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Observation of the Transport and Removal of Lipofuscin from the Mouse Myocardium using Transmission Electron Microscope

This study was performed to investigate whether the lipofuscin formed within cardiomyocytes can be excluded by the myocardial tissue. We have provided indicators that can be used for future studies on anti-aging interventions. In the present study, the heart of a 5-month-old BALB/c mouse was obtained for resin embedding and ultra-thin sectioning. The specimens were observed under a Hitach 7500 transmission electron microscope, and the images were acquired using an XR401 side-insertion device. Lipofuscin granules are found abundantly in myocardial cells. Cardiomyocytes can excrete lipofuscin granules into the myocardial interstitium using capsule-like protrusions that are formed on the sarcolemma. These granules enter the myocardial interstitium and can be de-aggregated to form “membrane-like garbage”, which can pass from the myocardial stroma into the lumen of the vessel through its walls in the form of soluble fine particles through diffusion or endocytosis of capillaries. Smaller lipofuscin granules can pass through the walls of the vessels and enter the blood vessel lumen through the active transport function of the capillary endothelial cells. When the extended cytoplasmic end of macrophages and fibroblasts fuse with the endothelial cells, the lipofuscin granules or clumps found in the cells of the myocardial interstitium are transported to the capillary walls, and then, they are released into the lumen of the blood vessel by the endothelial cells. The myocardial tissues of mice have the ability to eliminate the lipofuscin produced in the cardiomyocytes into the myocardial blood circulation. Although there are several mechanisms through which the myocardial tissues release lipofuscin into the bloodstream, it is mainly carried out in the form of small, fine, soluble, continuous transport.

This study was performed to investigate whether the lipofuscin formed within cardiomyocytes 23 can be excluded by the myocardial tissue. We have provided indicators that can be used for 24 future studies on anti-aging interventions. 25 In the present study, the heart of a 5-month-old BALB/c mouse was obtained for resin 26 embedding and ultra-thin sectioning. The specimens were observed under a Hitach 7500 27 transmission electron microscope, and the images were acquired using an XR401 28 side-insertion device. 29 Lipofuscin granules are found abundantly in myocardial cells. Cardiomyocytes can excrete 30 lipofuscin granules into the myocardial interstitium using capsule-like protrusions that are 31 formed on the sarcolemma. These granules enter the myocardial interstitium and can be 32 de-aggregated to form "membrane-like garbage", which can pass from the myocardial stroma 33 into the lumen of the vessel through its walls in the form of soluble fine particles through 34 diffusion or endocytosis of capillaries. Smaller lipofuscin granules can pass through the walls 35 of the vessels and enter the blood vessel lumen through the active transport function of the 36 capillary endothelial cells. When the extended cytoplasmic end of macrophages and 37 fibroblasts fuse with the endothelial cells, the lipofuscin granules or clumps found in the cells 38 of the myocardial interstitium are transported to the capillary walls, and then, they are 39 released into the lumen of the blood vessel by the endothelial cells. 40 The myocardial tissues of mice have the ability to eliminate the lipofuscin produced in the 41 cardiomyocytes into the myocardial blood circulation. Although there are several mechanisms Introduction 48 It is well known that one of most important research areas in in vivo anti-aging intervention is 49 to discover objective evaluation indicators. The content of lipofuscin in organs is an important 50 indicator for assessing the aging status. Lipofuscin is a yellowish-brown pigment composed 51 of highly oxidized proteins, lipids, and metals. Lipofuscin is widely observed in post-mitotic 52 cells, especially in cells with long life spans such as neurons and cardiomyocytes [1,2]. 53 Lipofuscin is often known as the "age pigment" and is considered a hallmark of aging. This is 3 54 not only because the amount of lipofuscin increases with age, showing an approximately 55 linear dependence, but also, and more importantly, because the rate of lipofuscin 56 accumulation correlates negatively with longevity[3-7]. 57 Lipofuscin, or age pigment, represents an intra-lysosomal polymeric material that cannot be 58 degraded by lysosomal hydrolases. Although its continuous accumulation over time in 59 post-mitotic cells, such as neurons and cardiomyocytes, has been known for more than 100 60 years, it has recently been suggested that accumulated lipofuscin may be hazardous to cellular 61 functions [6,8,9]. There are strong indications that progressive deposition of lipofuscin 62 ultimately decreases cellular adaptability and promotes the development of age-related 63 pathologies, including neuro-degenerative diseases, heart failure, and macular degeneration[8,  Hence, it is still not conclusive whether the non-dividing cells in tissues and organs possess a 84 mechanism for excluding lipofuscin. To ascertain whether the heart has a mechanism for 85 eliminating lipofuscin from the myocardium, we carried out investigations using the mouse 86 heart.

