Cardiac Muscle Bundles Research Articles

GH and IGF-2 Histological Effects on the Heart, Liver and Gizzard of Chickens were Studied

GH and IGF-2 were examined histologically in the present study on adult hens to learn more about the organs’ responses to GH and IGF-2. Cardiac protein synthesis is stimulated by GH and IGF-2, according to microscopic examination. The recent research found a considerable amount of adipose tissue in the cardiac muscle bundles, which is linked to the metabolic process. In addition, GH and IGF-2 were shown to promote protein synthesis and mitosis in liver and gizzard tissues, according to the research. In addition, the apoptosis, regeneration, and secretory activity of gizzard glands are increased by the aforementioned hormones.

Physiologic biomechanics enhance reproducible contractile development in a stem cell derived cardiac muscle platform

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling.

High efficiency preparation of skinned mouse cardiac muscle strips from cryosections for contractility studies.

What is the central question of this study? Can frozen cardiac papillary muscles and cryosectioning be used to reliably obtain uniform cardiac muscle strips with high yields? What is the main finding and its importance? A new method was developed using frozen cardiac papillary muscles and cryosectioning to reliably obtain uniform cardiac muscle strips with high yields. Experimental results demonstrate that this new methodology significantly increases the efficiency and application of quantitative biomechanical studies using skinned muscle fibres with an additional advantage of no need for transferring live animals. Skinned cardiac muscle preparations are widely used to study contractile function of myofilament proteins and pathophysiological changes. The current methods applied in these biomechanical studies include detergent permeabilization of freshly isolated papillary muscle, ventricular trabeculae, surgically dissected ventricular muscle strips, mechanically blended cardiac muscle bundles or myocytes, and enzymatically isolated single cardiomyocytes. To facilitate and expand the skinned cardiac muscle approach, we have developed an efficient and readily practical method for mechanical studies of skinned mouse cardiac papillary muscle strips prepared from cryosections. Longitudinal papillary muscle strips of 120-150µm width cut from 35-70µm-thick cryosections are mounted to a force transducer and chemically skinned for the studies of force-pCa and sarcomere length-tension relationship and rate of tension redevelopment. In addition to more effective skinning and perfusion than with whole papillary muscle and much higher yield of useful preparations than that from trabeculae, this new methodology has two more major advantages. One is to allow for the use of frozen cardiac muscle in storage to maximize the value of muscle samples, facilitating resource sharing among research institutions without the need of transferring live animals or fresh biopsies. The other is that the integrity of the muscle strips is well preserved during the preparation and mechanical studies, allowing coupled characterization of myofilament proteins. The combined power of biomechanics and protein biochemistry can provide novel insights into integrative physiological and pathophysiological mechanisms of cardiac muscle contraction while the high yield of high-quality muscle strips also provides an efficient platform for development of therapeutic reagents.

Molecular and Cellular Mechanisms of AtrialFibrosis in AtrialFibrillation.

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Atrial fibrosis has emerged as an important pathophysiological contributor and has been linked to AF recurrences, resistance to therapy and complications. Here, the author reviews the molecular and cellular mechanisms that control atrial fibrosis. It is important to note that not all tissue fibrosis is identical. For example, reactive (interstitial) fibrosis increases the amount of collagen between cardiac muscle bundles without fundamentally altering muscle bundle architecture. Replacement (reparative) fibrosis replaces dead cardiomyocytes with extracellular matrix tissue and fibroblasts, preserving tissue integrity at the expense of muscle bundle continuity. Replacement fibrosis may be much more disruptive to electric conduction and more difficult to reverse than reactive fibrosis. The author reviews the complex signaling systems that cause fibrosis, including those connected to connective tissue growth factor, angiotensin-II, platelet-derived growth factor, and transforming growth factor-β. The author then considers the molecular constitution of fibrous tissue, including the production and maturation of collagen and the roles of important extracellular matrix proteins such as fibronectin, tenascin-C, and thrombospodin-1. The author then discusses the evolving evidence for an important role of Ca2+ entry in the profibrotic activation of fibroblasts, along with evidence that dysregulation of Ca2+-transporting transient potential receptor channels and inward rectifier K+ channels in AF fibroblasts is profibrotic. Finally, the author reviews the evidence for micro-ribonucleic acid involvement in atrial fibrotic signaling and AF promotion. It is hoped that an improved understanding of the mechanisms controlling atrial fibrosis will open up new opportunities for AF prevention and management.

