Saranya P. Wyles

Modeling structural and functional deficiencies of RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells

Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 (RBM20), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20-dependent splice variants affected sarcomeric (TTN and LDB3) and calcium (Ca(2+)) handling (CAMK2D and CACNA1C) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length (RBM20: 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P < 0.0001) and decreased sarcomeric width (RBM20: 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P < 0.0001). Additionally, CMs showed defective Ca(2+) handling machinery with prolonged Ca(2+) levels in the cytoplasm as measured by greater area under the curve (RBM20: 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P < 0.05) and higher Ca(2+) spike amplitude (RBM20: 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P < 0.05). β-adrenergic stress induced with 10 µm norepinephrine demonstrated increased susceptibility to sarcomeric disorganization (RBM20: 86 ± 10.5% versus control: 40 ± 7%; P < 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool to dissect disease-relevant defects in RBM20 splicing as a global regulator of heart function.

Is Local Infiltration Analgesia Superior to Peripheral Nerve Blockade for Pain Management After THA: A Network Meta-analysis.

BackgroundLocal infiltration analgesia and peripheral nerve blocks are common methods for pain management in patients after THA but direct head-to-head, randomized controlled trials (RCTs) have not been performed. A network meta-analysis allows indirect comparison of individual treatments relative to a common comparator; in this case placebo (or no intervention), epidural analgesia, and intrathecal morphine, yielding an estimate of comparative efficacy.Questions/purposesWe asked, when compared with a placebo, (1) does use of local infiltration analgesia reduce patient pain scores and opioid consumption, (2) does use of peripheral nerve blocks reduce patient pain scores and opioid consumption, and (3) is local infiltration analgesia favored over peripheral nerve blocks for postoperative pain management after THA?MethodsWe searched six databases, from inception through June 30, 2014, to identify RCTs comparing local infiltration analgesia or peripheral nerve block use in patients after THA. A total of 35 RCTs at low risk of bias based on the recommended Cochrane Collaboration risk assessment tool were included in the network meta-analysis (2296 patients). Primary outcomes for this review were patient pain scores at rest and cumulative opioid consumption, both assessed at 24 hours after THA. Because of substantial heterogeneity (variation of outcomes between studies) across included trials, a random effect model for meta-analysis was used to estimate the weighted mean difference (WMD) and 95% CI. The gray literature was searched with the same inclusion criteria as published trials. Only one unpublished trial (published abstract) fulfilled our criteria and was included in this review. All other studies included in this systematic review were full published articles. Bayesian network meta-analysis included all RCTs that compared local infiltration analgesia or peripheral nerve blocks with placebo (or no intervention), epidural analgesia, and intrathecal morphine.ResultsCompared with placebo, local infiltration analgesia reduced patient pain scores (WMD, −0.61; 95% CI, −0.97 to −0.24; p = 0.001) and opioid consumption (WMD, −7.16 mg; 95% CI, −11.98 to −2.35; p = 0.004). Peripheral nerve blocks did not result in lower pain scores or reduced opioid consumption compared with placebo (WMD, −0.43; 95% CI, −0.99 to 0.12; p = 0.12 and WMD, −3.14 mg, 95% CI, −11.30 to 5.02; p = 0.45). However, network meta-analysis comparing local infiltration analgesia with peripheral nerve blocks through common comparators showed no differences between postoperative pain scores (WMD, −0.36; 95% CI, −1.06 to 0.31) and opioid consumption (WMD, −4.59 mg; 95% CI, −9.35 to 0.17), although rank-order analysis found local infiltration analgesia to be ranked first in more simulations than peripheral nerve blocks, suggesting that it may be more effective.ConclusionsUsing the novel statistical network meta-analysis approach, we found no differences between local infiltration analgesia and peripheral nerve blocks in terms of analgesia or opioid consumption 24 hours after THA; there was a suggestion of a slight advantage to peripheral nerve blocks based on rank-order analysis, but the effect size in question is likely not large. Given the slight difference between interventions, clinicians may choose to focus on other factors such as cost and intervention-related complications when debating which analgesic treatment to use after THA.Level of EvidenceLevel I, therapeutic study.

Inhibition of DNA Topoisomerase II Selectively Reduces the Threat of Tumorigenicity Following Induced Pluripotent Stem Cell-Based Myocardial Therapy

The advent of induced pluripotent stem cell (iPSC) technology creates new opportunities for transplant-based therapeutic strategies. The potential for clinical translation is currently hindered by the risk of dysregulated cell growth. Pluripotent stem cells reprogrammed by three-factor (Sox2, Klf, and Oct4) and four-factor (Sox2, Klf, Oct4, and c-Myc) strategies result in the capacity for teratogenic growth from residual pluripotent progeny upon in vivo transplantation. However, these pluripotent stem cells also have a stage-specific hypersensitivity to DNA-damaging agents that may allow separation of lineage-specific therapeutic subpopulation of cells. We aimed to demonstrate the selective effect of DNA topoisomerase II inhibitor, etoposide, in eliminating pluripotent cells in the early cardiac progenitor population thus decreasing the effect of teratoma formation. Immunodeficient murine hearts were infarcted and received implantation of a therapeutic dose of cardiac progenitors derived from partially differentiated iPSCs. Etoposide-treated cell implantation reduced mass formation in the intracardiac and extracardiac chest cavity compared with the same dose of iPSC-derived cardiac progenitors in the control untreated group. In vivo bioluminescence imaging confirmed the localization and engraftment of transplanted cells in the myocardium postinjection in both groups. Comparatively, the equivalent cell population without etoposide treatment demonstrated a greater incidence and size of teratoma formation. Hence, pretreatment with genotoxic etoposide significantly lowered the threat of teratogenicity by purging the contaminating pluripotent cells, establishing an adjunctive therapy to further harness the clinical value of iPSC-derived cardiac regeneration.

