![]() It is possible to isolate cells from fixed cardiac tissue, using 12.5 mol/L aqueous potassium hydroxide 6 however, the yield is low and the data generated does not match that obtained from enzymatically isolated cardiomyocytes from unfixed fresh tissue 7. Cellular isolation from fixed, archival tissue is difficult. Not only does this approach prevent the examination of the cardiomyocytes in situ, but there is also the possibility that the isolation process alters cell dimensions and volume. Obtaining enzymatically isolated cells usually requires fresh cardiac tissue. Enzymatic isolation is essential for studies employing cell culture, flow cytometry, or ex-vivo analysis of calcium signalling by confocal microscopy. In order to characterise the growth and properties of individual cardiomyocytes within the myocardium there are currently two methodological procedures that can be employed one is to enzymatically isolate cardiomyocytes from fresh unfixed heart tissue using a collagenase enzyme 5 and the other is to fix and embed tissue samples in paraffin, OCT (for frozen sectioning), or resin.Įnzymatically isolated cardiomyocytes can be used for the measurement of cell volume, nuclearity (number of nuclei per cell), and ploidy (number of genome copies per nucleus). Given that abnormal growth of the myocardium, particularly ventricular hypertrophy, is strongly linked to adverse cardiovascular events in later life 4, it is important to understand how cardiac muscle grows in early life and how it responds to cardiovascular disease and injury. Cardiomyocytes are replaced at a rate of 5% per year at 15 years and 0.5% at 60 years 2 however, the additional and replacement cardiomyocytes do not have the capacity to repair the heart after injury 3. Development of cardiac muscle is a far more dynamic process than previously realised in humans, the total number of cardiomyocytes triples between birth and 20 years of age 1. ![]() Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.Ĭardiomyocytes are the individual functional units of cardiac muscle, providing the contractile power of the heart. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. Previous methods have been restricted to thin sections (2–10 μm) and offer an incomplete picture of cardiomyocytes. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4′,6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. ![]() Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell).
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