Biology subjects

Grosberg, A.

Publications and source records attributed to Grosberg, A..

5 recordsLinked to original sources

bioRxiv2026

AN INTEGRATED HEART-ON-A-CHIP NAM RESOLVES DISTINCT CLINICAL PHENOTYPES OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY

Background: Reperfusion is required for myocardial salvage yet itself injures the heart, and cardioprotective interventions that reduce infarct size experimentally have repeatedly failed to improve outcomes in patients. These interventions are evaluated against viability or infarct size alone, whereas ischemia-reperfusion (I/R) injury produces structural, biochemical, and contractile dysfunction in the same tissue. No in vitro new approach methodology (NAM), however, resolves all three within one system. Methods: A NAM was created within a heart-on-a-chip platform, which was constructed by culturing neonatal rat ventricular myocytes (NRVMs) on muscular thin films. Cardiomyocytes were subjected to normoxia, 2 hours of ischemia, and ischemia followed by 2 hours of reperfusion. Sarcomeric architecture was quantified by immunofluorescence, cardiomyocyte injury by LIVE/DEAD staining and cardiac troponin I release into conditioned media, while contractile stress and spontaneous beat frequency were recorded on the same films before and after each condition. Results: Under ischemia, z-line density decreased while architectural metrics were preserved, indicating cell loss rather than sarcomere disorganization. Furthermore, viability decreased, troponin elevated, and every contractile stress component declined, while spontaneous beat frequency was unchanged. Reperfusion fractured this uniform depression. Cell death and troponin release increased further, yet structure was unchanged from ischemia, so every functional change occurred on a depleted but architecturally intact cardiomyocyte population. Systolic stress recovered partially, whereas diastolic stress exceeded baseline, leaving active stress suppressed despite oxygen recovery. Beat frequency increased with pronounced variability. Conclusions: This heart-on-a-chip NAM resolved structural, biochemical, and contractile injury that global indices conflate, distinguishing ischemic from reperfusion pathophysiology. Interestingly, individual readouts corresponded to distinct clinical syndromes of ischemic injury, positioning this platform for future testing of cardioprotective candidates against the full injury phenotype.

physiology↗

bioRxiv2025

In vitro Sex-specific Function-Structure Relationship in Neonatal Rat Cardiac Sheets

Even though in vivo rodent studies have been instrumental in investigating sex-specific differences in cardiac health, function, and pathology, they fall short in providing a fast and flexible platform for investigating sex differences of cardiac anisotropic monolayer in isolation. In vitro platforms offer an accessible and more controlled alternative to dissect and study the mechanisms by which male and female cardiac tissue sheets differ from one another. Here, we have shown on an in vitro heart-on-a-chip platform, primary neonatal rat ventricular myocytes can serve as a viable model showing sex chromosome-driven characteristics even in the absence of supplemented hormones. With controlled experimental conditions, the self-assembly of isolated cardiomyocytes resulted in morphological differences in the structure of the contractile apparatus. More importantly, the assembly of cardiac cells into confluent monolayers had a sex chromosome-driven divergence in both structure and the corresponding function. This work is the first characterization of the difference between sex-specific NRVMs in in vitro culture. Thus, this offers an avenue to investigate sex-based variations in cardiac function that are otherwise difficult to study.

bioengineering↗

bioRxiv2024

LMNA-Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-derived iPSC Differentiation Support Cell type and Lineage-specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency

LMNA-Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C (LMNA) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. Molecular mechanisms of disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of LMNA-Related DCM using induced pluripotent stem cells derived from a family with a heterozygous LMNA c.357-2A>G splice-site mutation. We differentiated one LMNA mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes (CM) and Epicardium-Derived Cells (EPDC). Data integration and comparative analyses of Patient and Control cells found cell type and lineage differentially expressed genes (DEG) with enrichment to support pathway dysregulation. Top DEG and enriched pathways included: 10 ZNF genes and RNA polymerase II transcription in Pluripotent cells (PP); BMP4 and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, CDH2 and EMT in CM; LMNA and epigenetic regulation and DDIT4 and mTORC1 signaling in EPDC. Top DEG also included: XIST and other X-linked genes, six imprinted genes: SNRPN, PWAR6, NDN, PEG10, MEG3, MEG8, and enriched gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs as proposed in other LMNA disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.

genomics↗

bioRxiv2023

Myofibrillar malformations that arise in mdx muscle fibers are driven by detyrosinated microtubules

In Duchenne muscular dystrophy (DMD), alterations in the myofibrillar structure of skeletal muscle fibers that impair contractile function and increase injury susceptibility arise as a consequence of dystrophic pathology. In murine DMD (mdx), myofibrillar alterations are abundant in advanced pathology (>4 months), an age where we formerly established the densification of microtubules (MTs) post-translationally modified by detyrosination (deTyr-MTs) as a negative disease modifier. Given the essential role of MTs in myofibrillar growth, maintenance, and repair, we examined the increased abundance of deTyr-MTs as a potential mechanism for these myofibrillar alterations. Here we find increased levels of deTyr-MTs as an early event in dystrophic pathology (4 weeks) with no evidence of myofibrillar alterations. At 16 weeks, we show the level of deTyr-MTs is significantly increased and co-localized to areas of myofibrillar malformation. Profiling the enzyme complexes responsible for deTyr-tubulin, we identify vasohibin 2 (VASH2) and small vasohibin binding protein (SVBP) significantly elevated in the mdx muscle at 4 wks. We then use the genetic increase in VASH2/SVBP expression in 4 wk wild-type mice and find densified deTyr-MTs that co-segregate with myofibrillar malformations similar to those in the 16 wk mdx. Given that no changes were identified in fibers expressing EGFP as a control, we conclude that disease dependent densification of deTyr-MTs underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers.

pathology↗

bioRxiv2021

Image quantification technique reveals novel lung cancer cytoskeletal phenotype with partial EMT signature

Epithelial-mesenchymal Transition (EMT) is a multi-step process that involves cytoskeletal rearrangement. Here, using novel image quantification tools, we have identified an intermediate EMT state with a specific cytoskeletal signature. We have been able to partition EMT into two steps: (1) initial formation of transverse arcs and dorsal stress fibers and (2) their subsequent conversion to ventral stress fibers with a concurrent alignment of fibers. Using the Orientational Order Parameter (OOP) as a figure of merit, we have been able to track EMT progression in live cells as well as characterize and quantify drug responses. Our technique has improved throughput and is non-destructive, making it a viable candidate for studying a broad range of biological processes. Further, owing to the increased stiffness (and hence invasiveness) of the intermediate phenotype compared to mesenchymal cells, our work can be instrumental in aiding the search for new treatment strategies that combat metastasis by specifically targeting the fiber alignment process.

biophysics↗