Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-β signaling

Abstract

Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.

Keywords: Cardiac development; Cardiomyocytes; Contractility; Gene regulation; Hypoplastic Left Heart Syndrome; Mechanical stretch.

Figure 1. Cyclic stretch increases EMCM proliferation, size, cardiac gene expression, and myofibrillar protein levels

A. EDU staining comparing the proliferation rates of static and stretched EMCMs. Stretched EMCMs have a 21.3% increase in relative proliferation compared to static controls (1.21 +/− 6% vs. 1 +/− 3.2%; p<0.001; n=8–9). B. EMCMs exposed to stretch are 33% larger compared to static controls as quantified by flow cytometry (1.42×10⁶ +/− 0.01 vs 1.07×10⁶ +/− 0.04; p<0.001; n=3). C. qPCR for key cardiac genes demonstrates increased expression in stretched EMCMs compared to static controls. Gapdh was used as control. n=4–6. D. Immunoblotting showing representative total sarcomeric myosin heavy chain expression and Titin isoform levels. Total myosin heavy chain levels are increased in stretched EMCMs. Titin N2BA is the predominate isoform in stretched EMCMs, as compared to Titin N2B as is seen in static controls. E. Stretched EMCMs have increased Myosin heavy chain levels (1.38 +/− 0.10 vs 1 +/− 0.03; p=0.014; n=4). F. Quantification of Titin isoforms. 80.8% +/− 8.6% of Titin in stretched EMCMs is of the N2BA isoform. In contrast, static cardiomyocytes contain 22.8% +/− 9.4% of the Titin N2BA isoform. (the rest is Titin N2B; n=4; *p<0.05, **p<0.005, ^p<0.001).

Figure 2. Cyclic stretch decreased Tgf-β expression and signaling

Since the GO-term associated with Tgf-β was modulated by stretch (p=3.95×10⁻⁵), the role of Tgf-β signaling in stretched EMCMs was examined. A. qPCR for Tgf-β expression demonstrates that cyclic stretch inhibits expression of Tgf-β2, Tgf-β3, and Tgfβr2. B. and C. Protein levels of Tgf-β ligands and receptors are decreased in stretched EMCMs. D and E. Western blotting for phospho-SMAD3 demonstrates decreased Tgf-β/SMAD signaling in stretched EMCMs. F. Global rVISTA analysis (which examines up to 5kb proximal to the transcriptional start site) predicts that 65% of stretch responsive myofibrillar (p=3.03×10⁻⁸) and 66% of cardiac development genes (p=4.96×10⁻³⁶) have SMAD binding sites within 5 kb. (n=3 panels A–E; *p<0.05, **p<0.005).

Figure 3. TGFβ2 modulates proliferation and growth of EMCMs

A. The addition of 1 ng/mg Tgf-β2 during stretch results in 20% reduction in proliferation (1 +/− 0.05 vs 0.80 +/− 0.05; p<0.05; n=3). B. Embryonic cardiomyocytes treated with ITD-1, a Tgfβr2 inhibitor, are 69% larger (1.40×10⁶ +/−0.10 vs 0.83×10⁶+/− 0.03; p<0.001; n=3).

Figure 4. Tgf-β2 is sufficient to reduce EMCM contractility

A. Bright field and fluorescent images of beating EMCMs obtained for DMFM. Fluorescence images at maximum contraction (green) and relaxation (red) are overlaid, leading to red/green patterns in regions of large deformation and yellow patterns in regions of small deformation. B. Mean squared deformations in the whole fluorescence field determined from tracking bead motion. Peaks correspond to maximum contraction while valleys correspond to relaxation. C. Model of the equilibrium principle used to determine intracellular stresses[40, 41]. D and E. Comparison of traction stresses (arrows) and intracellular stresses (heat map, in Pa) in EMCMs treated with 1ng/ml of Tgf-β2 compared to untreated controls, as measured by DMFM. Red represents higher intracellular stress. F. Boxplot showing that EMCMs treated with Tgf-β2 display decreased intracellular stress compared to controls (p<0.05, WT n=20, Tgf-β2 N=11). G. Boxplot showing that EMCMs treated with Tgf-β2 exhibit slower beating period compared to controls (p<0.001, WT n=15, Tgf-β2 n=11). Scale bars are 20 μm long.

Figure 5. Model of how stretch modulates Tgf-β signaling and cardiac development

Based upon our data, we propose that physiologic stretch of developing cardiomyocytes represses Tgf-β signaling, which in turn results in increased cardiomyocyte proliferation, size, gene expression, and contractile function.