Ultrastructural study of the myocardium of the sinus venosus and sinoatrial valve in the dogfish (Scyliorhinus canicula)

The myocardium of the sinus venosus of the dogfish ( Scyliorhinus canicula ) is located between a thick subepicardial collagen-rich layer and a subendocardial network of nerve fibres and ganglion cells. The sinoatrial valve consists of two transversal folds of the cardiac wall which are separated by connective tissue, except in their free margins.
The myocardium of the sinus venosus and the sinusal face of the sinoatrial valve is arranged in bundles which are surrounded by a 40 nm-thick basal lamina. The myocardial cells measure about 7-9 μm in diameter at the nuclear level. Nerve terminals are frequent in the centre of the bundles. Most of the sinusal myocardiocytes have a scarce amount of myofibrils which are randomly orientated. The sarcoplasmic reticulum is relatively well developed and consists of peripheral couplings, subsarcolemmal vesicles, circular and longitudinal tubules. The scarce intercalated discs show only fasciae adhaerentes . Gap junctions, desmosomes or specific granules are not observed in the sinusal myocardiocytes of the dogfish. In contrast, the atrial myocardiocytes are smaller, about 5-6 μm in diameter at the nuclear level. The cytoplasm is denser and the myofibrils are abundant and orientated in parallel directions. Specific granules are present. although scarce. Subsarcolemmal vesicles are less frequent, while the atrial intercalated discs are larger and more abundant than those of the sinus venosus. Neural elements are scarce in the atrium.
The differences observed between sinus venosus and atrium might be related to the morphological criteria to distinguish between the nodal tissue and the working myocardiocytes of higher vertebrates. On the other hand, we think that the connective tissue placed between sinus venom and atrium means that the contraction impulse generated in the sinus venosus must reach the atrium through the free margin of the valve. It might play a role in the sinoatrial valve function.     

Electrophysiological and structural studies on the heart muscle of the lobster Homarus americanus

The electrophysiological properties and structure of heart muscle fibres of the lobster myocardium have been investigated. Discontinuities were observed in the expected decrement of electrotonic potentials in a study of the passive membrane properties of the muscle. Injection of Procion yellow revealed transverse barriers to dye movement, identified in electron micrographs as intercellular junctions. Unlike the intercalated discs of the vertebrate myocardium, which these regions superficially resemble, nexus regions of ;close' or ;gap' junctions are absent. The intercellular cleft is generally 400 A in width, locally reduced to 75-100 A The functional implications of these findings are discussed. Cardiac myoneural junctions, which include regularly repeating structures extending into the synaptic cleft from the postsynaptic membrane, are described.     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

Cell junctions and their role in transmural diffusion in the embryonic chick heart

Summary Studies of cardiogenesis in the chick embryo focus attention upon the intercellular junctions of epicardial, myocardial, and endocardial cells, and the role they play in diffusion across the cardiac wall. Cell membranes of apposed epicardial cells approach as close together as 40 Å; those of the endocardium additionally form focal tight junctions. In the myocardium focal tight junctions are restricted to the apposed membranes of the superficial layer of cells. The majority of close appositions in all parts of the myocardium are 40 Å gap junctions. Desmosomes and fascia adherens are distributed throughout the myocardium.Diffusion of horseradish peroxidase through the epicardium and endocardium occurs primarily through the intercellular junctions. The width of the cleft between cells, 200–300 Å, also permits the diffusion between cells of the larger ferritin particles. Pinocytotic activity, responsible for ferritin transfer across mesothelial and endothelial cells in the adult, is not significant.Tracers injected into the pericardial cavity or vasculature can be observed passing through the heart in the direction of their respective diffusion gradients. Unlike the apical junctions of epithelial cells, to which they have been compared, membrane specializations of the superficial myocytes do not form a seal separating the pericardial cavity, or subepicardial space, from the extracellular spaces of the myocardium.Supported by the Medical Research Council of Canada.The author wishes to express his gratitude to Mrs. J. Blackbourn for her excellent technical assistance.     

