Kalziumhandling und Kalziumpufferung bei familiärer dilatativer Kardiomyopathie am Modell der induzierten pluripotenten Stammzellen
Calcium handling and calcium buffering in familial dilated cardiomyopathy in the induced pluripotent stem cell model
by Philipp Jung
Date of Examination:2024-02-06
Date of issue:2024-01-29
Advisor:Prof. Dr. Niels Voigt
Referee:Prof. Dr. Niels Voigt
Referee:Prof. Dr. Katrin Streckfuß-Bömeke
Referee:Prof. Dr. Ralf Dressel
Sponsor:Promotionskolleg für Medizinstudierende (Jacob-Henle-Programm) der Universitätsmedizin Göttingen
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EnglishDilated cardiomyopathy (DCM) is a heterogeneous disease characterised by left ventricular dilatation and a reduced ejection fraction. In addition to progressive left heart failure, the poor clinical prognosis is primarily due to the increased incidence of ventricular tachycardia, which is associated with a high risk of sudden cardiac death. Genetic mutations play a significant role in familial DCM. Mutations in at least 30 genes are associated with a familial clustering of DCM. It is interesting to note that patients with gene mutations in sarcomeric proteins are particularly frequently affected by cardiac arrhythmias. While previous work has focussed primarily on the molecular mechanisms of impaired contractile function, little is known about the underlying pathomechanism of cardiac arrhythmias in patients with DCM. To investigate this in more detail, cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) from DCM patients with an R173W point mutation (arginine to tryptophan) of cardiac troponin T (cTnT) were used in the present study. This mutation is known to be associated with a high incidence of ventricular arrhythmias. Using field stimulation experiments to measure cytosolic Ca2+ concentration ([Ca2+]i), TnT-R173W iPSC-CM were shown to have a lower threshold for [Ca2+]i-alternans compared to healthy control iPSC-CM of the same family. [Ca2+]i-alternans are known to be an indication of a proarrhythmogenic substrate of these cells. Patch-clamp studies were also performed to simultaneously measure Ca2+ currents and intracellular Ca2+ concentration. With the help of a caffeine protocol, the intracellular Ca2+ buffering could be further derived. This work shows for the first time an increased Ca2+ affinity of the intracellular buffers in TnT-R173W-CM, indicating an increased sensitivity of the myofilaments to Ca2+. Similarly, the use of EMD57033, a myofilament Ca2+ sensitiser, replicated the abnormal [Ca2+]i dynamics observed in TnT-R173W samples. EMD57033 also lowered the alternans threshold in field stimulation experiments. In contrast, application of a Ca2+ desensitizer (blebbistatin) to TnT-R173W iPSC-CM phenotypically rescued Ca2+ dynamics and minimised the occurrence of Ca2+ alternans at physiological frequencies. This finding suggests that increased Ca2+ buffering likely plays an important proarrhythmogenic role in DCM, particularly in patients with mutations in cardiac troponin T. Furthermore, this work provides evidence that modulation of myofilament Ca2+ sensitivity may be an effective antiarrhythmic target for pharmacological treatment of this disease.
Keywords: Calcium handling; Calcium buffering; Dilated cardiomyopathy; Troponin T; Arrhythmia; Patch clamp; iPSC-CM