Gross and microanatomical studies on the moderator bands (septomarginal trabecula) in the heart of mature Dromedary camel (Camelus dromedarius)

The current work was carried out to investigate the gross and microanatomical features of moderator bands (septomarginal trabecula) in camel heart. Ten hearts were collected from healthy mature dromedary camels. Anatomically, the moderator bands were present in both right and left ventricles. In right ventricle, the walls had one muscular moderator band which was extended from the interventricular septum to the opposite ventricular wall especially to the papillary muscle. In left ventricle, there were two bands; one extended from the interventricular septum to the papillary muscles, and the other one was present in various places especially in the apex running as a thin thread-like band across the left ventricular wall. Histological examination revealed that the moderator band consisted of two major layers; the central (core) myocardium and the peripheral endocardium, acting as band capsule. The myocardium had two bundles; the contractile cardiac muscle bundles and the Purkinje fiber bundles. The endocardium consisted of three layers; the endothelial layer of simple squamous epithelium, the subendothelial layer of loose connective tissue and the subendocardial layer, connecting the endocardium with the myocardium.

Arrhythmia and left ventricular ejection fraction: Once broken, always broken?

Left ventricular ejection fraction (LVEF) occupies a central place within current guidelines for implantable cardioveter-defibrillator (ICD) therapy in patients with nonischemic cardiomyopathy. The 2 largest trials recruited patients with an LVEF of ≤35% that was “stable” or “irreversible.” 1,2 Patients undergoing long-term treatment for nonischemic cardiomyopathy often have substantial improvements in LVEF, although the association with reduced arrhythmia risk has not been extensively studied. The Defibrillators in Non-Ischemic Cardiomyopathy Treatment Evaluation (DEFINITE) study included 458 patients with nonischemic cardiomyopathy, New York Heart Association class (NYHA) I–III symptoms, LVEF ≤35%, and ambient arrhythmias on Holter monitoring (ventricular ectopy or nonsustained ventricular tachycardia [NSVT]). All patients received optimal medical therapy and were randomized to receive an ICD or no device therapy. The trial reported a trend toward reduced mortality in the ICD arm, but did not achieve statistical significance. This may have been due to unexpectedly low mortality in the control arm. In this issue of HeartRhythm, Schliamser et al 3 investigate the relationship between changes in LVEF and clinical events in a post hoc analysis of the DEFINITE study. The protocol specified that LVEF should be assessed at yearly intervals, but only 17% patients had these data available. However, some repeated LVEF measurements were available in 197 of 449 (44%) patients and therefore these were studied. The patients were grouped according to the change in LVEF: 45% increase (group I), o5% change (group II), or 45% decrease (group III). An LVEF improvement was associated with substantial survival benefit (deaths in each group were 3.2%, 12.6%, and 33%, respectively). An improvement in NYHA class was also associated with improved survival. However, although group I had a tendency toward reduced arrhythmic events, this did not reach statistical significance. In 70 of 197 (36%) patients, LVEF increased to an absolute value of 435% but 4 of these patients went on to have an arrhythmic event after this improvement had occurred. The results of this retrospective analysis must be interpreted with caution. A large number of patients had to be excluded because follow-up LVEF assessments were not made. The patients in whom follow-up data were available had a higher incidence of ICD shocks, were more likely to be young and white, have diabetes, and have better 6-minute walk test results. Patients were categorized according to changes in LVEF of just 5%, with LVEF determination being performed according to the local hospital’s preference. Mostly, echocardiography was used, which has variable reproducibility and the assessors of LVEF were not blinded to other clinical information. While this is a technical limitation to the study, the data have been obtained within the context of standard clinical practice and, in this sense, may be more realistic. Why might recovery of LVEF not fully predict a reduction in arrhythmia risk? Nonischemic cardiomyopathy is an umbrella term that encompasses a wide range of pathological processes, with the end result being mechanical and electrical dysfunction. LVEF is just one marker of these disease processes. Electrolyte disturbances and stretch-induced arrhythmia may also contribute to arrhythmogenicity. Histological studies have shown increased fibrosis between cardiac muscle fibers and bundles that may enhance anisotropy and promote electrical uncoupling. This can provide the substrate for reentry during ventricular fibrillation. 4 In another small study of 29 patients undergoing ablation for ventricular arrhythmias in the presence of nonischemic cardiomyopathy, fibrosis was detected with magnetic resonance imaging in approximately half of the patients. 5 It is probable that fibrosis might persist even in patients whose LVEF improves. Other clinical parameters are also known to give an indication of arrhythmia risk. Left bundle branch block may be related to the presence of fibrosis.InthestudybySchliamser et al, there was a significantly lower proportion of patients with left bundle branch block in those with LVEF improvement. Other factors such as the presence of pulmonary hypertension, hyponatremia, NYHA class, and coexistent right-sided heart failure have prognostic significance. Logically, these additional pieces of information could contribute to risk stratification for

