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Purpose: Migraine, a severely debilitating condition, may be effectively managed with topiramate, known for its migraine prevention and weight loss properties due to changes in body muscle and fat composition and improved insulin sensitivity. However, the mechanism of topiramate in modulating insulin response in adipocytes and myocytes remains elusive. This study aims to elucidate these molecular mechanisms, offering insights into its role in weight management for migraine sufferers and underpinning its clinical application. Methods: Insulin resistance improvements were evaluated through glucose uptake measurements in C2C12 muscle cells and 3T3L-1 adipocytes, with Oil red O staining conducted on adipocytes. RNA-seq transcriptome analysis was used to identify the regulatory target genes of topiramate in these cells. The involvement of key genes and pathways was further validated through western blot analysis. Results: Topiramate effectively reduced insulin resistance in C2C12 and 3T3L-1 cells. In C2C12 cells, it significantly lowered SORBS1 gene and protein levels. In 3T3L-1 cells, topiramate upregulated CTGF and downregulated MAPK8 and KPNA1 genes. Changes were notable in nuclear cytoplasmic transport and circadian signaling pathways. Furthermore, it caused downregulation of MKK7, pJNK1/ JNK1, BMAL1, and CLOCK proteins compared to the insulin-resistant model. Conclusion: This study provides preliminary insights into the mechanisms through which topiramate modulates insulin resistance in C2C12 myocytes and 3T3L-1 adipocytes, enhancing our understanding of its therapeutic potential in managing weight and insulin sensitivity in migraine patients. [ABSTRACT FROM AUTHOR]

Background: It has been reported that botulinum toxin type A (BoNT-A) produces structural changes in masticatory muscles. However, not all histomorphometric parameters affected by BoNT-A parameters have been assessed. This study investigated the histomorphometric changes in the masseter muscle of rats after a single injection of BoNT-A. Methods: Forty-four adult animals were randomly divided into control group (n = 22) and BoNT-A group (n = 22). Controls received a single dose of 0.14 mL/kg of saline in masseter muscles, and the BoNT-A group received a 7 U/Kg of BoNT-A. The groups received the same volume of injected substances. Animals were sacrificed on 7th (n = 5), 14th (n = 5), 21st (n = 5), 28th (n = 4) and 90th (n = 3) days post-treatment. Histological masseter tissue slides were obtained from hematoxylin–eosin treatment and analyzed in optical microscopy regarding muscle cross-sectional area, amount of connective tissue and quantity and diameter of myocytes. For statistical analysis, generalized linear models were used to compare the data (ANOVA). In all test, the significance level of 5% was set. Results: BoNT-A values of cross-sectional area of the masseter muscle were significantly lower than controls (p < 0.01) throughout the study. Regarding myocytes quantity, BoNT-A subgroups presented higher values than controls (p < 0.0001) since the 14th day until the end of the study; however, the diameter of myocytes was smaller in all BoNT-A subgroups (p < 0.0001) in all assessment points. The amount of connective tissue was higher in BoNT-A subgroups (p < 0.0001) throughout the study. Conclusion: A single injection of BoNT-A altered the structure of masseter muscle of rats, regarding its histomorphometric parameters. No Level Assigned: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266. [ABSTRACT FROM AUTHOR]

Employing animal models to study heart failure (HF) has become indispensable to discover and test novel therapies, but their translatability remains challenging. Although cytoskeletal alterations are linked to HF, the tubulin signature of common experimental models has been incompletely defined. Here, we assessed the tubulin signature in a large set of human cardiac samples and myocardium of animal models with cardiac remodeling caused by pressure overload, myocardial infarction or a gene defect. We studied levels of total, acetylated, and detyrosinated α-tubulin and desmin in cardiac tissue from hypertrophic (HCM) and dilated cardiomyopathy (DCM) patients with an idiopathic (n = 7), ischemic (n = 7) or genetic origin (n = 59), and in a pressure-overload concentric hypertrophic pig model (n = 32), pigs with a myocardial infarction (n = 28), mature pigs (n = 6), and mice (n = 15) carrying the HCM-associated MYBPC32373insG mutation. In the human samples, detyrosinated α-tubulin was increased 4-fold in end-stage HCM and 14-fold in pediatric DCM patients. Acetylated α-tubulin was increased twofold in ischemic patients. Across different animal models, the tubulin signature remained mostly unaltered. Only mature pigs were characterized by a 0.5-fold decrease in levels of total, acetylated, and detyrosinated α-tubulin. Moreover, we showed increased desmin levels in biopsies from NYHA class II HCM patients (2.5-fold) and the pressure-overload pig model (0.2–0.3-fold). Together, our data suggest that desmin levels increase early on in concentric hypertrophy and that animal models only partially recapitulate the proliferated and modified tubulin signature observed clinically. Our data warrant careful consideration when studying maladaptive responses to changes in the tubulin content in animal models. [ABSTRACT FROM AUTHOR]

