Dataset Information

In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.

ABSTRACT: Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ systems. Our goal was to develop methods for intravital imaging of cardiac structural and calcium dynamics with microscopic resolution. Methods: Ventilated mice expressing GCaMP6f, a genetically encoded calcium indicator, received a thoracotomy to provide optical access to the heart. Vasculature was labeled with an injection of dextran-labeled dye. The heart was partially stabilized by a titanium probe with a glass window. Images were acquired at 30 frames per second with spontaneous heartbeat and continuously running, ventilated breathing. The data were reconstructed into three-dimensional volumes showing tissue structure, vasculature, and GCaMP6f signal in cardiomyocytes as a function of both the cardiac and respiratory cycle. Results: We demonstrated the capability to simultaneously measure calcium transients, vessel size, and tissue displacement in three dimensions with micrometer resolution. Reconstruction at various combinations of cardiac and respiratory phase enabled measurement of regional and single-cell cardiomyocyte calcium transients (GCaMP6f fluorescence). GCaMP6f fluorescence transients in individual, aberrantly firing cardiomyocytes were also quantified. Comparisons of calcium dynamics (rise-time and tau) at varying positions within the ventricle wall showed no significant depth dependence. Conclusion: This method enables studies of coupling between contraction and excitation during physiological blood perfusion and breathing at high spatiotemporal resolution. These capabilities could lead to a new understanding of normal and disease function of cardiac cells.

SUBMITTER: Jones JS

PROVIDER: S-EPMC6079295 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.

Jones Jason S JS Small David M DM Nishimura Nozomi N

Frontiers in physiology 20180731

Background: Understanding the microscopic dynamics of the beating heart has been challenging due to the technical nature of imaging with micrometer resolution while the heart moves. The development of multiphoton microscopy has made in vivo, cell-resolved measurements of calcium dynamics and vascular function possible in motionless organs such as the brain. In heart, however, studies of in vivo interactions between cells and the native microenvironment are behind other organ ...[more]

PMID: 30108510

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Project description:In vivo models of cardiac function maintain the complex relationship of cardiomyocytes with other heart cells, as well as the paracrine and mechanoelectrical feedback mechanisms. We aimed at imaging calcium transients simultaneously with heart contraction in zebrafish larvae. Methods: To image calcium in beating hearts, we generated a zebrafish transgenic line expressing the FRET-based ratiometric biosensor Twitch-4. Since emission ratioing canceled out the motion artifacts, we did not use myosin inhibitors or tnnt2a morpholinos to uncouple contraction from changes in calcium levels. We wrote an analysis program to automatically calculate kinetic parameters of the calcium transients. In addition, the ventricular diameter was determined in the fluorescence images providing a real-time measurement of contraction correlated with calcium. Results: Expression of Twitch-4 did not affect the force of contraction, the size of the heart nor the heart rate in 3- and 5-days post-fertilization (dpf) larvae. Comparison of 3 and 5 dpf larvae showed that calcium levels and transient amplitude were larger at 5 dpf, but the fractional shortening did not change. To validate the model, we evaluated the effect of drugs with known effects on cardiomyocytes. Calcium levels and the force of contraction decreased by the L-type calcium channel blocker nifedipine, whereas they increased with the activator Bay-K 8644. Caffeine induced bradycardia, markedly decreased ventricular diastolic calcium levels, increased the size of the calcium transients, and caused an escape rhythm in some larvae. Conclusions: The Tg(myl7:Twitch-4) line provides a physiological approach to image systolic and diastolic calcium levels in the heart of zebrafish larvae. Since the heart is beating, calcium levels and contraction can be correlated. This line will be a useful tool to address pathophysiological mechanisms in diseases like heart failure and arrhythmia, in cardiotoxicity studies and for drug screening.

Dataset Information

In Vivo Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.

Publications

<i>In Vivo</i> Calcium Imaging of Cardiomyocytes in the Beating Mouse Heart With Multiphoton Microscopy.

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