Mechano-Chemo-Transduction in Rabbit Cardiomyocytes Mediated by no Signaling

Abstract

Background: Increased mechanical stress under pathological conditions such as hypertension, infarction and fibrosis can cause arrhythmias and heart failure. However, little is known about Mechano-Chemo-Transduction (MCT) mechanisms that underlie heart disease development. We developed a novel Cell-in-Gel system to control mechanical loading on cardiomyocytes during excitation-contraction coupling. Here we investigate mechanical load effects on modulating Ca2+ signaling and contraction dynamics to test the hypothesis NO signaling mediates MCT.Methods: Freshly isolated adult rabbit ventricular myocytes were embedded in polyvinyl alcohol (10%) and crosslinked with tetravalent boronate-PEG (7.5%) to form a 3D viscoelastic hydrogel. The Cell-in-Gel system was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Cardiomyocyte contraction and Ca2+ transients were measured in-gel and compared with load-free cells in solution.Results: Cardiomyocytes contracting in-gel under mechanical load showed augmented systolic Ca2+ transients (Fura-2 fluorescence ratio 1.47 ± 0.07 in-gel vs 0.85 ± 0.03 load-free), revealing MCT transduces mechanical stress to increase intracellular Ca2+ release. NOS inhibition using 1 mM L-NAME reduced the in-gel Ca2+ transient by 33% (n=15), suggesting NO signaling mediates MCT. Cells in-gel showed 8% decrease in contraction (fractional shortening 13.5 ± 0.4% in-gel vs 14.7 ± 0.3% load-free). Cells in-gel treated with L-NAME demonstrated a further reduction in contraction (11.6 ± 0.8%) as a result of decreased MCT by NOS inhibition. Importantly, 12% of cells in-gel showed mechanical load-induced alternans, which was abolished with L-NAME treatment, suggesting NO signaling contributes to load-induced Ca2+ dysregulation.Conclusion: Our findings demonstrate MCT increases the systolic Ca2+ transient to enhance contractility in response to increased afterload, serving a compensatory mechanism to autoregulate contractility. MCT is attenuated by inhibiting NOS. These results suggest NOS mediates increased Ca2+ signaling in cardiomyocytes under mechanical load, providing a mechanistic basis for the Anrep effect.