RESUMO
Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1wt/V65MCS mice, with IC50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2wt/A478V mice were less sensitive to ZOL inhibition, showing an IC50 of ~500 nM and a slope of ~0.3. In homozygous SUR2A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 µM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.
Assuntos
Músculo Esquelético , Ácido Zoledrônico , Animais , Camundongos , Trifosfato de Adenosina , Modelos Animais de Doenças , Glibureto/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ácido Zoledrônico/farmacologia , Canais KATP/efeitos dos fármacos , Canais KATP/metabolismo , Receptores de Sulfonilureias/efeitos dos fármacos , Receptores de Sulfonilureias/metabolismoRESUMO
(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10-5 M in SUR2wt/AV and 8.6 ± 0.4 × 10-6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] "knock-in" mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.
Assuntos
Cardiomegalia/genética , Cardiomegalia/metabolismo , Hipertricose/genética , Hipertricose/metabolismo , Complexo Mediador/metabolismo , Músculo Esquelético/metabolismo , Mutação/genética , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Animais , Atrofia/patologia , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Humanos , Camundongos , FenótipoRESUMO
Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.
Assuntos
Cardiomegalia , Hipertricose , Osteocondrodisplasias , Receptores de Sulfonilureias/genética , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Células Cultivadas , Feminino , Humanos , Hipertricose/genética , Hipertricose/metabolismo , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Miócitos Cardíacos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismoRESUMO
Cantù syndrome (CS) arises from mutations in ABCC9 and KCNJ8 genes that lead to gain of function (GOF) of ATP-sensitive potassium (KATP) channels containing SUR2A and Kir6.1 subunits, respectively, of KATP channels. Pathological consequences of CS have been reported for cardiac and smooth muscle cells but consequences in skeletal muscle are unknown. Children with CS show muscle hypotonia and adult manifest fatigability. We analyzed muscle properties of Kir6.1[V65M] CS mice, by measurements of forelimb strength and ultrasonography of hind-limb muscles, as well as assessing KATP channel properties in native Flexor digitorum brevis (FDB) and Soleus (SOL) fibers by the patch-clamp technique in parallel with histopathological, immunohistochemical and Polymerase Chain Reaction (PCR) analysis. Forelimb strength was lower in Kir6.1wt/VM mice than in WT mice. Also, a significant enhancement of echodensity was observed in hind-limb muscles of Kir6.1wt/VM mice relative to WT, suggesting the presence of fibrous tissue. There was a higher KATP channel current amplitude in Kir6.1wt/VM FDB fibers relative to WT and a reduced response to glibenclamide. The IC50 of glibenclamide to block KATP channels in FDB fibers was 1.3 ± 0.2 × 10-7 M in WT and 1.2 ± 0.1 × 10-6 M in Kir6.1wt/VM mice, respectively; and it was 1.2 ± 0.4 × 10-7 M in SOL WT fibers but not measurable in Kir6.1wt/VM fibers. The sensitivity of the KATP channel to MgATP was not modified in Kir6.1wt/VM fibers. Histopathological/immunohistochemical analysis of SOL revealed degeneration plus regressive-necrotic lesions with regeneration, and up-regulation of Atrogin-1, MuRF1, and BNIP3 mRNA/proteins in Kir6.1wt/VM mice. Kir6.1wt/VM mutation in skeletal muscle leads to changes of the KATP channel response to glibenclamide in FDB and SOL fibers, and it is associated with histopathological and gene expression changes in slow-twitch muscle, suggesting marked atrophy and autophagy.
RESUMO
Cantu syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) KATP channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knockin mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved by genetic downregulation of KATP channel activity specifically in VSM, and by chronic administration of the clinically used KATP channel inhibitor, glibenclamide. These findings demonstrate that VSM KATP channel GoF underlies CS cardiac enlargement and that CS-associated abnormalities are reversible, and provide evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS.
Assuntos
Cardiomegalia , Glibureto/farmacologia , Hipertricose , Canais KATP , Osteocondrodisplasias , Receptores de Sulfonilureias , Animais , Cardiomegalia/tratamento farmacológico , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Humanos , Hipertricose/tratamento farmacológico , Hipertricose/genética , Hipertricose/metabolismo , Hipertricose/patologia , Canais KATP/genética , Canais KATP/metabolismo , Camundongos , Camundongos Transgênicos , Osteocondrodisplasias/tratamento farmacológico , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismoRESUMO
Dramatic cardiomegaly arising from gain-of-function (GoF) mutations in the ATP-sensitive potassium (KATP) channels genes, ABCC9 and KCNJ8, is a characteristic feature of Cantú syndrome (CS). How potassium channel over-activity results in cardiac hypertrophy, as well as the long-term consequences of cardiovascular remodeling in CS, is unknown. Using genome-edited mouse models of CS, we therefore sought to dissect the pathophysiological mechanisms linking KATP channel GoF to cardiac remodeling. We demonstrate that chronic reduction of systemic vascular resistance in CS is accompanied by elevated renin-angiotensin signaling, which drives cardiac enlargement and blood volume expansion. Cardiac enlargement in CS results in elevation of basal cardiac output, which is preserved in aging. However, the cardiac remodeling includes altered gene expression patterns that are associated with pathological hypertrophy and are accompanied by decreased exercise tolerance, suggestive of reduced cardiac reserve. Our results identify a high-output cardiac hypertrophy phenotype in CS which is etiologically and mechanistically distinct from other myocardial hypertrophies, and which exhibits key features of high-output heart failure (HOHF). We propose that CS is a genetically-defined HOHF disorder and that decreased vascular smooth muscle excitability is a novel mechanism for HOHF pathogenesis.
