Stem cell models of inherited arrhythmias.

Inherited arrhythmias are a heterogeneous group of conditions that confer risk of sudden death. Many inherited arrhythmias have been linked to pathogenic genetic variants that result in ion channel dysfunction, although current genetic testing panels fail to identify variants in many patients, potentially secondary to their underlying substrates being oligogenic or polygenic. Here we review the current state of knowledge surrounding the cellular mechanisms of inherited arrhythmias generated from stem cell models with a focus on integrating genetic and mechanistic data. The utility and limitations of human induced pluripotent stem cell models in disease modeling and drug development are also explored with a particular focus on examples of pharmacogenetics and precision medicine. We submit that progress in understanding inherited arrhythmias is likely to be made by using human induced pluripotent stem cells to model probable polygenic cases as well as to interrogate the diverse and potentially complex molecular networks implicated by genome-wide association studies.

Emerging Targeted Therapies for Inherited Cardiomyopathies and Arrhythmias.

Inherited cardiomyopathy and arrhythmia syndromes are associated with significant morbidity and mortality, particularly in young people. Medical management of these conditions has primarily been limited to agents previously developed for more common forms of heart disease and not tailored to their distinct pathophysiology. As our understanding of their underlying genetics and disease mechanisms has improved, an era of targeted therapies for these rare conditions has begun to emerge. In recent years, several novel agents have been developed and tested in preclinical models and, in some cases, have advanced to both the clinical trial and clinical approval stages with exciting results. These new treatments are derived from multiple classes of therapeutics, including small molecules, antisense oligonucleotides, small interfering RNAs, adeno-associated virus-mediated gene therapies, and in vivo gene editing. Collectively, they carry the promise of revolutionizing management of affected patients and their families.

A Case of Pericarditis and Pericardial Masses Associated With Mycobacterium Paragordonae.

Tuberculosis is a common cause of pericarditis worldwide and has been associated with pericardial masses. Non-tuberculous mycobacteria are uncommonly associated with cardiac disease, having primarily been described in cases of endocarditis. Here we describe a case of an immunocompetent patient with Mycobacterium paragordonae infection causing pericarditis with a large effusion containing pericardial masses. The patient presented with chest pain, hypoxia and biochemical evidence of inflammation (CRP 216.1 mg/L). This report illustrates a rare case of pericarditis with pericardial masses associated with non-tuberculous mycobacteria and the first example of pericarditis associated with M. paragordonae.

New-Onset Atrial Fibrillation After Transcatheter Aortic Valve Replacement: A Systematic Review and Meta-Analysis.

OBJECTIVES: The authors aimed to identify risk factors and outcomes associated with new-onset atrial fibrillation (NOAF) after transcatheter aortic valve replacement (TAVR). BACKGROUND: NOAF is a common complication after TAVR, although estimates of the precise occurrence are variable. This study sought to quantify the occurrence of NOAF after TAVR and to explore the outcomes and predictors associated with this complication. METHODS: We searched Medline, EMBASE, and the Cochrane database from 2016 to 2020 for articles that reported NOAF after TAVR. We extracted data for studies published before 2016 from a previous systematic review. We pooled data using a random effects model. RESULTS: We identified 179 studies with 241,712 total participants (55,271 participants with pre-existing atrial fibrillation (AF) were excluded) that reported NOAF from 2008 to 2020. The pooled occurrence of NOAF after TAVR was 9.9% (95% CI: 8.1%-12%). NOAF after TAVR was associated with a longer index hospitalization (mean difference = 2.66 days; 95% CI: 1.05-4.27), a higher risk of stroke in the first 30 days (risk ratio [RR]: 2.35; 95% CI: 2.12-2.61), 30-day mortality (RR: 1.76; 95% CI: 1.12-2.76), major or life-threatening bleeding (RR: 1.60; 95% CI: 1.39-1.84), and permanent pacemaker implantation (RR: 1.12; 95% CI: 1.05-1.18). Risk factors for the development of NOAF after TAVR included higher Society of Thoracic Surgeons score, transapical access, pulmonary hypertension, chronic kidney disease, peripheral vascular disease, and severe mitral regurgitation, suggesting that the risk for NOAF is highest in more comorbid TAVR patients. CONCLUSIONS: NOAF is common after TAVR. Whether AF after TAVR is a causal factor or a marker of sicker patients remains unclear.

