Adult Cardiac Fibroblasts Regulate Naive Cardiomyocytes Transformation Into Mature Cardiomyocytes

Background and objectiveAcute myocardial infarction is most commonly caused by coronary artery atherosclerosis.Atherosclerotic plaque rupture can lead to blocking of the left anterior descending artery,causing reduced blood supply to,and therefore necrosis.In recent years,stem cell therapy has become a promising option for cardiac regenerative medicine.However,due to the immature nature of pluripotent stem cell-derived cardiomyocytes,they often produce serious side effects,such as arrhythmia and teratoma formation.Hence,the study of cardiac maturation not only forms the core of understanding postnatal heart development and certain cardiac diseases,but also provide important clues to enhance cardiomyocyte maturity in vitro.Although the role of the cardiomyocyte microenvironment has been extensively studied,little is known regarding its dynamics,and contributions to heart maturation.Thus,the goal of this study is to elucidate regulatory roles of non-cardiomyocytes in cardiac maturation to serve a therapeutic purpose.MethodsFirstly,we performed single-cell RNA sequencing on heart cells isolated from P1,P4,P7,P14 and P56 postnatal mice.Bioinformatic analysis identified several known cell types,and further clustered them into subtypes.Changes in cell subtype composition,corresponding gene expression,and putative signaling pathways revealed cardiac fibroblasts as a major player in cardiac maturation.To this end,we performed co-cultures of adult(P56)cardiac fibroblasts with neonatal(PI)cardiomyocytes for 3.5 days to observe the effect of the former on the maturation status of the latter.Relative expression of cardiac maturation genes and proliferation genes were assessed by real-time PCR.Expression of proliferation markers AURKB,MKI67 and pH3 were detected by immunofluorescence.Structural features of mature cardiomyocytes,such as gap junctions(GJA1)and T-tubules,were measured by staining.Further,maturity grading of cardiomyocytes was based on phalloidin staining of actin.Patch clamping was used to detect action potentials and ion channel currents(sodium,potassium,calcium)of cardiomyocytes in co-culture,whereas changes in calcium transients were detected by Fluo-4 AM.In addition,we performed RNA sequencing and single-cell RNA sequencing on co-cultured cells to identify pathways critical for fibroblast-induced cardiomyocyte maturation.Based on the preceding information,we used chemokine receptor inhibitor Plerixafor,cytokine inhibitor Linifanib,and ECM receptor inhibitor BP-1-102,to individually inhibit pathways predicted to be important to cardiomyocyte maturation.Proliferation and maturation status of cardiomyocytes were determined via staining.To assess their effects in vivo,we applied them separately to neonatal,adult and infarcted mice for 14 days and 21 days,and cardiac function was detected by cardiac ultrasonography.Proliferation and formation of gap junctions were determined by immunofluorescence.Finally,we co-cultured human embryonic stem cell-derived cardiomyocytes with adult cardiac fibroblasts for 7 days,and examined proliferation and maturation status via various techniques.ResultsThese results illustrated the maturation of postnatal cardiomyocytes,in which cardiac fibroblasts played a central regulatory role,mainly through inter-cell type crosstalk via cytokines,chemokines and ECM receptor signaling pathways.In particular,a subset of cardiac fibroblasts marked with ENTPD2+played a significant role in this process(p<0.05).In the co-culture system,adult fibroblasts(AFs)induced a series of changes in cardiomyocytes,including decreased proliferation and increased maturation grading(p<0.05).In electrophysiological experiments,AF co-culture promoted a significant increase in APD50 and APD90 in cardiomyocytes.Changes in sodium,potassium,calcium channels and calcium transients all exhibited signs of cardiac maturation.Markers of mature cardiomyocytes,the number of T-tubules and GJA1 expression,both significantly increased(p<0.05).Further analysis showed that genes elevated in the co-culture system were similar to high-expressing genes in mature cardiomyocytes,while those decreased were similar to low-expressing ones.GSEA showed that upregulated genes in the co-culture system were highly enriched on the P56 side(mature)of cardiomyocytes,while downregulated genes were significantly enriched on the P1 side(neonatal)of cardiomyocytes.Single-cell sequencing results validated the evolution from naive to mature gene changes of cardiomyocytes in the co-culture system.KEGG analysis of both in vitro and in vivo maturating cardiomyocytes showed 9 overlapping signaling pathways,including cytokine,chemokine receptor and ECM receptor interaction signaling pathways.Interestingly,application of individual inhibitors to those pathways suppressed morphological maturation of cardiomyocytes in co-culture(p<0.05),but did not promote proliferation(p>0.05).Drug administration to neonatal mice retarded cardiomyocyte proliferation,evidenced by increased expression of proliferation markers at 14,but not 21,days after treatment with Plerixafor or BP-1-102.Such changes were absent in adult mice.Administration of Plerixafor and BP-1-102 to the myocardial infarction model gave rise to proliferating cardiomyocytes in the area around the infarct after 14 days(but not 21 days),marked by significant increases in the expression of proliferating proteins AURKB,MKI67 and pH3(p<0.05).Importantly,co-culture with adult cardiac AFs decreased the expression of AURKB,MKI67 and pH3 in human embryonic stem cell-derived cardiomyocytes,with the majority graded as CI,II(mature).At the electrophysiological level,AFs promoted significant increases in APD50 and APD90 of cardiomyocytes(p<0.05).Similar changes towards maturation upon co-culture with AFs were also manifested by changes in calcium transients,EFP and impedance.ConclusionsOur study revealed that cardiac fibroblasts act as a major component of cardiac microenvironment to drive cardiac maturation,and that signaling pathways involved in ligand-receptor interactions with cardiomyocytes may regulate cardiomyocyte maturation in heart disease and cardiac regeneration.