Telocytes form networks in normal cardiac tissues.

Telocytes (TC) are a class of interstitial cells present in heart. Their characteristic feature is the presence of extremely long and thin prolongations (called telopodes). Therefore, we were interested to see whether or not TCs form networks in normal cardiac tissues, as previously suggested. Autopsy samples of cardiac tissues were obtained from 13 young human cadavers, without identifiable cardiac pathology and with a negative personal history of cardiovascular disease. Immunohistochemistry on formalin-fixed paraffin-embedded tissues was performed using monoclonal antibodies for CD117/c-kit. Additionally, ventricular samples from 5 Sprague-Dawley rats were ultrastructurally evaluated under transmission electron microscopy. We found c-kit positive cells with TC features in subepicardium, as well in subepicardial arteries and in subepicardial fat. TCs were also present in the subendocardium. Light and electron microscopy revealed the existence of intramyocardial networks built up by bipolar TCs. Larger c-kit positive multipolar TCs were found between cardiac muscle bundles. Our results support the existence of a cardiac network of telocytes.

Gene Therapy for Repair of Cardiac Fibrosis

Because of the increase in life expectancy and a high survival rate after myocardial infarction, cardiac fibrosis is becoming one of the most important problems in cardiology. It is characterized by excessive accumulation of fibrillar collagen in the extracellular space, either because of a loss of cardiomyocytes (replacement fibrosis) and/or as an interstitial response to various chronic cardiovascular diseases such as hypertension, myocarditis, and congestive heart failure (reactive fibrosis).1 In humans, cardiac fibrosis is universal in the aging heart. Activated fibroblasts play a pivotal role in the formation and maintenance of fibrous tissue by the production of various extracellular-matrix proteins, including collagen and fibronectin. It is now recognized that even in areas with long-standing fibrosis, such as postinfarction scars, fibroblasts remain metabolically active.2 Their activity is regulated by various autocrine and paracrine factors, such as angiotensin II, aldosterone, endothelins, cytokines, and growth factors.3 The renin-angiotensin-aldosterone system is considered to be of major importance for the regulation of cardiac fibrosis. Increased tissue levels of angiotensin II have shown to promote, and ACE inhibitors are able to delay the development of, cardiac fibrosis.4,5 See p 394 Interstitial fibrosis reduces the electrical coupling between cardiac myocytes because fibroblasts produce smaller or larger collagenous septa, which electrically insulate cardiac cells or muscle bundles. As a result, the normal myocardial architecture becomes disrupted and is transformed into a pathological substrate characterized by the presence of multiple insulating barriers, which force the depolarization wave to spread nonuniformly. As predicted by computer simulations and demonstrated in cell cultures, in such a medium, long local conduction delays occur that may slow the effective conduction velocity to very low values. Unlike a decrease of the rapid sodium current, which will result in conduction failure when conduction velocity is depressed to approximately 30% of control, slow …

Implantation of Immature Neonatal Cardiac Cells Into the Wall of the Aorta in Rats