Enzyme histochemical studies on the Purkinje fibres of canine, bovine and porcine hearts

Synopsis Whereas in ungulates the Purkinje fibres of the atrioventricular conducting system are highly characteristic cells, those in the canine heart are poorly differentiated and accordingly they cannot always be readily identified in histological sections. Consequently in this paper the results of various histochemical tests on bovine and porcine hearts have been compared with the view of evaluating them as dependable methods for identifying Purkinje fibres that are microscopically poorly differentiated.It appeared that, histochemically, canine Purkinje fibres differ consistently in similar ways and as markedly from the common myocardial fibres as the morphologically typical conducting fibres in bovine and porcine hearts. The conducting fibres distinguish themselves from the myocardium proper in containing more glycogen and fewer lipids, in possessing higher activities of the enzymes -glucan phosphorylase,l-glycerol-3-phosphate:menadione oxidoreductase, myosin adenosine triphosphatase and monoamine oxidase, as well as in possessing lower activities of several dehydrogenases, cytochrome oxidase, peroxidase and mitochondrial adenosine triphosphatase. The relatively high activity of -glucan phosphorylase in particular is striking. As the activity of this enzyme persists during periods of up to 20 min after death, the staining method for this enzyme provides a valuable technique for identifying Purkinje fibres even if they are cytologically poorly differentiated.It is of interest in relation to electrophysiological data that the histochemical properties are similar in Purkinje fibres derived from widely differing levels of the conducting system. From the present histochemical findings it may be assumed that, as compared with the myocardium proper, the Purkinje fibres have a higher rate of anaerobic and a lower rate of aerobic metabolism. Furthermore, it is pointed out that histochemically the differences between Purkinje fibres and common myocardial cells on the one hand, and those between white (Type II) and red (Type I) striated muscle fibres on the other, are essentially similar.     

Endocardial endothelium in the rat: junctional organization and permeability

Selective permeability of endocardial endothelium has been suggested as a mechanism underlying the modulation of the performance of subjacent myocardium. In this study, we characterized the organization and permeability of junctional complexes in ventricular endocardial endothelium in rat heart. The length of intercellular clefts viewed en face per unit endothelial cell surface area was lower, and intercellular clefts were deeper in endocardial endothelium than in myocardial vascular endothelium, whereas tight junctions had a similar structure in both endothelia. On this basis, endocardia endothelium. might be less permeable than capillary endothelium. However, confocal scanning laser microscopy showed that intravenously injected dextran 10000 coupled to Lucifer Yellow penetrated first the endocardial endothelium and later the myocardial capillary endothelium. Penetration of dextran 10000 in myocardium occurred earlier through subepicardial capillary endothelium than through subendocardial capillary endothelium. Penetration of tracer might thus be influenced by hydrostatic pressure. Dextran of MW 40000 did not diffuse through either endocardial endothelium or capilary endothelium. The ultrastructure of endocardial endothelium may constitute an adaptation to limit diffusion driven by high hydrostatic pressure in the heart. Differences in paracellular diffusion of dextran 10000 between endocardial endothelium and myocardial vessels, may result from differing permeability properties of the endocardium and underlying myocardium.     

A heart for fibrillin: spatial arrangement in adult wild-type murine myocardial tissue

Fibrillins are major constituents of microfibrils, which are essential components of the extracellular matrix of connective tissues where they contribute to the tissue homeostasis. Although it is known that microfibrils are abundantly expressed in the left ventricle of the heart, limited data are available about the presence of microfibrils in the other parts of the myocardial tissue and whether there are age or sex-related differences in the spatial arrangement of the microfibrils. This basic knowledge is essential to better understand the impact of fibrillin-1 pathogenic variants on the myocardial tissue as seen in Marfan related cardiomyopathy. We performed histological analyses on wild-type male and female murine myocardial tissue collected at different time-points (1, 3 and 6 months). Fibrillin-1 and -2 immunofluorescence stainings were performed on cross-sections at the level of the apex, the mid-ventricles and the atria. In addition, other myocardial matrix components such as collagen and elastin were also investigated. Fibrillin-1 presented as long fibres in the apex, mid-ventricles and atria. The spatial arrangement differed between the investigated regions, but not between age groups or sexes. Collagen had a similar broad spatial arrangement to that of fibrillin-1, whereas elastic fibres were primarily present in the atria and the vessels. In contrast to fibrillin-1, limited amounts of fibrillin-2 were observed. Fibrillin-rich fibres contribute to the architecture of the myocardial tissue in a region-dependent manner in wild-type murine hearts. This knowledge is helpful for future experimental set-ups of studies evaluating the impact of fibrillin-1 pathogenic variants on the myocardial tissue.     

