A brief guide to cardiovascular ultrastructural findings by transmission electon microscopy.
Ultrastructural examination of cardiovascular tissues is an infrequent, yet sometimes important, part of the diagnostic arsenal for generating the correct diagnosis of some cardiovascular diseases. Historically, transmission electron microscopy (TEM or EM) was a frequently used tool prior to the wide usage of immunohistochemical stains to diagnose tumor types. Now EM is primarily used by renal pathologists and less frequently by other groups. It has been recommended by a consensus group that a piece of heart tissue be taken for EM from essentially all native heart biopsies. This specialized method need not be run in every case, but it is useful to have this material based on the clinical history and findings on light microscopy. Tissue to be used for EM should be collected in fresh gluteraldeyde fixative.
In the sections below, this tutorial will provide a brief guide of both normal and abnormal findings at EM. This guide does not cover every possible entity or appearance of entities. It should provide the user with some general knowledge from which they can start to build expertise in this area.
Thick sections are stained with toluidine blue and can be useful for identifying some processes. This stain is useful to note artifactual changes to the tissue, contraction-bands related to processing, and certain disease processes. Both myelin figures, seen in hydroxychloroquine cardiotoxicity and Fabry disease, and amyloid can be seen on thick sections. In some institutions, multiple thick sections are assessed to select an area to perform EM.
Myofibrils are the main structural component of the cardiomyocyte. They are notable for sarcomeres which are defined as the structure between two Z-lines (or Z-bands). The Z-lines are the most electron dense part of the myofibril that run perpendicular to the direction of the myofibril. A healthy cardiomyocte has very distinct Z-lines (yellow arrow). As cardiomyocytes undergo degenerative changes the Z-line can be noted to increase in width and decrease in sharpness. Additionally, in degeneration, the myofibers may represent a smaller proportion of the cytoplasm as compared to a healthy cardiomyocyte. It is important to know that a myofibril artifactual change can occur by placing the cardiac tissue in cold (refrigerated) gluteraldeyde solution. The myofibers can tighten or relax akin to a contraction band. This is not pathologic.
Mitochondria are expected to be frequent in cardiomyocytes. Although we think of mitochondria as solitary ovoid structures, there is some evidence that they can exist in a dynamic syncytium. By usual appearance, they are typically seen as round, to ovoid, to elongated structures. They normally are with 1-2 fold size of each other. Healthy mitochondria have numerous cristae that can extend from one side of the mitochondria to the other or just across part of the structure.
A number of other cell types can be found in EM sections. The most obvious are red blood cells (yellow arrow) notable for their homogenous coloring with various shades of grey. These are often found in small capillaries, of which the endothelial cell nuclei (purple arrow) are often apparent with a thin layer of cytoplasm surrounding the lumen. Fibroblasts, lymphocytes, dendritic cells, macrophages, and other rare inflammatory cells can be seen in the interstitial space. Collagens appear as thick fibrils of sinuoidal or curving structures that align together (red arrow).
Lipid droplets are a feature of degenerative changes to a myocyte. They tend to be few in number, but can increase dramatically in more severe myocardial disease. They are usually aligned with sarcomeres and in the boundaries of the sarcoplasmic reticulum. They measure 0.5 to 1 µm (a relaxed sarcomere measures ~2 µm). These lipid droplets are homogeneous grey electron-dense with a slightly more pale edge. They may relate to fat accumulation within cardiomyocytes, a feature notable by an Oil Red O stain.
In contrast, cardiac microvesicular steatosis shows pleomorphic vacuoles variable in size. Commonly larger than the span of a sarcomere and are definitively visible in H&E, confirmed by oil-red-O. Ultrastructurally they are identical in electron-density to the lipid vacuoles but distinctly larger and variable in size.
