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L-type [Ca.sup.2+] channels play a critical role in regulating [Ca.sup.2+]-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type [Ca.sup.2+] channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type [Ca.sup.2+] channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for [[beta].sub.2]-adrenergic receptor (AR) regulation of [I.sub.Ca,L-] Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel ([Ca.sub.v]1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-[Ca.sub.v]1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, [Ca.sub.v]1.2, [[beta].sub.2]-AR (not [[beta].sub.1]-AR), G protein [[alpha].sub.s], adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized [[beta].sub.2]-AR/[Ca.sub.v]1.2 signaling complex, [[beta].sub.2]-AR stimulation (salbutamol plus atenolol) of [I.sub.Ca,L] was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of [I.sub.Ca,L] in response to [[beta].sub.2]-AR activation was eliminated by disruption of caveolae with 10 mM methyl [beta]-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas [[beta].sub.1]-AR stimulation (norepinephrine plus prazosin) of [I.sub.Ca,L] was not altered. These findings demonstrate that subcellular localization of L-type [Ca.sup.2+] channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways. caveolae | electrophysiology | ventricular myocyte

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices. [ABSTRACT FROM AUTHOR]

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel, a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e., glycosaminoglycans and integrins), the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation, peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast, surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling, which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK), which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate. [ABSTRACT FROM AUTHOR]

L-type Ca2+ channels play a critical role in regulating Ca2+-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca2+ channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca2+ channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for β2-adrenergic receptor (AR) regulation of ICa,L. Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Cav1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Cav1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Cav1.2, β2-AR (not β1-AR), G protein αs, adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized β2-AR/Cav1.2 signaling complex, β2-AR stimulation (salbutamol plus atenolol) of ICa,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of ICa,L in response to β2-AR activation was eliminated by disruption of caveolae with 10 mM methyl β-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas β1-AR stimulation (norepinephrine plus prazosin) of ICa,L was not altered. These findings demonstrate that subcellular localization of L-type Ca2+ channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways. [ABSTRACT FROM AUTHOR]

The article focuses on canonical Wnt signaling which guides the differentiation of human pluripotent stem cells (hPSCs) into functional human cardiomyocytes. It states that this was used to design a scalable growth factor-free process for the production of pure cardiomyocyte population from hPSCs without the use of cell sorting or selection techniques. Also other paracrine signaling pathways can be induced by small molecules that are used to modulate the signaling of Wnt genes.

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction ( n = 16) compared to nonfailing donor hearts ( n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease., Competing Interests: Competing interest statement: T.J.K. is a consultant for Fujifilm Cellular Dynamics Incorporated., (Copyright © 2020 the Author(s). Published by PNAS.)

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices., Competing Interests: A.Z.A. is the sole member of VistaMotif, LLC, and founder of the nonprofit WINStep Forward.

Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.