Adult Isoforms Research Articles

Rescue of Scn5a mis-splicing does not improve the structural and functional heart defects of a DM1 heart mouse model.

Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant multisystemic disorder for which cardiac features, including conduction delays and arrhythmias, are the second leading cause of disease mortality. DM1 is caused by expanded CTG repeats in the 3' untranslated region of the DMPK gene. Transcription of the expanded DMPK allele produces mRNAs containing long tracts of CUG repeats, which sequester the Muscleblind-Like family of RNA binding proteins, leading to their loss-of-function and the dysregulation of alternative splicing. A well-characterized mis-regulated splicing event in the DM1 heart is the increased inclusion of SCN5A exon 6A rather than the mutually exclusive exon 6B that normally predominates in adult heart. As previous work showed that forced inclusion of Scn5a exon 6A in mice recapitulates cardiac DM1 phenotypes, we tested whether rescue of Scn5a mis-splicing would improve the cardiac phenotypes in a DM1 heart mouse model. We generated mice lacking Scn5a exon 6A to force the expression of the adult SCN5A isoform including exon 6B and crossed these mice to our previously established CUG960 DM1 heart mouse model. We showed that correction Scn5a mis-splicing does not improve the DM1 heart conduction delays and structural changes induced by CUG repeat RNA expression. Interestingly, we found that in addition to Scn5a mis-splicing, Scn5a expression is reduced in heart tissues of CUG960 mice and DM1-affected individuals. These data indicate that Scn5a mis-splicing is not the sole driver of DM1 heart deficits and suggest a potential role for reduced Scn5a expression in DM1 cardiac disease.

Structural switch in acetylcholine receptors in developing muscle.

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres1. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

Abstract 5904: Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways

Abstract Pancreatic cancer (PaCa) ranks as the fourth leading cause of cancer-related deaths in the United States and is among the top five worldwide. Its hallmark characteristics include early invasion, metastasis, resistance to chemotherapy and radiotherapy, and aggressive tumor progression. Evaluating new generation treatments with significant potential in this ongoing battle against cancer is crucial. Voltage-gated sodium channels (VGSC), long detected in various metastatic cell lines and tissue samples beyond breast, lung, and prostate cancer, are of particular interest. This study aimed to explore the therapeutic effects of inhibiting NaV1.7 and its neonatal variant, nNaV1.7, in pancreatic cancer cells. The study utilized human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2. It investigated the roles and effects of adult and neonatal isoforms of the NaV1.7 channel in pancreatic cancer proliferation, invasion, and metastasis. The findings revealed significantly higher adult and neonatal NaV1.7 mRNA expressions in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells. Specific siRNAs effectively downregulated channel mRNA and protein expressions. Notably, NaV1.7 siRNA treatments significantly inhibited cell proliferation and colony-forming capacity in PANC-1 and MiaPaCa-2 cells, without cytotoxic effects on HaCaT cells. Combining gemcitabine with specific NaV1.7 siRNAs enhanced drug efficacy in the PANC-1 cell line. Furthermore, NaV1.7 siRNAs significantly reduced cell invasion, migration, and wound healing in both cell lines. These siRNAs also induced apoptosis and G1 arrest in the cell cycle. They inhibited protein expressions involved in key signaling pathways, including P-Src/P-Fak/Integrin β1, EF2K, P-Akt, P-mTOR, PARP, Caspase-3/9, Cyclin D1/E1. This study is pioneering, as literature lacks research on the expression and function of NaV1.7 and nNaV1.7 in pancreatic cancer cells or tissue samples. In conclusion, NaV1.7 channel isoforms play a significant role in the development, progression, and metastasis of pancreatic cancer and present as a potential therapeutic target in PaCa. Citation Format: Mumin Alper Erdogan, Ayse Caner. Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5904.

