Mouse embryonic stem cells (mESc) and human induced pluripotent stem cells (hiPSc) are commonly exploited in research for their ability to potentially differentiate into any cell subtype. Regarding cardiac differentiation, today's existing protocols are well advanced in recapitulating and promoting cardiomyocytes (CMs) differentiation, making CMs derived from mESc and hiPSc a good in vitro model for analyzing heart pathologies. In this thesis, these models have been used to characterize and better comprehend the functional output caused by genetic alterations. In the first part, mESc-derived cardiomyocytes help to elucidate the electrical dysregulation produced by the knock-out of Striatin (STRN), a scaffold protein with a role in organizing subcellular multiprotein complexes. STRN loss has been found in patients with dilated cardiomyopathy (DCM) and its mutation linked to DCM and ARVC in dogs. However, STRN physiological role in the cardiac context is not yet clear, for this reason the electrical characterization, by patch-clamp analysis, of mouse embryonic stem cells mESc-derived cardiomyocytes (mCMs) knock out for the STRN gene (STRN-KO) may help elucidating STRN role and its pathological implications. In this context, action potential (AP) analysis showed a higher rate and upstroke velocity of STRN-KO mCMs compared to its isogenic WT control. Accordingly, a significantly larger INa current density, responsible of the phase 0, was found in STRN-KO than in WT mCMs, while the other currents analyzed (If, ICaL and IKr) were similar. Literature data show that downregulation of STRN destabilize microtubules, and their disassembling increases sodium current in neonatal cardiomyocytes. Here is shown that treating STRN-KO mCMs with the microtubule stabilizer Taxol (10 μM) reverted INa density to values similar to WT cells. These data lead to the conclusion that STRN loss affects the electrical properties of mCMs likely acting on cytoskeleton stability and consequently on sodium channels trafficking and/or activity, creating a substrate more prone to develop arrhythmias. In the second part of the thesis, hiPSc-derived cardiomyocytes, obtained from a patient with atrial fibrillation (AF)carrying a heterozygous GOF point mutation in the PITX2 gene, are used to distinguish if this genetic condition predispose or AF. In GWAS studies, PITX2 is the genetic locus most associated with AF, it is often dysregulated in AF patients and its presence or absence correlates with electrical alterations. Moreover, PITX2 loss of function in zebrafish has been linked to perturbations of metabolic pathways forerunning AF-like phenotypes. APs recorded from small hiPSc-derived CMs (hpCMs) clusters reveals that PITX2-hpCMs are bradycardic, with a larger action potential amplitude and a shorter action potential duration than to three unrelated controls. Moreover, preliminary data obtained from atrial hiPSc-derived CMs, used to further elucidate the consequence of this mutation in the atrial background, showed no differences in ICaL and If densities; however, activation curve of If is negatively shifted with a higher inverse slope factor in PITX2 atrial hiPSc-derived CMs, implying a minor contribution of this current during the slow diastolic depolarization. Seahorse metabolic analysis indicates that PITX2-haCMs generates more ATP compared to isogenic control, and this difference is due to increased oxidative phosphorylation in mitochondria. In conclusion, although a more detailed characterization will be required, the PITX2 GOF mutation seems to enhance oxidative phosphorylation and a slower beating rate, altering not only the electrical activity but also the metabolism of cardiomyocytes. Overall, these in vitro models of cardiomyocytes were ideal to identify the functional outcome of genetic alteration, either to investigate the electrical mechanism behind arrhythmias or to characterize the role of a specific protein in the specific cardiac context.

HIPS AND MES AS A CELLULAR MODEL TO STUDY THE MECHANISMS BEHIND ARRHYTHMIAS / F. Giannetti ; tutor: A.F. Barbuti ; coordinator: C. Sforza. - : . Dipartimento di Bioscienze, 2022 Mar 18. ((34. ciclo, Anno Accademico 2021.

HIPS AND MES AS A CELLULAR MODEL TO STUDY THE MECHANISMS BEHIND ARRHYTHMIAS.