Cardiomyocyte nuclei are notable for their electron dense borders and often prominent nucleolus. Their nuclear membranes can have sharp angulations and tortuosity or be more rounded. The nucleus space tends to be hetereogeneous shades of grey, likely relating to the presence of chromosomes. The nucleus is a useful landmark as much cellular material including lipofuscin collects in the perinuclear area. The nucleus can be indented by inclusions or other material. In general, there are no specific cardiomyocyte nuclei pathologies one should be aware of.
The extend of collagen in the heart is variable by location and disease entity. In general, identifying collagen by TEM is not necessarily meaningful to the larger heart, but it is useful to distinguish collagen from other materials such as amyloid fibrils. Collagen forms long (300nm), thin (1.5nm) strands that tend to move together. They often have sinusoidal or curved shapes. Each collagen helix is distinct from adjacent collagen strands with a small amount of space between them.
Degenerative changes are amongst the most common changes observed by EM on heart failure biopsies. Some of these features include a loss of myofibrils, increased widening of the Z-bands, an increase in lipid droplets, increased glycogen, increased lipofuscin, and increased lysosomes. If the cells have developed contraction bands (which can be noted on the thick/toluidine blue) sections, then one must be careful to assess myofibril loss as that may just be an artifact of processing. Z-band changes and lipid droplets were covered previously. Lipofuscin, also known as heart failure pigment, are electron dense, generally round collections of undigested proteins. They are generally slightly larger than mitochondria. They increase with aging and can increase in long-standing heart failure among younger individuals.
In the absence of a specific disease, it is useful to describe the extent of degeneration of the myocytes. A simple scoring scheme could describe the degenerative changes as mild, moderate, or severe and be based on the presence of these changes in a few or most of the myocytes. Additionally, the extent of these changes within each cell can be evaluated. As degeneration becomes more severe, the appearance of the cardiomyocyte deviates significantly from the typical look shown in the normal myofibril image above.
Amyloidosis is a relatively frequent diagnosis that can be made on endomyocardial biopsy. Typically, the diagnosis can be made on the light microscopy material with a pattern of amorphous pink material interdigitating between cardiac myocytes or within small blood vessel walls. A congo red stain, demonstrating “apple-green” birefringence is the classic confirmatory stain.
While not typically needed, EM can be used when the amyloid is scant or questionable in the biopsy. Amyloid fibrils appear as individual and haphazard fibrils of ~14 nm diameter. They have been referred to as “pick up sticks” for their disorganized relationship to each other, in sharp contrast to collagen fibrils. Amyloid fibrils are found in the extracellular space, not classically within cardiomyocytes.
Amyloid typing, essential for treatment and prognosis, can be performed by immuno-electron microscopy where mass-spectrometry is not used. Immuno-labelling techniques, through primary antibodies to anti-lambda and kappa light chains and transthyretin (TTR) reveal amyloid type by the presence of colloidal gold particles amongst the amyloid material.
Abnormal mitochondria most frequently occur due to genetic mutations in mitochondrial genes. However, they have been seen in other genetic diseases or even associated with hydroxychloroquine use. Therefore, they are suggestive, but not specific, of an entity. Abnormal mitochondria are usual enlarged, the so-called ‘megamitochondria,’ or ring-shaped with all cristae connecting between an inner and outer ring. Another abnormal shape is the ‘targetoid’ appearance where the cristae resemble a bulls-eye.
Oversized mitochondria are generally best compared to the size of normal mitochondria as there is typically a mixture of normal and enlarged mitochondria. Megamitochondria can be from 5-20x larger than a typical mitochondria.