The review discusses the role of metabolic disorders (in particular, insulin resistance) in the development of age-related diseases and normal aging with special emphasis on the changes in postmitotic cells of higher organisms. Caloric restriction helps to prevent such metabolic disorders, which could probably explain its ability to prolong the lifespan of laboratory animals. Maintaining metabolic homeostasis is especially important for the highly differentiated long-lived body cells, whose lifespan is comparable to the lifespan of the organism itself. Normal functioning of these cells can be ensured only upon correct functioning of the cytoplasm clean-up system and availability of all required nutrients and energy sources. One of the central problems in gerontology is the age-related disruption of glucose metabolism leading to obesity, diabetes, metabolic syndrome, and other related pathologies. Along with the adipose tissue, skeletal muscles are the main consumers of insulin; hence the physical activity of muscles, which supports their energy metabolism, delays the onset of insulin resistance. Insulin resistance disrupts the metabolism of cardiomyocytes, so that they fail to utilize the nutrients to perform their functions even being surrounded by a nutrient-rich environment, which contributes to the development of age-related cardiovascular diseases. Metabolic pathologies also alter the nutrient sensitivity of neurons, thus disrupting the action of insulin in the central nervous system. In addition, there is evidence that neurons can develop insulin resistance as well. It has been suggested that affecting nutritional sensors (e.g., AMPK) in postmitotic cells might improve the state of the entire multicellular organism, slow down its aging, and increase the lifespan. [ABSTRACT FROM AUTHOR]

In recent years, considerable evidence is accumulated pointing to participation of gamma-aminobutyric acid (GABA) in intercellular signaling in the peripheral nervous system, including, in particular, neuromuscular transmission. However, where in the neuromuscular synapse GABA is synthesized remains not quite clear. We used histochemical methods to detect GABA and l-glutamate decarboxylase (GAD) in developing skeletal muscle fibers and in cultured motor neurons. We found that GABA can be detected already in myocytes, but with further muscle maturation, GABA synthesis gradually attenuates and completely ceases in early postnatal development. We found also that formation of GABA in muscle tissue does not depend on activity of GAD, but presumably proceeds through some other, alternative pathways. In motor neurons, GABA and GAD can be detected at the early stage of development (prior to synapse formation). Our data support the hypothesis that GABA and GAD, which are detectable in adult neuromuscular junctions, have neuronal origin. The mechanism of GABA production and its role in developing muscle tissue need further clarification. [ABSTRACT FROM AUTHOR]

