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Vascular Electrophysiology and Pathogenic Consequences of Cardiovascular KATP Channel Mutations
Vascular Electrophysiology and Pathogenic Consequences of Cardiovascular KATP Channel Mutations
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211151439
- ISBN
- 9798382551715
- DDC
- 574
- 서명/저자
- Vascular Electrophysiology and Pathogenic Consequences of Cardiovascular KATP Channel Mutations
- 발행사항
- [Sl] : Washington University in St Louis, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 131 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-11, Section: B.
- 주기사항
- Advisor: Nichols, Colin.
- 학위논문주기
- Thesis (Ph.D.)--Washington University in St. Louis, 2024.
- 초록/해제
- 요약The complex cardiovascular disorder Cantu Syndrome (CS) arises from gain-of-function (GoF) mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of cardiovascular ATP-sensitive potassium (KATP) channels, respectively. CS involves an array of cardiovascular pathologies, including cardiac hypertrophy and hypercontractility, low systemic vascular resistance, and excessively compliant, dilated, and tortuous vessels. Together, the latter features exemplify the hypomyotonic and hyperelastic components of CS vasculopathy. It was recently established that CS vasculopathy drives the associated cardiac pathologies, which are observed even in the absence of cardiac KATP GoF. In this thesis, I carried out experiments to determine the molecular mechanisms by which KATP function is altered by several pathogenic CS mutations in distinct structural domains of the TMD2 domain of SUR2: Y985S (YS), G989E (GE), M1060I (MI), R1154Q (RQ), and R1154W (RW). I showed that the cluster of YS/GE/MI substitutions, as well as RQ and RW, augmented Mg2+-nucleotide activation of the KATP channel. I also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. RQ and RW, which are the two most common CS-associated mutations, significantly decreased glibenclamide potency. CRISPR/Cas9 genome engineering was used to introduce SUR2[R1150Q], the equivalent of human SUR2[R1154Q], to the mouse ABCC9 gene. As previously seen in mice carrying the CS-associated SUR2[A478V] and Kir6.1[V65M] mutations, both heterozygous and homozygous RQ animals exhibited enlarged hearts, elevated cardiac output, and hypotension, but, surprisingly, there was almost complete loss of SUR2-dependent KATP in homozygous RQ ventricles. The introduced mutation is located in a putative exon splicing enhancer site at the 3' end of exon 27. Sequencing of SUR2 cDNA from mouse tissues revealed not only the full-length ABCC9 transcript, but also a novel in-frame deletion of 93 bases (corresponding to the 31 amino acids encoded by exon 28), the latter being present in ~40% and ~90% of transcripts from hetero- and homozygous tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in non-functional channels. To determine whether this phenomenon is present in humans, I used RQ and RW CS patient-derived human induced pluripotent stem cells (hiPSCs) to generate novel hiPSC-cardiomyocyte (hiPSC-CM) and hiPSC-vascular smooth muscle cell (hiPSC-VSMC) models for CS. hiPSC-CMs and hiPSC-VSMCs carrying the RQ mutation showed only full-length ABCC9 transcripts. This was consistent with my analysis of ABCC9 RNA from primary tissues that had been surgically removed from an RQ patient. Together, these data suggest that aberrant ABCC9 splicing is specific to the murine model. I then carried out the first electrophysiological analysis of control hiPSC-VSMCs, demonstrating that membrane potential and functional expression of voltage-gated K+ (Kv) and L-type Ca2+ currents (LTCCs) are very similar to those I measured in native mouse arterial VSMCs, validating hiPSC-VSMCs as an electrical model of human VSMCs. Functional KATP expression in hiPSC-VSMCs was also consistent with previous studies on native mouse VSMCs, and pinacidil sensitivity demonstrated SUR2 expression. However, both basal and pinacidil-activated KATP currents were considerably larger in RQ and RW hiPSC-VSMCs. Consistent with lack of cell-autonomous modulation of Kv and LTCCs that I demonstrated in native arterial VSMCs isolated from CS mice, KATP GoF in hiPSC-VSMCs resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Consistent with the hyperelastic component of CS, increased compliance and dilation was observed in isolated aortae from CS mice, which was associated with increased elastin mRNA expression in these vessels. I then found increased elastin mRNA in CS hiPSC-VSMCs. These results show that increased elastogenesis is driven by genetic KATP overactivity in the context of CS vasculopathy, which is therefore a cell-autonomous consequence of membrane hyperpolarization.
- 일반주제명
- Cellular biology
- 일반주제명
- Pharmacology
- 일반주제명
- Physiology
- 일반주제명
- Molecular biology
- 키워드
- Cantu Syndrome
- 키워드
- KATP mutations
- 기타저자
- Washington University in St. Louis Biology & Biomedical Sciences (Molecular Cell Biology)
- 기본자료저록
- Dissertations Abstracts International. 85-11B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■020 ▼a9798382551715
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■1001 ▼aHanson, Alex Michael.▼0(orcid)0000-0002-4931-0590
■24510▼aVascular Electrophysiology and Pathogenic Consequences of Cardiovascular KATP Channel Mutations
■260 ▼a[Sl]▼bWashington University in St Louis▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a131 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-11, Section: B.
