The effect of mechanical stretch on phospholamban mutated (PLN R14del) cardiomyocytes

Background/Introduction:
The cellular effects of mechanical stretch on adherent cells is generally two-fold: in the short-term, heterochromatin-mediated mechanosensing drives calcium-dependent nuclear softening, while on the long-term cellular re-orientation is induced, perpendicular to the experienced stretch (i.e. strain avoidance response). In epidermal progenitor cells it has been demonstrated that a disruption in the calcium homeostasis results in failure to elicit the strain avoidance response leading to nuclear rupture and cell death. Interestingly, immature and human induced pluripotent stem cell (hiPSC) derived cardiomyocytes generally do not show strain avoidance response.
In cardiomyocytes, phospholamban (PLN) controls calcium homeostasis by regulating the function of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a). A deletion of Arginine 14 (R14del) in PLN leads to an inhibition of SERCA2a, and a disruption in the intracellular calcium homeostasis. In patients this has been associated with the development of cardiomyopathy. However, it remains unknown to what extend the PLN R14del mutation effects the mechano-responsiveness of cardiomyocytes, and how this affects the development and progression of the disease.

Purpose:
In this project we aimed at elucidating how the PLN R14del mutation affects the mechano-responsiveness of cardiomyocytes when exposed to mechanical stretch.

Methods and Results:
We generated hiPSCs from patients with the PLN R14del mutation and healthy controls, as well as an isogenic corrected PLN R14del mutated line by means of CRISPR/Cas9 (PLN corrected). Cardiomyocytes derived from these three hiPSC lines were exposed to short-term (30 min and 6 hrs) and long-term (24 hrs and 48 hrs) mechanical stretch (4% uniaxial strain, 1 Hz frequency), using a FlexCell culture system. Cellular injury was assessed by determining cell apoptosis (cleaved caspase-3 and TUNEL positive nuclei) and nuclear damage (nuclear bodies). PLN R14del mutated cardiomyocytes demonstrated a significant increase in apoptosis, upon both short- and long-term mechanical stretch. A response that was not observed in the cardiomyocytes derived from the healthy controls or the PLN corrected hiPSC. Interestingly, the increased levels of apoptosis in the PLN R14del cardiomyocytes correlated with an increased presence of nuclear bodies, suggesting an increase in nuclear damage. Finally, we observed that the PLN R14del cardiomyocytes undergo the strain avoidance response, as measured by changes in nuclear and α-actinin fiber orientations, while cardiomyocytes derived from the healthy controls or the PLN corrected hiPSC did not show strain avoidance.

Conclusion(s):
PLN R14del mutated cardiomyocytes are more susceptible towards mechanical stretch and have a lower cell viability. These cardiomyocytes try to reduce mechanical damage by undergoing the strain avoidance response, an effect usually not observed in hiPSC derived or immature cardiomyocytes.