Microtubules have many roles in non-differentiating cardiomyocytes, including structural stability, morphological integrity, and protein trafficking. However, these proteins had never been visualized. Using adeno-associated viral vectors expressing the microtubule-associated protein EB3 tagged with EGFP, we were able to perform live imaging and capture microtubule dynamics in cardiomyocytes in real time. We quantified growth rate and distance, shrink rate and distance, catastrophe rate, rescue rate, and directionality. We also quantified the temperature dependence of microtubule dynamics. The vast majority of microtubules move perpendicular to T-tubules, and the microtubules were more stable on the Z-lines. Hydrogen peroxide increased the rate of catastrophe among microtubules. Previous studies have shown that heart failure increases microtubule density; however, little work had been done examining microtubules immediately after myocardial infarction (MI). We quantified microtubule dynamics and found an increase in the catastrophe rate of microtubules three days after MI. Moreover, this increase in catastrophe was replicated by exposing myocytes to a concentration of hydrogen peroxide inducing levels of oxidative stress similar those seen in cardiomyocytes after MI. Transient outward current (Ito) density, which constitutes phase I of the action potential in cardiomyocytes, is reduced after MI, resulting in prolonged action potentials. The mechanism of this reduction in Ito current was unknown. Using electrophysiology, we recorded Ito currents and found a similar decrease among MI cardiomyocytes and cardiomyocytes treated with hydrogen peroxide, colchicine, or both. Lastly, we performed a surface biotinylation blot on these cells and found decreases in surface expression of Kv4.2 and 4.3 channels, the channels responsible for Ito, in these conditions compared to control cells. We conclude that oxidative stress after MI disrupts microtubule dynamics, leading to a decrease in trafficking Kv4.2 and 4.3 channels to the membrane, thus decreasing Ito.
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