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Five-month-old BALB/c mice were purchased from Laboratory Animal Center of our 90 institution. All applicable international, national and institutional guidelines for the care and 91 use of animals were followed. The animals were anesthetized using ether. The chest was 92 opened and the heart was removed. Small tissue blocks were obtained from the anterior wall 93 of the left ventricle and were fixed with 1.5% glutaraldehyde. The fixed tissue blocks were 94 further treated with 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. The  104 Microscopic observation of myocardial tissue revealed clearly visible lipofuscin deposits that 105 were scattered around the myocardial fibers (cardiac cells) and myocardial interstitium. These 106 lipid-containing, brown deposits were granular or agglomerated; widely varied in size and 107 form; displayed high or medium electron density; uniform or uneven internal structure; or 108 lamellar or lipid droplets; with or without membrane coating; or dispersed or aggregated into 109 large clumps. 110 Lipofuscin found in the myocardial fibers was mainly distributed in the cytoplasm-rich 111 regions at both ends of the nucleus and was interspersed between a large numbers of 112 mitochondria. The distribution of lipofuscin granules was lower in the myofibrils that were far 113 from the nucleus (Fig 1). Lipofuscin granules could also be seen at the edges of myocardial 114 fibers and below the sarcolemma (Fig 2, 15, 16). electron density (Fig 5, 7, 9). can be seen, and the granule on the right seems to be partially exposed outside the endothelium. In the middle of

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In the interstitial matrix of the myocardial space, a large number of non-cellular structured 256 membrane-like structures can be seen, which can be of different sizes and electron density. 257 They form loose, irregular sac-like structures with single or several layers, or dense myelin 258 figures with multiple layers, creating complex structures that greatly vary in size, density, 259 morphology and number of membrane layers (Fig 6, 8, 10-14). We termed these complex 260 membrane-like structures as "membrane-like garbage". These "membrane-like garbage" are 261 widely and irregularly distributed in the matrix of the myocardial interstitium, either sparsely 262 or densely.

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The image shows the longitudinal section of the myocardial fibers. Wider space is seen between the two 265 myocardial fibers. A large number of randomly distributed "membrane-like garbage" can be seen in the space. In the interstitium, some free lipofuscin particles can also be seen that vary widely in size. 285 Some are in the form of small granules, while others form large masses, which might be either 286 coated or uncoated at the periphery (Fig 14). Large clumps formed by the aggregation of 287 several lipofuscin granules are sometimes seen (Fig 15). Some lipofuscin granules have low 288 density and a relatively loose structure. The shape is similar to the high-density 289 "membrane-like garbage," and there is no obvious morphological boundary as that seen in the 290 "membrane garbage," which is denser in structure (Fig 10-14).

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There are many capillaries in the myocardial interstitium, which are continuous in nature. 302 These capillaries can be roughly divided into two types: high density and low density 303 endothelial cell types. 304 High-density endothelial cell-type capillary vessels have thick walls and high cytoplasmic 305 density. Endothelial cells can form tight junctions. In addition to the scattered mitochondria 306 inside the cytoplasm of the endothelial cells, they contain abundant pinocytotic vesicles and 307 exocytosis vesicles. These vesicles are often highly dense, indicating that active transport 308 occurs through their walls. High electron density granules and fine particles are found 10 309 abundantly in the cytoplasm (Fig 2, 3, 5, 8, 10-12, 16, and S1 Fig). accompanying blood vessels (Fig 2, 3  stroma into the lumen (Fig 18).

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The figure shows four possible pathways through which the lipofuscin found in the myocardial interstitium