α-Synuclein mutation impairs processing of endomembrane compartments and promotes exocytosis and seeding of α-synuclein pathology.

Neuronal loss in Parkinson's disease (PD) is associated with impaired proteostasis and accumulation of α-syn microaggregates in dopaminergic neurons. These microaggregates promote seeding of α-synuclein (α-syn) pathology between synaptically linked neurons. However, the mechanism by which seeding is initiated is not clear. Using human pluripotent stem cell (hPSC) models of PD that allow comparison of SNCA mutant cells with isogenic controls, we find that SNCA mutant neurons accumulate α-syn deposits that cluster to multiple endomembrane compartments, specifically multivesicular bodies (MVBs) and lysosomes. We demonstrate that A53T and E46K α-syn variants bind and sequester LC3B monomers into detergent-insoluble microaggregates on the surface of late endosomes, increasing α-syn excretion via exosomes and promoting seeding of α-syn from SNCA mutant neurons to wild-type (WT) isogenic controls. Finally, we show that constitutive inactivation of LC3B promotes α-syn accumulation and seeding, while LC3B activation inhibits these events, offering mechanistic insight into the spread of synucleinopathy in PD.

Prevalence of Frailty in Patients Referred to the Kidney Transplant Waitlist.

Background: Comparisons between frailty assessment tools for waitlist candidates are a recognized priority area for kidney transplantation. We compared the prevalence of frailty using three established tools in a cohort of waitlist candidates. Methods: Waitlist candidates were prospectively enrolled from 2016 to 2020 across five centers. Frailty was measured using the Frailty Phenotype (FP), a 37-variable frailty index (FI), and the Clinical Frailty Scale (CFS). The FI and CFS were dichotomized using established cutoffs. Agreement was compared using κ coefficients. Area under the receiver operating characteristic (ROC) curves were generated to compare the FI and CFS (treated as continuous measures) with the FP. Unadjusted associations between each frailty measure and time to death or waitlist withdrawal were determined using an unadjusted Cox proportional hazards model. Results: Of 542 enrolled patients, 64% were male, 80% were White, and the mean age was 54±14 years. The prevalence of frailty by the FP was 16%. The mean FI score was 0.23±0.14, and the prevalence of frailty was 38% (score of ≥0.25). The median CFS score was three (IQR, 2-3), and the prevalence was 15% (score of ≥4). The κ values comparing the FP with the FI (0.44) and CFS (0.27) showed fair to moderate agreement. The area under the ROC curves for the FP and FI/CFS were 0.86 (good) and 0.69 (poor), respectively. Frailty by the CFS (HR, 2.10; 95% CI, 1.04 to 4.24) and FI (HR, 1.79; 95% CI, 1.00 to 3.21) was associated with death or permanent withdrawal. The association between frailty by the FP and death/withdrawal was not statistically significant (HR, 1.78; 95% CI, 0.79 to 3.71). Conclusion: Frailty prevalence varies by the measurement tool used, and agreement between these measurements is fair to moderate. This has implications for determining the optimal frailty screening tool for use in those being evaluated for kidney transplant.

Frailty and the Kidney Transplant Wait List: Protocol for a Multicenter Prospective Study.

