Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy

Barefield, David; Puckelwartz, Megan J.; Kim, Ellis Y.; Wilsbacher, Lisa D.; Vo, Andy H.; Waters, Emily A.; Earley, Judy U.; Hadhazy, Michele; Dellefave‐Castillo, Lisa; Pesce, Lorenzo L.; McNally, Elizabeth M.

doi:10.1161/circulationaha.117.028585

Cited by 35 publications

(47 citation statements)

References 51 publications

(50 reference statements)

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“…Cardiomyocyte differentiation and replating. Reprogrammed iPSCs were differentiated to cardiomyocytes according to previously described protocol (58), with the starting densities between 100,000-320,000 cells/ cm 2 . Differentiated cardiomyocytes were replated 5-7 days after beating was observed to 12 mm No.…”

Section: Methodsmentioning

confidence: 99%

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

Kim

Barefield

et al. 2019

JCI Insight

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Section: Methodsmentioning

confidence: 99%

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

Kim

Barefield

et al. 2019

JCI Insight

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“…Transthoracic echocardiography was performed prior to sacrifice at the indicated time points, as previously described ( 26 , 27 ). Briefly, the rats were anesthetized with 2.0% isoflurane via inhalation.…”

Section: Methodsmentioning

confidence: 99%

O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

Huang

Yuan

et al. 2018

Int J Mol Med

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The O-linked β-N-acetylglucosamine (O-GlcNAc) modification and autophagy are associated with diabetic myocardial injury, however, the molecular mechanisms between the two processes remain to be fully elucidated. The purpose of the present study was to elucidate the molecular regulation of autophagy by O-GlcNAc-modified synaptosomal-associated protein 29 (SNAP29) in diabetic myocardial injury. A rat model of type I diabetes was established via intraperitoneal injection of streptozotocin (STZ; 55 mg/kg). Significant increases in the O-GlcNAc modification and accumulation of the autophagy markers microtubule-associated protein 1 light chain 3α II/I and P62, which suggest that autophagic flux is inhibited, were observed in rats 8 weeks following STZ induction. Subsequently, the selective O-GlcNAcase inhibitor, thiamet G, increased the level of O-GlcNAc modification, which further disrupted autophagic flux; deteriorated cardiac diastolic function, as indicated by an increased left ventricular filling peak velocity/atrial contraction flow peak velocity ratio shown by echocardiography; and exacerbated myocardial abnormalities, as characterized by cardiomyocyte disorganization and fat and interstitial fibrosis accumulation. By contrast, 6-diazo-5-oxo-L-norleucine, an inhibitor of glucosamine fructose-6-phosphate aminotransferase isomerizing 1, acted as an O-GlcNAc antagonist and reduced the level of O-GlcNAc modification, which maintained autophagic flux and improved cardiac diastolic function. In vitro, high glucose (25 mM) was used to stimulate primary neonatal rat cardiomyocytes (NRCMs). Consistent with the myocardium of diabetic rats, it was also shown in the NRCMs that O-GlcNAc modification of SNAP29 negatively regulated autophagic flux. The application of the short hairpin RNA interference lysosome-associated membrane protein (LAMP2) and the autophagy inhibitor 3-methyladenine demonstrated that high glucose inhibited autophagy-mediated degradation rather than affected the initial stage of autophagy. Finally, co-immunoprecipitation was used to determine the role of the O-GlcNAc-modified substrate protein SNAP29, which acted as an SNAP29-syntaxin-17 (STX17)-vesicle-associated membrane protein 8 (VAMP8) complex during disease progression. The present study is the first, to the best of our knowledge, to demonstrate that SNAP29 is an O-GlcNAc substrate and that an increase in O-GlcNAc-modified SNAP29 inhibits SNAP29-STX17-VAMP8 complex formation, thereby inhibiting the degradation of autophagy and exacerbating myocardial injury in type I diabetic rats.

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“…In vivo cardiac function was assessed by echocardiography and was conducted under anesthesia (0.8 liters/min, 1% vaporized isoflurane in 100% O 2 ) as described previously (Barefield et al, 2017). Echocardiography was performed using a Visual Sonics Vevo 2100 imaging system with a MS550D 22-55 MHz solid-state transducer (FujiFilm).…”

Section: Echocardiographymentioning

confidence: 99%

A gene-edited mouse model of limb-girdle muscular dystrophy 2C for testing exon skipping

Demonbreun

Wyatt

Fallon

et al. 2019

Disease Models &Amp; Mechanisms

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Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan (SGCG) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg. These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.

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Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy

Cited by 35 publications

References 51 publications

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

O-GlcNAc-modified SNAP29 inhibits autophagy-mediated degradation via the disturbed SNAP29-STX17-VAMP8 complex and exacerbates myocardial injury in type I diabetic rats

A gene-edited mouse model of limb-girdle muscular dystrophy 2C for testing exon skipping

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