Assuntos
Mutação com Ganho de Função , Canais KATP , Camundongos , Animais , Canais KATP/genética , Mutação com Ganho de Função/genética , Remodelação Ventricular , Receptores de Sulfonilureias/genética , Cardiomegalia/genética , Trifosfato de AdenosinaRESUMO
Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1 G > A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.
Assuntos
Trifosfato de Adenosina/metabolismo , Canalopatias/metabolismo , Predisposição Genética para Doença/genética , Deficiência Intelectual/metabolismo , Doenças Musculares/metabolismo , Mutação , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Linhagem Celular , Criança , Modelos Animais de Doenças , Fácies , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Coração , Cardiopatias/genética , Cardiopatias/metabolismo , Homozigoto , Humanos , Hipertricose/genética , Hipertricose/metabolismo , Deficiência Intelectual/parasitologia , Masculino , Complexo Mediador/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Doenças Musculares/genética , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/fisiopatologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Linhagem , Fenótipo , Rubídio , Sequenciamento Completo do Genoma , Adulto Jovem , Peixe-ZebraRESUMO
We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.
Assuntos
Oxirredutases do Álcool/metabolismo , Saccharomyces cerevisiae/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Aldeído Redutase , Aldo-Ceto Redutases , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Mutação , Fenótipo , Saccharomyces cerevisiae/genéticaRESUMO
Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1-dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.
Assuntos
Cardiomegalia/fisiopatologia , Ventrículos do Coração/fisiopatologia , Hipertricose/fisiopatologia , Canais KATP/metabolismo , Osteocondrodisplasias/fisiopatologia , Receptores de Sulfonilureias/metabolismo , Animais , Cardiomegalia/diagnóstico , Cardiomegalia/genética , Modelos Animais de Doenças , Ecocardiografia , Acoplamento Excitação-Contração/genética , Feminino , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Ventrículos do Coração/diagnóstico por imagem , Humanos , Hipertricose/diagnóstico , Hipertricose/genética , Canais KATP/genética , Masculino , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiopatologia , Miócitos Cardíacos , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/genética , Técnicas de Patch-Clamp , Receptores de Sulfonilureias/genética , Vasodilatação/genética , Remodelação Ventricular/genéticaRESUMO
Human aldose reductase (AKR1B1) has been implicated as a factor in the pathogenesis of diabetic complications. However, little is known about the physiological role of this enzyme or of related aldo-keto reductases in human tissues. In mammalian systems, a gene knock out approach is often employed as an experimental strategy to probe for gene function. However, in the murine system, phenotypic characterization of an aldose reductase (AKR1B3) knock out is likely to be complicated due to functional compensation by redundant AKRs including AKRs 1A (aldehyde reductase), 1B7 (FR-1) and 1B8 (MVDP). As an alternate strategy, we are examining the budding yeast Saccharomyces cerevisiae as a model system for a functional genomics study of AKRs. A distinct advantage of this system centers on the ability to readily ablate multiple targeted genes in a single strain. In addition to providing insights into functional redundancy, this system allows us to use a genetic approach to study possible effector pathways associated with one or more individual genes. Yeast open reading frames (ORFs) encoding AKRs with functional similarity to human aldose reductase (AKR1B1) were identified by BLAST analysis and were functionally validated by studies of recombinant proteins. By ablating three of the yeast AKR genes most functionally similar to AKR1B1, we have created a unique strain of S. cerevisiae that shows enhanced sensitivity to stress. Ongoing studies with oligonucleotide arrays show that the triple null strain has an altered transcription profile consistent with an enhanced stress response in comparison with the parental strain. These data indicate that AKR-null strains may provide new insights into signaling mechanisms involving this family of proteins.
Assuntos
Oxirredutases do Álcool/metabolismo , Estresse Oxidativo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Aldeído Redutase , Aldo-Ceto Redutases , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: KATP channels, assembled from pore-forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina-like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. METHODS AND RESULTS: We generated transgenic mice expressing wild-type (WT), ATP-insensitive Kir6.1 [Gly343Asp] (GD), and ATP-insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD-QR) subunits, under Cre-recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter-driven tamoxifen-inducible Cre-recombinase (SMMHC-Cre-ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD-QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant-negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD-QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil-activated conductance were elevated in GD but not in WT myocytes. CONCLUSIONS: KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.
Assuntos
Pressão Sanguínea , Hipotensão/metabolismo , Canais KATP/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Relação Dose-Resposta a Droga , Predisposição Genética para Doença , Hipotensão/genética , Hipotensão/fisiopatologia , Canais KATP/genética , Potenciais da Membrana , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Mutação , Fenótipo , Potássio/metabolismo , Vasoconstrição , Vasodilatação , Vasodilatadores/farmacologiaRESUMO
Aldose reductase (AKR1B1) is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway. Accelerated glucose metabolism through this pathway has been implicated in diabetic cataract and retinopathy. Some human tissues contain AKR1B1 as well as AKR1B10, a closely related member of the aldo-keto reductase gene superfamily. This opens the possibility that AKR1B10 may also contribute to diabetic complications. The goal of the current study was to characterize the expression profiles of AKR1B1 and AKR1B10 in the human eye. Using quantitative reverse transcriptase-PCR and immunohistochemical staining, we observed expression of both AKR genes in cornea, iris, ciliary body, lens, and retina. Expression of AKR1B1 was the highest in lens and retina, whereas AKR1B10 was the highest in cornea. Lenses from transgenic mice designed for overexpression of AKR1B10 were not significantly different from nontransgenic controls, although a significant number developed a focal defect in the anterior lens epithelium following 6 months of experimentally induced diabetes. However, lenses from AKR1B10 mice remained largely transparent following longterm diabetes. These results indicate that AKR1B1 and AKR1B10 may have different functional properties in the lens and suggest that AKR1B10 does not contribute to the pathogenesis of diabetic cataract in humans.