BACKGROUND: Understanding how frailty affects patients listed for transplantation has been identified as a priority research need. Frailty may be associated with a high risk of death or wait-list withdrawal, but this has not been evaluated in a large multicenter cohort of Canadian wait-listed patients. OBJECTIVE: The primary objective is to evaluate whether frailty is associated with death or permanent withdrawal from the transplant wait list. Secondary objectives include assessing whether frailty is associated with hospitalization, quality of life, and the probability of being accepted to the wait list. DESIGN: Prospective cohort study. SETTING: Seven sites with established renal transplant programs that evaluate patients for the kidney transplant wait list. PATIENTS: Individuals who are being considered for the kidney transplant wait list. MEASUREMENTS: We will assess frailty using the Fried Phenotype, a frailty index, the Short Physical Performance Battery, and the Clinical Frailty Scale at the time of listing for transplantation. We will also assess frailty at the time of referral to the wait list and annually after listing in a subgroup of patients. METHODS: The primary outcome of the composite of time to death or permanent wait-list withdrawal will be compared between patients who are frail and those who are not frail and will account for the competing risks of deceased and live donor transplantation. Secondary outcomes will include number of hospitalizations and length of stay, and in a subset, changes in frailty severity over time, change in quality of life, and the probability of being listed. Recruitment of 1165 patients will provide >80% power to identify a relative hazard of ≥1.7 comparing patients who are frail to those who are not frail for the primary outcome (2-sided α = .05), whereas a more conservative recruitment target of 624 patients will provide >80% power to identify a relative hazard of ≥2.0. RESULTS: Through December 2019, 665 assessments of frailty (inclusive of those for the primary outcome and all secondary outcomes including repeated measures) have been completed. LIMITATIONS: There may be variation across sites in the processes of referral and listing for transplantation that will require consideration in the analysis and results. CONCLUSIONS: This study will provide a detailed understanding of the association between frailty and outcomes for wait-listed patients. Understanding this association is necessary before routinely measuring frailty as part of the wait-list eligibility assessment and prior to ascertaining the need for interventions that may modify frailty. TRIAL REGISTRATION: Not applicable as this is a protocol for a prospective observational study.

CONTEXTE: La compréhension de l'incidence de la fragilité sur les patients en attente d'une greffe rénale a été désignée comme un besoin prioritaire de recherche. La fragilité pourrait être associée à un risque élevé de mortalité ou de se voir retiré de la liste d'attente pour une transplantation, mais elle n'a jamais été évaluée dans une vaste cohorte multicentrique de patients canadiens en attente d'une greffe. OBJECTIFS: Le principal objectif consiste à déterminer si la fragilité d'un patient l'expose à un plus grand risque de décès ou de retrait permanent de la liste d'attente pour une greffe. Nous souhaitons également vérifier s'il existe un lien entre la fragilité et le nombre d'hospitalisations, la qualité de vie et la probabilité d'être accepté sur la liste d'attente. TYPE D'ÉTUDE: Étude de cohorte prospective. CADRE: Sept sites disposant d'un programme de transplantation rénale évaluant les patients en vue de leur inscription sur la liste d'attente pour une greffe. SUJETS: Des candidats à la liste d'attente pour une transplantation rénale. MESURES: La fragilité sera évaluée à l'aide du Phénotype de Fried (un indice de la fragilité), du test SPPB (Short Physical Performance Battery) et de l'échelle Clinical Frailty Scale au moment de l'inscription sur la liste d'attente pour une transplantation. Nous mesurerons la fragilité des patients de leur orientation vers le programme jusqu'à leur inscription sur la liste, puis sur une base annuelle après leur inclusion dans un sous-groupe de patients. MÉTHODOLOGIE: Le résultat principal, soit un composite du délai avant le décès ou le retrait permanent de la liste, sera comparé entre les patients fragiles et non fragiles, et tiendra compte des risques concurrents découlant de la transplantation selon que l'organe provient d'un donneur vivant ou décédé. Les résultats secondaires comprendront le nombre d'hospitalisations et leur durée, les variations de la fragilité et de la qualité de vie au fil du temps (pour un sous-groupe de patients), de même que les probabilités d'être inscrit sur la liste d'attente. Le recrutement de 1 165 patients nous permettrait d'obtenir un risque relatif d'au moins 1,7 dans plus de 80 % des cas lors de la comparaison des patients fragiles à ceux qui ne le sont pas pour le résultat principal (double erreur alpha = 0,05), alors que ce risque relatif serait de 2,0 avec un objectif de recrutement plus conservateur de 624 patients. RÉSULTATS: Un total de 665 évaluations de la fragilité (tant pour le résultat primaire que pour les résultats secondaires, y compris les mesures répétitives) a été complété en décembre 2019. LIMITES: Les résultats et leur analyse devront tenir compte des possibles variations entre les différents sites en ce qui concerne les processus d'aiguillage et d'inscription sur les listes d'attente pour une greffe. CONCLUSION: Cette étude fournira une compréhension détaillée de l'association entre la fragilité et les résultats cliniques pour les patients en attente d'une greffe. La compréhension de cette association est nécessaire avant d'inclure systématiquement la mesure de la fragilité au processus d'évaluation de l'admissibilité à la liste d'attente et avant d'établir le besoin de procéder à des interventions susceptibles de modifier la fragilité du patient.

