Approaches to High-Throughput Analysis of Cardiomyocyte Contractility

Abstract

The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca 2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points

Local hyperactivation of L-type Ca2+ channels increases spontaneous Ca2+ release activity and cellular hypertrophy in right ventricular myocytes from heart failure rats

“…Cardiomyocytes attached to laminin were loaded with 1uM of Fura-2AM. CytoCypher system setup and operation has been previously described 41 . Cells were paced at 0.5 Hz and sarcomere shortening and calcium transient were recorded for multiple cells selected.…”

Small Molecules Acting on Myofilaments as Treatments for Heart and Skeletal Muscle Diseases

“…Nebivolol selectively desensitised permeabilised cardiac muscle from Mybpc3-targeted knock-in (KI) cardiomyopathy mouse model without resulting in any significant change in contractility [ 95 ]. The drug also had a negative impact on shortening in the cardiomyocyte, while causing a slower contraction and relaxation [ 96 ].…”

Feature Detection to Segment Cardiomyocyte Nuclei for Investigating Cardiac Contractility

“…Cardiomyocytes are ellipsoid shaped units that occupy significant myocardial architectural volume and are fundamentally responsible for myocardial contractility 33,34 . Precise contractile evaluation of the myocardial contractility requires isolation of the individual cardiomyocytes from the surrounding tissue 34 .…”

“…Precise contractile evaluation of the myocardial contractility requires isolation of the individual cardiomyocytes from the surrounding tissue 34 . Current myocardial deformation methods involving cardiomyocyte volume isolation by heart extraction of rat, mouse, or guinea pig models suffer from the limited investigation into tissue due to histological section thickness 33 . Consequently, statistical significance is hard to prove due to the limited sampling size and lifetime of viable cells 33 .…”

“…Current myocardial deformation methods involving cardiomyocyte volume isolation by heart extraction of rat, mouse, or guinea pig models suffer from the limited investigation into tissue due to histological section thickness 33 . Consequently, statistical significance is hard to prove due to the limited sampling size and lifetime of viable cells 33 . However, recent advancements in non-invasive cardiac optical sectioning microscopy 11,13,18,35 and transgenic zebrafish generation have enabled accurate in vivo 4D cardiac contractility quantification by cardiomyocyte nuclei tracking analysis 18,35 .…”