Hydroxychloroquine / chloroquine cardiotoxicity
Hydroxychloroquine is widely used for various clinical conditions such as malarial prevention, treatment of rheumatoid arthritis, systemic lupus erythematosus and other inflammatory disorders. Hydroxychloroquine is a weak base and accumulates in lysosomes and other acidic (inflamed) tissues. The drug interferes with lysosomal activity and autophagy, and inhibits immune activation by reducing cytokine production via the Toll-like receptor pathway mechanism (Schrezenmeier et al. Nature Reviews Rheumatology 2020). Prolonged use has been associated with cardiac conduction disturbances that include atrioventricular block, bundle-branch block, and less frequently ventricular arrhythmias mainly with azithromycin. An endomyocardial biopsy specimen may be obtained to diagnose hydroxychloroquine-induced cardiac toxicity. Light microscopy demonstrates myocytes with numerous cytoplasmic vacuoles with an absence of an inflammatory process. A periodic acid–schiff stain shows the vacuoles are negative. Pathognomonic cytoplasmic inclusion bodies (myeloid and curvilinear myeloid) can be seen on ultrastructural evaluation. These findings represent lysosomal dysfunction and the accumulation of metabolic products.
Anthracyclines such as doxorubicin and daunorubicin are commonly used in chemotherapeutic regimens for various malignancies including breast, lung, and lymphoproliferative neoplasms. These toxic agents promote DNA damage and oxidative stress, which lead to membrane lipid peroxidation, myocyte vacuolation, and ultimately cell death. Historically, endomyocardial biopsy was used to monitor cardiotoxicity of these drugs and guide patient management. Dr. Margaret Billingham developed a 3 tier grading system of anthracycline toxicity based on EM findings. Advancing grades were associated with increasing levels of myofibrillar loss and/or sarcotubular vacuolization. Cardiotoxicity classically exhibits diffuse myocyte vacuolization on standard H&E-stained light microscopy, which can be further appreciated on toluidine blue-stained semi-thick sections. Myofibrillar lysis is a characteristic finding on toluidine-blue stained semi-thick sections which, along with sarcotubular swelling can be detected on ultrastructural analysis with electron microscopy.
Light chain deposition disease
Light chain deposition disease (LCDD) is a disease of amyloid fibrils related to amyloidosis. LCDD is more commonly observed in the kidney and is a rare diagnosis in the heart. It is notable for the deposition of monoclonal light chains at the basement membrane of cardiomyocytes in the absence of typical polarizing and dense amyloid material. LCDD is seen in patients with multiple myeloma. By EM, LCDD is notable for amorphous material collecting alongside cardiomyocytes or other cells. The material, like that seen in amyloidosis, is much thinner and more disorganized than collagen fibrils, making it difficult to see “strands”. LCDD can appear as small “cotton balls” gathering alongside the cardiomyocytes.
Although a rare entity, LCDD cannot typically be diagnosed on routine histology slides. Therefore, cases of suspected amyloidosis in patients with known myeloma should also be reviewed by EM for LCDD if amyloidosis is absent. Also, of note, LCDD is a rare disease that can recur in a transplanted heart.
Fabry disease is an X chromosome-linked lysosomal storage disease. It is caused by mutations and deficiencies in alpha-galactosidase A. Subtle or overt clues to Fabry disease can be noted on light microscopic slides based on the presence of myocyte vacuolization and amorphous material within myocytes which is similar, but often larger than, lipofuscin.
Thick EM sections stained with Toluidine Blue can better identify granular material. By EM, the diagnostic finding is the presence of myelin figures (zebroid bodies) within the cardiomyocytes. Note that these same figures can be seen in hydroxychloroquine disease and rarely in other long term drug toxicities. Typically, in Fabry disease numerous myelin figures are seen in most cells, as a solitary myelin figure may be seen rarely in a cell, but is of likely no consequence.
Danon disease, caused by LAMP2 mutations, is a type IIb glycogen storage disease. It generally causes a hypertrophic cardiomyopathy. By EM, Danon is notable for having large autophagic vacuoles with sarcolemmal features (AVSF). These can contain cytoplasmic debris, myeloid bodies and electron dense materials. The number of myeloid bodies tend to be much fewer than those reported in Fabry disease or hydroxychloroquine cardiotoxicity. Additional, general degenerative changes can be observed as well.