Intravital imaging of cardiac function at the single-cell level

Abstract

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.intravital micoscopy | molecular imaging | cardiovascular imaging | fluorescence | pacing W ith knowledge of the molecular bases for cardiovascular diseases expanding rapidly, a considerable void exists in our ability to phenotype heart function at the subcellular scale in vivo and in real time. Understanding how the fundamental unit of myocardial function, the cardiomyocyte, responds, adapts, and ultimately fails in response to stress, both individually and in a network of cells contributing to whole-organ function, is central to unraveling mechanisms of disease and designing novel therapies aimed at molecular pathways. The ability to measure single cardiomyocyte contractile function in vivo in the native environment is not possible using existing techniques.Optical microscopy has potential to assess cardiomyocyte structure and function in rodent models (1). Intravital confocal and two-photon microscopy have been used in combination with fluorescence molecular imaging probes in cancer research, immunology, and the neurosciences to reveal biological processes at the cellular level in living organisms (2). Application of intravital techniques for imaging the beating heart in rodent models has been significantly limited by motion from cardiac contraction and respiration, and most studies as a result have used noncontracting Langendorf heart preparations (3-10) or transplanted heart models (11). These model systems do not allow investigation of cardiomyocyte biology in the native heart under physiologic conditions. A few studies have achieved intravital imaging in orthotopic hearts at relatively modest spatial and temporal resolutions that prevent visualization of subcellular structures. Essential to these techniques are methods of macrostabilization such as affixing the heart with sutures (12), compressing the heart with a coverslip (11, 13), bonding the heart with a mechanical stabilizer (14), or suction-based devices (15,16). Although tissue stabilization methods alone can facilitate very-low-resolution cardiac imaging (e.g., microvasculature, cellular recruitment, and flow), they do not achieve the necessary temporal or spatial resolution for subcellular imaging of cardiomyocytes throughout the cardiac cycle. To f...

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