Phosphorylation differentially regulates tau isoforms

AbstractBackgroundSix major tau isoforms are alternatively spliced in an age‐dependent manner: 3R tau (3‐repeat domain) is expressed in development, while both 3R and 4R (4‐repeat domain) tau isoforms are expressed in adult brain. While FTDP‐17‐causing point mutations in tau show isoform‐dependent effects on aggregation and microtubule stabilization, there are not tau mutations linked to Alzheimer’s disease. Meanwhile, effects of phosphorylation on different tau isoforms remain largely unexplored.MethodWe use a combination of recombinant proteins, mouse aging studies and iPSC‐derived neurons to test for isoform‐dependent effects of tau phosphorylation on its function and dysfunction.ResultPhosphorylation at certain sites selectively diminishes microtubule polymerization activity by the fetal tau isoform while a comparable adult isoform was largely unaffected. Conversely, phosphorylation at these same sites accelerated in vitro aggregation of an adult isoform compared to the fetal isoform.ConclusionPhosphorylation to tau has isoform‐dependent effects on its function and dysfunction, thus emphasizing the importance of distinguishing pathological and regulatory phospho‐tau species in future therapeutic efforts.

Evolving prion-like tau conformers differentially alter postsynaptic proteins in neurons inoculated with distinct isolates of Alzheimer’s disease tau

ObjectivesAlthough accumulation of misfolded tau species has been shown to predict cognitive decline in patients with Alzheimer’s disease (AD) and other tauopathies but with the remarkable diversity of clinical manifestations, neuropathology profiles, and time courses of disease progression remaining unexplained by current genetic data. We considered the diversity of misfolded tau conformers present in individual AD cases as an underlying driver of the phenotypic variations of AD and progressive loss of synapses.MethodsTo model the mechanism of tau propagation and synaptic toxicity of distinct tau conformers, we inoculated wild-type primary mouse neurons with structurally characterized Sarkosyl-insoluble tau isolates from the frontal cortex of six AD cases and monitored the impact for fourteen days. We analyzed the accumulation rate, tau isoform ratio, and conformational characteristics of de novo-induced tau aggregates with conformationally sensitive immunoassays, and the dynamics of synapse formation, maintenance, and their loss using a panel of pre-and post-synaptic markers.ResultsAt the same concentrations of tau, the different AD tau isolates induced accumulation of misfolded predominantly 4-repeat tau aggregates at different rates in mature neurons, and demonstrated distinct conformational characteristics corresponding to the original AD brain tau. The time-course of the formation of misfolded tau aggregates and colocalization correlated with significant loss of synapses in tau-inoculated cell cultures and the reduction of synaptic connections implicated the disruption of postsynaptic compartment as an early event.ConclusionsThe data obtained with mature neurons expressing physiological levels and adult isoforms of tau protein demonstrate markedly different time courses of endogenous tau misfolding and differential patterns of post-synaptic alterations. These and previous biophysical data argue for an ensemble of various misfolded tau aggregates in individual AD brains and template propagation of their homologous conformations in neurons with different rates and primarily postsynaptic interactors. Modeling tau aggregation in mature differentiated neurons provides a platform for investigating divergent molecular mechanisms of tau strain propagation and for identifying common structural features of misfolded tau and critical interactors for new therapeutic targets and approaches in AD.

Maturation of iPSC-derived cardiomyocytes in a heart-on-a-chip device enables modeling of dilated cardiomyopathy caused by R222Q-SCN5A mutation

To better understand sodium channel (SCN5A)-related cardiomyopathies, we generated ventricular cardiomyocytes from induced pluripotent stem cells obtained from a dilated cardiomyopathy patient harbouring the R222Q mutation, which is only expressed in adult SCN5A isoforms. Because the adult SCN5A isoform was poorly expressed, without functional differences between R222Q and control in both embryoid bodies and cell sheet preparations (cultured for 29–35 days), we created heart-on-a-chip biowires which promote myocardial maturation. Indeed, biowires expressed primarily adult SCN5A with R222Q preparations displaying (arrhythmogenic) short action potentials, altered Na+ channel biophysical properties and lower contractility compared to corrected controls. Comprehensive RNA sequencing revealed differential gene regulation between R222Q and control biowires in cellular pathways related to sarcoplasmic reticulum and dystroglycan complex as well as biological processes related to calcium ion regulation and action potential. Additionally, R222Q biowires had marked reductions in actin expression accompanied by profound sarcoplasmic disarray, without differences in cell composition (fibroblast, endothelial cells, and cardiomyocytes) compared to corrected biowires. In conclusion, we demonstrate that in addition to altering cardiac electrophysiology and Na+ current, the R222Q mutation also causes profound sarcomere disruptions and mechanical destabilization. Possible mechanisms for these observations are discussed.

The emerging spectrum of fetal acetylcholine receptor antibody-related disorders (FARAD).

