Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes

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.

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 I_Ca,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 I_Ca,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.