Origin of the brushborder in the differentiating midgut of Melasoma saliceti (Chrysomelidae, Coleoptera) embryos

The embryonic development of Melasoma saliceti takes eight days at room temperature. At the beginning of the 5th day the endoderm cells have already formed a unilayered epithelium of the midgut primordium. The midgut epithelium is formed by flat cells that are not connected by specialized intercellular junctions. Large vesicles can be seen in dilated intercellular spaces of the epithelium. Cytoplasmic projections, similar to microvilli, appear in the vesicles. During the 5th day ofdevelopment, the vesicles grow and become enclosed by the intercellular junctions of a zonula adherens type. During the 6th day of development the cell junctions surrounding the vesicles become transformed into a septate type. On the 8th day of development the vesicles come close to the apical sides of the midgut cells and open towards the yolk. At the same time the microvilli spread over the apical surface of the midgut primordium to form the regular brushborder of the larval midgut. In the species studied the vesicles appear to "prefabricate" the apical surfaces of the future midgut epithelium.     

Relation between expression of TNF alpha, iNOS, VEGF mRNA and development of heart failure after experimental myocardial infarction in rats.

An injury to the heart due to myocardial infarction (MI) may progress to heart failure. Among factors, whose interactions promote remodeling of ischemic myocardium, the increased expression of tumor necrosis factor alpha (TNFalpha), inducible nitric oxide synthase (iNOS) and Vascular Endothelial Growth Factor (VEGF) was found. However, little is known about the temporal and spatial relation between expression of iNOS, cytokine TNFalpha, and growth factor VEGF during pathological process of development of heart failure after the myocardial infarction. Male Sprague-Dawley rats were used for experimental myocardial infarction. The procedure was performed by anterolateral thoracotomy and snearing LAD with the metal clip. The hemodynamic measurements were done with the Langendorff preparation converted into a working heart system. The hemodynamic parameters were recorded at day 6, 11, 28, 40 and the myocardium for gene expression was collected at day 1, 4, 11, 28, 40. Control group was sham operated rats. The VEGF, TNFalpha, iNOS, and GAPDH genes were detected by RT-PCR assay from samples taken at border zone of myocardial infarction. Expression of isoform VEGF120 was found at day 1 and 4 after MI, whereas isoforms VEGF164 and VEGF188 along with expression of TNFalpha and iNOS was found at day 1, 4, 11, 28, 40. No expression of examined genes was detected in the myocardium of control rats. The expression of studied factors was parallel with development of heart failure after myocardial infarction assessed by hemodynamic measurements. These findings confirm the postulated involvement of TNFalpha, iNOS and growth factor VEGF in the remodeling of the myocardium and development of heart failure after experimental myocardial infarction.     