In utero exposure to maternal antibodies targeting the fetal acetylcholine receptor isoform (fAChR) can impair fetal movement, leading to arthrogryposis multiplex congenita (AMC). Fetal AChR antibodies have also been implicated in apparently rare, milder myopathic presentations termed fetal acetylcholine receptor inactivation syndrome (FARIS). The full spectrum associated with fAChR antibodies is still poorly understood. Moreover, since some mothers have no myasthenic symptoms, the condition is likely underreported, resulting in failure to implement effective preventive strategies. Here we report clinical and immunological data from a multicentre cohort (n = 46 cases) associated with maternal fAChR antibodies, including 29 novel and 17 previously reported with novel follow-up data. Remarkably, in 50% of mothers there was no previously established myasthenia gravis (MG) diagnosis. All mothers (n = 30) had AChR antibodies and, when tested, binding to fAChR was often much greater than that to the adult AChR isoform. Offspring death occurred in 11/46 (23.9%) cases, mainly antenatally due to termination of pregnancy prompted by severe AMC (7/46, 15.2%), or during early infancy, mainly from respiratory failure (4/46, 8.7%). Weakness, contractures, bulbar and respiratory involvement were prominent early in life, but improved gradually over time. Facial (25/34; 73.5%) and variable peripheral weakness (14/32; 43.8%), velopharyngeal insufficiency (18/24; 75%) and feeding difficulties (16/36; 44.4%) were the most common sequelae in long-term survivors. Other unexpected features included hearing loss (12/32; 37.5%), diaphragmatic paresis (5/35; 14.3%), CNS involvement (7/40; 17.5%) and pyloric stenosis (3/37; 8.1%). Oral salbutamol used empirically in 16/37 (43.2%) offspring resulted in symptom improvement in 13/16 (81.3%). Combining our series with all previously published cases, we identified 21/85 mothers treated with variable combinations of immunotherapies (corticosteroids/intravenous immunoglobulin/plasmapheresis) during pregnancy either for maternal MG symptom control (12/21 cases) or for fetal protection (9/21 cases). Compared to untreated pregnancies (64/85), maternal treatment resulted in a significant reduction in offspring deaths (P < 0.05) and other complications, with treatment approaches involving intravenous immunoglobulin/ plasmapheresis administered early in pregnancy most effective. We conclude that presentations due to in utero exposure to maternal (fetal) AChR antibodies are more common than currently recognized and may mimic a wide range of neuromuscular disorders. Considering the wide clinical spectrum and likely diversity of underlying mechanisms, we propose 'fetal acetylcholine receptor antibody-related disorders' (FARAD) as the most accurate term for these presentations. FARAD is vitally important to recognize, to institute appropriate management strategies for affected offspring and to improve outcomes in future pregnancies. Oral salbutamol is a symptomatic treatment option in survivors.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/β myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81mm/s, NTG: 338.7 ± 28.07mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

The role of Limch1 alternative splicing in skeletal muscle function.

Postnatal skeletal muscle development is a highly dynamic period associated with widespread alternative splicing changes required to adapt tissues to adult function. These splicing events have significant implications because the reversion of adult mRNA isoforms to fetal isoforms is observed in forms of muscular dystrophy. LIMCH1 is a stress fiber-associated protein that is alternatively spliced to generate uLIMCH1, a ubiquitously expressed isoform, and mLIMCH1, a skeletal muscle-specific isoform containing six additional exons simultaneously included after birth in the mouse. CRISPR/Cas9 was used to delete the six alternatively spliced exons of LIMCH1 in mice, thereby forcing the constitutive expression of the predominantly fetal isoform, uLIMCH1. mLIMCH1 knockout mice had significant grip strength weakness in vivo, and maximum force generated was decreased ex vivo. Calcium-handling deficits were observed during myofiber stimulation that could explain the mechanism by which mLIMCH1 knockout leads to muscle weakness. In addition, LIMCH1 is mis-spliced in myotonic dystrophy type 1, with the muscleblind-like (MBNL) family of proteins acting as the likely major regulator of Limch1 alternative splicing in skeletal muscle.

Muscleblind-like 2 knockout shifts adducin 1 isoform expression and alters dendritic spine dynamics of cortical neurons during brain development.