Morphological and functional development of implanted neonatal cardiac cells in the wall of the abdominal aorta in rats was investigated. Cardiomyocytes from neonatal Fischer rats (both sexes) or medium were injected into the wall of the abdominal aorta in female Fischer rats (n=22 in each group). Two or 6 weeks later, the grafted site was exposed and fixed for histological and immunohistological examination. Polymerase chain reaction analysis of the SRY gene to identify male cells was performed in the treated aortas. Seven of 10 cell-treated aortas but none of 10 medium-treated aortas showed spontaneous rhythmic beating at the grafted site after excision of the heart at 2 weeks. Polymerase chain reaction of the SRY gene was positive in 3 cell-treated aortas and none of 3 medium-treated aortas at 6 weeks. Hematoxylin-and-eosin staining showed viable grafts in 9 of 10 aortas at 2 weeks and 9 of 9 aortas at 6 weeks in the cell-treated group but in none of the aortas receiving medium. Neonatal cardiomyocytes in the graft formed compact, longitudinally oriented cardiac muscle bundles and had cross-striations and vascularization. Immunohistochemical staining for sarcomeric actin was positive in 4 of 10 aortas at 2 weeks and 9 of 9 aortas at 6 weeks in the cell group but in none of the aortas in the medium group. Grafted neonatal cardiomyocytes survive, differentiate, grow, develop a blood supply, and spontaneously contract within the wall of the aorta in rats.

Tissue engineering of a differentiated cardiac muscle construct.

Cardiac tissue engineering is an emerging field. The suitability of engineered heart tissue (EHT) for both in vitro and in vivo applications will depend on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro, contractile function, and electrophysiological properties. Here, we demonstrate that cardiac myocytes from neonatal rats, when mixed with collagen I and matrix factors, cast in circular molds, and subjected to phasic mechanical stretch, reconstitute ring-shaped EHTs that display important hallmarks of differentiated myocardium. Comparative histological analysis of EHTs with native heart tissue from newborn, 6-day-old, and adult rats revealed that cardiac cells in EHTs reconstitute intensively interconnected, longitudinally oriented, cardiac muscle bundles with morphological features resembling adult rather than immature native tissue. Confocal and electron microscopy demonstrated characteristic features of native differentiated myocardium; some of these features are absent in myocytes from newborn rats: (1) highly organized sarcomeres in registry; (2) adherens junctions, gap junctions, and desmosomes; (3) a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and (4) a basement membrane surrounding cardiac myocytes. Accordingly, EHTs displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong beta-adrenergic inotropic response. Action potential recordings demonstrated stable resting membrane potentials of -66 to -78 mV, fast upstroke kinetics, and a prominent plateau phase. The data indicate that EHTs represent highly differentiated cardiac tissue constructs, making EHTs a promising material for in vitro studies of cardiac function and tissue replacement therapy.

Localization of GABA- and glutamate-like immunoreactivity in the cardiac ganglion of the lobster Panulirus argus.

Wholemount immunohistochemical methods were used to examine the localization of gamma-aminobutyric acid (GABA) and glutamate within the cardiac system of the Caribbean spiny lobster Panulirus argus. All of the GABA-like immunoreactivity (GABAi) in the cardiac ganglion originated from a single bilateral pair of fibers that entered the heart via the two dorsal nerves. Each GABAi axon bifurcated upon entering the ganglion and gave rise to varicose fibers that surrounded the somata and initial segments of the five large motor neurons. The four small posterior cells did not appear to receive somatic contacts. Double-labeling experiments in which individual motor neurons were injected with Neurobiotin showed that their dendritic processes, which project to muscle bundles adjacent to the ganglion and are thought to respond to stretch, were also accompanied by branches of the GABAi fibers. Glutamate-like immunoreactivity (GLUi) was present in each of the motor neuron cell bodies. In some preparations, GLUi was also detected in large caliber fibers in the major ganglionic nerves. These fibers gave rise to more slender branches that innervated the cardiac muscle bundles. GLUi was also found in the small cell bodies and in fibers surrounding motor neuron somata. Taken together, these findings support previous electrophysiological, pharmacological and anatomical studies indicating that GABA mediates extrinsic inhibition and that glutamate acts as a neuromuscular and intraganglionic transmitter in this system. While axosomatic contacts may play a major role in both transmitter systems, the GABAergic inhibition also appears to involve substantial axodendritic synaptic signaling.