Cellular Development of the American Lobster Heart

The heart of the American lobster, Homarus americanus, representsa relatively simple nerve-muscle system. A nine-celled cardiacganglion functions to initiate the contractions of the transverselystriated myocardium. To describe the development of this nerve-musclesystem, hearts of lobster embryos in different stages of developmenthave been examined with an electron microscope. The heart wallconsists of an outer adventitial layer of fibroblast cells andan inner layer of transversely striated myocardial cells. Adjacentfibroblast cells form fasciae adherent and gap junctions. Intercalateddiscs and membrane regions of close apposition (4 nm) occurbetween adjacent myocardial cells. A cardiac ganglion that containsnine neurons and synaptic neuropil is present. Neuromuscularsynapses occur in the myocardium. Cells that we have termedstorage cells contain dense cytoplasmic inclusions that resembleyolk material and form gap junctions with myocardial cells.The heart at six weeks is about 200 µm long and 160 µmwide; at six months it is 300 and 250 /m respectively. The myocardiumgrows by an increase in the size and in the number of myocardialcells. The myocardium contains pre-myoblast, myoblast and well-differentiatedmyocardial cells. Sarcomere formation occurs first adjacentto the inner surface of the sarcolemma where the myofilamentsalign and anchor. Distinct A and I bands appear first, followedby the Z lines. The myocardium is sparsely innervated when theinitial heartbeats occur; a preliminary quantitative analysishas shown that three days later, the density of innervationhas increased six-fold.     

Enzyme histochemical studies on the conducting system of the human heart

Summary In this communication, the results of applying various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases in the human heart are presented. The Purkinje fibres of the atrioventricular conducting system of the human heart differ from the myocardium proper in containing a slightly higher activity of most of the glycolytic and gluconeogenetic enzymes investigated. The relatively higher activity of 6-phosphofructokinase, the key enzyme in anaerobic carbohydrate metabolism, is especially noteworthy. On the other hand, the activities of some of the enzymes that play a part in the aerobic energy metabolism is slightly less than those in the myocardium fibres.As for the activity of the NADPH regenerating enzymes, the activity of 6-phosphogluconate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating) is somewhat higher, and the activity of glucose-6-phosphate dehydrogenase similar, in the Purkinje fibres compared to that in the myocardial fibres. The activity of myosin ATPase is similar for both types of fibre. Likewise, the fibres of the conducting system and of the myocardium show a similar activity of acid phosphatase, -glucuronidase, non-specific naphthylesterase and peroxidase. The neurogenic function of the conducting system of the human heart was demonstrated by the high activity of acetylcholinesterase in the Purkinje fibres and in the atrioventricular node. All these histochemical findings in Purkinje fibres are similar at widely differing levels of the conducting system.     

Gap junctions in the heart of the adult Protopterus aethiopicus

In thin sections and in freeze-fracture replicas small and sparse gap junctions appear to be developed on the longitudinal plasma membrane of Protopterus cardiac cells near a macula or fascia adhaerens. By thin-section electron microscopy, they had septalaminar profiles with a length between 0.042 and 0.260 micron. In freeze-fracture images they appear on the P-fracture face as maculate particle aggregations with complementary pits on the E-fracture face. Particles with a central intercellular channel could be observed. The average center-to-center distance between neighbouring particles or pits is 10.05 +/- 1.87 nm (N = 2429). The diameter of the junctional maculae in replicas lies between 0.037 and 0.229 nm. The particle packing density increases in larger maculate aggregations, while particle-free areas emerge which could be related to the degradation or reformation of gap junctions Atypical configurations of gap junctions observed in the myocardium of lower vertebrates are rarely encountered in this primitive vertebrate.     

A G‐CSF functionalized scaffold for stem cells seeding: a differentiating device for cardiac purposes

Myocardial infarction and its consequences represent one of the most demanding challenges in cell therapy and regenerative medicine. Transfer of skeletal myoblasts into decompensated hearts has been performed through intramyocardial injection. However, the achievements of both cardiomyocyte differentiation and precise integration of the injected cells into the myocardial wall, in order to augment synchronized contractility and avoid potentially life‐threatening alterations in the electrical conduction of the heart, still remain a major target to be pursued. Recently, granulocytes colony‐stimulating factor (G‐CSF) fuelled the interest of researchers for its direct effect on cardiomyocytes, inhibiting both apoptosis and remodelling in the failing heart and protecting from ventricular arrhythmias through the up‐regulation of connexin 43 (Cx43). We propose a tissue engineering approach concerning the fabrication of an electrospun cardiac graft functionalized with G‐CSF, in order to provide the correct signalling sequence to orientate myoblast differentiation and exert important systemic and local effects, positively modulating the infarction microenvironment. Poly‐(l ‐lactide) electrospun scaffolds were seeded with C2C12 murine skeletal myoblast for 48 hrs. Biological assays demonstrated the induction of Cx43 expression along with morphostructural changes resulting in cell elongation and appearance of cellular junctions resembling the usual cardiomyocyte arrangement at the ultrastructural level. The possibility of fabricating extracellular matrix‐mimicking scaffolds able to promote myoblast pre‐commitment towards myocardiocyte lineage and mitigate the hazardous environment of the damaged myocardium represents an interesting strategy in cardiac tissue engineering.     

Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo

Embryonic chick myocardium (stages 8+ to 12?) was studied by light and electron microscopy. The myocardium, which is initially comprised of radially oriented cells with large intercellular spaces gradually becomes more tightly packed. Intercellular spaces decrease and the cells assume a circumferential orientation. Myocardial cells remain epithelial throughout formation of the functional tubular heart and specialized epithelial junctions (apical junctional complex or terminal bars) undergo modification to form intercalated discs. Embryonic myocardial cells contain large amounts of free ribosomes and particulate glycogen, the latter often associated with portions of granular reticulum. Unlike developing skeletal muscle. The amount of granular reticulum contained in the myocardial cell cytoplasm is large and, along with a hypertrophied Golgi apparatus, suggests that these cells may have a secretory function. These organelles persist during the initial period of fibril formation. Myofibrils apparently form from non filamentous precursor material and not by alignment of sequentially synthesized components.     

Age-related characteristics of the electrical activity of the cardiac fibers in the human embryo

Transmembrane action potential and intercellular electric coupling have been studied in isolated heart during various stages of intrauterine growth of human embryos as well as in heart preparations of human adults. Formation of electrical properties of ventricular myocardium was slower than that of atrial fibres and was found to be fully completed in the first 14 weeks of intrauterine growth. Intercellular electrical coupling enhanced in the course of the embryonic development as indicated by a drop in the input resistance and an increase in electronic length.     

Enzyme histochemical studies on the Purkinje fibres of the arrioventricular system of the bovine and porcine hearts

Synopsis In this communication the results of applying various histochemical semipermeable membrane techniques to the localization of several enzymes in bovine and procine heart are presented. The Purkinje fibres of the atrioventricular conducting system of the bovine heart differ from the myocardium proper in containing a greater activity of the glycolytic and gluconeogenetic enzymes—lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase, hexokinase, glucosephosphate isomerase and phosphoglucomutase, and less activity of the aerobic enzymes-NADH: nitroBT oxidoreductase and isocitrate dehydrogenase (NADP⁺). The metabolic reactions obtained with Purkinje fibres of the porcine heart are less pronounced. These histochemical findings are in accordance with the impression that Purkinje fibres, compared with the common myocardial fibres, have a higher rate of anaerobic metabolism and a lower rate of aerobic metabolism.The activity of the NADPH regenerating enzymes, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating), and the activity of acid hydrolases such as non-specific esterase and acid phosphatase is higher in the Purkinje fibres of both the bovine and porcine heart.     

Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum

In Ambystoma mexicanum, recessive mutant gene c results in an absence of embryonic heart function because of altered influences from surrounding tissues (Humphrey, 1972). The present light and electron microscope study compares heart development in normal and mutant embryos from Harrison stage 34 or 6 days (at which normal heart beat initiates) through stage 41 or 25 days (at which mutant embryos die). The hearts display increasing differences as development progresses, and by stage 41 mutant abnormalities are striking. The normal myocardium shows organized sarcomeres at stage 34 and numerous intercalated discs subsequently appear. By stage 41, the normal myocardium is composed of highly differentiated muscle cells and shows extensive trabeculation. The mutant myocardium throughout development remains only one cell layer thick with no indication of developing trabeculae. Mutant cells at stage 34 have a few 140 Å and 60 Å filaments along with what appear to be Z bodies. A partial organization of myofibrillar components is occasionally noted at stages 38–41; however, distinct sarcomeres are not apparent and intercalated discs are rarely seen. In general the mutant cells appear less differentiated than usual and in many respects are reminiscent of pre-heart-beat normal cells. Although most mutant cells show images characteristic of pathological conditions (e.g., pleomorphic mitochondria, membranous whorls, and numerous autophagic vacuoles), selective myocardial cell death, a phenomenon associated with normal trabeculation, is not evident. It is clear that gene c, in homozygous condition, results in an altered pattern of heart cell differentiation. The mutation, by way of abnormal inductive processes, appears to affect the synthesis and organization of heart contractile proteins.     