Muscleblind-like 2 (MBNL2) plays a crucial role in regulating alternative splicing during development and mouse loss of MBNL2 recapitulates brain phenotypes in myotonic dystrophy (DM). However, the mechanisms underlying DM neuropathogenesis during brain development remain unclear. In this study, we aim to investigate the impact of MBNL2 elimination on neuronal development by Mbnl2 conditional knockout (CKO) mouse models. To create Mbnl2 knockout neurons, cDNA encoding Cre-recombinase was delivered into neural progenitors of Mbnl2flox/flox mouse brains by in utero electroporation. The morphologies and dynamics of dendritic spines were monitored by confocal and two-photon microscopy in brain slices and live animals from the neonatal period into adulthood. To investigate the underlying molecular mechanism, we further detected the changes in the splicing and molecular interactions of proteins associated with spinogenesis. We found that Mbnl2 knockout in cortical neurons decreased dendritic spine density and dynamics in adolescent mice. Mbnl2 ablation caused the adducin 1 (ADD1) isoform to switch from adult to fetal with a frameshift, and the truncated ADD1 failed to interact with alpha-II spectrin (SPTAN1), a critical protein for spinogenesis. In addition, expression of ADD1 adult isoform compensated for the reduced dendritic spine density in cortical neurons deprived of MBNL2. MBNL2 plays a critical role in maintaining the dynamics and homeostasis of dendritic spines in the developing brain. Mis-splicing of downstream ADD1 may account for the alterations and contribute to the DM brain pathogenesis.

Not young but still immature: a HIF-1α-mediated maturation checkpoint in regenerating muscle.

Muscle fibers express particular isoforms of contractile proteins, depending on the fiber's function and the organism's developmental stage. In the adult, after a muscle injury, newly generated fibers transition through embryonic and neonatal myosins, prior to selecting their distinctive adult myosin isoform. In this issue of the JCI, Wang et al. discover a checkpoint that regulates the neonatal-to-adult myosin isoform transition. They found that HIF-1α regulated this checkpoint, with elevated HIF-1α levels blocking progression, while HIF-1α knockout accelerated the transition. They further related these findings to centronuclear myopathy, a disease in which HIF-1α is similarly elevated and neonatal myosin expression is maintained. These findings highlight a maturation checkpoint that impacts the skeletal muscle regeneration following ischemic injury, providing a pharmacologically accessible pathway in injury and diseases such as centronuclear myopathy.

A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer’s disease

PurposeAlzheimer’s disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.MethodiPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.ResultsCells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.ConclusionOur data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

Maturation of hiPSC-derived cardiomyocytes in cardiac microtissues promotes adult alternative splicing of sodium channel revealing mutation effects associated with cardiac arrhythmia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Marie Skłodowska-Curie Actions - Individual Fellowship Background The cardiac sodium channel encoded by SCN5A gene undergoes fetal-to-adult isoform switch postnatally, regulated by alternative splicing. Various mutations in SCN5A associated with severe cardiac arrhythmia have been previously studied using cardiomyocytes derived from human induced pluripotent stem-cells (hiPSC-CMs), demonstrating the importance of a human cardiac cellular context for such analysis. Mutations in the adult isoform of SCN5A are however of difficult evaluation in hiPSC-CMs, because of their typical immature/fetal-like characteristics. Combining hiPSC-CMs in 3-dimensional microtissues together with hiPSC-derived cardiac fibroblasts and endothelial cells induced maturation in gene expression and functional properties, including electrophysiology. Purpose We investigated whether the maturation promoted by the microtissue system could reveal the functional phenotype of a mutation in the adult isoform of SCN5A. Methods We derived hiPSC-CMs from a cardiac arrhythmia patient carrying two compound mutations in SCN5A: p.W156X in exon 4 and p.R225W in the adult splicing variant of exon 6 (exon 6B). Using CRISPR/Cas9, we corrected exon 4 mutation to investigate specific effects of exon 6B mutation on sodium current by single-cell patch clamp. HiPSC-CMs were matured in tricell-type cardiac microtissues and analysed after dissociation. The mechanism underlying exon 6B expression was investigated by overexpressing or knocking-out the alternative splicing regulator MBNL1 in hiPSC-CMs. Results ddPCR analysis showed low expression of exon 6B-containing transcripts in hiPSC-CMs, accompanied by no effect of exon 6B mutation on the sodium current. Instead, maturing hiPSC-CMs in cardiac microtissues promoted SCN5A exon 6B expression and revealed the contribution of both SCN5A mutations to the diseased cellular electrophysiology. MBNL1 was upregulated in microtissues and indeed its overexpression was sufficient to increase exon 6B levels in hiPSC-CMs, while lack of MBNL1 prevented the switch of SCN5A splicing isoforms. Conclusions Our data demonstrate that a maturation of hiPSC-CMs is required to express the adult SCN5A isoform. This was achieved in the microtissue system via upregulation of MBNL1, and allowed to reveal SCN5A mutation contributions, by dissecting changes in ionic current that cause adult arrhythmic disease phenotypes in humans.

