Mechanics of the Cardiovascular System
A small and specific subpopulation of cardiomyocytes of the myocardium, termed "pacemaker cells," has the ability to generate action potentials locally in the sinoatrial . Once initiated this depolarization signal is propagated, traveling down the atrium, to the AV node, where it is able to pass into the bundle of His and finally into the subendocardium via a specialized collection of conducting tissue called the "Purkinje fibers.” The Purkinje fibers are essential to heart conduction, maintaining a consistent rhythm, as they are able to conduct cardiac action potentials more efficiently and faster than their smaller cardiomyocyte counterparts . This is due to their unique structural organization. In comparison to cardiomyocytes, Purkinje fibers have a larger number of mitochondria and fewer myofibrils. Electrocardiography serves as a medical tool to monitor patterns in heart conduction and can be broken down into four distinct phases: the P wave, representing atrial depolarization; a QRS complex, which is characteristic of ventricular depolarization; the T wave, which shows ventricular repolarization; and finally the U wave, a pattern that denotes papillary muscle (ventricle muscles] repolarization [21, 31]. Diastolic and systolic cycles act in concert for blood perfusion. The left and right atria of the heart are filled by veins in the first stage of diastole, while the left and right ventricles are relaxed. In the second stage, contraction of the right and left atria by the myocardium results in the pumping of blood into the right and left ventricles via the AV valves. In the first stage of systole, both ventricles contract simultaneously, pushing blood, where pulmonary arteries are fed by the right ventricle, while the aorta is fed by the left ventricle. Systolic and diastolic blood pressure are measured from the second stage of the systole and the diastole, or the pressure of the heart at rest. Normal systolic and diastolic pressure ranges are between 80-120 and 60-80, respectively .
Blood is composed of three components: red blood cells (or erythrocytes); white blood cells, containing leukocytes and platelets; and plasma, an aqueous solution that contains ionic solution, water, and proteins. The total blood constituents can be fractioned as follows: 45% red blood cells, 0.18% white blood cells, and the remaining plasma. Since 90% of the plasma is made up of water, the non-Newtonian fluid properties of blood can be attributed mainly to erythrocytes, deformable biconcave disks that have an average diameter of approximately 8 pm. Direct measurement of plasma rheological properties has been achieved, although with varying results in that the specific concentrations of albumin, fibrinogen, and globulins do fluctuate. H owever, general guidelines of plasma protein concentration demonstrate that it consists of ~50% albumin, ~45% globulins, and ~5% fibrinogen . The Newtonian properties of plasma have been measured—with a fluid viscosity of 1.2 centipoise (cP)—making blood a viscous fluid with non-Newtonian behavior.
Cardiovascular Extracellular Matrix Composition
The feedback between physical and chemical cues within the extracellular cardiac niche play an important role in the structure and function of heart and vascular tissue. Bidirectional, inside-out signaling from the cell to the ECM is achieved via transmembrane integrin proteins [34, 35]. The dimerization of integrin a and P subunits physically links intracellular actin-linked proteins such as zyxin, paxillin, talin, vinculin, and a-actinin to ECM residues. The complexity of integrin motifs extends to the ability of multiple or singular ECM ligands to bind to a single integrin receptor. Furthermore, several integrin heterodimers can adhere to a single ECM ligand. Nonetheless, once activated, integrin complexes regulate GTPases such as Rho or Rac, which promotes the assembly of focal adhesion complexes, or kinases such as src kinase and focal adhesion kinase .
In general, there are over 20 known members of the integrin family constructed by 8p and 18a subunits. Some of the integrins resident to cardiomyocytes include binding motifs for collagen type I (a3pi), laminin (aipi, a3pl, and a.7pl), and fibronectin (a3pl and a.5pi) . While ECs are anchored to the vessel wall by nearly 20 different ECM proteins, the 2D mesh primarily contains collagen IV, laminin, fibronectin, and proteoglycan perlecan . A thin membrane called the tunica intima, composed of collagen VI and VIII, separates ECs from the tunica media. The tunica media, which is typically thickened in arteries, contains supportive mural cell populations, namely smooth muscle cells, which are found in large arteries and veins, and pericytes, which reside in small capillaries. The specific ECM contribution to vasculature is dependent on tissue function. Vascular tone is mediated, in part, by the elastic nature of the ECM proteins fibrillin and elastin. Where vessel elasticity is not essential, as in the case of vascularized muscle tissues, whose role is to distribute blood to different organs, the ECM is enriched with concentric layers of smooth muscle cell sheaths.
Experiments utilizing atomic force microscopy demonstrated that developing hearts in chicken embryos, specifically 36 to 408 hours after fertilization, show a steady increase in elastic moduli, ranging from 0.9 kPa to 8 kPa, during development . These changes in stiffness of the developing heart coincide with changes in the ECM composition, identified through time-traced studies using quantitative polymerase chain reaction. Here, heart development was preceded by increased collagen expression, with maturation leading to decreasing laminin and fibronectin expression.
