RESUMO
In this issue of Cell, Hulsmans et al. identify a subset of macrophages residing within the cardiac conduction system, which orchestrates cardiac rhythm. Macrophages directly couple with cardiomyocytes, and their perturbation alters cardiac conduction, suggesting that pharmacological manipulation of resident macrophages might represent a new strategy to combat cardiac arrhythmias.
Assuntos
Sistema de Condução Cardíaco , Macrófagos , Arritmias Cardíacas , Humanos , Miócitos CardíacosRESUMO
BACKGROUND: Many human gene mutations have been linked to congenital heart disease (CHD), yet CHD remains a major health issue worldwide due in part to an incomplete understanding of the molecular basis for cardiac malformation. RESULTS: Here we identify the orthologous mouse Pou6f1 and zebrafish pouC as POU homeodomain transcription factors enriched in the developing heart. We find that pouC is a multi-functional transcriptional regulator containing separable activation, repression, protein-protein interaction, and DNA binding domains. Using zebrafish heart development as a model system, we demonstrate that pouC knockdown impairs cardiac morphogenesis and affects cardiovascular function. We also find that levels of pouC expression must be fine-tuned to enable proper heart formation. At the cellular level, we demonstrate that pouC knockdown disrupts atrioventricular canal (AVC) cardiomyocyte maintenance, although chamber myocyte specification remains intact. Mechanistically, we show that pouC binds a bmp4 intronic regulatory element to mediate transcriptional activation. CONCLUSIONS: Taken together, our study establishes pouC as a novel transcriptional input into the regulatory hierarchy that drives AVC morphogenesis in zebrafish. We anticipate that these findings will inform future efforts to explore functional conservation in mammals and potential association with atrioventricular septal defects in humans. Developmental Dynamics 248:173-188, 2019. © 2018 Wiley Periodicals, Inc.
Assuntos
Proteína Morfogenética Óssea 4/genética , Regulação da Expressão Gênica no Desenvolvimento , Septos Cardíacos/crescimento & desenvolvimento , Fatores do Domínio POU/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Proteína Morfogenética Óssea 4/metabolismo , Coração/embriologia , Coração/crescimento & desenvolvimento , Defeitos dos Septos Cardíacos , Septos Cardíacos/embriologia , Camundongos , Fatores do Domínio POU/metabolismo , Ligação Proteica , Fatores de Transcrição , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoAssuntos
Cardiomiopatias , Cromatina , Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Cromatina/genética , Humanos , AnamneseRESUMO
Efficient intracellular cargo delivery is a key hurdle for the translation of many emerging stem cell and cellular reprogramming therapies. Recently, a microfluidic-based device constructed from silicon was shown to transduce macromolecules into cells via shear-induced formation of plasma membrane pores. However, the scalability and widespread application of the current platform is limited since physical deformation-mediated delivery must be optimized for each therapeutic application. Therefore, we sought to create a low-cost, versatile device that could facilitate rapid prototyping and application-specific optimization in most academic research labs. Here we describe the design and implementation of a microfluidic device constructed from Polydimethylsiloxane (PDMS) that we call Cyto-PDMS (Cytoplasmic PDMS-based Delivery and Modification System). Using a systematic Cyto-PDMS workflow, we demonstrate intracellular cargo delivery with minimal effects on cellular viability. We identify specific flow rates at which a wide range of cargo sizes (1-70 kDa) can be delivered to the cell interior. As a proof-of-principle for the biological utility of Cyto-PDMS, we show (i) F-actin labeling in live human fibroblasts and (ii) intracellular delivery of recombinant Cre protein with appropriate genomic recombination in recipient fibroblasts. Taken together, our results demonstrate that Cyto-PDMS can deliver small-molecules to the cytoplasm and biologically active cargo to the nucleus without major effects on viability. We anticipate that the cost and versatility of PDMS can be leveraged to optimize delivery to a broad array of possible cell types and thus expand the potential impact of cellular therapies.
Assuntos
Fibroblastos/metabolismo , Dispositivos Lab-On-A-Chip , Actinas/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Dimetilpolisiloxanos , Portadores de Fármacos/química , Desenho de Equipamento , Fibroblastos/citologia , Humanos , Camundongos , Peso MolecularRESUMO
Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.
Assuntos
Diferenciação Celular/fisiologia , Indução Embrionária/fisiologia , Fibroblastos/citologia , Coração/embriologia , Miócitos Cardíacos/fisiologia , Fatores de Transcrição/metabolismo , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Primers do DNA/genética , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Imuno-Histoquímica , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genéticaRESUMO
The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Sistema de Condução Cardíaco/crescimento & desenvolvimento , Animais , Nó Atrioventricular/crescimento & desenvolvimento , Nó Atrioventricular/metabolismo , Genótipo , Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Cardiopatias/genética , Cardiopatias/fisiopatologia , Humanos , Fenótipo , Ramos Subendocárdicos/crescimento & desenvolvimento , Ramos Subendocárdicos/metabolismo , Nó Sinoatrial/crescimento & desenvolvimento , Nó Sinoatrial/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity.
