Culturing of Cardiac Fibroblasts in Engineered Heart Matrix Reduces Myofibroblast Differentiation but Maintains Their Response to Cyclic Stretch and Transforming Growth Factor ß1.

Isolation and culturing of cardiac fibroblasts (CF) induces rapid differentiation toward a myofibroblast phenotype, which is partly mediated by the high substrate stiffness of the culture plates. In the present study, a 3D model of Engineered Heart Matrix (EHM) of physiological stiffness (Youngs modulus ~15 kPa) was developed using primary adult rat CF and a natural hydrogel collagen type 1 matrix. CF were equally distributed, viable and quiescent for at least 13 days in EHM and the baseline gene expression of myofibroblast-markers alfa-smooth muscle actin (Acta2), and connective tissue growth factor (Ctgf) was significantly lower, compared to CF cultured in 2D monolayers. CF baseline gene expression of transforming growth factor-beta1 (Tgfß1) and brain natriuretic peptide (Nppb) was higher in EHM-fibers compared to the monolayers. EHM stimulation by 10% cyclic stretch (1 Hz) increased the gene expression of Nppb (3.0-fold), Ctgf (2.1-fold) and Tgfß1 (2.3-fold) after 24 h. Stimulation of EHM with TGFß1 (1 ng/mL, 24 h) induced Tgfß1 (1.6-fold) and Ctgf (1.6-fold). In conclusion, culturing CF in EHM of physiological stiffness reduced myofibroblast marker gene expression, while the CF response to stretch or TGFß1 was maintained, indicating that our novel EHM structure provides a good physiological model to study CF function and myofibroblast differentiation.

Left atrial remodeling in mitral regurgitation: A combined experimental-computational study.

AIMS: Progressive changes to left atrial (LA) structure and function following mitral regurgitation (MR) remain incompletely understood. This study aimed to demonstrate potential underlying mechanisms using experimental canine models and computer simulations. METHODS: A canine model of MR was created by cauterization of mitral chordae followed by radiofrequency ablation-induced left bundle-branch block (LBBB) after 4 weeks (MR-LBBB group). Animals with LBBB alone served as control. Echocardiography was performed at baseline, acutely after MR induction, and at 4 and 20 weeks, and correlated with histology and computer simulations. RESULTS: Acute MR augmented LA reservoir and contractile strain (40±4 to 53±6% and -11±5 to -22±9% respectively, p<0.05). LA fractional area change increased significantly (47±4 to 56±4%, p<0.05) while LA end-systolic area remained unchanged (7.2±1.1 versus 7.9±1.1 cm2 respectively, p = 0.08). LA strain 'pseudonormalized' after 4 weeks and decompensated at 20 weeks with both strains decreasing to 25±6% and -3±2% respectively (p<0.05) together with a progressive increase in LA end-systolic area (7.2±1.1 to 14.0±6.3 cm2, p<0.05). In the LBBB-group, LA remodeling was less pronounced. Histology showed a trend towards increased interstitial fibrosis in the LA of the MR-LBBB group. Computer simulations indicated that the progressive changes in LA structure and function are a combination of progressive eccentric remodeling and fibrosis. CONCLUSION: MR augmented LA strain acutely to supranormal values without significant LA dilation. However, over time, LA strain gradually decreases (pseudornormal and decompensated) with LA dilation. Histology and computer simulations indicated a correlation to a varying degree of LA eccentric remodeling and fibrosis.

Piezo1 Mechanosensitive Ion Channel Mediates Stretch-Induced Nppb Expression in Adult Rat Cardiac Fibroblasts.

In response to stretch, cardiac tissue produces natriuretic peptides, which have been suggested to have beneficial effects in heart failure patients. In the present study, we explored the mechanism of stretch-induced brain natriuretic peptide (Nppb) expression in cardiac fibroblasts. Primary adult rat cardiac fibroblasts subjected to 4 h or 24 h of cyclic stretch (10% 1 Hz) showed a 6.6-fold or 3.2-fold (p < 0.05) increased mRNA expression of Nppb, as well as induction of genes related to myofibroblast differentiation. Moreover, BNP protein secretion was upregulated 5.3-fold in stretched cardiac fibroblasts. Recombinant BNP inhibited TGFß1-induced Acta2 expression. Nppb expression was >20-fold higher in cardiomyocytes than in cardiac fibroblasts, indicating that cardiac fibroblasts were not the main source of Nppb in the healthy heart. Yoda1, an agonist of the Piezo1 mechanosensitive ion channel, increased Nppb expression 2.1-fold (p < 0.05) and significantly induced other extracellular matrix (ECM) remodeling genes. Silencing of Piezo1 reduced the stretch-induced Nppb and Tgfb1 expression in cardiac fibroblasts. In conclusion, our study identifies Piezo1 as mediator of stretch-induced Nppb expression, as well as other remodeling genes, in cardiac fibroblasts.