Stem Cells: The Pursuit of Genomic Stability

Stem cells harbor significant potential for regenerative medicine as well as basic and clinical translational research. Prior to harnessing their reparative nature for degenerative diseases, concerns regarding their genetic integrity and mutation acquisition need to be addressed. Here we review pluripotent and multipotent stem cell response to DNA damage including differences in DNA repair kinetics, specific repair pathways (homologous recombination vs. non-homologous end joining), and apoptotic sensitivity. We also describe DNA damage and repair strategies during reprogramming and discuss potential genotoxic agents that can reduce the inherent risk for teratoma formation and mutation accumulation. Ensuring genomic stability in stem cell lines is required to achieve the quality control standards for safe clinical application.

Pharmacological Modulation of Calcium Homeostasis in Familial Dilated Cardiomyopathy: An In Vitro Analysis From an RBM20 Patient‐Derived iPSC Model

For inherited cardiomyopathies, abnormal sensitivity to intracellular calcium (Ca2+), incurred from genetic mutations, initiates subsequent molecular events leading to pathological remodeling. Here, we characterized the effect of β‐adrenergic stress in familial dilated cardiomyopathy (DCM) using human‐induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes (CMs) from a patient with RBM20 DCM. Our findings suggest that β‐adrenergic stimulation accelerated defective Ca2+ homeostasis, apoptotic changes, and sarcomeric disarray in familial DCM hiPSC‐CMs. Furthermore, pharmacological modulation of abnormal Ca2+ handling by pretreatment with β‐blocker, carvedilol, or Ca2+‐channel blocker, verapamil, significantly decreased the area under curve, reduced percentage of disorganized cells, and decreased terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL)‐positive apoptotic loci in familial DCM hiPSC‐CMs after β‐adrenergic stimulation. These translational data provide patient‐based in vitro analysis of β‐adrenergic stress in RBM20‐deficient familial DCM hiPSC‐CMs and evaluation of therapeutic interventions to modify heart disease progression, which may be personalized, but more importantly generalized in the clinic.

Calreticulin secures calcium-dependent nuclear pore competency required for cardiogenesis

Calreticulin deficiency causes myocardial developmental defects that culminate in an embryonic lethal phenotype. Recent studies have linked loss of this calcium binding chaperone to failure in myofibrillogenesis through an as yet undefined mechanism. The purpose of the present study was to identify cellular processes corrupted by calreticulin deficiency that precipitate dysregulation of cardiac myofibrillogenesis related to acquisition of cardiac phenotype. In an embryonic stem cell knockout model, calreticulin deficit (crt(-/-)) compromised nucleocytoplasmic transport of nuclear localization signal-dependent and independent pathways, disrupting nuclear import of the cardiac transcription factor MEF2C. The expression of nucleoporins and associated nuclear transport proteins in derived crt(-/-) cardiomyocytes revealed an abnormal nuclear pore complex (NPC) configuration. Altered protein content in crt(-/-) cells resulted in remodeled NPC architecture that caused decreased pore diameter and diminished probability of central channel occupancy versus wild type counterparts. Ionophore treatment of impaired calcium handling in crt(-/-) cells corrected nuclear pore microarchitecture and rescued nuclear import resulting in normalized myofibrillogenesis. Thus, calreticulin deficiency alters nuclear pore function and structure, impeding myofibrillogenesis in nascent cardiomyocytes through a calcium dependent mechanism. This essential role of calreticulin in nucleocytoplasmic communication competency ties its regulatory action with proficiency of cardiac myofibrillogenesis essential for proper cardiac development.

Regenerative Therapy Prevents Heart Failure Progression in Dyssynchronous Nonischemic Narrow QRS Cardiomyopathy

Background Cardiac resynchronization therapy using bi-ventricular pacing is proven effective in the management of heart failure (HF) with a wide QRS-complex. In the absence of QRS prolongation, however, device-based resynchronization is reported unsuitable. As an alternative, the present study tests a regenerative cell-based approach in the setting of narrow QRS-complex HF. Methods and Results Progressive cardiac dyssynchrony was provoked in a chronic transgenic model of stress-triggered dilated cardiomyopathy. In contrast to rampant end-stage disease afflicting untreated cohorts, stem cell intervention early in disease, characterized by mechanical dyssynchrony and a narrow QRS-complex, aborted progressive dyssynchronous HF and prevented QRS widening. Stem cell-treated hearts acquired coordinated ventricular contraction and relaxation supporting systolic and diastolic performance. Rescue of contractile dynamics was underpinned by a halted left ventricular dilatation, limited hypertrophy, and reduced fibrosis. Reverse remodeling reflected a restored cardiomyopathic proteome, enforced at systems level through correction of the pathological molecular landscape and nullified adverse cardiac outcomes. Cell therapy of a dyssynchrony-prone cardiomyopathic cohort translated prospectively into improved exercise capacity and prolonged survivorship. Conclusions In narrow QRS HF, a regenerative approach demonstrated functional and structural benefit, introducing the prospect of device-autonomous resynchronization therapy for refractory disease.

