Functional studies on the mechanosensitive ion channel PIEZO1 in human induced pluripotent stem cell-derived cardiomyocytes

Der Herzmuskel muss sich einer dynamischen und sich mechanisch verändernden Umgebung anpassen. Die Mechanosignaltransduktion ermöglicht es Zellen mechanischen Kräfte zu erfassen und durch nachgeschaltete biochemische Signalkaskaden darauf zu reagieren. Obwohl verschiedene Gewebestrukturen und Proteine damit in Verbindung gebracht wurden, wie das Herz die mechanischen Kräfte wahrnimmt, ist unser Verständnis der kardialen Mechanosignaltransduktion unvollständig. Durch Dehnung aktivierte Ionenkanäle spielen eine wichtige Rolle bei der mechanosensitiven Autoregulation des Herzens. Um die funktionelle Rolle von PIEZO1 in Kardiomyozyten zu untersuchen, habe ich daher PIEZO1 in induzierten pluripotenten Stammzellen mittels Genomeditierung deletiert. Die PIEZO1-/- Zellen wurden dann in lebensfähige, herzähnlich schlagende Kardiomyozyten differenziert. In phänotypische Analysen der elektrophysiologischer Eigenschaften, Zellmorphologie und der herzähnlichen Schlagaktivität habe ich den Effekt der PIEZO1-deletion in genomeditierten Kardiomyozyten untersucht. Die Deletion von PIEZO1 zeigte zum ersten Mal, dass es PIEZO1-abhängige dehnungsaktivierte und Kalzium-Ströme in vom Menschen stammenden differenzierten Kardiomyozyten gibt. Dies legt nahe, dass PIEZO1 eine Rolle in der Mechanosignaltransduction in Herzzellen spielt. Darüber hinaus zeigte eine RNA-Sequenz Analyse, dass der Verlust von PIEZO1 in vom Menschen stammenden differenzierten Kardiomyozyten mit der Herunterregulation von Proteinen korreliert, die für die extrazellulärer Matrix von Bedeutung sind. Diese Daten unterstreichen die Rolle von PIEZO1 in Kardiomyozyten und legen seine Bedeutung für die Organisation und Struktur der extrazellulären Matrix nahe.The cardiac muscle has to adapt in a highly dynamic mechanical environment. Mechanotransduction is the process that allows cells to sense the mechanical forces and respond by downstream biochemical signaling cascades. Although different tissue structures and proteins have been implicated in how the heart senses the mechanical forces, yet our understanding in cardiac mechanotransduction is incomplete. Stretch-activated channels (SACs) have been suggested to play an important role in the mechanosensitive autoregulation of the heart. PIEZO1 is a stretch-activated channel and has been involved in vascularization, erythrocyte volume homeostasis and regulation of the baroreceptor reflex, yet its role in cardiac mechanotransduction has not been described. To study the functional role of PIEZO1 in cardiomyocytes I have generated a PIEZO1 knockout (KO) human induced pluripotent cell (hiPSC) line using genome editing technology. The genome edited cells were then differentiated into viable, beating cardiomyocytes. Different phenotypic analyses were conducted, including the evaluation of electrophysiological characteristics, observation of cell morphology and beating activity of the genome edited hiPSC-derived cardiomyocytes. With this approach the aim was to gain more insight into PIEZO1 function in cardiomyocytes using a reliable, efficient and reproducible human cellular model system. For the first time PIEZO1-dependent calcium transients and stretch-activated currents were observed in hiPSC-derived cardiomyocytes (hiPSC-CMs). This proposes a possible role of PIEZO1 as a cardiac mechanotransducer. Furthermore, RNA-seq analysis revealed that loss of PIEZO1 in hiPSC-CMs is associated with downregulation of the expression of extracellular matrix-associated proteins. These data highlight the role of PIEZO1 in cardiomyocytes and suggest its implication in extracellular matrix organization and structure

Left Ventricular Unloading Using an Impella CP Improves Coronary Flow and Infarct Zone Perfusion in Ischemic Heart Failure

Background-Delivering therapeutic materials, like stem cells or gene vectors, to the myocardium is difficult in the setting of ischemic heart failure because of decreased coronary flow and impaired microvascular perfusion (MP). The aim of this study was to determine if mechanical left ventricular (LV) unloading with the Impella increases coronary flow and MP in a subacute myocardial infarction. Methods and Results-Anterior transmural myocardial infarction (infarct size, 26.0 +/- 3.4\%) was induced in Yorkshire pigs. At 2 weeks after myocardial infarction, 6 animals underwent mechanical LV unloading by Impella, whereas 4 animals underwent pharmacological LV unloading using sodium nitroprusside for 2 hours. LV unloading with Impella significantly reduced end-diastolic volume (-16 +/- 11mL, P=0.02) and end-diastolic pressure (EDP; -32 +/- 23 mm Hg, P=0.03), resulting in a significant decrease in LV end-diastolic wall stress (EDWS) (infarct: 71.6 +/- 14.7 to 43.3 +/- 10.8 kdynes/cm(2) [P=0.02]; remote: 66.6 +/- 20.9 to 40.6 +/- 13.3 kdynes/cm(2) [P=0.02]). Coronary flow increased immediately and remained elevated after 2 hours in Impella-treated pigs. Compared with the baseline, MP measured by fluorescent microspheres significantly increased within the infarct zone (109 +/- 81\%, P=0.003), but not in the remote zone. Although sodium nitroprusside effectively reduced LV-EDWS, 2 (50\%) of sodium nitroprusside-treated pigs developed profound systemic hypotension. A significant correlation was observed between the infarct MP and EDWS (r(2)=0.43, P=0.03), suggesting an important role of EDWS in regulating MP during LV unloading in the infarcted myocardium. Conclusions-LV unloading using an Impella decreased EDWS and increased infarct MP without hemodynamic decompensation. Mechanical LV unloading is a novel and efficient approach to increase infarct MP in patients with subacute myocardial infarction.This study was partly supported by a research grant from ABIOMED Inc (Danvers, MA) (Ishikawa); National Institutes of Health (NIH) grants R01 HL139963 (Ishikawa) and HL117505, HL119046, HL129814, 128072, HL131404, R01HL135093, and P50 HL112324 (Hajjar); American Heart Association-Scientist Development Grant 17SDG33410873 (Ishikawa); and 2 Transatlantic Fondation Leducq grants. Bikou was supported by the Deutsche Herzstiftung. We would like to acknowledge the Gene Therapy Resource Program of the National Heart, Lung, and Blood Institute, NIH.S