{"id":{"repo_id":"milano","oai_identifier":"oai:air.unimi.it:2434/1023131"},"canonical_url":"https://search.dev.ndltd.org/etd/milano/oai:air.unimi.it:2434/1023131","repository":{"repo_id":"milano","name":"Università degli Studi di Milano","base_url":"https://air.unimi.it/oai/request"},"display":{"title":"MES-DERIVED CARDIOMYOCYTES AS CELLULAR MODEL TO STUDY CARDIAC DISEASES: ALTERATIONS IN STRN AND GNB5 GENES","abstract":"Striatin (Strn), a scaffold protein expressed in many tissues, among which in cardiomyocytes (CMs), whose altered expression has been found in various cardiac diseases. Here we studied the role(s) of cardiac STRN by comparing the electrophysiological properties of CMs, generated from STRN-KO and isogenic WT mouse embryonic stem cell (mESC) lines. 10-12-day old beating mESC-CMs were analyzed by Patch-clamp, motion video tracking, Ca2+ dynamics and immunofluorescence analysis. STRN-KO cells have a higher spontaneous beating rate and faster action potential depolarization (dV/dt) than WT, correlated with a larger fast INa conductance. Since in HEK cells downregulation of STRN was reported to destabilize microtubules and increase INa, immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in KO CMs. Motion video tracking analysis highlighted an altered contraction in STRN-KO CMs, and this was associated with a global increase in intracellular Ca2+. This was likely due to an increased late Na+ current (INaL) and a reduction of Ca2+extrusion through the Na+/Ca2+ exchanger (NCX). Other currents such as ICaL, If and IKr were not altered in STRN-KO cells. Incubation of STRN-KO CMs with the microtubule stabilizer Taxol, induced a reduction of INa conductance toward WT levels. In conclusion, loss of STRN alters CMs electrical and contractile profile and affects cell functionality by a disarrangement of multi-protein complexes leading to the impairment of microtubules dynamics and trafficking of Na+ channels.","abstract_html":"Striatin (Strn), a scaffold protein expressed in many tissues, among which in cardiomyocytes (CMs), whose altered expression has been found in various cardiac diseases. Here we studied the role(s) of cardiac STRN by comparing the electrophysiological properties of CMs, generated from STRN-KO and isogenic WT mouse embryonic stem cell (mESC) lines. 10-12-day old beating mESC-CMs were analyzed by Patch-clamp, motion video tracking, Ca2+ dynamics and immunofluorescence analysis. STRN-KO cells have a higher spontaneous beating rate and faster action potential depolarization (dV/dt) than WT, correlated with a larger fast INa conductance. Since in HEK cells downregulation of STRN was reported to destabilize microtubules and increase INa, immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in KO CMs. Motion video tracking analysis highlighted an altered contraction in STRN-KO CMs, and this was associated with a global increase in intracellular Ca2+. This was likely due to an increased late Na+ current (INaL) and a reduction of Ca2+extrusion through the Na+/Ca2+ exchanger (NCX). Other currents such as ICaL, If and IKr were not altered in STRN-KO cells. Incubation of STRN-KO CMs with the microtubule stabilizer Taxol, induced a reduction of INa conductance toward WT levels. In conclusion, loss of STRN alters CMs electrical and contractile profile and affects cell functionality by a disarrangement of multi-protein complexes leading to the impairment of microtubules dynamics and trafficking of Na+ channels.","abstract_has_math":false,"creators":["COSPITO, ALESSANDRO"],"institution":"Università degli Studi di Milano","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["tutor: A. Barbuti ; phd school coordinator: R. Mantovani","A. Cospito","BARBUTI, ANDREA FRANCESCO","MANTOVANI, ROBERTO"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-01-24","date_published":"2024-01-24","updated_at":"2026-07-27T20:18:46Z","subjects":["Striatin","sodium current","arrhythmia","microtubule","gnb5","IDDCA"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dx.doi.org/10.13130/cospito-alessandro_phd2024-01-24","10.13130/cospito-alessandro_phd2024-01-24"],"render_values":[{"text":"http://dx.doi.org/10.13130/cospito-alessandro_phd2024-01-24","href":"http://dx.doi.org/10.13130/cospito-alessandro_phd2024-01-24","code":true},{"text":"10.13130/cospito-alessandro_phd2024-01-24","href":"https://doi.org/10.13130/cospito-alessandro_phd2024-01-24","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2434/1023131","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["tutor: A. Barbuti ; phd school coordinator: R. Mantovani","A. 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Here we studied the role(s) of cardiac STRN by comparing the electrophysiological properties of CMs, generated from STRN-KO and isogenic WT mouse embryonic stem cell (mESC) lines. 10-12-day old beating mESC-CMs were analyzed by Patch-clamp, motion video tracking, Ca2+ dynamics and immunofluorescence analysis. STRN-KO cells have a higher spontaneous beating rate and faster action potential depolarization (dV/dt) than WT, correlated with a larger fast INa conductance. Since in HEK cells downregulation of STRN was reported to destabilize microtubules and increase INa, immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in KO CMs. Motion video tracking analysis highlighted an altered contraction in STRN-KO CMs, and this was associated with a global increase in intracellular Ca2+. This was likely due to an increased late Na+ current (INaL) and a reduction of Ca2+extrusion through the Na+/Ca2+ exchanger (NCX). Other currents such as ICaL, If and IKr were not altered in STRN-KO cells. Incubation of STRN-KO CMs with the microtubule stabilizer Taxol, induced a reduction of INa conductance toward WT levels. In conclusion, loss of STRN alters CMs electrical and contractile profile and affects