The combinatorial roles of elastic fibrous proteins such as fibrin and collagen, as well as adhesive glycoproteins such as laminin and fibronectin, in the basal laminae aid in the proper configuration of cells within the heart. For example, the orientation of cardiomyocytes is dictated by the fibrillar architecture of the surrounding collagen as assembled primarily by cardiac fibroblasts, ensuring regularity in adjacent sarcomere organization, which supports systolic performance . In addition, collagen serves as an electrically conductive material, allowing action potential propagation extending from adjacent cardiomyocytes to the cells of the AV node, bundle branches, and Purkinje fibers.
Increasing research has outlined the role of glycosaminoglycans (GAGs) and matrix proteoglycans in forming the early structures of the heart [1, 41-43]. Gradients of hyaluronic acid (HA) and fibronectin 1 (Fnl) in particular help guide heart field migration, formation of early heart tubes, and chamber septation [1, 42, 44- 46]. Gradually, as migration and proliferation occur, heart structures stabilize, and postnatal hemodynamics begins to put biomechanical strain on the myocardium. As a result, cardiac fibroblasts and other supporting cell types exchange the softer, growth-factor-rich gels of HA with stiffer, highly cross-linked, aligned fibers of collagens I and III, supported by collagen IV and laminin in particular. The adult heart is highly fibrillar (particularly in the ventricles), guiding anisotropic conduction, but relatively poor in fibronectin, GAGs, and other proteoglycans, which guide effective wound healing in other tissues, leading to the fatty and scar-like responses to insults such as infarction [47, 48].
Cardiac p-adrenergic responses, influencing action potential morphology, calcium metabolism, force development, and tension, are all mediated by laminin-mediated integrin activation . Integrin signaling is equally important in the functionality of other cell types within the heart, such as cardiac fibroblasts [37, 43, 47, 50]. Fibronectin and osteopontin adhesion are mediated by a5pl expression by cardiac fibroblasts. Additionally, the expression of a5pl, a.5p3, and oc5p5 integrins drives cardiac fibroblastadhesion to vitronectin, fibronectin, and osteopontin. Novel in vitro and in vivo experiments have begun to uncover the role of integrin expression and ECM composition in cardiac development. In comparison to adult cardiac fibroblasts, embryonic cardiac fibroblasts are able to stimulate the proliferation of cardiomyocytes in an ECM-dependent manner . Using messenger RNA profiling techniques, Ieda et al. found embryonic cardiac fibroblasts have enhanced expression of proteins, such as fibronectin, and collagens tenascin C and periostin when compared to adult cardiac fibroblasts. In coculture experiments, where cardiomyocytes were cultured in the presence embryonic cardiac fibroblasts, inhibition of cardiac proliferation was achieved when fibroblast expression of Fnl and/or collagen (Col3al) was temporarily knocked down through small interfering RNA treatment. Similarly, when cardiac pi integrin expression was inhibited, proliferation was suppressed .
Engineering Approaches to Studying Mechanotransduction in Cardiovascular Development
Progressive heart failure results from infarction, due to the lack of functional regeneration of the myocardium, leading to aneurysmal thinning and scarring. This can be attributed to the lack of sternness within cardiomyocytes and their inability to proliferate. Tissue engineering approaches to augment the function of damaged cardiac tissue include implementing cellular grafts that either induce an angiogenic response or supplement the mechanical requirement of the infarcted wall. Both of these approaches are key in re-establishing the structure for proper ventricular function. After infarction, engrafted cardiomyocytes of fetal or neonatal origin have been shown to generate new functional tissue separate from the injured scar tissue. In addition to the lack of functional integration in diseased sites, many of the implanted cardiomyocytes died upon implantation, clearly demonstrating the need for structural support for viable engraftment .
The limited regenerative capacity of adult heart muscle, attributed variously to insufficient proliferation and lack of sternness in the resident populations of the myocardium, continues to motivate technologies for cardiac regeneration. Bare injection or implantation of preparations of differentiated cardiomyocytes (such as injections, clusters, aggregates, spheroids, or cell sheets) for heart regeneration is hampered due to diminished postimplantation viability and lack of functionality. Cardiomyocytes differentiated from human stem cells currently reach only a fetal-like maturation phenotype, with poor sarcomere organization, immature calcium cycling, pacemaker-like automaticity, and both low expression and poor localization of appropriate gap junctions and mature potassium channels in particular. This leads to the poor functional integration that is often seen between stem cell-derived cardiomyocytes and remaining viable native tissue, with poor electromechanical cell-cell coupling, and also increases concerns that therapy with these preparations might potentially be arrhythmogenic in and of themselves. To this end, engineering approaches to the in vitro conditioning and in vivo delivery of cardiovascular cell populations have been highlighted as among the most promising strategies to improve viability, engraftment, and functionality of cardiomyocytes and other necessary cell types; once surgically implanted, these biomaterials also facilitate host cell infiltration, further ensuring that transplanted cells can functionally integrate into and augment the function of the diseased tissue (Fig. 4.1).
Figure 4.1 Engineering tools to mechanically drive cardiovascular specification from pluripotent stem cells.