the left and right side of the figure, respectively. Arrowhead in the magnified contact zone highlights a fibroblast expressing Nav1.5. Scale bars = 100 µm. (B) Fluorescence intensity of Nav1.5/Dapi. (C–E; C’–E’; F) Representative images and quantification of the protein expression of the VCS markers NAV1.5, Contactin-2 and Connexin 40 in the cardiomyocyte-enriched area (cardio) and the contact zone (Contact) of the same co-cultures. The relative protein expression in the contact zone was defined as the fold induction of the cardiomyocyte-enriched area from the same cover slip. Results are disposed as a mean ± SEM. Data were subjected to paired Student’s t-test against the cardio group; p < 0.05 was considered significant. N = 6–12 (four different experiments). Scale bars = 20 µm.

Figure 2

Cardiac fibroblasts influence calcium handling of adjacent cardiomyocytes. Cardiomyocytes were plated in the center of the delimited co-culture system and either cardiac fibroblasts or mesenchymal stem cells were plated on the outside area. The calcium transients of contractile cells in the contact zones and cardiomyocyte-enriched areas were recorded and measured. (A) CaT mean curves for each experimental group. (B) Heatmap analysis of CaT parameters for each experimental group. (C) Normalized CaT amplitudes. (D) Time to reach 50% of calcium decay (CaD50). (E) Time to reach 90% of calcium decay (CaD90). (F) Time to reach the maximum peak of calcium per beating cycle (Time to Peak). (G) Time to reach basal levels of Calcium after maximum peak (Ca decay time). (H) The Resting interval or the time between the basal level reached after contraction until the new CaT. The cardiomyocyte-enriched area in the center of the cardiomyocyte-only coverslip was used as the control. p < 0.05, **p < 0.01, ***p < 0.001 after Bonferroni post-hoc test (alpha 0.05). Sample sizes were 15 cells of Cardiomyocytes (center), 12 cells of Cardio/Fibro (contact) and 10 cells of Cardio/MSCs (contact) from three different experiments.

Figure 3

Electrical properties of cardiomyocytes exposed to fibroblast conditioned media (fCM). (A) Representative traces of action potential in control (red) and fCM exposed cardiomyocytes (blue) groups. (B) Time required to reach 10%, 50%, and 90% of full AP repolarization. APR represents action potential repolarization, and n the number of cells. *p < .05 using Two-sample t-Test.

Figure 4

The paracrine effect of fibroblast-secreted mediators is sufficient to regulate key genes of the VCS genetic program. (A–D;A’–D’) Immunofluorescence imaging of cardiomyocyte-enriched cultures and co-cultures seven days after plating. Cell nuclei (DAPI), cardiomyocytes (cardiac Troponin I positive cells, Red), and cardiac fibroblasts (Vimentin positive cells, Green). Scale bars = 20 µm. (E) Cellular composition of cardiomyocyte-enriched cultures and co-cultures by flow cytometry. N = 3–5. (F–P) Relative expression of key cardiac genes determined by real time RT-PCR seven days after plating. (F–L) Total RNA was isolated from whole extracts of cardiomyocyte-enriched cultures, cardiomyocyte-cardiac fibroblast co-cultures, and cardiac fibroblast cultures. After fold induction calculation, relative gene expression was normalized by the number of cardiomyocytes to avoid bleaching effects due to the augmented proportion of cardiac fibroblasts in the co-cultures. (M–P) Total RNA was isolated from whole extracts of cardiomyocyte-enriched cultures treated with control, cardiac fibroblast- (fCM) or MSC-conditioned media (mCM). Cyclophilin and GAPDH were used as housekeeping genes and the results are displayed as 2^-ΔΔCt , mean ± SEM. Data were subjected to one-way ANOVA, followed by multiple comparisons against the cardiomyocyte-enriched culture group, used as control; p < 0.05 was considered significant. N = 3–7 (three different experiments).