cell functionality by a disarrangement of multi-protein complexes leading to the impairment of microtubules dynamics and trafficking of Na+ channels.","IDDCA is an autosomal recessive syndrome, characterized by global developmental delay and cardiac abnormalities, most commonly sick sinus syndrome (SSS), with sinus node bradycardia. The disease is characterized by loss of function mutations in GNB5, that encode for the G protein subunit β5. To study the GNB5 role in development and function of sinus node cells (SAN), we used two lines of mouse embryonic stem cells (mESC) one wild-type and one GNB5 knock-out. mESC were differentiated into cardiomyocytes using a well-established hanging drops method and selection of CD166+ SAN precursors sorted at day 8 of differentiation. The proportion of CD166+ precursors was similar in both lines, and we did not observe any significant difference in SAN developmental markers such as Shox2 and Tbx18, suggesting that GNB5 is not required for SAN development. We found Hcn4 more expressed in KO cells while TnnI3 and Myh7 were less expressed in KO than in WT cells. Patch clamp analysis revealed that on average KO CD166+ SAN-like cells showed a higher intrinsic beating rate (2.5±0.1 Hz) than WT cells (1.5±0.04 Hz) and displayed an irregular pattern with pauses. We treated cells with isoproterenol (1μM) and carbachol (100nM), to mimic the stimulation by the autonomous nervous system. We found that both lines responded similarly to isoproterenol: carbachol induced a mild effect on WT cells while stopped the spontaneous activity of KO cell. Further analysis is necessary to elucidate the detailed molecular pathways beneath the cardiac manifestation caused by GNB5 dysfunction."]},{"key":"dc:title","label":"Title","values":["MES-DERIVED CARDIOMYOCYTES AS CELLULAR MODEL TO STUDY CARDIAC DISEASES: ALTERATIONS IN STRN AND GNB5 GENES"]}]}],"canonical_facts":{"dc:contributor":["tutor: A. Barbuti ; phd school coordinator: R. Mantovani","A. Cospito","BARBUTI, ANDREA FRANCESCO","MANTOVANI, ROBERTO"],"dc:creator":["COSPITO, ALESSANDRO"],"dc:date":["2024-01-24"],"dc:description":["Striatin (Strn), a scaffold protein expressed in many tissues, among which in cardiomyocytes (CMs), whose altered expression has been found in various cardiac diseases. Here we studied the role(s) of cardiac STRN by comparing the electrophysiological properties of CMs, generated from STRN-KO and isogenic WT mouse embryonic stem cell (mESC) lines. 10-12-day old beating mESC-CMs were analyzed by Patch-clamp, motion video tracking, Ca2+ dynamics and immunofluorescence analysis. STRN-KO cells have a higher spontaneous beating rate and faster action potential depolarization (dV/dt) than WT, correlated with a larger fast INa conductance. Since in HEK cells downregulation of STRN was reported to destabilize microtubules and increase INa, immunofluorescence analysis confirmed the higher Na+ channel expression and a more dynamic microtubule network in KO CMs. Motion video tracking analysis highlighted an altered contraction in STRN-KO CMs, and this was associated with a global increase in intracellular Ca2+. This was likely due to an increased late Na+ current (INaL) and a reduction of Ca2+extrusion through the Na+/Ca2+ exchanger (NCX). Other currents such as ICaL, If and IKr were not altered in STRN-KO cells. Incubation of STRN-KO CMs with the microtubule stabilizer Taxol, induced a reduction of INa conductance toward WT levels. In conclusion, loss of STRN alters CMs electrical and contractile profile and affects cell functionality by a disarrangement of multi-protein complexes leading to the impairment of microtubules dynamics and trafficking of Na+ channels.","IDDCA is an autosomal recessive syndrome, characterized by global developmental delay and cardiac abnormalities, most commonly sick sinus syndrome (SSS), with sinus node bradycardia. The disease is characterized by loss of function mutations in GNB5, that encode for the G protein subunit β5. To study the GNB5 role in development and function of sinus node cells (SAN), we used two lines of mouse embryonic stem cells (mESC) one wild-type and one GNB5 knock-out. mESC were differentiated into cardiomyocytes using a well-established hanging drops method and selection of CD166+ SAN precursors sorted at day 8 of differentiation. The proportion of CD166+ precursors was similar in both lines, and we did not observe any significant difference in SAN developmental markers such as Shox2 and Tbx18, suggesting that GNB5 is not required for SAN development. We found Hcn4 more expressed in KO cells while TnnI3 and Myh7 were less expressed in KO than in WT cells. Patch clamp analysis revealed that on average KO CD166+ SAN-like cells showed a higher intrinsic beating rate (2.5±0.1 Hz) than WT cells (1.5±0.04 Hz) and displayed an irregular pattern with pauses. We treated cells with isoproterenol (1μM) and carbachol (100nM), to mimic the stimulation by the autonomous nervous system. We found that both lines responded similarly to isoproterenol: carbachol induced a mild effect on WT cells while stopped the spontaneous activity of KO cell. Further analysis is necessary to elucidate the detailed molecular pathways beneath the cardiac manifestation caused by GNB5 dysfunction."],"dc:identifier":["https://hdl.handle.net/2434/1023131","http://dx.doi.org/10.13130/cospito-alessandro_phd2024-01-24","10.13130/cospito-alessandro_phd2024-01-24"],"dc:language":["eng"],"dc:publisher":["Università degli Studi di Milano"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:subject":["Striatin","sodium current","arrhythmia","microtubule","gnb5","IDDCA"],"dc:title":["MES-DERIVED CARDIOMYOCYTES AS CELLULAR MODEL TO STUDY CARDIAC DISEASES: ALTERATIONS IN STRN AND GNB5 GENES"],"dc:type":["info:eu-repo/semantics/doctoralThesis"]},"updated_at":"2026-07-27T20:18:46Z"}