Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes

Abstract

New Findings What is the central question of this study?Can imipramine, an antidepressant agent that is a cationic amphiphilic drug that interferes with the phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) interactions with proteins maintaining the tubular system, be validated as a new detubulating tool? What is the main finding and its importance?Imipramine was validated as a more efficient and less toxic detubulating agent of cardiomyocytes than formamide. New insights are provided on how PI(4,5)P2 is crucial to maintaining T‐tubule attachment to the cell surface and on the cardiotoxic effects of imipramine overdoses. Abstract Cardiac T‐tubules are membrane invaginations essential for excitation–contraction coupling (ECC). Imipramine, like other cationic amphiphilic drugs, interferes with phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) interactions with proteins maintaining the tubular system connected to the cell surface. Our main purpose was to validate imipramine as a new detubulating agent in cardiomyocytes. Staining adult rat ventricular myocytes (ARVMs) with di‐4‐ANEPPS, we showed that unlike formamide, imipramine induces a complete detubulation with no impact on cell viability. Using the patch‐clamp technique, we observed a ∼40% decrease in cell capacitance after imipramine pretreatment and a reduction of ICa,L amplitude by ∼72%. These parameters were not affected in atrial cells, excluding direct side effects of imipramine. β‐Adrenergic receptor (β‐AR) stimulation of the remaining ICa,L with isoproterenol (Iso) was still effective. ECC was investigated in ARVMs loaded with Fura‐2 and paced at 1 Hz, allowing simultaneous measurement of the Ca2+ transient (CaT) and sarcomere shortening (SS). Amplitude of both CaT and SS was decreased by imipramine and partially restored by Iso. Furthermore, detubulated cells exhibited Ca2+ homeostasis perturbations. Real‐time cAMP variations induced by Iso using a Förster resonance energy transfer biosensor revealed ∼27% decreased cAMP elevation upon β‐AR stimulation. To conclude, we validated a new cardiomyocyte detubulation method using imipramine, which is more efficient and less toxic than formamide. This antidepressant agent induces the hallmark effects of detubulation on ECC and its β‐AR stimulation. Besides, we provide new insights on how an imipramine overdose may affect cardiac function and suggest that PI(4,5)P2 is crucial for maintaining T‐tubule structure.

Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation

“…To verify our method, we investigated the effect of detubulation induced by imipramine ( Bourcier et al, 2019 ). We first confirmed the detubulation effect of imipramine by measuring the inward rectifier potassium tail current ( I K1,tail ) in Tyrode solution in both control ventricular myocytes and in ventricular myocytes pre-treated with 225 μM imipramine in which I K1,tail was strongly suppressed, as expected ( Supplementary Figure S4A ).…”

“…In contrast, studies using the often-used detubulation techniques report lower values of f t in rat ventricular myocytes. Most data were obtained using hyperosmotic shock (e.g., f t = 0.264 in Kawai et al, 1999 , 0.29 in Brette and Orchard 2006 , 0.32 in Brette et al, 2006 , 0.315 in Bryant et al, 2015 ); later studies report values obtained from hypoosmotic shock (0.27 in Moench et al, 2013 ) or from the effect of imipramine (∼0.4 in Bourcier et al, 2019 ). The value f t = 0.337 ± 0.017 estimated by our new method ( Figure 3C ) fits well with the range of values obtained so far by detubulation approaches.…”

“…To partially detubulate ventricular myocytes, the isolated cells were treated for 15 min with 75, 150, and 225 μM imipramine (according to Bourcier et al, 2019 , but lower concentrations were used in our study to prevent the complete detubulation) and then they were centrifuged and the imipramine was washed using the control Tyrode solution.…”

“…The hard-to-determine fraction of the t-tubules that resist detubulation can impair measurement accuracy as well ( Pásek et al, 2008a ). Recently, a new method of detubulation using antidepressant imipramine has emerged that allows complete detubulation to be achieved ( Bourcier et al, 2019 ).…”

Distinct functions of cardiac β-adrenergic receptors in the T-tubulevs.outer surface membrane

“…This network is called the transverse (T−) tubules (TT) system, although the invaginations may often bifurcate in the axial direction or form branches (36). These tubular structures are found mostly in adult ventricular myocytes, where they represent about 30% of the total cell membrane (12, 17), and occur near the sarcomeric z-discs (18), allowing functional junction with the SR called dyad. They are critical for EC coupling by concentrating LTCCs and positioning them at close proximity of RyR2 clusters at the junction of SR to form Ca 2+ release units (31, 63).…”