Axonal pathology in hPSC-based models of Parkinson's disease results from loss of Nrf2 transcriptional activity at the Map1b gene locus.

While mutations in the SNCA gene (α-synuclein [α-syn]) are causal in rare familial forms of Parkinson's disease (PD), the prevalence of α-syn aggregates in the cortices of sporadic disease cases emphasizes the need to understand the link between α-syn accumulation and disease pathogenesis. By employing a combination of human pluripotent stem cells (hPSCs) that harbor the SNCA-A53T mutation contrasted against isogenic controls, we evaluated the consequences of α-syn accumulation in human A9-type dopaminergic (DA) neurons (hNs). We show that the early accumulation of α-syn in SNCA-A53T hNs results in changes in gene expression consistent with the expression profile of the substantia nigra (SN) from PD patients, analyzed post mortem. Differentially expressed genes from both PD patient SN and SNCA-A53T hNs were associated with regulatory motifs transcriptionally activated by the antioxidant response pathway, particularly Nrf2 gene targets. Differentially expressed gene targets were also enriched for gene ontologies related to microtubule binding processes. We thus assessed the relationship between Nrf2-mediated gene expression and neuritic pathology in SNCA-A53T hNs. We show that SNCA-mutant hNs have deficits in neuritic length and complexity relative to isogenic controls as well as contorted axons with Tau-positive varicosities. Furthermore, we show that mutant α-syn fails to complex with protein kinase C (PKC), which, in turn, results in impaired activation of Nrf2. These neuritic defects result from impaired Nrf2 activity on antioxidant response elements (AREs) localized to a microtubule-associated protein (Map1b) gene enhancer and are rescued by forced expression of Map1b as well as by both Nrf2 overexpression and pharmaceutical activation in PD neurons.

Comparison of the effects of standard vs low-dose prolonged-release tacrolimus with or without ACEi/ARB on the histology and function of renal allografts.

Targeting the renin-angiotensin system and optimizing tacrolimus exposure are both postulated to improve outcomes in renal transplant recipients (RTRs) by preventing interstitial fibrosis/tubular atrophy (IF/TA). In this multicenter, prospective, open-label controlled trial, adult de novo RTRs were randomized in a 2 × 2 design to low- vs standard-dose (LOW vs STD) prolonged-release tacrolimus and to angiotensin-converting enzyme inhibitors/angiotensin II receptor 1 blockers (ACEi/ARBs) vs other antihypertensive therapy (OAHT). There were 2 coprimary endpoints: the prevalence of IF/TA at month 6 and at month 24. IF/TA prevalence was similar for LOW vs STD tacrolimus at month 6 (36.8% vs 39.5%; P = .80) and ACEi/ARBs vs OAHT at month 24 (54.8% vs 58.2%; P = .33). IF/TA progression decreased significantly with LOW vs STD tacrolimus at month 24 (mean [SD] change, +0.42 [1.477] vs +1.10 [1.577]; P = .0039). Across the 4 treatment groups, LOW + ACEi/ARB patients exhibited the lowest mean IF/TA change and, compared with LOW + OAHT patients, experienced significantly delayed time to first T cell-mediated rejection. Renal function was stable from month 1 to month 24 in all treatment groups. No unexpected safety findings were detected. Coupled with LOW tacrolimus dosing, ACEi/ARBs appear to reduce IF/TA progression and delay rejection relative to reduced tacrolimus exposure without renin-angiotensin system blockade. ClinicalTrials.gov identifier: NCT00933231.

Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson's disease.

Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function in dopaminergic (DA) neurons of the substantia nigra pars compacta. An association has been reported between PD onset and exposure to mitochondrial toxins, including the agrochemicals paraquat (PQ), maneb (MB), and rotenone (Rot). Here, with the use of a patient-derived stem cell model of PD, allowing comparison of DA neurons harboring a mutation in the α-synuclein (α-syn) gene ( SNCA-A53T) against isogenic, mutation-corrected controls, we describe a novel mechanism whereby NO, generated from SNCA-A53T mutant neurons exposed to Rot or PQ/MB, inhibits anterograde mitochondrial transport through nitration of α-tubulin (α-Tub). Nitration of α-Tub inhibited the association of both α-syn and the mitochondrial motor protein kinesin 5B with the microtubules, arresting anterograde transport. This was, in part, a result of nitration of α-Tub in the C-terminal domain. These effects were rescued by inhibiting NO synthesis with the NOS inhibitor Nω-nitro-L-arginine methyl ester. Collectively, our results are the first to demonstrate a gene by environment interaction in PD, whereby agrochemical exposure selectively triggers a deficit in mitochondrial transport by nitrating the microtubules in neurons harboring the SNCA-A53T mutation.-Stykel, M. G., Humphries, K., Kirby, M. P., Czaniecki, C., Wang, T., Ryan, T., Bamm, V., Ryan, S. D. Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinson's disease.

Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein.

Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA-transgenic mice, we show that SNCA-mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA-mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA-mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn.

Muscle-relevant genes marked by stable H3K4me2/3 profiles and enriched MyoD binding during myogenic differentiation.

Post-translational modifications of histones play a key role in the regulation of gene expression during development and differentiation. Numerous studies have shown the dynamics of combinatorial regulation by transcription factors and histone modifications, in the sense that different combinations lead to distinct expression outcomes. Here, we investigated gene regulation by stable enrichment patterns of histone marks H3K4me2 and H3K4me3 in combination with the chromatin binding of the muscle tissue-specific transcription factor MyoD during myogenic differentiation of C2C12 cells. Using k-means clustering, we found that specific combinations of H3K4me2/3 profiles over and towards the gene body impact on gene expression and marks a subset of genes important for muscle development and differentiation. By further analysis, we found that the muscle key regulator MyoD was significantly enriched on this subset of genes and played a repressive role during myogenic differentiation. Among these genes, we identified the pluripotency gene Patz1, which is repressed during myogenic differentiation through direct binding of MyoD to promoter elements. These results point to the importance of integrating histone modifications and MyoD chromatin binding for coordinated gene activation and repression during myogenic differentiation.

TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells.

Change in the identity of the components of the transcription pre-initiation complex is proposed to control cell type-specific gene expression. Replacement of the canonical TFIID-TBP complex with TRF3/TBP2 was reported to be required for activation of muscle-gene expression. The lack of a developmental phenotype in TBP2 null mice prompted further analysis to determine whether TBP2 deficiency can compromise adult myogenesis. We show here that TBP2 null mice have an intact regeneration potential upon injury and that TBP2 is not expressed in established C2C12 muscle cell or in primary mouse MuSCs. While TFIID subunits and TBP are downregulated during myoblast differentiation, reduced amounts of these proteins form a complex that is detectable on promoters of muscle genes and is essential for their expression. This evidence demonstrates that TBP2 does not replace TBP during muscle differentiation, as previously proposed, with limiting amounts of TFIID-TBP being required to promote muscle-specific gene expression.

HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles.

Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC-myomiR-BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a "latent" myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles.

Testosterone enhances cardiomyogenesis in stem cells and recruits the androgen receptor to the MEF2C and HCN4 genes.