Assuntos
Sistema de Condução Cardíaco , Proteínas de Homeodomínio , Miócitos Cardíacos , Proteínas com Domínio T , Animais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Sistema de Condução Cardíaco/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Animais Recém-Nascidos , Análise de Célula Única , Transcriptoma , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/fisiologia , Nó Atrioventricular/metabolismo , Nó Sinoatrial/metabolismo , Fascículo Atrioventricular/metabolismoAssuntos
Coração/fisiologia , Regeneração , Animais , Linhagem da Célula , Reprogramação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismoRESUMO
Damage to the atrioventricular conduction system (AVCS), the main electrical connection between the atrial and ventricular chambers, can result in a variety of cardiac conduction disorders. Here, we provide a protocol for selective damage of the mouse AVCS to study its response during injury. We describe tamoxifen-induced cellular ablation, detection of AV block through electrocardiography, and quantification of histological and immunofluorescence markers to analyze the AVCS. This protocol can be used to study mechanisms associated with AVCS injury repair and regeneration. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).1.
Assuntos
Nó Atrioventricular , Eletrocardiografia , Animais , Camundongos , Nó Atrioventricular/cirurgiaRESUMO
Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHDs). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.
Assuntos
Cardiopatias Congênitas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Diferenciação Celular/genética , Cardiopatias Congênitas/genéticaRESUMO
Comprehensive cis-regulatory landscapes are essential for accurate enhancer prediction and disease variant mapping. Although cis-regulatory element (CRE) resources exist for most tissues and organs, many rare - yet functionally important - cell types remain overlooked. Despite representing only a small fraction of the heart's cellular biomass, the cardiac conduction system (CCS) unfailingly coordinates every life-sustaining heartbeat. To globally profile the mouse CCS cis-regulatory landscape, we genetically tagged CCS component-specific nuclei for comprehensive assay for transposase-accessible chromatin-sequencing (ATAC-Seq) analysis. Thus, we established a global CCS-enriched CRE database, referred to as CCS-ATAC, as a key resource for studying CCS-wide and component-specific regulatory functions. Using transcription factor (TF) motifs to construct CCS component-specific gene regulatory networks (GRNs), we identified and independently confirmed several specific TF sub-networks. Highlighting the functional importance of CCS-ATAC, we also validated numerous CCS-enriched enhancer elements and suggested gene targets based on CCS single-cell RNA-Seq data. Furthermore, we leveraged CCS-ATAC to improve annotation of existing human variants related to cardiac rhythm and nominated a potential enhancer-target pair that was dysregulated by a specific SNP. Collectively, our results established a CCS-regulatory compendium, identified novel CCS enhancer elements, and illuminated potential functional associations between human genomic variants and CCS component-specific CREs.
Assuntos
Núcleo Celular , Cromatina , Sistema de Condução Cardíaco , Contração Miocárdica , Animais , Humanos , Camundongos , Núcleo Celular/genética , Cromatina/genética , Redes Reguladoras de Genes , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Fatores de Transcrição/genética , Sistema de Condução Cardíaco/fisiologiaRESUMO
The cardiac conduction system (CCS) ensures regular contractile function, and injury to any of its components can cause cardiac dysrhythmia. Although all cardiomyocytes (CMs) originate from common progenitors, the CCS is composed of biologically distinct cell types with unique functional and developmental characteristics. In contrast to ventricular cardiomyocytes, which continue to proliferate after birth, most CCS cells terminally exit the cell cycle during fetal development. Although the CCS should thus provide a poor substrate for postnatal injury repair, its regenerative capacity remains untested. Here, we describe a genetic system for ablating CMs that reside within the atrioventricular conduction system (AVCS). Adult mouse AVCS ablation resulted in regenerative failure characterized by persistent atrioventricular conduction defects and contractile dysfunction. In contrast, AVCS injury in neonatal mice led to recovery in a subset of these mice, thus providing evidence for CCS plasticity. Furthermore, CM proliferation did not appear to completely account for the observed functional recovery, suggesting that mechanisms regulating recovery from dysrhythmia are likely to be distinct from cardiac regeneration associated with ventricular injury. Taken together, we anticipate that our results will motivate further mechanistic studies of CCS plasticity and enable the exploration of rhythm restoration as an alternative therapeutic strategy.
Assuntos
Nó Atrioventricular/lesões , Miócitos Cardíacos/fisiologia , Regeneração/fisiologia , Animais , Nó Atrioventricular/fisiologia , Plasticidade Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The cardiac conduction system initiates and propagates each heartbeat. Specialized conducting cells are a well-conserved phenomenon across vertebrate evolution, although mammalian and avian species harbor specific components unique to organisms with four-chamber hearts. Early histological studies in mammals provided evidence for a dominant pacemaker within the right atrium and clarified the existence of the specialized muscular axis responsible for atrioventricular conduction. Building on these seminal observations, contemporary genetic techniques in a multitude of model organisms has characterized the developmental ontogeny, gene regulatory networks, and functional importance of individual anatomical compartments within the cardiac conduction system. This review describes in detail the transcriptional and regulatory networks that act during cardiac conduction system development and homeostasis with a particular emphasis on networks implicated in human electrical variation by large genome-wide association studies. We conclude with a discussion of the clinical implications of these studies and describe some future directions.
