|Ph.D Student||Mekies Lucy Naomi|
|Subject||Investigating the Pathophysiological Features of Induced|
Pluripotent Stem Cell (iPSC) - Derived
Cardiomyocytes from Duchenne Muscular
Dystrophy (DMD) and Titin...
|Department||Department of Medicine||Supervisor||PROFESSOR EMERITUS Ofer Binah|
|Full Thesis text|
Duchenne Muscular Dystrophy (DMD) caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females. DMD is characterized by progressive muscle degeneration, loss of ambulation and death mostly by late 20's. Dilated Cardiomyopathy (DCM) which is a key pathological feature, is a major cause of morbidity and mortality. To decipher the mechanism(s) underlying the depressed ventricular function in DMD patients, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. The hypothesis was tested by 2 Aims: (1) Investigate the ionotropic responses to isoproterenol and elevated [Ca2]out; (2) Investigate the SR Ca2 handling machinery in DMD cardiomyocytes. [Ca2]i transients and contractions were recorded from control and DMD cardiomyocytes, using the IonOptix Calcium and Contractility system. While in healthy cardiomyocytes isoproterenol caused a concentration-dependent positive inotropic effect, DMD iPSC-CMs displayed a blunted inotropic response. To determine whether the blunted response was due to dysfunctional β-adrenergic cascade or impaired downstream elements mediating common positive inotropic interventions, we investigated the effect of elevated [Ca2]out. Like isoproterenol, in healthy iPSC-CMs, elevated [Ca2]out caused positive inotropic and lusitropic effects, while DMD iPSC-CMs were unresponsive. Next, we tested the functionality of the sarcoplasmic reticulum (SR, the common downstream denominator mediating inotropic interventions) by measuring caffeine-induced Ca2 release. In healthy iPSC-CMs caffeine caused an abrupt increase in [Ca2]i followed by a gradual decline in [Ca2]i level. In contrast, DMD iPSC-CMs exhibited a reduced caffeine-induced Ca2 signal amplitude and recovery time. In support of impaired SR-Ca2 discharge capacity in DMD cardiomyocytes, ryanodine (RyR blocker) and cyclopiazonic acid (CPA, SERCA inhibitor) caused a smaller negative inotropic effect in DMD, compared to healthy cardiomyocytes. Our findings demonstrate that DMD iPSC-CMs exhibit blunted β-adrenergic inotropic response likely caused by depleted SR Ca2 stores.
DCM, a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in decreased ventricular function. Titin (TTN) - a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that iPSC-CMs generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 2 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions. iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CMs showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CMs exhibited diminished inotropic and lusitropic responses to -adrenergic stimulation with isoproterenol, increased [Ca2]out and angiotensin-II. Additionally, mutated iPSC-CMs displayed prolonged recovery in response to caffeine. These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.