Since a previous study (Goldman-Johnson et al., 2008 [4]) has shown that androgens can stimulate increased differentiation of mouse embryonic stem (mES) cells into cardiomyocytes using a genomic pathway, the aim of our study is to elucidate the molecular mechanisms regulating testosterone-enhanced cardiomyogenesis. Testosterone upregulated cardiomyogenic transcription factors, including GATA4, MEF2C, and Nkx2.5, muscle structural proteins, and the pacemaker ion channel HCN4 in a dose-dependent manner, in mES cells and P19 embryonal carcinoma cells. Knock-down of the androgen receptor (AR) or treatment with anti-androgenic compounds inhibited cardiomyogenesis, supporting the requirement of the genomic pathway. Chromatin immunoprecipitation (ChIP) studies showed that testosterone enhanced recruitment of AR to the regulatory regions of MEF2C and HCN4 genes, which was associated with increased histone acetylation. In summary, testosterone upregulated cardiomyogenic transcription factor and HCN4 expression in stem cells. Further, testosterone induced cardiomyogenesis, at least in part, by recruiting the AR receptor to the regulatory regions of the MEF2C and HCN4 genes. These results provide a detailed molecular analysis of the function of testosterone in stem cells and may offer molecular insight into the role of steroids in the heart.

ß-catenin is essential for efficient in vitro premyogenic mesoderm formation but can be partially compensated by retinoic acid signalling.

Previous studies have shown that P19 cells expressing a dominant negative ß-catenin mutant (ß-cat/EnR) cannot undergo myogenic differentiation in the presence or absence of muscle-inducing levels of retinoic acid (RA). While RA could upregulate premyogenic mesoderm expression, including Pax3/7 and Meox1, only Pax3/7 and Gli2 could be upregulated by RA in the presence of ß-cat/EnR. However, the use of a dominant negative construct that cannot be compensated by other factors is limiting due to the possibility of negative chromatin remodelling overriding compensatory mechanisms. In this study, we set out to determine if ß-catenin function is essential for myogenesis with and without RA, by creating P19 cells with reduced ß-catenin transcriptional activity using an shRNA approach, termed P19[shß-cat] cells. The loss of ß-catenin resulted in a reduction of skeletal myogenesis in the absence of RA as early as premyogenic mesoderm, with the loss of Pax3/7, Eya2, Six1, Meox1, Gli2, Foxc1/2, and Sox7 transcript levels. Chromatin immunoprecipitation identified an association of ß-catenin with the promoter region of the Sox7 gene. Differentiation of P19[shß-cat] cells in the presence of RA resulted in the upregulation or lack of repression of all of the precursor genes, on day 5 and/or 9, with the exception of Foxc2. However, expression of Sox7, Gli2, the myogenic regulatory factors and terminal differentiation markers remained inhibited on day 9 and overall skeletal myogenesis was reduced. Thus, ß-catenin is essential for in vitro formation of premyogenic mesoderm, leading to skeletal myogenesis. RA can at least partially compensate for the loss of ß-catenin in the expression of many myogenic precursor genes, but not for myoblast gene expression or overall myogenesis.

Myosin phosphatase modulates the cardiac cell fate by regulating the subcellular localization of Nkx2.5 in a Wnt/Rho-associated protein kinase-dependent pathway.