Maturation of hiPSC-derived cardiomyocytes promotes adult alternative splicing of SCN5A and reveals changes in sodium current associated with cardiac arrhythmia.

Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

Spliced isoforms of the cardiac Nav1.5 channel modify channel activation by distinct structural mechanisms.

Alternative splicing is an important cellular mechanism that fine tunes the gating properties of both voltage- and ligand-gated ion-channels. The cardiac voltage-gated sodium channel, Nav1.5, is subject to alternative splicing of the DI S3-S4 linker, which generates two types of channels with different activation properties. Here, we show that the gating differences between the adult (mH1) and neonatal (Nav1.5e) isoforms of Nav1.5 are mediated by two amino acid residues: Thr/Ser at position 207 and Asp/Lys at position 211. Electrophysiological experiments, in conjunction with molecular dynamics simulations, revealed that each residue contributes equally to the overall gating shifts in activation, but that the underlying structural mechanisms are different. Asp/Lys at position 211 acts through electrostatic interactions, whereas Thr/Ser at position 207 is predicted to alter the hydrogen bond network at the top of the S3 helix. These distinct structural mechanisms work together to modify movement of the voltage-sensitive S4 helix to bring about channel activation. Interestingly, mutation of the homologous Asp and Thr residues of the skeletal muscle isoform, Nav1.4, to Lys and Ser, respectively, confers a similar gating shift in channel activation, suggesting that these residues may fulfill a conserved role across other Nav channel family members.

Abstract P2-03-02: Detection of insulin receptor isoforms by mass spectrometry in breast cancer

Abstract Breast cancer cells express Type 1 Insulin-like Growth Factor Receptor (IGF1R) and Insulin Receptor (IR). These receptors have a role in breast cancer biology and mediate metabolism, proliferation, survival, and motility. IR gene undergoes differential splicing that generates two IR isoforms, IRA (exclusion of exon 11) and IRB. High levels of IRA expression in prenatal growth and development are observed, whereas IRB expression is more highly expressed in adult insulin responsive tissues. IRA overexpression is the predominant receptor isoform expressed in breast cancer specimens obtained from patients with endocrine resistant disease. To measure levels of IRA and IRB mRNA expression, we utilized IR isoform specific primers in quantitative reverse transcription PCR (qRT-PCR). The validation of IRA and IRB primers was determined via using IRA and IRB overexpressing clones of MCF7 cells. IRA and IRB mRNA expression was also studied in human adipose and liver tissues that majorly express the adult IRB isoform. Over 40 breast cancer cell lines and 20 ER+ patient tumor samples were studied to determine IRA and IRB mRNA expression. Total IR, IGF1R, IRS1, and IRS2 were also analyzed in cell lines and patient samples. Across the ATCC breast cancer cell lines and ER+ patient tumors, heterogeneity was found among all targeted genes. Using MCF-7 cells as control, we found one of the breast cancer cell lines, Du4475, has very high IRA mRNA expression level (200-fold of that in MCF-7). Using mass spectrometry technology, we quantified levels of IR isoform expression in Du4475 cells. Briefly, IR immunoprecipitates were subjected to SDS-PAGE gels and the IRA protein gel band was submitted for mass spectrometry. Since IRA does not contain exon 11, the identified unique peptide consistent with its expression is TFEDYLHNVVFVPRPS. After this IRA specific peptide was determined, the heavy-labeled (C13- and N15-containing) IRA peptide was used as an internal injection control to quantify the IRA level. Quantitative results of IRA peptode sequence were proportional to the mRNA levels previously detected in Du4475 cell lysate. Using MCF-7 IRB overexpression cell line MCF7L-IRB-C5, we identified two IRB peptides, KTSSGTGAEDPRPSR and TSSGTGAEDPRPSR at exon 11 region by mass spectrometry. Cell membrane and total cell lysates could also be used to detect IR protein isoforms by mass spectrometry analysis. Further measurement of IRA and IRB level on other breast cancer cell lines is ongoing. In summary, we are the first to use mass spectrometry to validate protein expression of IR isoforms at levels that correlate with mRNA expression. IRA mRNA and protein expression levels in breast cancer cell lines may serve as biomarkers to screen patients for targeted therapy. Citation Format: Xihong Zhang, LeeAnn Higgins, Todd Markowski, Tzu-Yi Yang, Jacob Wragge, Kevin Murray, Bruce Witthuhn, Albert Barrios, Douglas Yee. Detection of insulin receptor isoforms by mass spectrometry in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-03-02.

