Mechano-Chemotransduction in the Single Cardiac Myocyte Contracting in 3D Elastic Gel

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Objective: Mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause heart failure and arrhythmias. However, little is known about the mechanotransduction mechanisms that underlie heart disease development due to previous lack of practical techniques to control the mechanical stress on single myocytes necessary for investigating at cellular and molecular levels. Here we use this system to study the mechanical load effects on modulating myocyte Ca2+ signaling and contraction dynamics. Methods: Recently, we developed a novel Cell-in-Gel system that allows control of mechanical load on single rabbit myocytes during excitation-contraction coupling in a 3D elastic gel matrix composed of polyvinyl alcohol (PVA) and tetravalent boronate-PEG crosslinker. The mechanical load can be controlled by adjusting gel stiffness with different mixing ratios of PVA and crosslinker. Results: Myocyte contraction and calcium transients were measured in-gel and compared with load-free cells. Contracting cells in-gel showed a significantly lower fractional shortening (12.6 ± 0.9 in-gel vs. 18.2 ± 0.9 load-free, p<0.001), demonstrating a “knock-down factor” of 31% when the myocyte is pulling mechanical load. Contraction departure and return velocities were significantly slower in-gel than in the load-free state as expected. However, the systolic calcium transient was greater in-gel than load-free (Fura-2 fluorescence ratio peak height 1.47 ± 0.09 in-gel vs. 0.87 ± 0.04 load-free, p<0.0001), revealing the mechano-chemotransduction that translates external stress to intracellular Ca2+ increase. The calcium transient departure and return velocities were also significantly higher in-gel than load-free. Conclusions: Our newly-developed versatile Cell-in-Gel system provides a novel experimental method to control mechanical stress at the single cell level for investigating mechano-chemotransduction pathways in intact myocytes. The above experimental results are consistent with our modeling predictions, demonstrating the mechanical load effects on altering myocyte Ca2+ handling and contraction dynamics.