Assuntos
Sistema de Condução Cardíaco/embriologia , Sistema de Condução Cardíaco/fisiologia , Animais , Sistema Nervoso Autônomo/fisiologia , Eletrocardiografia , Junções Comunicantes/fisiologia , HumanosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.
Assuntos
Núcleo Celular/química , Separação Celular/métodos , Animais , Núcleo Celular/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Imunoprecipitação , Magnetismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Miocárdio/metabolismoRESUMO
The atrioventricular node (AVN) coordinates the timing of atrial and ventricular contraction to optimize cardiac performance. To study this critical function using mouse genetics, however, new reagents are needed that allow AVN-specific manipulation. Here we describe a novel Gjd3-CreEGFP mouse line that successfully recombines floxed alleles within the AVN beginning at E12.5. These mice have been engineered to express CreEGFP under the control of endogenous Gjd3 regulatory elements without perturbing native protein expression. Detailed histological analysis of Gjd3-CreEGFP mice reveals specific labeling of AVN cardiomyocytes and a subset of cardiac endothelial cells. Importantly, we show that Gjd3-CreEGFP mice have preserved cardiac mechanical and electrical function. In one application of our newly described mouse line, we provide a three-dimensional (3D) view of the AVN using tissue clearing combined with confocal microscopy. With this 3D model as a reference, we identify specific AVN sub-structures based on marker staining characteristics. In addition, we use our Gjd3-CreEGFP mice to guide microdissection of the AVN and construction of a single-cell atlas. Thus, our results establish a new transgenic tool for AVN-specific recombination, provide an updated model of AVN morphology, and describe a roadmap for exploring AVN cellular heterogeneity.
Assuntos
Potenciais de Ação , Nó Atrioventricular/citologia , Nó Atrioventricular/fisiologia , Conexinas/fisiologia , Células Endoteliais/citologia , Receptores ErbB/metabolismo , Miócitos Cardíacos/citologia , Animais , Células Endoteliais/metabolismo , Receptores ErbB/genética , Técnicas de Introdução de Genes , Átrios do Coração/citologia , Átrios do Coração/fisiopatologia , Integrases/metabolismo , Camundongos , Miócitos Cardíacos/metabolismoRESUMO
Ectopic expression of transcription factors (TFs) can reprogram cell state. However, because of the large combinatorial space of possible TF cocktails, it remains difficult to identify TFs that reprogram specific cell types. Here, we develop Reprogram-Seq to experimentally screen thousands of TF cocktails for reprogramming performance. Reprogram-Seq leverages organ-specific cell-atlas data with single-cell perturbation and computational analysis to predict, evaluate, and optimize TF combinations that reprogram a cell type of interest. Focusing on the cardiac system, we perform Reprogram-Seq on MEFs using an undirected library of 48 cardiac factors and, separately, a directed library of 10 epicardial-related TFs. We identify a combination of three TFs, which efficiently reprogram MEFs to epicardial-like cells that are transcriptionally, molecularly, morphologically, and functionally similar to primary epicardial cells. Reprogram-Seq holds promise to accelerate the generation of specific cell types for regenerative medicine.
Assuntos
Reprogramação Celular/genética , Biologia Computacional/métodos , Pericárdio/citologia , Pericárdio/metabolismo , Análise de Célula Única/métodos , Fatores de Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , RNA-Seq , Software , Fatores de Transcrição/genéticaRESUMO
Direct reprogramming of fibroblasts into cardiomyocytes is a promising approach for cardiac regeneration but still faces challenges in efficiently generating mature cardiomyocytes. Systematic optimization of reprogramming protocols requires scalable, objective methods to assess cellular phenotype beyond what is captured by transcriptional signatures alone. To address this question, we automatically segmented reprogrammed cardiomyocytes from immunofluorescence images and analyzed cell morphology. We also introduce a method to quantify sarcomere structure using Haralick texture features, called SarcOmere Texture Analysis (SOTA). We show that induced cardiac-like myocytes (iCLMs) are highly variable in expression of cardiomyocyte markers, producing subtypes that are not typically seen in vivo. Compared to neonatal mouse cardiomyocytes, iCLMs have more variable cell size and shape, have less organized sarcomere structure, and demonstrate reduced sarcomere length. Taken together, these results indicate that traditional methods of assessing cardiomyocyte reprogramming by quantifying induction of cardiomyocyte marker proteins may not be sufficient to predict functionality. The automated image analysis methods described in this study may enable more systematic approaches for improving reprogramming techniques above and beyond existing algorithms that rely heavily on transcriptome profiling.
Assuntos
Reprogramação Celular , Fibroblastos/citologia , Processamento de Imagem Assistida por Computador/métodos , Miócitos Cardíacos/citologia , Análise de Célula Única/métodos , Algoritmos , Animais , Células Cultivadas , CamundongosRESUMO
Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.