Takotsubo syndrome (TTS) is characterised by acute left ventricular dysfunction in the absence of obstructive coronary lesions.1 TTS is a life-threatening disease with a mortality rate of up to 8% in the acute phase. Furthermore, 21.8% of patients show in-house complications, such as cardiogenic shock or ventricular tachycardia.2 Although enhanced β-adrenergic signalling and higher sensitivity to catecholamine-induced stress toxicity were identified as mechanisms associated with the TTS phenotype in the authors’ former study, the pathogenesis of TTS is still not completely understood.3 The aim of the study was to prove the hypothesis of a phosphodiesterase (PDE)-dependent regulation of 3’,5’-cyclic adenosine monophosphate (cAMP) signalling in TTS under catecholamine stress.
METHODS AND RESULTS
Functional TTS-induced pluripotent stem cell-derived cardiomyocytes (TTS-iPSC-CM) were generated from six patients and the cells were treated with catecholamines to mimic a TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, it was observed that β-adrenergic receptor (β-AR) stimulations led to stronger FRET responses in the cytosol of TTS-CM compared to controls. In addition to β-AR, PDE are key molecules involved in cAMP signalling in CM. At basal level, TTS-CM show a significantly higher PDE3A and a reduced PDE4D protein expression in the TTS-CM compared to control. In addition, FRET experiments show that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of control cells were significantly decreased in TTS-CM. This is in line with previously described reduction of the overall cytosolic PDE4 activity in severely hypertrophied and failing rat and mouse.4 By analysing PDE-dependent cAMP downstream effects as PKA-dependent phosphorylation, an additional increase of phospholamban (PLN) phosphorylation (PLN-S16) was observed, especially in the control group, when treating iPSC-CM with a combination of isoprenaline and PDE4 inhibitor. In contrast, in TTS-iPSC-CM the contribution of the PDE-families PDE2, 3, or 4 to phosphorylation of PLN-S16 was increased in comparison to isoprenaline alone. This suggests that different PDE in TTS and control are involved in functional segregation of the sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) microdomain from the cytosol in terms of cAMP downstream effects. To directly address the hypothesis that local cAMP dynamics might be altered in TTS, a SERCA microdomain targeted FRET based cAMP sensor was used. In contrast to the cytosolic cAMP regulation, the contribution of PDE2 and PDE3 to local cAMP degradation was increased in both TTS and control. Nevertheless, the strong PDE4 inhibitor effects in control cells are still reduced in TTS in the SERCA domain.
The data, for the first time, shows alterations of local cAMP signalling in healthy and diseased TTS-iPSC-CM. The PDE-specific contribution to cAMP degradation in the cytosol of TTS iPSC-CM is lost in the SERCA domain. Additionally, the results uncover a PDE-dependent altered β-adrenergic signalling as a potential disease cause. Furthermore, the data highlight that TTS-iPSC-CM can be used to provide a versatile tool for evaluating new treatment options for TTS as therapeutic targets.