Systems biology surveillance decrypts pathological transcriptome remodeling.

BackgroundPathological cardiac development is precipitated by dysregulation of calreticulin, an endoplasmic reticulum (ER)-resident calcium binding chaperone and critical contributor to cardiogenesis and embryonic viability. However, pleiotropic phenotype derangements induced by calreticulin deficiency challenge the identification of specific downstream transcriptome elements that direct proper cardiac formation. Here, differential transcriptome navigation was used to diagnose high priority calreticulin domain-specific gene expression changes and decrypt complex cardiac-specific molecular responses elicited by discrete functional regions of calreticulin.MethodsWild type (WT), calreticulin-deficient (CALR−/−), and calreticulin truncation variant (CALR−/−-NP and CALR−/−-PC) pluripotent stem cells were used to investigate molecular remodeling underlying a model of cardiopathology. Bioinformatic deconvolution of isolated transcriptomes was performed to identify predominant expression trends, gene ontology prioritizations, and molecular network features characteristic of discrete cell types.ResultsStem cell lines with wild type (WT), calreticulin-deficient (CALR−/−) genomes, as well as calreticulin truncation variants exclusively expressing either the chaperoning (CALR−/−-NP) or the calcium binding (CALR−/−-PC) domain exhibited characteristic molecular signatures determined by unsupervised agglomerative clustering. Kohonen mapping of RNA expression changes identified transcriptome dynamics that segregated into 12 discrete gene expression meta-profiles which were enriched for regulation of Eukaryotic Initiation Factor 2 (EIF2) signaling. Focused examination of domain-specific gene ontology remodeling revealed a general enrichment of Cardiovascular Development in the truncation variants, with unique prioritization of “Cardiovascular Disease” exclusive to the cohort of down regulated genes of the PC truncation variant. Molecular cartography of genes that comprised this cardiopathological category revealed uncharacterized and novel gene relationships, with identification of Pitx2 as a critical hub within the topology of a CALR−/− compromised network.ConclusionsDiagnostic surveillance, through an algorithm that integrates pluripotent stem cell transcriptomes with advanced high throughput assays and computational bioinformatics, revealed collective gene expression network changes that underlie differential phenotype development. Stem cell transcriptomes provide a deep collective molecular index that reflects ad hoc robustness of the pluripotent gene network. Remodeling events such as monogenic lesions provide a background by which high priority candidate disease effectors and regulators can be identified, demonstrated here by a molecular profiling algorithm that decrypts pluripotent wild type versus disrupted genomes.

Myosin-1E interacts with FAK proline-rich region 1 to induce fibronectin-type matrix

Significance Focal adhesion kinase (FAK) is an intensely studied protein involved in many medically relevant biological processes, including cancer. Despite the large interest in FAK, a promising strategy to target FAK therapeutically is elusive. Here, we show that a region within the FAK protein that contains autophosphorylation site tyrosine (Y) 397 is essential for FAK activity in vivo. Myosin-1E (MYO1E), an actin-dependent molecular motor protein, directly interacts with FAK to induce Y397 autophosphorylation, which, in turn, causes changes in gene expression commonly observed in aggressive cancer. Our findings are significant because they further delineate FAK function in vivo and identify the MYO1E–FAK interaction as a possible Achilles’ heel for cancer. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)–kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.

Systems-Based Technologies in Profiling the Stem Cell Molecular Framework for Cardioregenerative Medicine

Over the last decade, advancements in stem cell biology have yielded a variety of sources for stem cell-based cardiovascular investigation. Stem cell behavior, whether to maintain its stable state of pluripotency or to prime toward the cardiovascular lineage is governed by a set of coordinated interactions between epigenetic, transcriptional, and translational mechanisms. The science of incorporating genes (genomics), RNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics) data in a specific biological sample is known as systems biology. Integrating systems biology in progression with stem cell biologics can contribute to our knowledge of mechanisms that underlie pluripotency maintenance and guarantee fidelity of cardiac lineage specification. This review provides a brief summarization of OMICS-based strategies including transcriptomics, proteomics, and metabolomics used to understand stem cell fate and to outline molecular processes involved in heart development. Additionally, current efforts in cardioregeneration based on the “one-size-fits-all” principle limit the potential of individualized therapy in regenerative medicine. Here, we summarize recent studies that introduced systems biology into cardiovascular clinical outcomes analysis, allowing for predictive assessment for disease recurrence and patient-specific therapeutic response.