All mammals experience a slowdown of cardiac pacemaker rate with aging. The main mechanisms to explain that phenomenon are related to alterations in the ionic currents that underlie the diastolic depolarization phase of the action potential. We have previously reported that pacemaker cells from old mice have reduced L-type calcium currents. We further explore the mechanism underlying that reduction, testing cell hypertrophy and alteration in the scaffolding of L-type calcium channels as potential mechanisms. To test for cell hypertrophy, we combined immunostaining and high-resolution imaging to map the HCN4-positive pacemaker region of isolated upper heart explants from young and old mice. We compared cell length, width, and area between young and old cells. We also determined these morphological parameters in HCN4-positive enzymatically dissociated pacemaker cells. We found no significant difference in cell dimensions or area between ages, ruling out hypertrophy as a potential mechanism. We used mass spectrometry to identify expression changes in scaffolding proteins essential for calcium channel organization at the plasma membrane. Through this approach, we identified a large reduction of caveolin 3 as a possible mechanism. Caveolin is a protein essential to forming signaling microdomains between calcium channels and other proteins. Using western blotting, we confirmed a 50% reduction of caveolin 3 in isolated pacemaker tissues from old animals. Using proximity ligation assay and super-resolution microscopy, we showed altered recruitment of L-type calcium channels into caveolae. Our findings suggest that the age-associated decrease of L-type calcium current is caused by a reduced insertion of these channels in caveolae.

The heart's primary function is to pump blood to supply oxygen and nutrients to the body. The biomechanical principles of the heart are determined by specializations at the organ, tissue, cellular, and molecular levels. Little is known about how these specializations have adapted to sustain high heart rates in animals with extreme biology, as is the case of the hummingbird, whose heart rate above 1000 bpm makes it the endotherm with the highest heart rate observed in nature. We hypothesize that the hummingbird heart has evolved several adaptations at all the abovementioned levels to i) generate fast firing rates, ii) optimize electrical-contraction coupling, and iii) sustain fast contraction-relaxation cycles. Using different histological and imaging approaches, we have started to characterize the architecture of the hummingbird’s heart for the first time in a research lab. To describe the overall dimensions and structure of the hummingbird heart, we generated CT scans and 3D reconstructions of iodine-labeled Calypte anna hummingbird hearts. To characterize the organization of the tissue, we present data using hematoxylin-eosin and lectin stainings in fixed paraffin-embedded slices of the hummingbird heart. Our preliminary results showed that hummingbird ventricles have a cell density of 110 cells per 5000 µm2, around 7-fold larger than mouse ventricles. Ventricular cells in the hummingbird are 8-fold smaller with a cross-sectional area of 41 ± 4 µm2. Hummingbird hearts also have a higher capillary density with 18.0 ± 0.6 capillaries per 2500 µm2. Our results provide a foundation for structural and functional characterization of the hummingbird heart at an organ, tissue, and cellular level while opening avenues for further investigation of extreme cardiac physiology.

Organs maintain consistent shape, form, and volume through complex processes, one of which is cell-cell adhesion. E-Cadherin, a key cell-cell junction protein, is critical for cell shape, arrangement, and tissue structure. In this study, I investigate the role of E-Cadherin in the morphogenesis of the Drosophila Malpighian tubules, a model system where I can manipulate E-Cadherin expression and use fluorescence microscopy to observe the effects on organ growth. Previous work involved fixing and staining embryos to track E-Cadherin localization using fluorescent imaging to measure its intensity. I will further analyze E-Cadherin localization spatiotemporally by constructing a fluorescent fly line for live imaging during development. I expect E-Cadherin concentration to increase during elongation and to be enriched in looped regions of the tubules. To assess the requirement of E-Cadherin in organ formation, I will reduce its expression using RNAi and degradFP, expecting significant developmental defects due to the protein's vital role in morphogenesis. These defects will be quantified by comparing changes in cell and organ shape in control and E-Cadherin-reduced tubules. Additionally, I will help develop Python tools for 3D image analysis, including cell segmentation, creating a 3D model of E-Cadherin in tubular cells, and extracting protein intensity. Developing these tools not only enables our work in these tubular organs but also allows for comprehensive image analysis of other tubular 3D organ forms. Elucidating the precise mechanisms behind cell behavior, shape, and cell-cell interaction has important human health implications and will enable work in many other fields such as cancer, regenerative treatments, tissue growth, and organ synthesis.