Figure 5

Cardiac fibroblast-conditioned media reduces the proportion of cells expressing working myocyte characteristics. Working myocyte phenotypic analysis by high content screening. Phenotypic parameters were retrieved for each single-cell identified and only cardiomyocytes were included in the analysis. (A–D’) Control and fCM-treated group representative immunofluorescence images, respectively. Cell nuclei were stained with (DAPI), cardiomyocytes (cardiac Troponin I positive cells, Red), and connexin 43 (Green). Asterisk symbol illustrates working myocyte expressing connexin 43. Arrows indicate smaller troponin I positive cells which do not express connexin 43. Scale bars = 20 µm. (E) Mean cardiomyocyte area in micrometers (μm²) given by the average of total troponin I area surrounding a valid nucleus. (F) Mean expression of connexin 43 per cardiomyocyte given by the average of connexin 43 fluorescence intensity in the cardiomyocyte population. (G) Percentage of cardiomyocytes connexin 43 positive (Cx43 +) in the cardiomyocyte population. Results are disposed as a mean ± SEM. Data were subjected to unpaired Student’s t-test against the control group; p < 0.05 was considered significant. N = 6 (each consisting of more than 500 cells). (H–I) Relative protein expression of connexin 40 and connexin 43 determined by Western Blotting. Gels were cut to keep the groups of interest together and full-length blots are presented in Supplementary Figure S6A-B. Protein samples were isolated from whole extracts of cardiomyocyte-enriched cultures treated with the control or cardiac fibroblast-conditioned media (fCM). GAPDH was used as housekeeping protein, and the results are disposed as the relative expression to the control group, mean ± SEM. Data were subjected to unpaired Student’s t test, against the control group; p < 0.05 was considered significant. N = 6.

Figure 6

Fibroblast-secreted mediators induce 3-dimensional patterning of VCS markers. Evaluation of the influence of fibroblast-secreted factors on the expression of VCS markers in neonatal rat cardiomyocyte organoids using automated high content screening analysis. Phenotypic parameters were retrieved for each single-cell identified in 12 Z-planes and only cardiomyocytes were included in the analysis. (A) Illustration of cardiomyocyte organoid analysis (B) Total number of cells per z plane. N = 6–8. (C–J’) Control and fCM-treated organoids representative z-stack immunofluorescence images from summed intensities. Cell nuclei (DAPI), cardiomyocytes (cardiac Troponin T positive cells, Red), connexin 40 and NAV1.5 (Green). Scale bars = 100 µm. (K–N’) Single z plane view of representative outer surface regions of control and fCM-treated organoids. (O–P) Mean expression of connexin 40 and NAV1.5 per cardiomyocyte given by the average of fluorescence intensity in the cardiomyocyte population. The mean fluorescence intensity was measured in three different z-positions for the organoid outer (z4–z6) and inner surfaces (z8–z10). Results are disposed as a mean ± SEM. Data were subjected to a two-way ANOVA followed by a post test comparing each group to every other group; p < 0.05 was considered significant. N = 9–12.

Figure 7

Notch1 pathway has a central role in the fCM-mediated induction VCS-like cells. Protein samples were isolated from total extracts of cardiomyocyte-enriched cultures treated with control media (CTL), fibroblast-conditioned media (fCM), conditioned media of fibroblasts previously inhibited with DAPT (fCMi) or fibroblast-conditioned media with the concomitant addition of DAPT inhibitor (fCM + i). Relative protein expression was analyzed by Western Blot. (A) Illustrative scheme of treatments. Each group was cultured for seven days and had its media changed every 48 h. (B) Relative protein expression levels of NOTCH1 receptor in cardiomyocytes according to the defined treatments, N = 5 Gel was cut to keep the groups of interest together and full-length blots are presented in Supplementary Figure S6C. (C) Relative protein expression levels of the activated-NOTCH1 receptor (NICD, Notch IntraCellular Domain) in cardiomyocytes. N = 6–8. (D) Relative protein expression levels of connexin 40 in cardiomyocytes according to treatments, N = 3–5. The GAPDH protein was used as housekeeping protein, and the results are disposed as the relative expression to the control group for each gel, mean ± SEM. Data were submitted to the one-way ANOVA test and Dunnett's posthoc test against the control group; p < 0.05 was considered significant.

Figure 8

Cardiac fibroblast-mediated patterning of VCS-like cells in vitro. We have shown that cardiac fibroblasts from neonate rats induce molecular and functional changes characteristic of fast-conducting fibers in adjacent cardiomyocytes (E.g. increased expression of Cx40, Irx3, Nkx2.5, Scn5a and CONTACTIN 2, and longer calcium transients) and reduce characteristics of working cardiomyocytes (E.g. decreased cell area, Cx43 expression, and faster calcium transients). The cardiac fibroblast-conditioned media was sufficient to activate the NOTCH1 pathway and induce the expression of ventricular conduction system markers in cardiomyocytes. Interestingly, the secretion of these factors was dependent on the activation of the NOTCH pathway in fibroblasts; which sheds light on the central role that the NOTCH1 pathway plays in the intercommunication between fibroblasts and cardiomyocytes and the consequent induction of VCS-like phenotype in cardiomyocytes in a juxtacrine manner in vitro.