Introduction

The heart is a four-chambered organ responsible for pumping throughout the body. It receives deoxygenated blood from the body, sends it to the lung, receives oxygenated blood from the lungs, and then distributes the oxygenated blood throughout the body. At the histological level, the cellular features of the heart play a vital role in the normal function and adaptations of the heart.

Issues of Concern

The cells that constitute the heart are unique. It can initiate and propagate electricity throughout each cardiac cell. This physiology allows the heart to contract synchronously, permitting optimal function of circulating blood to the lungs and the rest of the distal organs.

Structure

The fibrous skeleton, cardiac muscle, and impulse conduction system constitute the basic framework of the heart. The base of the heart contains a highly dense structure known as the fibrous or cardiac skeleton. Functions of the fibrous skeleton include providing a strong framework for cardiomyocytes, anchoring the valvular leaflets, and acting as electrical insulation separating the conduction in the atria and ventricles.[1]

The wall of the heart separates into the following layers: epicardium, myocardium, and endocardium. These three layers of the heart are embryologically equivalent to the three layers of blood vessels: tunica adventitia, tunica media, and tunica intima, respectively. A double-layer, fluid-filled sac known as the pericardium surrounds the heart. The two layers of the pericardium are called the outer fibrous/parietal pericardium and the inner serous/visceral pericardium. The epicardium constitutes the visceral pericardium, underlying fibro-elastic connective tissue, and adipose tissue.[2] Coronary arteries and veins, lymphatic vessels, and nerves run below the epicardium. The endocardium is composed of the endothelium and the subendothelial connective tissue layer. The subendocardium is found between the endocardium and myocardium and contains the impulse-conducting system.

The impulse conducting system has specialized cardiac cells for the conduction of electrical impulses throughout the heart.  Electrical impulses initiate at the sinoatrial (SA) node, situated at the junction of the superior vena cava and right atrium. These impulses travel throughout the atria until it reaches the atrioventricular (AV) node, located between the interatrial and interventricular septum. As the fibers travel inferiorly, it penetrates the central fibrous body of the cardiac skeleton to form the bundle of His. These fibers are the Purkinje fibers after they divide within the interventricular septum and branch into the ventricles.

Valves are an important component of the heart. Not only do they act as an exit gate, but they also prevent backflow into the chamber. The aortic valve, separating the aorta from the left ventricle, and the pulmonic valve, separating the pulmonary artery from the right ventricle, are known as semilunar valves. The two atrioventricular (AV) valves are the tricuspid and mitral valves. The tricuspid valve marks the separation between the right atrium and right ventricle, while the mitral valve separates the left atrium from the left ventricle. A unique aspect of the AV valves is their attachments to the ventricles with the assistance of chordae tendinae inserted onto the papillary muscle of the ventricles. 

Function

The heart's main function is to pump blood throughout the body. Cardiac function can be best represented by cardiac output, the amount of blood pumped out of the heart per minute. Many factors determine cardiac output. The product of stroke volume and heart rate equals cardiac output. Hence, cardiac output is directly alterable through variations in these two factors. Stroke volume is the blood volume ejected after ventricular contraction, calculated by taking the difference between end-diastolic volume and end-systolic volume. Contractility, afterload, and preload can change stroke volume.

Preload is the amount of stress placed on cardiomyocytes by the end-diastolic volume before systole. The end-diastolic volume is the best way to measure preload. On the other hand, afterload is the total tension the ventricle must overcome during systole. The law of LaPlace is the foundation for the definition of afterload. Therefore, changes in pressure, radius, or wall thickness directly affect afterload.[3][4]

Tissue Preparation

Histological and cytological studies of the heart are necessary for diagnostic purposes, assessment of allograft rejection after a cardiac transplant, or evaluation of the effect of drug toxicity on the heart. An endomyocardial biopsy obtains cardiac tissue to be analyzed.

During an endomyocardial biopsy procedure, 1 to 2 mm3 of endocardium and myocardium are taken from the right ventricle.[5] Peripheral proximity to the venous entry of the bioptome and a thicker wall relative to the atrium makes the right ventricle an ideal location for a biopsy. The cardiac sample is then placed into a fixative, such as formalin, to preserve the tissue. These preserved samples are then placed into cassettes, embedded in paraffin wax, thinly sliced, and mounted onto glass slides. Hematoxylin and eosin is an initial, basic stains for visualization of the heart tissue under light microscopy.

