Biology subjects

Mack, D.

Publications and source records attributed to Mack, D..

5 recordsLinked to original sources

bioRxiv2026

Ryanodine receptor 1 ( RYR1 ) patient-derived muscle cells recapitulate disease phenotypes in 2D and 3D culture models

The Ryanodine Receptor 1 (RyR1) is the major Ca2+ release channel in skeletal muscle and plays a crucial role in excitation-contraction coupling. Pathogenic variants in RYR1 are the most common cause of congenital myopathy, for which there are no approved treatments. Patient-centric disease models may help to facilitate the design and screening of novel therapeutics in a human genomic context. In this report, we describe the differentiation of five dominant RYR1-related myopathy patient-derived induced pluripotent stem cell (iPSC) lines into muscle progenitor cells (MPCs), and subsequently into multinucleated myotubes in 2- and 3- Dimensional (D) culture models. In 2D, we show significantly reduced Ca2+ release in a patient line compared to a healthy control following stimulation with caffeine. In 3D engineered muscle tissues (EMTs), patient-relevant phenotypes including reduced twitch amplitude, delayed relaxation and altered force-frequency relationships were observed in a patient line compared to two healthy controls. We also show that the 2D cultures are a suitable platform for screening the efficacy and cellular toxicity of antisense oligonucleotide therapeutics. Together, these results suggest that iPSC-derived skeletal muscle cultures are useful models for understanding the pathobiology of RYR1-related myopathies and as a testbed for emerging treatments.

Cell Biology↗

bioRxiv2023

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration

RationaleOver 200 mutations in the sarcomeric protein {beta}-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. ObjectiveThis study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. MethodsWe developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. ResultsAt the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7WT/G256E) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. ConclusionsMYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

molecular biology↗

bioRxiv2021

Elevated colonic microbiota-associated paucimannosidic and truncated N-glycans in pediatric ulcerative colitis

Pediatric ulcerative colitis (UC) is a distinct type of inflammatory bowel disease with severe disease activity and rapid progression, which can lead to detrimental life-long consequences. The pathogenesis of pediatric UC remains unclear, although dysbiosis of the gut microbiota has been considered an important factor. In this study, we used mass spectrometry-based glycomic approaches to examine the N-glycans that were associated with the intestinal mucosal-luminal interface microbiota cells of treatment-naive pediatric UC or control patients. We observed abundant paucimannosidic and other truncated N-glycans that were associated with the microbiota and found that the pediatric UC microbiota samples contained significantly higher levels of these atypical N-glycans compared to those of controls. This study indicates that intestinal N-glycans may be used as novel UC biomarker candidates and the aberrant metabolism of glycans by gut microbiota may be involved in the pathogenesis of UC in children.

systems biology↗

bioRxiv2019

Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Metagenomic and metaproteomic approaches have been used to study the composition and functions of the microbiota. However, no studies have examined post-translational modifications (PTM) on human microbiome proteins at the metaproteome level, and it remains unknown whether the microbial PTM is altered or not in patient microbiome. Herein we used anti-acetyl-lysine (Kac) antibody enrichment strategy and mass spectrometry to characterize the protein lysine acetylation in human microbiome, which successfully identified 35,200 Kac peptides corresponding to 31,821 Kac sites from the microbial or host proteins in human gut microbiome samples. The gut microbial proteins exhibited Kac motifs that were distinct from those of human proteins. Functional analysis showed that microbial Kac proteins were significantly enriched in energy production and abundant in enzymes related to transferases and oxidoreductases. Applying to the analysis of pediatric Crohns disease (CD) patient microbiome identified 52 host and 136 microbial protein Kac sites that were differentially abundant in CD versus controls. Interestingly, most of the decreased Kac sites in CD were derived from Firmicutes and most of the increased sites were derived from Bacteroidetes. Forty-six out of the 52 differentially abundant human protein Kac sites were increased in CD patients, including those on calprotectin, lactotransferrin and immunoglobulins. Taken together, this study provides an efficient approach to study the lysine acetylation in microbiome and revealed taxon-specific alterations in the lysine acetylome as well as changes in host protein acetylation levels in intestinal samples during the on-set of disease in CD patients.

systems biology↗

bioRxiv2018

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

bioengineering↗