Purpose: Rotator cuff (RC) tears result not only in functional impairment but also in RC muscle atrophy, muscle fattening and eventually to muscle fibrosis. We hypothesized that allogenic bone marrow derived mesenchymal stem cells (MSC) and myocytes can be utilized to improve the rotator cuff muscle fattening and increase the atrophied muscle mass in a rat model.Methods: The right supraspinatus (SSP) tendons of 105 inbred rats were detached and muscle fattening was provoked over 4 weeks; the left side remained untouched (control group). The animals (n = 25) of the output group were euthanized after 4 weeks for reference purposes. The SSP-tendon of one group (n = 16) was left unoperated to heal spontaneously. The SSP-tendons of the remaining 64 rats (4 groups with n = 16) were repaired with transosseous sutures. One group received a saline solution injection in the SSP muscle belly, two other groups received 5 × 106 allogenic myocytes and 5 × 106 allogenic MSC injections from donor rats, respectively, and one group received no additional treatment. After 4 weeks of healing, the supraspinatus muscle mass was compared quantitatively and histologically to all the treated groups and to the untreated contralateral side.Results: In the end of the experiments at week 8, the myocyte and MCS treated groups showed a significantly higher muscle mass with 0.2322 g and 0.2257 g, respectively, in comparison to the output group (0.1911 g) at week 4 with p < 0.05. There was no statistical difference between the repaired, treated, or spontaneous healing groups at week 8. Supraspinatus muscle mass of all experimental groups of the right side was significantly lower compared to the untreated contralateral muscle mass.Conclusion: This defect model shows that the injection of allogenic mycocytes and MSC in fatty infiltrated SSP muscles is better than no treatment and can partially improve the SSP muscle belly fattening. Nevertheless, a full restoration of the degenerated and fattened rotator cuff muscle to its original condition is not possible using myocytes and MSC in this model. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na+/K+-ATPase (NKA) has been found that it could not only regulate intracellular Na+ levels but also control the intracellular Ca2+ ([Ca2+]i) level through Na+/Ca2+-exchanger (NCX). Recent studies have reported that klotho could affect [Ca2+]i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca2+]i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca2+]i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca2+]i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression. [ABSTRACT FROM AUTHOR]

Salicylates, one of the oldest medicinal compounds known to humans, have been reported to show anti-inflammatory effects via cyclooxygenase (COX) inhibition. However, the pathophysiological role of COX-2 in the heart is conflicting, and the role of sodium salicylate in the regulation of cardiac inflammation has not yet been elucidated. We aimed to investigate the effect of salicylate on COX-2 expression and its associated prostaglandin production using cultured neonatal rat cardiomyocytes. The cells were incubated in the presence or absence of sodium salicylate (8 mM). Treatment with sodium salicylate significantly increased COX-2 expression at both mRNA and protein levels, induced prostaglandin D2 release, and increased TNF-α mRNA expression in cardiomyocytes. In addition, salicylate treatment induced cardiomyocyte hypertrophy. Taken together, we demonstrated that salicylate induced COX-2 expression, which in turn resulted in the regulation of expression of several inflammatory mediators in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. The objective of this study was to investigate the expression profile of zinc regulated transporter like- and iron-regulated transporter-like proteins (ZIPs) and zinc transporter proteins (ZnTs) in cardiomyocytes and their modulation in response to hypoxia and reoxygenation. Adult rat ventricular myocytes (ARVMs) were subjected to 6 h of hypoxia, followed by 18 h of reoxygenation. Intracellular and extracellular zinc concentrations were determined using Fluozin-3 and Newport Green fluorescence, respectively. Expression of ZnTs 1, 2, 5, and 9 along with ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14 was detectable in the cardiomyocytes by real-time reverse transcriptase polymerase chain reaction. Hypoxia elicited accumulation of intracellular free zinc, but subsequent reoxygenation resulted in striking loss of intracellular free zinc and decreased the cardiomyocyte viability. Concomitantly, extracellular zinc levels dropped rapidly during hypoxia, but increased after reoxygenation. Immunoblotting analysis revealed that hypoxia increased the expression of ZnT1, but reoxygenation significantly increased the expression of ZnTs 2 and 5. Neither hypoxia nor reoxygenation altered the levels of ZnT9. Increased intracellular zinc at the end of hypoxia is related to enhanced expression of ZIPs, whereas decreased intracellular zinc during reoxygenation appears to be due to lowered expression of all ZIPs, in addition to elevated levels of ZnTs 2 and 5. These results thus suggest that there is impaired accumulation of intracellular zinc during reoxygenation, due to overexpression of specific ZnTs and downregulation of ZIP expression. [ABSTRACT FROM AUTHOR]