Depending on the purpose of the endomyocardial biopsy, the method to prepare the tissue may vary. For instance, if viral myocarditis is on the differential, a frozen sample is needed for the identification of the virus through a polymerase chain reaction.[6]

Histochemistry and Cytochemistry

The study of cells and tissue (histochemistry) and intracellular activities (cytochemistry) is useful for narrowing down the correct diagnosis. Immunohistochemistry uses antibodies to target specific antigens in a specimen. The antibody-antigen complex can then be stained to appreciate the presence of the particular antigen. This test can aid in the diagnosis of acute allograft rejection, amyloidosis, neoplasms, and cardiomyopathy.[5] T-lymphocytes, seen in myocarditis, can also be identified with the help of immunohistochemistry. Immunofluorescence is very similar to immunohistochemistry. However, the antibodies contain a fluorescent dye which is visible when the antibody is attached to an antigen.  Immunofluorescence can assist with the diagnosis of allograft rejection and certain cardiomyopathies.[7] 

Special stains highlight specific components in a specimen that might be difficult to visualize using hematoxylin and eosin. The Congo red and methyl violet stains are useful to detect amyloid deposits in tissue. In myocarditis or allograft rejection, methyl green-pyronine stain can spot if lymphocytes. Masson's elastic trichrome stains connective tissue, such as elastic fiber and collagen. In patients with iron-overload cardiomyopathy, possibly due to hemochromatosis, any iron deposition in the tissue can be stained using the Prussian blue stain.[5]

Microscopy, Light

Histologically, the heart is mainly composed of cardiomyocytes and connective tissue. Dense connective tissue with elastic fibers is present in the cardiac/fibrous skeleton. Certain stains, such as Masson's elastic trichrome stains, can help visualize these components. The pericardium subdivides into two layers, a superficial fibrous layer, and a deeper serous layer. The fibrous layer is composed of fibrous connective tissue. The serous layer further divides into two layers, an outer layer inseparable from the fibrous pericardium and an inner layer overlying the myocardium. Both of these layers are histologically the same, composed of a continuous layer of mesothelial cells with microvilli facing the pericardial cavity. The fibrous pericardium and the outer serous pericardium combined are known as the parietal pericardium. The inner serous pericardium, known as the visceral pericardium, is also part of the epicardium. In between the outer and inner serous layer is a potential space known as the pericardial cavity containing pericardial fluid, which is produced and reabsorbed by the microvilli on the mesothelial cells.

A large part of the cardiac wall is made up of the myocardium. Cardiomyocytes join together to make up this layer. These cardiomyocytes are striated like myocytes found in skeletal muscle. However, unlike skeletal muscle cells, they are branched, contain intercalated disks, and are usually mononucleated. They are also unable to regenerate. After an insult, such as a myocardial infarction, the necrotic area gets replaced by scar tissue. This histological change is evident under light microscopy as the fibrous component of the scar tissue is stained blue with Masson's elastic trichrome stain.

The endocardium consists of a single layer of endothelial cells lining the chambers of the heart. Occasionally, small amounts of smooth muscle can also be in the endocardium. Compared to the right atrium, the left atrium has a thicker endocardium because of high pressure from the pulmonary veins. The subendothelial layer, between the myocardium and endocardium, contain loose elastic tissue, collagen bundles, nerves, and occasionally blood vessel.

The conduction system consists of specialized myocardial cells and fibers that allow for the initiation and propagation of impulses. The SA node is composed of nodal (P) cells and transitional (T) cells. These cells are looks similar to myocardial cells but contain fewer myofibrils. Dense connective tissue insulates and separates this area from the rest of the atria. The atrioventricular (AV) node, situated next to the fibrous skeleton of the heart, has specialized muscle fibers that receive impulses from the SA node. Purkinje fibers, branches from the atrioventricular nodes, can be located within the epicardium. These fibers are rich in glycogen and also contain fewer myofibrils.[8]

Valves have three layers: spongiosa, fibrosa, and ventricularis. Identifying these layers can help orient the valves on microscopy. The spongiosa is on the atrial side of atrioventricular valves or the arterial side of semilunar valves. Large amounts of proteoglycans, such as glycosaminoglycan, and loose connective tissue, are characteristic of the spongiosa layer.[9] The fibrosa, an extension of the cardiac skeleton, contains dense irregular connective tissue. The ventricularis, located on the ventricular side of the valve, has elastic fibers and an endothelial lining. In the AV valves, branches of the ventricularis form the chordae tendineae. The chordae tendineae is predominantly made up of dense regular connective tissue, along with collagen elastic fibers, to restrain these valves from high pressure.

