Abstract
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.
Highlights
Cardiomyocytes have limited regenerative capacity in the adult heart, and following a myocardial infarction many cardiomyocytes are irreversibly lost[1]
Cell–sarcomere misalignment was lower in the more elongated CMs, a relationship not seen in the induced cardiac-like myocytes (iCLMs). This is possibly due to a coordinated effort in endogenous cardiomyocyte development to place new sarcomeres along the leading edge of the cell[26]. These methods introduce a new framework for using multiple immunofluorescence channels to automatically segment cells and analyze both morphological and cytoskeletal features of neonatal mouse and fibroblast-reprogrammed cardiomyocytes
The segmentation algorithm can be used without prior manual cell type classification, as it was with the reprogrammed cardiomyocytes
Summary
Cardiomyocytes have limited regenerative capacity in the adult heart, and following a myocardial infarction many cardiomyocytes are irreversibly lost[1]. We developed a method, based on offset distance–angle distributions of Haralick texture features, called SarcOmere Texture Analysis (SOTA) Using this method, we found that current methods of direct reprogramming generate cardiomyocytes with less organized sarcomeres, shorter sarcomere lengths, as well as an apparent lack of coordination between cellular elongation and sarcomere alignment. We found that current methods of direct reprogramming generate cardiomyocytes with less organized sarcomeres, shorter sarcomere lengths, as well as an apparent lack of coordination between cellular elongation and sarcomere alignment These new automated image analysis methods may facilitate quantitative screening of experimental protocols that further enhance the efficiency and fidelity of cardiomyocyte reprogramming
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