Since the discovery of a novel mechanism of cell death that differs from traditional necrosis, i.e., apoptosis, there have been numerous studies concluding that increased apoptosis augments myocardial infarction and heart failure and that limiting apoptosis protects the heart. Importantly, the vast majority of cells in the heart are non-myocytes with only roughly 30 % myocytes, yet almost the entire field studying apoptosis in the heart has disregarded non-myocyte apoptosis, e.g., only 4.7 % of 423 studies on myocardial apoptosis in the past 3 years quantified non-myocyte apoptosis. Accordingly, we reviewed the history of apoptosis in the heart focusing first on myocyte apoptosis, followed by the history of non-myocyte apoptosis in myocardial infarction and heart failure. Apoptosis of several of the major non-myocyte cell types in the heart (cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, macrophages and leukocytes) may actually be responsible for affecting the severity of myocardial infarction and heart failure. In summary, even though it is now known that the majority of apoptosis in the heart occurs in non-myocytes, very little work has been done to elucidate the mechanisms by which non-myocyte apoptosis might be responsible for the adverse effects of apoptosis in myocardial infarction and heart failure. The goal of this review is to provide an impetus for future work in this field on non-myocyte apoptosis that will be required for a better understanding of the role of apoptosis in the heart. [ABSTRACT FROM AUTHOR]

Trekrezan activated the in vivo expression of the PGC-1α gene in rat muscle tissues. Quantification of mitochondria in tissues and gene expression analysis for genes involved in regulatory energy metabolism cascades revealed that the mechanisms of the energy metabolism regulation are probably coupled with protein biosynthesis as well as with innate immunity. The immunostimulatory and adaptogenic drug trekrezan was found to induce concerted in vivo stimulation of the expression of the gene coding tryptophanyl-tRNA synthetase and its activity in the expression of the PGC-1α gene. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the anti-apoptotic potency of TNFR:Fc gene in ischemia/reperfusion-induced myocardial cell injury and hypoxia/reoxygenation-induced H9c2 rat cardiomyocytes injury. Rats were randomly divided into the following groups ( n = 8): (1) sham operation group; (2) ischemia-reperfusion (I/R) rats treated with rAAV-EGFP; (3) I/R rats treated with rAAV-TNFR:Fc group. rAAV-EGFP or rAAV-TNFR:Fc was injected intra-myocardial at four sites on the anterior and posterior walls of left ventricle immediately after the construction of I/R-induced AMI model in rats. The effects of TNFR:Fc on apoptosis and cardiacfunction were observed after 72 h of coronary reperfusion. In the in vitro study, apoptosis was analyzed in H9c2 rat cardiomyocytes treated either with nomoxia alone, or hypoxia/reoxygenation in the presence of rAAV-GFP or rAAV-TNFR:Fc. We found that (1) TNFR:Fc gene improved cardiac function (EF, LVESP, LVEDP and d p/d t) post I/R-induced AMI; (2) TNFR:Fc gene inhibited I/R-induced apoptosis and attenuated the level of TNF-α in serum and cardiac tissue; (3) TNFR:Fc gene prevented apoptosis in hypoxia/reoxygenation-induced H9c2 rat cardiomyocytes associated with inhibition of caspase-3 activation and normalization of ratio of the Bcl-2/Bax. We concluded that TNFR:Fc gene transfection has anti-apoptotic potency in ischemia/reperfusion-induced myocardial cell injury. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is a major and growing cause of morbidity and mortality. Despite initial successes, there have been few recent therapeutic advances. A better understanding of HF pathophysiology is needed with renewed focus on the myocardium itself. A new imaging technique is now available that holds promise. T1 mapping is a cardiovascular magnetic resonance (CMR) technique for non-invasive myocardial tissue characterization. T1 alters with disease. Pre-contrast (native) T1 changes with a number of processes such as fibrosis, edema and infiltrations. If a post contrast scan is also done, the extracellular volume fraction (ECV) can be measured, a direct measure of the interstitium and its reciprocal, the cell volume. This dichotomy is fundamental - and now measurable promising more targeted therapy and new insights into disease biology. [ABSTRACT FROM AUTHOR]

Insulin is the main glucoregulator that promotes the uptake of glucose by tissues and the subsequent utilization of glucose as an energy source. In this paper, we describe a novel glucoregulator, the alpha-synuclein (SNCA) protein, that has previously been linked to Parkinson's disease. Treatment with recombinant SNCA promotes glucose uptake in vitro in preadipocytes and in vivo in the adipose tissues and skeletal muscles of mice through the LPAR2/Gab1/PI3K/Akt pathway; these effects occur independently of the insulin receptor. This function of SNCA represents a new mechanistic insight that creates novel avenues of research with respect to the process of glucose regulation. [ABSTRACT FROM AUTHOR]

