Opravy

Basic research project

• The advance in regenerative medicine, especially in the field of cardiology, is due, among other factors, to obtaining induced pluripotency stem cells (iPS), which makes it possible to study diseases such as anthracycline-induced cardiotoxicity (CIA ).

• A class of more effective chemotherapeutics used as a therapeutic measure to treat neoplasms are anthracyclines.

• Its antineoplastic effects are considered to involve DNA strand intercalation, stabilization of the topoisomerase IIα cleavage complex and imbalance in the redox system, generating an accumulation of toxic and reactive oxygen radicals, inducing apoptosis.

• Despite the benefit generated in the treatment of cancer, its clinical use is hampered by the risk of cardiotoxicity and it is still not known what predisposes the individual to develop such a side effect.

• Due to cardiotoxic side effects, there is considerable effort to develop cardioprotective drugs, but so far dexrazoxane (DXZ) is the only drug approved for the prevention of ASD.

• Its probable cardioprotective effect is related to the removal of intracellular iron, preventing the production of the anthracycline-iron complex and subsequent production of toxic radicals, in addition to antagonizing the association of anthracyclines with topoisomerase IIβ, an isoform present in non-proliferating cells, such as cardiomyocytes.

• It is speculated that, in this way, this cardioprotectant reduces the damage induced by anthracyclines to the DNA of cardiomyocytes.

• Thus, the use of patient-specific cardiomyocytes differentiated from iPS (iPS-CM) represents a promising platform to perform pharmacological tests and study the mechanisms of cardiotoxicity induction.

• The protocols that most efficiently mimic cardiomyogenesis use a base medium composed of RPMI 1640 plus B27, a supplement containing 21 components, originally developed for the culture of hippocampal neurons and recently adapted for differentiation into iPS-CM.

• As most of the B27 component molecules are antioxidants, it is possible that their addition causes a cardioprotective effect on the iPS-CM generated in vitro, possibly altering their maturation and the observed results.

• Thus, the objective of this work is to establish a protocol that presents high efficiency of cardiac differentiation (CO), but that does not make use of the supplement B27.

• In this work, we used two strains of iPS: one obtained from a patient treated with cumulative doses of anthracyclines (240 mg/m2 doxorubicin and 300 mg/m2 epirubicin) and who developed heart failure (ejection fraction = 32.9%) after 7 years of treatment, and another from a healthy donor (control).

• Patient-specific iPS underwent CD using two different protocols: P1 – using B27 (RPMI 1640 and supplement B27 1X serum free or B27 minus insulin), and P2 – CDM3 (RPMI 1640, 213 μg/mL L-ascorbic acid 2 -phosphate, 500 μg/mL bovine serum albumin).

• Starting from the eighth day, the spontaneous contractility of the iPS-CM was evaluated by optical phase contrast microscopy.

• After thirty days, we evaluated by flow cytometry the efficiency of the two protocols by cardiac troponin T labeling and obtained efficiency >90%.

• With this, we were able to obtain iPS-CM through an efficient and reproducible protocol, using a medium without B27.