analysis-cardiac-chamber-development-during-mouse-embryogenesis-using

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

assessing-cardiac-reprogramming-using-high-content-imaging-analysis

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Vanderbilt University Medical Center

Vanderbilt University

The Promise of Cardiac Regeneration by In Situ Lineage Conversion.

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.

High content analysis identifies unique morphological features of reprogrammed cardiomyocytes.

MLC-2v is a myosin light chain regulatory protein which is specifically expressed in ventricular cardiomyocytes and slow twitch skeletal muscle cells. MLC-2v plays critical roles in ventricular maturation during heart development. Mice lacking MLC-2v are embryonic lethal due to heart failure associated with abnormal myofibrillar organization of ventricular cardiomyocytes. To study the development of ventricular cardiac muscle and slow twitch skeletal muscle, we generated a new MLC-2v reporter mouse line by knocking-in a tdTomato reporter cassette into 3' UTR of the MLC-2v gene without disrupting the endogenous gene. Our results demonstrated specific MLC-2v-tdTomato knock-in reporter expression in ventricular cardiomyocytes and slow twitch muscle during myogenesis, precisely recapitulating the spatiotemporal expression pattern of endogenous MLC-2v. No tdTomato expression was observed in the atria, fast twitch muscle or other organs throughout development into adulthood. Isolated neonatal and adult ventricular cardiomyocytes uniformly express tdTomato. Taken together, MLC-2v-tdTomato knock-in reporter mouse model described in this article will serve as a valuable tool to study cardiac chamber and skeletal muscle specification during development and regeneration by overcoming the pitfalls of transgenic strategies.

Generation of MLC-2v-tdTomato knock-in reporter mouse line.

Nppa is a cardiac hormone which plays critical roles in regulating salt-water balance. Its expression is restricted to the atria of the healthy post-natal heart. During heart development, spatio-temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re-expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking-in a tagBFP reporter cassette into 3'-UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa-tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa-tagBFP reporter knock-in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.

Generation of Nppa-tagBFP reporter knock-in mouse line for studying cardiac chamber specification.

Previous studies have shown that forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs). This cardiac reprogramming approach suggests a potential strategy for cardiomyocyte regeneration. However, a major challenge of this approach remains the low conversion rate. Here, we showed that ensuring expression of four cardiogenic transcription factors (i.e. Gata4 (G), Hand2 (H), Mef2c (M), and Tbx5 (T)) in individual fibroblasts is an initial bottleneck for cardiac reprogramming. Following co-transduction of three or four retroviral vectors encoding individual cardiogenic transcription factors, only a minor subpopulation of cells indeed expressed all three (GMT) or four (GHMT) factors. By selectively analyzing subpopulations of cells expressing various combinations of reprogramming factors, we found that co-expression of GMT in individual fibroblasts is sufficient to induce sarcomeric proteins. However, only a small fraction of those cells expressing GMT were able to develop organized sarcomeric structures and contractility. In contrast, ensuring expression of GHMT markedly enhanced the development of contractile cardiac structures and functions in fibroblasts, although its incremental effect on sarcomeric protein induction was relatively small. Our findings provide new insights into the mechanistic basis of inefficient cardiac reprogramming and can help to devise efficient reprogramming strategies.

Ensuring expression of four core cardiogenic transcription factors enhances cardiac reprogramming.

Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown.

Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.

Reprogramming of fibroblasts to induced cardiomyocyte-like cells (iCMs) offers potential strategies for new cardiomyocyte generation. However, a major challenge of this approach remains its low efficiency for contractile iCMs. Here, we showed that controlled stoichiometric expression of Gata4 (G), Hand2 (H), Mef2c (M), and Tbx5 (T) significantly enhanced contractile cardiomyocyte reprogramming over previously defined stoichiometric expression of GMT or uncontrolled expression of GHMT. We generated quad-cistronic vectors expressing distinct relative protein levels of GHMT within the context of a previously defined splicing order of M-G-T with high Mef2c level. Transduction of the quad-cistronic vector with a splicing order of M-G-T-H (referred to as M-G-T-H) inducing relatively low Hand2 and high Mef2c protein levels not only increased sarcomeric protein induction, but also markedly promoted the development of contractile structures and functions in fibroblasts. The expressed Gata4 and Tbx5 protein levels by M-G-T-H transduction were relatively higher than those by transductions of other quad-cistronic vectors, but lower than those by previously defined M-G-T tri-cistronic vector transduction. Taken together, our results demonstrate the stoichiometric requirement of GHMT expression for structural and functional progresses of cardiomyocyte reprogramming and provide a new basic tool-set for future studies.

ArticleTotal : 2	Year
Publication title Cited by 1	2019
Publication title Cited by 4	2020

Article	Year
The Promise of Cardiac Regeneration by In Situ Lineage Conversion. Circulation\| PubMed ID: 28264888	2017
High content analysis identifies unique morphological features of reprogrammed cardiomyocytes. Scientific reports\| PubMed ID: 29352247	2018
Generation of MLC-2v-tdTomato knock-in reporter mouse line. Genesis (New York, N.Y. : 2000)\| PubMed ID: 30307112	2018
Generation of Nppa-tagBFP reporter knock-in mouse line for studying cardiac chamber specification. Genesis (New York, N.Y. : 2000)\| PubMed ID: 30920727	2019
Ensuring expression of four core cardiogenic transcription factors enhances cardiac reprogramming. Scientific reports\| PubMed ID: 31019236	2019
Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling. Circulation\| PubMed ID: 31581792	2019
Stoichiometric optimization of Gata4, Hand2, Mef2c, and Tbx5 expression for contractile cardiomyocyte reprogramming. Scientific reports\| PubMed ID: 31628386	2019

Young-Jae Nam

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