F. Giannetti
2022-03-18

Abstract

Mouse embryonic stem cells (mESc) and human induced pluripotent stem cells (hiPSc) are commonly exploited in research for their ability to potentially differentiate into any cell subtype. Regarding cardiac differentiation, today's existing protocols are well advanced in recapitulating and promoting cardiomyocytes (CMs) differentiation, making CMs derived from mESc and hiPSc a good in vitro model for analyzing heart pathologies. In this thesis, these models have been used to characterize and better comprehend the functional output caused by genetic alterations. In the first part, mESc-derived cardiomyocytes help to elucidate the electrical dysregulation produced by the knock-out of Striatin (STRN), a scaffold protein with a role in organizing subcellular multiprotein complexes. STRN loss has been found in patients with dilated cardiomyopathy (DCM) and its mutation linked to DCM and ARVC in dogs. However, STRN physiological role in the cardiac context is not yet clear, for this reason the electrical characterization, by patch-clamp analysis, of mouse embryonic stem cells mESc-derived cardiomyocytes (mCMs) knock out for the STRN gene (STRN-KO) may help elucidating STRN role and its pathological implications. In this context, action potential (AP) analysis showed a higher rate and upstroke velocity of STRN-KO mCMs compared to its isogenic WT control. Accordingly, a significantly larger INa current density, responsible of the phase 0, was found in STRN-KO than in WT mCMs, while the other currents analyzed (If, ICaL and IKr) were similar. Literature data show that downregulation of STRN destabilize microtubules, and their disassembling increases sodium current in neonatal cardiomyocytes. Here is shown that treating STRN-KO mCMs with the microtubule stabilizer Taxol (10 μM) reverted INa density to values similar to WT cells. These data lead to the conclusion that STRN loss affects the electrical properties of mCMs likely acting on cytoskeleton stability and consequently on sodium channels trafficking and/or activity, creating a substrate more prone to develop arrhythmias. In the second part of the thesis, hiPSc-derived cardiomyocytes, obtained from a patient with atrial fibrillation (AF)carrying a heterozygous GOF point mutation in the PITX2 gene, are used to distinguish if this genetic condition predispose or AF. In GWAS studies, PITX2 is the genetic locus most associated with AF, it is often dysregulated in AF patients and its presence or absence correlates with electrical alterations. Moreover, PITX2 loss of function in zebrafish has been linked to perturbations of metabolic pathways forerunning AF-like phenotypes. APs recorded from small hiPSc-derived CMs (hpCMs) clusters reveals that PITX2-hpCMs are bradycardic, with a larger action potential amplitude and a shorter action potential duration than to three unrelated controls. Moreover, preliminary data obtained from atrial hiPSc-derived CMs, used to further elucidate the consequence of this mutation in the atrial background, showed no differences in ICaL and If densities; however, activation curve of If is negatively shifted with a higher inverse slope factor in PITX2 atrial hiPSc-derived CMs, implying a minor contribution of this current during the slow diastolic depolarization. Seahorse metabolic analysis indicates that PITX2-haCMs generates more ATP compared to isogenic control, and this difference is due to increased oxidative phosphorylation in mitochondria. In conclusion, although a more detailed characterization will be required, the PITX2 GOF mutation seems to enhance oxidative phosphorylation and a slower beating rate, altering not only the electrical activity but also the metabolism of cardiomyocytes. Overall, these in vitro models of cardiomyocytes were ideal to identify the functional outcome of genetic alteration, either to investigate the electrical mechanism behind arrhythmias or to characterize the role of a specific protein in the specific cardiac context.
BARBUTI, ANDREA FRANCESCO
SFORZA, CHIARELLA
hiPSc; mESc; cardiomyocytes; electrophysiology; PITX2; Striatin; atrial differentiation
Settore BIO/09 - Fisiologia
HIPS AND MES AS A CELLULAR MODEL TO STUDY THE MECHANISMS BEHIND ARRHYTHMIAS / F. Giannetti ; tutor: A.F. Barbuti ; coordinator: C. Sforza. - : . Dipartimento di Bioscienze, 2022 Mar 18. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/903405
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