Microscopy, Electron

Electron microscopy allows for the visualization of the heart's ultrastructure that is not visible through light microscopy. Fabry disease, cardiac myxoma, cardiomyopathy, and amyloidosis are a few examples of diseases diagnosed with the assistance of electron microscopy.[5]

Individual sarcomeres of the myofibrils are clearly viewable with the help of transmission electron microscopy. Critical areas of the sarcomere include the Z-lines at the ends, the central H-zone, myosin-rich A-band, and the actin-rich I-band.[10] Cellular structures unique to cardiomyocytes are also appreciated. Cardiomyocyte’s dense endomysium, abundant mitochondria between myofibrils, intercalated disks, and T-tubules (present on the Z-lines) are visible on electron microscopy.

Two unusual characteristics that can be present within the cardiac myocytes are lipofuscin granules and atrial dense-core bodies. Lipofuscin granules are a result of lysosomal digestion. The number of lipofuscin granules increases with age. On the other hand, atrial dense-core bodies are found in the atrium and are visually opaquer than lipofuscin granules.

Pathophysiology

In hypertrophic cardiomyopathy, changes at the cellular level have a significant impact on the gross structure and physiology of the heart. There are several causes for hypertrophic cardiomyopathy. Some examples include genetic mutation, hypertension, and aortic stenosis. Key histologic findings are cardiomyocyte disarray (hypertrophied and disorganized myocytes) and interstitial fibrosis of the left ventricle and interventricular septum.[11] These cellular changes can be due to increased afterload placed on the left ventricle or mutations in sarcomere proteins, such as the B-myosin heavy chain (MYH7) and the cardiac myosin binding protein C (MYBPC3) genes.[12] Under electron microscopy, abnormal myocytes and sarcomeres are visible in areas of myocyte disarray. Masson's elastic trichrome stain can help identify areas of fibrosis. Further hypertrophy of the myocardium causes diastolic dysfunction and heart failure.

Clinical Significance

A variety of pathologies can affect every area of the heart. The fibrous skeleton is near the cardiac valves and conduction fibers. During a valvectomy, portions of the fibrous skeleton may undergo accidental removal. Complications, such as arrhythmias, can occur if the conduction fibers are damaged.[1]

Constrictive pericarditis and cardiac tamponade are two serious pathologies involving the pericardium. Occasionally, a pericardiectomy is needed to allow for the heart to function seamlessly. The pericardium is now a specimen that must undergo evaluation. Fibrosis and chronic inflammation are present in constrictive pericarditis. Granulomatous infection and metastasis are not common but must be ruled out.  Epicardial fat can be an incidental finding in these specimens.

The myocardium occupies the bulk of the heart. Restrictive cardiomyopathy, viral myocarditis, and cardiac allograft rejection can have uncommon histological findings. In restrictive cardiomyopathy, the histology depends on the underlying etiology. Some examples of causes of restrictive cardiomyopathy include sarcoidosis, amyloidosis, iron-overload cardiomyopathy, and neoplasms. In viral myocarditis, the virus causes lymphocytes to infiltrate the myocardium, and infected cardiomyocytes undergo necrosis. Shortly after the cardiac transplant, cardiac allograft rejection may occur and must be diagnosed with an endomyocardial biopsy. Findings include abnormal myocytes and the presence of lymphocytes.[5]

Aging can cause significant changes to the valves and conduction system. Increased collagen fibers and calcification results in valvular abnormalities. An example of valvular changes in the elderly is aortic stenosis. If the valve is not replaced, severe aortic stenosis can cause syncope, angina, and, eventually, death.[13] Fibrosis of the SA node and bundle branch is also a result of aging. Consequently, patients can develop a left bundle branch block or other related arrhythmias.