High-throughput functional analysis of human Nav1.6 variants expressed in adult and neonatal isoforms

SCN8A encodes the voltage-gated sodium channel NaV1.6 expressed at axon initial segments and nodes of Ranvier in central and peripheral neurons. SCN8A variants are associated with epileptic encephalopathy and related neurodevelopmental disorders. Similar to other NaV channels, SCN8A undergoes developmentally-regulated alternative mRNA splicing. Because splice isoforms of other NaV channels exhibit distinct functional properties, the SCN8A isoform used for variant expression may affect observed pathogenicity.

Contractility and myosin heavy chain content of skeletal muscle engineered from adult and aged rats

Functional three-dimensional skeletal muscle constructs (myooids) were engineered from myogenic cells harvested from the muscles of adult and aged rats. Myooids formed by self-organization of myogenic cells in the absence of an artificial scaffold and were maintained in culture. Myooid excitability and contractility were measured at days 1, 3, 7, and 24 after myooid formation. Skeletal muscle myosin heavy chain (MHC) content was measured and MHC isoforms were separated on SDS-PAGE gels and quantified. Adult and aged rat myooids had only ~35-60% of the skeletal muscle MHC content of control skeletal muscle from rats, the remaining MHC content consisting of isoforms found in cultured fibroblasts but not in control skeletal muscle. In addition, myooids expressed only developmental isoforms of skeletal muscle MHC, known to generate less specific force than adult isoforms. Myooids from aged rats generated greater normalized force than myooids from adult rats, but only 1-3% of that for adult skeletal muscle controls. Normalizing for skeletal muscle MHC content and isoform expression predicts myooid forces of ~6-18% of control adult skeletal muscles. We hypothesize that the remaining force deficit is due to cellular and sub-cellular disorganization—Myooids lack the density and organization of sarcomeric arrays seen in skeletal muscle.

Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis.

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Epigenetic Regulation of Cardiac Troponin Genes in Pediatric Patients with Heart Failure Supported by Ventricular Assist Device.

Ventricular Assist Device (VAD) therapy is considered as a part of standard care for end-stage Heart Failure (HF) children unresponsive to medical management, but the potential role of miRNAs in response to VAD therapy on molecular pathways underlying LV remodeling and cardiac function in HF is unknown. The aims of this study were to evaluate the effects of VAD on miRNA expression profile in cardiac tissue obtained from HF children, to determine the putative miRNA targets by an in-silico analysis as well as to verify the changes of predicated miRNA target in the same cardiac samples. The regulatory role of selected miRNAs on predicted targets was evaluated by a dedicated in vitro study. miRNA profile was determined in cardiac samples obtained from 13 HF children [median: 29 months; 19 LVEF%; 9 Kg] by NGS before VAD implant (pre-VAD) and at the moment of heart transplant (Post-VAD). Only hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-1246 were differentially expressed at post-VAD when compared to pre-VAD, and validated by real-time PCR. Putative targets of the selected miRNAs were involved in regulation of sarcomere genes, such as cardiac troponin (cTns) complex. The expression levels of fetal ad adult isoforms of cTns resulted significantly higher after VAD in cardiac tissue of HF pediatric patients when compared with HF adults. An in vitro study confirmed a down-regulatory effect of hsa-miR-19a-3p on cTnC expression. The effect of VAD on sarcomere organization through cTn isoform expression may be epigenetically regulated, suggesting for miRNAs a potential role as therapeutic targets to improve heart function in HF pediatric patients.