不整脈原性右室心筋症（ＡＲＶＣ）が疑われた突然死の１例 心肺蘇生に伴う右心室破裂合併例

A 49-year-old woman was found submerged in the bathtub at home. Closed chest compression was performed first by her husband for approximately 10 minutes, and then by ambulance personnel for 37 minutes. She was transported to our hospital's emergency room while cardiopulmonary resuscitation was continued. She received external chest compression for 19 minutes and then open-chest direct cardiac massage for 45 minutes. Major autopsy findings were hemorrhage (approximately 400ml) in the pericardial sac and a large longitudinal 3.5cm laceration of the right ventricle. Histopathological examination of the right ventricle revealed marked fatty infiltrations, which had partially replaced the major cardiac muscle layer. Some of cardiac muscle bundles in the right ventricle were disarrayed with fibrosis and fat. Infiltrations of fat were also seen in the left ventricular wall and septum near the conducting system. These autopsy findings suggest that the major cause of death in this case might have been a lethal arrhythmia due to arrhythmogenic right ventricular cardiomyopathy (ARVC), and that the ruptured right ventricular wall and cardiac tamponade were complications of the vigorous resuscitative efforts. In conclusion, the softened myocardium with massive fatty infiltration in ARVC may be susceptible to CPR-related injuries.

Ion-specific protein destabilization of the contractile proteins of cardiac muscle fibers.

We investigated the inhibitory effects of increased salt concentrations on maximal calcium-activated force (Fmax) of rabbit cardiac papillary muscle bundles skinned with Triton X-100. While other studies have reported a lack of ion-specific effects on Fmax of cardiac muscle, we clearly demonstrated the presence of such effects when a wider variety of salts was investigated. In addition, like skeletal muscle, cardiac muscle was found to be sensitive to ionic strength and not to ionic equivalence. In support of our hypothesis that the ion-specific effects are due to protein destabilization, we found that a protein stabilizer (trimethylamine N-oxide, TMAO) completely abolished the ion-specific effects on Fmax. The ion-specific effect is probably due to binding of ions to the contractile proteins. The general ionic effect is most likely due to electrostatic shielding that remains in the presence of TMAO. Neither 300 mM sucrose nor TMAO significantly altered Fmax at physiological ionic strength indicating that the ion-specific depression of Fmax was not due to a colligative/osmotic effect. Furthermore, adding sucrose to solutions with a supraphysiological ionic strength caused a further decrease in Fmax indicating that certain osmolytes can alter Fmax if the contractile proteins are initially destabilized.

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade.

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with antihypertensive and non-antihypertensive doses of the angiotensin II receptor AT1 antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.

Osmotic compression of skinned cardiac and skeletal muscle bundles: Effects on force generation, Ca 2+ sensitivity and Ca 2+ binding

Length-dependence of myofilament Ca2+ sensitivity is now considered to be an important component of the steep relationship between active force and sarcomere length along the ascending limb of the cardiac force-length curve. Studies with skinned cardiac muscle preparations have demonstrated that Ca(2+)-troponin C affinity is significantly increased as sarcomere length is increased over the range 1.7-2.3 microns. Increase in sarcomere length is accompanied by a reduction in interfilament spacing. In skinned fiber preparations from both cardiac and skeletal muscle osmotic compression of the filament lattice enhances myofilament Ca2+ sensitivity. This study was undertaken to evaluate the hypothesis that a change in filament separation may contribute to the length-dependent activation seen in cardiac muscle. Moderate reduction in interfilament spacing caused by exposure to Dextran T-500 (5-10%) produced an increase in force generation in both maximally activated and partially activated preparations of skinned bovine ventricular muscle. With fiber bundles of mean sarcomere length 1.7 microns the addition of 5% Dextran T-500 produced an increase in Ca2+ sensitivity of about 0.25 pCa units and a significant increase in Ca2+ binding in the pCa range (6.0-5.0) in which the single regulatory site of cardiac troponin C is titrated. This concentration of Dextran T-500 produced a reduction in fiber width equivalent to that produced by stretching fibers from sarcomere length 1.7 microns to sarcomere length 2.3 microns Osmotic compression of skinned rabbit psoas muscle fibers also enhanced Ca2+ sensitivity but there was no significant change in Ca(2+)-troponin C affinity. These data suggest that 1) an important component of length-dependent Ca2+ sensitivity in both cardiac and skeletal muscle is the change in interfilament spacing, and 2) in cardiac muscle a reduction in spacing, like increase in length, leads to a specific increase in Ca(2+)-troponin C affinity. Thus both filament overlap and filament separation contribute to the length dependence of Ca2+ sensitivity and Ca2+ binding in cardiac muscle.