■500 ▼aAdvisor: Nichols, Colin.
■5021 ▼aThesis (Ph.D.)--Washington University in St. Louis, 2024.
■520 ▼aThe complex cardiovascular disorder Cantu Syndrome (CS) arises from gain-of-function (GoF) mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of cardiovascular ATP-sensitive potassium (KATP) channels, respectively. CS involves an array of cardiovascular pathologies, including cardiac hypertrophy and hypercontractility, low systemic vascular resistance, and excessively compliant, dilated, and tortuous vessels. Together, the latter features exemplify the hypomyotonic and hyperelastic components of CS vasculopathy. It was recently established that CS vasculopathy drives the associated cardiac pathologies, which are observed even in the absence of cardiac KATP GoF. In this thesis, I carried out experiments to determine the molecular mechanisms by which KATP function is altered by several pathogenic CS mutations in distinct structural domains of the TMD2 domain of SUR2: Y985S (YS), G989E (GE), M1060I (MI), R1154Q (RQ), and R1154W (RW). I showed that the cluster of YS/GE/MI substitutions, as well as RQ and RW, augmented Mg2+-nucleotide activation of the KATP channel. I also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. RQ and RW, which are the two most common CS-associated mutations, significantly decreased glibenclamide potency. CRISPR/Cas9 genome engineering was used to introduce SUR2[R1150Q], the equivalent of human SUR2[R1154Q], to the mouse ABCC9 gene. As previously seen in mice carrying the CS-associated SUR2[A478V] and Kir6.1[V65M] mutations, both heterozygous and homozygous RQ animals exhibited enlarged hearts, elevated cardiac output, and hypotension, but, surprisingly, there was almost complete loss of SUR2-dependent KATP in homozygous RQ ventricles. The introduced mutation is located in a putative exon splicing enhancer site at the 3' end of exon 27. Sequencing of SUR2 cDNA from mouse tissues revealed not only the full-length ABCC9 transcript, but also a novel in-frame deletion of 93 bases (corresponding to the 31 amino acids encoded by exon 28), the latter being present in ~40% and ~90% of transcripts from hetero- and homozygous tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in non-functional channels. To determine whether this phenomenon is present in humans, I used RQ and RW CS patient-derived human induced pluripotent stem cells (hiPSCs) to generate novel hiPSC-cardiomyocyte (hiPSC-CM) and hiPSC-vascular smooth muscle cell (hiPSC-VSMC) models for CS. hiPSC-CMs and hiPSC-VSMCs carrying the RQ mutation showed only full-length ABCC9 transcripts. This was consistent with my analysis of ABCC9 RNA from primary tissues that had been surgically removed from an RQ patient. Together, these data suggest that aberrant ABCC9 splicing is specific to the murine model. I then carried out the first electrophysiological analysis of control hiPSC-VSMCs, demonstrating that membrane potential and functional expression of voltage-gated K+ (Kv) and L-type Ca2+ currents (LTCCs) are very similar to those I measured in native mouse arterial VSMCs, validating hiPSC-VSMCs as an electrical model of human VSMCs. Functional KATP expression in hiPSC-VSMCs was also consistent with previous studies on native mouse VSMCs, and pinacidil sensitivity demonstrated SUR2 expression. However, both basal and pinacidil-activated KATP currents were considerably larger in RQ and RW hiPSC-VSMCs. Consistent with lack of cell-autonomous modulation of Kv and LTCCs that I demonstrated in native arterial VSMCs isolated from CS mice, KATP GoF in hiPSC-VSMCs resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Consistent with the hyperelastic component of CS, increased compliance and dilation was observed in isolated aortae from CS mice, which was associated with increased elastin mRNA expression in these vessels. I then found increased elastin mRNA in CS hiPSC-VSMCs. These results show that increased elastogenesis is driven by genetic KATP overactivity in the context of CS vasculopathy, which is therefore a cell-autonomous consequence of membrane hyperpolarization.
■590 ▼aSchool code: 0252.
■650 4▼aCellular biology
■650 4▼aPharmacology
■650 4▼aPhysiology
■650 4▼aMolecular biology
■653 ▼aElectrophysiology
■653 ▼aCantu Syndrome
■653 ▼aKATP mutations
■690 ▼a0379
■690 ▼a0419
■690 ▼a0719
■690 ▼a0307
■71020▼aWashington University in St. Louis▼bBiology & Biomedical Sciences (Molecular Cell Biology).
■7730 ▼tDissertations Abstracts International▼g85-11B.
■790 ▼a0252
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17161746▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
