| Abstract: | Atrial myocytes are continuously exposed to mechanical forces including shear stress. However, in atrial myocytes, the effects of shear stress are poorly understood, particularly with respect to its effect on ion channel function. Here, we report that shear stress activated a large outward current from rat atrial myocytes, with a parallel decrease in action potential duration. The main ion channel underlying the increase in current was found to be Kv1.5, the recruitment of which could be directly observed by total internal reflection fluorescence microscopy, in response to shear stress. The effect was primarily attributable to recruitment of intracellular pools of Kv1.5 to the sarcolemma, as the response was prevented by the SNARE protein inhibitor N-ethylmaleimide and the calcium chelator BAPTA. The process required integrin signaling through focal adhesion kinase and relied on an intact microtubule system. Furthermore, in a rat model of chronic hemodynamic overload, myocytes showed an increase in basal current despite a decrease in Kv1.5 protein expression, with a reduced response to shear stress. Additionally, integrin beta1d expression and focal adhesion kinase activation were increased in this model. This data suggests that, under conditions of chronically increased mechanical stress, the integrin signaling pathway is overactivated, leading to increased functional Kv1.5 at the membrane and reducing the capacity of cells to further respond to mechanical challenge. Thus, pools of Kv1.5 may comprise an inducible reservoir that can facilitate the repolarization of the atrium under conditions of excessive mechanical stress.The electrical properties of the myocardium are governed by the interplay of ion channels, whose expression and regulation determines the precise electrical responses of the tissue. The activity of ion channels can be regulated in a variety of ways: for example, interaction with accessory subunits (1), phosphorylation (2), oxidation state (3), and gene expression (4). Recently, increased attention has been focused on trafficking as a means to regulate ion channel function, notably by modulating the number of active channels present at the plasma membrane (5, 6). This regulation is a complex process derived from a balance between trafficking of newly synthesized channel, endocytosis, and recycling/degradation. Trafficking of ion channels is known to be a dynamically regulated process that depends on Rab-GTPases as well as dynamin motors (7, 8). Indeed, certain antiarrhythmogenic drugs have been shown to exert their activity by modifying the number of channels at the plasma membrane (9). In this context, we previously showed that ion channels are recruited from a submembranous pool in response to cholesterol depletion (10). In addition, several ion channels are regulated by mechanical forces, which directly affect the gating of the channel (11) or indirectly activate intracellular signaling pathways to alter channel properties (12).The myocardium is subjected to a variety of forces with each contraction and therefore must adapt to the various associated mechanical stresses. The response to stretch has been well-studied and includes gene regulation (13), activation of stretch-activated ion channels (14–17), and the release of atrial natriuretic peptide (ANP) (18–21). In addition, it has been shown that direct stretch of β1 integrins activates ICl,swell as well as a cation current (22). Less well-studied are the responses of cardiomyocytes to shear stress. Shear forces in the myocardium arise from blood flow and the relative movement of sheets of myocytes, causing cell deformation as the myocardial layers slide against each other with each heart beat (23, 24). Although the effect of shear stress upon cardiomyocytes has not been extensively explored, it has been shown that increased shear stress stimulates intracellular calcium transients (25, 26), induces an increase in the beating rate of neonatal ventricular myocytes (27), and triggers propagating action potentials (APs) in monolayers of ventricular myocytes (28). Thus far, the response to shear stress remains relatively unknown, particularly with regard to ion channel regulation. Ion channel activity determines both the shape of the AP and the firing frequency of excitable cells. Therefore, the response of cardiomyocytes to shear stress is important for normal cardiac excitability and could be central in pathological conditions in which the working conditions of the myocardium are altered.In this study, we investigate the response of native adult rat cardiomyocytes to shear stress, reproduced in vitro by laminar flow. Using a combination of whole-cell patch-clamp and single-channel recordings, high spatial resolution 3-dimensional and total internal reflection fluorescence (TIRF) microscopy, we show that shear stress induces an increase in outward current and shortens AP duration within the range of a few minutes. This phenomenon is saturable and reversible, and is caused by Kv1.5 exocytosis from the recycling endosome. We identify the mechanotransduction pathway of this recruitment, which involves integrin/focal adhesion kinase (FAK) signaling. Finally, the response to shear stress is altered in chronically hemodynamically overloaded and dilated atria. |
