Three lenses into the cardiac dyad: calcium signaling, microtubule trafficking, and junctional sarcoplasmic reticular mobility
Drum, Benjamin Mark Loren
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The cardiac dyad, the intersection between the sarcoplasmic reticulum and the sarcolemma in cardiac myocytes, is critically important for cellular contraction and function. It is the hub of calcium signaling where ryanodine receptors on the sarcoplasmic reticulum and L-type calcium channels on the sarcolemma interact. If the geometric distance between these two proteins is disrupted, calcium signaling will be impaired. In addition, these proteins are trafficked and maintained by microtubules. In this dissertation, I take a three-pronged approach to understanding the dyad. First, I use a mouse model of Timothy syndrome (TS), a disease created by an overactive L-type calcium channel, to examine the effects of the physiological calcium environment on calcium signaling. We find that the resting level of calcium in the cytosol is increased by 1.6-fold, calcium transients are increased by 1.8-fold, and sarcoplasmic reticulum load is increased by 1.5-fold in TS, leading to arrhythmogenicity. Secondly, I use an adeno-associated virus serotype 9 expressing a plus-end microtubule binding protein (AAV9-EB3-EGFP) to visualize microtubules in real time. I quantify microtubule dynamics in ventricular myocytes and find that oxidative stress in myocardial infarction disrupts microtubules, leading to a decrease in the surface expression of KV4.2 and KV4.3 and decrease in Ito, also leading to arrhythmia by prolonging the action potential. I also show that microtubules and molecular motors, specifically kinesin-1, are responsible for trafficking the β2 subunit of CaV1.2, and that disruption of kinesin-1 results in a loss in current density of CaV1.2 current. I also examine microtubule dynamics in atrial myocytes and use this research to probe the role of BIN1 in linking microtubule dynamics to the cardiac dyad. Lastly, I use an adeno-associated virus expressing triadin, a protein localized to the junctional sarcoplasmic reticulum (jSR) tagged with a photo-activatable GFP (AAV9-TRD-PAGFP) to directly monitor the movement of the jSR under normal physiology and find that 8% of jSR segments exert mobility. This movement is produced by molecular motors and is increased in the setting of myocardial infarction. By exploring the cardiac dyad through the lenses of calcium signaling, microtubule trafficking, and direct jSR movement, I am able to elucidate a multifaceted look at the regulation and function of the dyad as well as its importance in cardiac physiology.