Development and Program Implementation of an Algorithm to Estimate the Mean Sarcomere Length of a Cardiomyocyte

Myachina, Tatiana; Butova, Xenia; Lookin, Oleg

doi:10.1134/s0006350919050178

Cited by 8 publications

(7 citation statements)

References 15 publications

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“…Observed data demonstrate pronounced regional differences in the amplitude and the time course of calcium transient and sarcomere shortening between the chambers consistently with [10] , [11] , [12] ( Fig. 5 ).…”

Section: Reagentssupporting

confidence: 60%

“…The emitted fluorescence was collected every 1–3 ms in the continuous scanning mode at 493–575 nm in a narrow (3 pixels high, 200–500 pixels long) region horizontally oriented along the long axis in the center of a cardiomyocyte. The change in fluorescence intensity (ΔF/F 0 , where F 0 is the initial fluorescence) was calculated and used as an index of the change in cell cytosolic calcium concentration using custom-made software EqapAll 6 [10] .…”

Section: Reagentsmentioning

confidence: 99%

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A combined Langendorff-injection technique for simultaneous isolation of single cardiomyocytes from atria and ventricles of the rat heart

2021

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Single cardiomyocytes are widely used for investigations of the cellular and molecular mechanisms of regulation and modulation of cardiac performance. Intact cardiomyocytes allow one to study in detail cell function avoiding the effects of extracellular matrix and neighboring cells. The most established protocols of cardiomyocyte isolation are based on the isolated heart perfusion using a Langendorff-apparatus or on intraventricular perfusion using a syringe. However, the yield of single cardiomyocytes obtained by these methods may be low due to the cell injury following non-uniform enzyme digestion of connective tissue in different heart chambers. Moreover, isolation of atrial cardiomyocytes is challenging because of their small size and complex geometric shape. Here we present a new protocol for simultaneous isolation of high quality cardiomyocytes from the atria, ventricular free walls and interventricular septum. The protocol is based on the combination of the Langendorff perfusion method with the intraventricular and intra-atrial injection technique taking into account the collagen content variation between the different heart chambers. Obtained cells demonstrate rod-shaped morphology, a clear and regular sarcomere striation pattern and rat-specific frequency-dependence of contraction and calcium transient parameters. Our protocol provides gentle cell isolation that increases the yield of single cardiomyocytes suitable for biophysical researches .

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Section: Reagentssupporting

confidence: 60%

Section: Reagentsmentioning

confidence: 99%

A combined Langendorff-injection technique for simultaneous isolation of single cardiomyocytes from atria and ventricles of the rat heart

2021

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“…For measurements of sarcomere length (SL), the image intensity profile in a selected narrow area (3 pixel high), horizontally oriented along a cell long axis, was recorded every 1–3 msec in the optical channel. To capture changes in SL during cell contraction/relaxation, a custom-made software (EqapAll6) was used [ 59 , 60 ]. Cells with obvious sarcolemmal blebs or spontaneous contractions were not used for mechanical recording.…”

Section: Methodsmentioning

confidence: 99%

The Acute Effects of Leptin on the Contractility of Isolated Rat Atrial and Ventricular Cardiomyocytes

Khokhlova

Myachina

Butova

et al. 2022

IJMS

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Leptin is a pleiotropic peptide playing an important role in the regulation of cardiac functions. It is not clear whether leptin directly modulates the mechanical function of atrial cardiomyocytes. We compared the acute effects of leptin on the characteristics of mechanically non-loaded sarcomere shortening and cytosolic Ca2+ concentration ([Ca2+]i) transients in single rat atrial and ventricular cardiomyocytes. We also studied the functional properties of myosin obtained from cardiomyocytes using an in vitro motility assay and assessed the sarcomeric protein phosphorylation. Single cardiomyocytes were exposed to 5, 20, and 60 nM leptin for 60 min. In ventricular cardiomyocytes, 60 nM leptin depressed sarcomere shortening amplitude and decreased the rates of shortening and relaxation. These effects were accompanied by a decrease in the phosphorylation of cMyBP-C, an increase in Tpm phosphorylation, and a slowdown of the sliding velocity of thin filaments over myosin in the in vitro motility assay. In contrast, in atrial cardiomyocytes, the phosphorylation of cMyBP-C and TnI increased, and the characteristics of sarcomere shortening did not change. Leptin had no effect on the characteristics of [Ca2+]i transients in ventricular cardiomyocytes, while 5 nM leptin prolonged [Ca2+]i transients in atrial cardiomyocytes. Thus, leptin-induced changes in contractility of ventricular cardiomyocytes may be attributed to the direct effects of leptin on cross-bridge kinetics and sarcomeric protein properties rather than changes in [Ca2+]i. We also suggest that the observed differences between atrial and ventricular cardiomyocytes may be associated with the peculiarities of the expression of leptin receptors, as well as signaling pathways in the atrial and ventricular myocardium.

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“…Prior to the measurement, a narrow region of scanning was selected on the cell image and the intensity profile was recorded. Data signal was then processed and the mean sarcomere length was derived from the striation pattern based on Discrete Fourier Transform using custom made software [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Generative adversarial networks for construction of virtual populations of mechanistic models: simulations to study Omecamtiv Mecarbil action

Parikh

Rumbell

Butova

et al. 2021

J Pharmacokinet Pharmacodyn

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Biophysical models are increasingly used to gain mechanistic insights by fitting and reproducing experimental and clinical data. The inherent variability in the recorded datasets, however, presents a key challenge. In this study, we present a novel approach, which integrates mechanistic modeling and machine learning to analyze in vitro cardiac mechanics data and solve the inverse problem of model parameter inference. We designed a novel generative adversarial network (GAN) and employed it to construct virtual populations of cardiac ventricular myocyte models in order to study the action of Omecamtiv Mecarbil (OM), a positive cardiac inotrope. Populations of models were calibrated from mechanically unloaded myocyte shortening recordings obtained in experiments on rat myocytes in the presence and absence of OM. The GAN was able to infer model parameters while incorporating prior information about which model parameters OM targets. The generated populations of models reproduced variations in myocyte contraction recorded during in vitro experiments and provided improved understanding of OM’s mechanism of action. Inverse mapping of the experimental data using our approach suggests a novel action of OM, whereby it modifies interactions between myosin and tropomyosin proteins. To validate our approach, the inferred model parameters were used to replicate other in vitro experimental protocols, such as skinned preparations demonstrating an increase in calcium sensitivity and a decrease in the Hill coefficient of the force–calcium (F–Ca) curve under OM action. Our approach thereby facilitated the identification of the mechanistic underpinnings of experimental observations and the exploration of different hypotheses regarding variability in this complex biological system.

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Development and Program Implementation of an Algorithm to Estimate the Mean Sarcomere Length of a Cardiomyocyte

Cited by 8 publications

References 15 publications

A combined Langendorff-injection technique for simultaneous isolation of single cardiomyocytes from atria and ventricles of the rat heart

A combined Langendorff-injection technique for simultaneous isolation of single cardiomyocytes from atria and ventricles of the rat heart

The Acute Effects of Leptin on the Contractility of Isolated Rat Atrial and Ventricular Cardiomyocytes

Generative adversarial networks for construction of virtual populations of mechanistic models: simulations to study Omecamtiv Mecarbil action

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