Introduction:

Mitochondria are the main energy producing organelles in cells. They are crucial for sustaining the continuous heart’s function because of its extensive energy demand. The effect of contractility on cellular physiology and mitochondria is however poorly understood. Using chemogenetics we reversibly inhibited contractility in beating engineered heart tissue (EHT) to study the interplay between contractility and cardiomyocyte physiology, particularly focused on lysosomal degradation of mitochondria. This can be of relevance for studies on the beneficial cellular effects of unloading of the left ventricle in heart failure.

Methods

We genetically engineered human induced pluripotent stem cells (hiPSCs) and introduced an inhibitory pharmacologically selective actuator module fused to the glycine receptor chloride-selective ion pore domain (PSAM-GlyR). Cardiomyocytes derived from PSAM-GlyR-iPSCs were cast in engineered heart tissue (EHT). After an EHT maturation phase of 21 days, the pharmacologically selective effector molecule 89S (PSEM^89S) was applied to the beating EHTs for either 24 hours, 7 or 21 days. The PSEM^89S induced chloride flux stopped the EHT contractility via depolarization block. This effect was reversible upon wash-out of PSEM^89S. Physiological characterization of the EHTs was performed using video-optical analysis. To assess cellular changes as well as mitophagy, we performed immunostainings, proximity ligation assay (PLA) and transmission electron microscopy (TEM). 

Results

The localization and morphology of mitochondria, sarcomeres and the cytoskeleton upon inhibition of contractility was substantially changed within 24 hours, as revealed by immunostainings. Cardiomyocytes displayed a decrease in size and a more roundish cell shape. Cessation of EHT contractions lead to sarcomere disassembly and subsequent disorganization of the mitochondria previously aligned within the sarcomeres. The lysosome size was increased and lysosomes, mitochondria and microtubules showed a perinuclear localization. Proximity ligation assay revealed an interaction between lysosomes and mitochondria, indicative of mitochondrial degradation. These results were further corroborated by TEM. Notably, EHTs started to contract immediately after PSEM^89S wash-out and the observed cellular changes were fully reversed within seven days. 

Discussion

We have developed a model to study the effect of contractility on cardiomyocyte biology in a three-dimensional environment in vitro. With this model we demonstrate substantial morphological cellular changes upon contractility inhibition. PSAM-GlyR-EHTs provide versatile opportunities for future studies to assess the effect of cardiac work on cardiomyocyte biology.