Impedance analysis applicable to cardiac muscle and smooth muscle bundles

An electrical equivalent circuit was constructed to represent a chain of five myocardial cells in a cardiac muscle bundle with various degrees of cell-to-cell coupling, and an impedance analysis was performed. The impedance across the entire network was measured at frequencies ranging from 10(1) to 10(6) Hz. The Bode plots were nearly superimposable for 1, 10, and 100 tunnels; for 10(3), 10(4), and 10(5) tunnels, the absolute zeta at 10 Hz was lower: e.g., 9.82 M omega for 1 tunnel compared to 6.64 M omega for 10(5) tunnels. The delta zeta 1/2 values were shifted to the left in the well-coupled cases: e.g., for 1 tunnel, f1/2 was 37.8 kHz, and for 10(5) tunnels, f1/2 was 1.2 kHz. For high coupling, the Bode plots contained a double component due to the end membranes. When Ro was increased by eight times, zeta increased by 7.47 fold (for 1 tunnel, 10 Hz), and by 3.72 fold (for 10(5) tunnels, 10 Hz). Raising Ro to x 12, x 100, and x 1000 produced a further and further shift to the left of the Bode plots. The total tissue resistivity (Rt) increased as a function Ro. Thus, in low coupling cases, almost all of the applied current passes through the interstitial space; e.g., at 1 tunnel (10 Hz), 1.0% of the current passes through the cell pathway (Rcell). The ratio of impedances at 10 kHz to 10 Hz (zeta 10kHz/zeta 10Hz) decreased with increasing tunnels (for Ro x 1). The ratio of resistivities at Ro x 8 to Ro x 1 (Rt'/Rt) was 7.47 for 1 tunnel. In contrast, the ratio at 10(5) tunnels was 3.73. It is concluded that it is difficult to determine the degree of cell coupling from such impedance analysis, unless the same tissue can be used for its own control, i.e., before and after a large change in cell coupling is introduced.

Bound calcium and force development in skinned cardiac muscle bundles: Effect of sarcomere length

There is evidence that the steep ascending limb of the force-length curve in cardiac muscle (Frank-Starling relation) is based on a length-dependence of myofilament Ca2+ sensitivity. Previous work from this laboratory has indicated that in the sarcomere length range corresponding to the ascending limb of the cardiac force length curve (1.7 to 2.3 microns) the Ca2+-troponin C affinity is length-dependent. In this study Ca2+ binding to chemically skinned bovine cardiac muscle bundles was measured during ATP-induced force generation with fiber bundles having sarcomere lengths of 2.2 to 2.4 microns and 1.6 to 1.8 microns. A double isotope technique was used to make concurrent determinations of the force-pCa and bound Ca2+-pCa relationships. At the longer sarcomere lengths the fibers bound, at saturation, an amount of Ca2+ equivalent to approximately 3 mol Ca2+/mol troponin C. Force development appeared to be coupled to titration of the single, low-affinity Ca2+-specific site. In the pCa range 7.0 to 6.0 sarcomere length had no effect on Ca2+ binding. In the pCa range 6.0 to 5.0, in which force increased steeply, there was, in addition to a decreased relative force, a significant reduction in bound Ca2+ at the shorter sarcomere length. Thus sarcomere length appears to influence the Ca2+ binding properties of the regulatory site on troponin C. These data provide direct evidence that length-dependent modulation of Ca2+-troponin C affinity may make a major contribution to the force-length relationship in cardiac muscle.

Effect of length and cross-bridge attachment on Ca2+ binding to cardiac troponin C.