On the spatial relationship between fibroblasts and myogenic cells during early development of skeletal muscles

The spatial relation between fibroblasts and myoblasts as well as myotubes in the extensor muscles of the forearm of mouse embryos was investigated with routine electron microscopic techniques. From day 13 of embryonic development onwards, fibroblasts and myogenic cells may be distinguished by their ultrastructure from each other. Above all the fibroblasts are characterized by their long cell processes. Specialized cell contacts between them are visible. Fibroblasts often encircle some myogenic cells indicating the division of the muscles into smaller bundles. Over long distances, the intercellular gap between both cell types is narrow and measures about 15 nm. Sometimes specialized intercellular junctions can be seen. They resemble simple desmosomes and seem to be mechanical cell junctions. After the establishment of the basal lamina around the myogenic cells such specialized cell contacts are no longer visible. It seems possible that the fibroblasts act as mechanical counterpart for the organization of the myogenic cells.     

Ultrastructural morphometry of the myocardium of Thunnus alalunga

The common ventricle in the heart of the Thunnus alalunga was studied. The ventricular myocardium consists of an outer compact layer and a thick inner spongy layer. The compact layer has slightly larger cells (4-6 microns diameter) than the spongy layer (2.5-5 microns diameter). Ultrastructurally the myocardium displays normal arrangements of myofibrils and mitochondria. The sarcoplasmic reticulum is poorly developed. The intercalated discs are simple with the fascia adherens being the most frequent junctional type observed; occasionally a desmosome was seen. Nexus type junctions are present but are unassociated with the intercalated discs. There are no t-tubules evident but the plasmalemma exhibits numerous caveolae which rarely form couplings with the sarcoplasmic reticulum. A morphometric analysis of the volume percent of mitochondria and myofibrils showed that the myocardial cells in the spongy layer of the heart have a significantly greater volume percentage of mitochondria than the compact layer. No significant differences were found between myocardial regions when the volume percentages of myofibrils were compared. The physiological studies revealed that the albacore tuna has heart rates (120 bpm) and ventricular blood pressures (100 mmHg) that are among the highest reported for fish.     

Pathophysiologic role of myocardial apoptosis in post-infarction left ventricular remodeling

Left ventricular (LV) remodeling and heart failure (HF) complicate acute myocardial infarction (AMI) even weeks to months after the initial insult. Apoptosis may represent an important pathophysiologic mechanism causing progressive myocardiocyte loss and LV dilatation even late after AMI. This review will discuss the role of apoptosis according to findings in animal experimental data and observational studies in humans in order to assess clinical relevance, determinants, and mechanisms of myocardial apoptosis and potential therapeutic implications. More complete definition of the impact of myocardiocyte loss on prognosis and of the mechanisms involved may lead to improved understanding of cardiac remodeling and possibly improved patients' care. Mitochondrial damage and bcl-2 to bax balance play a central role in ischemia-dependent apoptosis while angiotensin II and beta(1)-adrenergic-stimulation may be major causes of receptor-mediated apoptosis. Benefits due to treatment with ACE-inhibitors and beta-blockers appear to be in part due to reduced myocardial apoptosis. Moreover, infarct-related artery patency late after AMI may be a major determinant of myocardial apoptosis and clinical benefits deriving from an open artery late post AMI (the "open artery hypothesis") may be, at least in part, due to reduced myocardiocyte loss.