RATIONALE: Nkx2.5 is a transcription factor that regulates cardiomyogenesis in vivo and in embryonic stem cells. It is also a common target in congenital heart disease. Although Nkx2.5 has been implicated in the regulation of many cellular processes that ultimately contribute to cardiomyogenesis and morphogenesis of the mature heart, relatively little is known about how it is regulated at a functional level. OBJECTIVE: We have undertaken a proteomic screen to identify novel binding partners of Nkx2.5 during cardiomyogenic differentiation in an effort to better understand the regulation of its transcriptional activity. METHODS AND RESULTS: Purification of Nkx2.5 from differentiating cells identified the myosin phosphatase subunits protein phosphatase 1ß and myosin phosphatase targeting subunit 1 (Mypt1) as novel binding partners. The interaction with protein phosphatase 1 ß/Mypt1 resulted in exclusion of Nkx2.5 from the nucleus and, consequently, inhibition of its transcriptional activity. Exclusion of Nkx2.5 was inhibited by treatment with leptomycin B and was dependent on an Mypt1 nuclear export signal. Furthermore, in transient transfection experiments, Nkx2.5 colocalized outside the nucleus with phosphorylated Mypt1 in a manner dependent on Wnt signaling and Rho-associated protein kinase. Treatment of differentiating mouse embryonic stem cells with Wnt3a resulted in enhanced phosphorylation of endogenous Mypt1, increased nuclear exclusion of endogenous Nkx2.5, and a failure to undergo terminal cardiomyogenesis. Finally, knockdown of Mypt1 resulted in rescue of Wnt3a-mediated inhibition of cardiomyogenesis, indicating that Mypt1 is required for this process. CONCLUSIONS: We have identified a novel interaction between Nkx2.5 and myosin phosphatase. Promoting this interaction represents a novel mechanism whereby Wnt3a regulates Nkx2.5 and inhibits cardiomyogenesis.

Generic immunosuppression in solid organ transplantation: a Canadian perspective.

The introduction of generic immunosuppressant medications may present an opportunity for cost savings in solid organ transplantation if equivalent clinical outcomes to the branded counterparts can be achieved. An interprofessional working group of the Canadian Society of Transplantation was established to develop recommendations on the use of generic immunosuppression in solid organ transplant recipients (SOTR) based on a review of the available data. Under current Health Canada licensing requirements, a demonstration of bioequivalence with the branded formulation in healthy volunteers allows for bridging of clinical data. Cyclosporine, tacrolimus, and sirolimus are designated as "critical dose drugs" and are held to stricter criteria. However, whether this provides sufficient guarantee of therapeutic equivalence in SOTR remains controversial, and failure to maintain an appropriate balance of immunosuppression may have serious consequences, including rejection, graft loss, and death. Published evidence supporting therapeutic equivalence of generic formulations in SOTR is lacking. Moreover, in the setting of multiple generic formulations the potential for uncontrolled product switching is a major concern, since generic preparations are not required to demonstrate bioequivalence with each other. Although close monitoring is recommended with any change in formulation, drug product switches are likely to occur without prescriber knowledge and may pose a significant patient safety risk. The advent of generic immunosuppression will require new practices including more frequent therapeutic drug and clinical monitoring, and increased patient education. The additional workload placed on transplant centers without additional funding will create challenges and could ultimately jeopardize patient outcomes. Until more robust clinical data are available and adequate regulatory safeguards are instituted, caution in the use of generic immunosuppressive drugs in solid organ transplantation is warranted.

Retinoic acid enhances skeletal myogenesis in human embryonic stem cells by expanding the premyogenic progenitor population.

Human embryonic stem cells (hESCs) are a potential source of material for cell therapy of muscle diseases. To date, it has proven difficult to generate skeletal muscle from hESCs in high yields and within a reasonable timeframe. Further, a hESC-derived Pax3/7-positive skeletal muscle progenitor population has not yet been described. Previous studies have shown that Pax3/7-positive progenitor cells can repopulate the satellite cell niche, indicating the importance of this population for therapy. We sought to optimize the differentiation of hESCs into skeletal muscle in order to characterize myogenesis at a molecular level and shorten the time course. We treated hESCs with retinoic acid (RA) and found an enhancement of skeletal myogenesis, and the expression of the myogenic regulatory factors (MRFs) MyoD and myogenin by day 25. Furthermore, we found that RA treatment expanded the muscle progenitor pool, which occurred as a distinct Pax3(+ve) population prior to MRF expression. Non-skeletal muscle tissue types were not significantly affected. Therefore, we have identified a differentiation pathway in hESCs that provides a skeletal muscle progenitor population which can undergo myogenesis more efficiently. We propose that RA could fit into a directed culture method for deriving skeletal muscle from hESCs.