The sensitivity of skinned cardiac muscle bundles to Ca2+ is a function of sarcomere length. Ca2+ sensitivity is increased as fiber length is extended along the ascending limb of the force-length curve and it has been suggested that this phenomenon makes a major contribution to the steep force-length relationship that exists in living cardiac muscle. To gain greater insight into the mechanism behind the length dependence of Ca2+ sensitivity isotopic measurements of Ca2+ binding to detergent-extracted bovine, ventricular muscle bundles were made under conditions in which troponin C was the only major Ca2+ binding species. Experiments were designed to determine whether 1) Ca2+-troponin C affinity varies in the sarcomere length range corresponding to the ascending limb of the force-length curve, and 2) Ca2+ binding correlates with length per se or with changes in the number of length-dependent cross-bridge attachments. Measurements were made of Ca2+ binding in the rigor and relaxed states. The latter state was produced by suppressing actin-myosin interaction with the phosphate analogue, sodium vanadate. After vanadate treatment it is possible to obtain a complete Ca2+ saturation curve in the presence of physiological MgATP concentrations and at constant sarcomere length. The results show that the binding of Ca2+ to the regulatory site of cardiac troponin C is length dependent but this length dependence is actually a dependence on the number of attached cross bridges.

Histochemical and ultrastructural study on the innervation of human and porcine atrio-ventricular valves.

The presence of nerve fibers was investigated in porcine and human atrio-ventricular valves by AChE technique, formaldehyde-induced fluorescence, en bloc silver and gold chloride impregnation and electron microscopy. Elaborate nerve plexuses were observed in every leaflet and in some chordae tendineae of all the samples examined, without significant species differences in the pattern of innervation. The presence of a sensory innervation was inferred from the demonstration, in whole mount samples processed for acetylcholinesterase, of thick myelinated nerve fibers and of endings with dot-like or brush-like appearance. Moreover the results of the combined histochemical and ultrastructural methods showed the existence of both cholinergic and adrenergic efferent nerve fibers. Nerve varicosities with clear or dense-cored vesicles were frequently observed in proximity to blood vessels and to cardiac and smooth muscle bundles, which therefore can be considered as the targets of the efferent nerve supply. The complex pattern of the innervation herein demonstrated suggests the existence of a nervous control of valvular function through the regulation of contractile elements.

Ratio of transverse to longitudinal resistivities of isolated cardiac muscle fiber bundles

The ratio of transverse to longitudinal resistivities (рt/р1) of isolated cat cardiac muscle bundles (papillary muscles and ventricular trabeculae) composed of parallel fibers was measured and compared with frog sartorius muscles. Similar measurements made on a physical model representing such bundles of parallel fibers, consisting of layers of glass rods orderly packed with Ringer solution filling the interstices, gave a рt/р1 of 7. This ratio averaged 11 for cardiac muscle and 21 for skeletal muscle, indicating that in both muscles the transverse conductivity and effective diffusion coefficient are much less than the longitudinal values. Equilibration of cardiac muscle in ten-fold diluted Ringer (sucrose to isotonicity) to raise the resistance of the interstitial fluid (ISF) 8.5-fold raised р1 6.3-fold, but hardly affected р1, so that рt/р1 was markedly reduced to 2.0. In sartorius, the high-resistance solution had much less of an effect on р1 or on рt/р1. Equilibration in hypertonic Ringer (2 X isotonicity with sucrose) raised р1 and lowered рt slightly in both types of muscles, resulting in a small decrease in рt/р1. Calculations of the ratio of the resistance of the cellular pathway to that of the extracellular pathway averaged 20 for cardiac muscle compared to only 0.82 for skeletal muscle. The data indicate that the longitudinal current path through the myocardial cells is high in resistance compared to that through the ISF, suggesting that the intercalated disks are high-resistance membranes. The resistivity of the disk membranes averages 576 Ω-cm2, assuming a 6.4-fold increase in surface area due to convolutions; this value approaches that of the sarcolemma, and is much higher than the maximum (2 Ω-cm2) allowable for significant DC electrical coupling to occur between contiguous cells. The fact that the extracellular fluid of a parallel-fibered muscle bundle itself acts as a cable could account for the observation that measurements of the tissue length constant by extracellular application of current give large values; thus, such measurements cannot be used to determine the resistance of the cell junctions in muscles composed of short cells.