Protein kinase G type I (PKGI) plays a critical role in survival signaling of pre- and postconditioning downstream of cardiac cGMP. However, it is unclear whether PKGI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) metabolism can target cardiomyocyte mitochondria independently of cGMP/PKGI. We tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation and inhibition, adenosine A receptor (AAR) agonist, phosphodiesterase type-5 (PDE-5) inhibitor, or mitochondria-targeted S-nitrosothiol (MitoSNO) was affected by a cardiomyocyte-specific ablation of the PKGI gene in the mouse (CMG-KO). In situ hearts underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in CMG-CTRs all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10-s reperfusion and 10-s coronary occlusion) with or without treatment with the sGC inhibitor ODQ, treatment with the specific sGC activator BAY58-2667 (BAY58), the selective AAR agonist BAY60-6583 (BAY60), PDE-5 inhibitor sildenafil, and MitoSNO. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO· and S-nitrosates thiol proteins. In contrast, the hearts of CMG-KO animals were not protected by BAY58 and sildenafil, whereas the protective effects of IPost, IPost with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI is important for the protection against ischemia reperfusion injury afforded by sGC activation or PDE-5 inhibition. However, the beneficial effects of IPost, activation of the AAR, as well as the direct effects via mitochondrial S-nitrosation do not depend on PKGI in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that β-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs) . β-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. β-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ~50% below basal levels at 3 h after β-AR stimulation. This decline in eIF-2α phosphorylation was prevented by β1-AR, not by β2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited β-AR-stimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited β-AR-stimulated and thapsigargin-induced apoptosis. In vivo, β-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased β-AR-stimulated apoptosis in the heart. Thus, β-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role. [ABSTRACT FROM AUTHOR]

The phosphoinositide phospholipid PtdIns5 P has previously been implicated in insulin-stimulated translocation of the glucose transporter GLUT4 into the plasma membrane of adipocytes, but its potential role in glucose transport in muscle has not been explored. The involvement of PtdIns5 P in insulin-stimulated glucose uptake was therefore investigated in myotubes of the skeletal muscle cell line L6. Stimulation with insulin produced a transient increase in PtdIns5 P, which was abolished by the over-expression of the highly active PtdIns5 P 4-kinase PIP4Kα. PIP4Kα over-expression also abolished both the enhanced glucose uptake and the robust peak of PtdIns(3,4,5) P production stimulated by insulin in myotubes. Delivery of exogenous PtdIns5 P into unstimulated myotubes increased Akt phosphorylation, promoted GLUT4 relocalisation from internal membrane to plasma membrane fractions and its association with plasma membrane lawns and also stimulated glucose uptake in a tyrosine kinase and phosphoinositide 3-kinase (PI 3-kinase)-dependent fashion. Our results are consistent with a role for insulin-stimulated PtdIns5 P production in regulating glucose transport by promoting PI 3-kinase signalling. [ABSTRACT FROM AUTHOR]

The heart is composed of both cellular and acellular components that act in a dynamic fashion from birth to death. The cellular components consist of myocytes, fibroblasts, and vascular cells, including endothelium and smooth muscle. Changes in these components are intimately associated with function by altering the mechanical, chemical, and electrical properties of the heart. In future investigations, it will be important to examine these interactions as dynamic changes in response to physiological signals. [ABSTRACT FROM AUTHOR]

Embryonic stem cell derived cardiomyocytes have been widely investigated for stem cell therapy or in vitro model systems. This study examines how two specific biophysical stimuli, collagen I and cell alignment, affect the phenotypes of embryonic stem cell derived cardiomyocytes in vitro. Three phenotypic indicators are assessed: sarcomere organization, cell elongation, and percentage of binucleation. Murine embryonic stem cells were differentiated in a hanging drop assay and cardiomyocytes expressing GFP-α-actinin were isolated by fluorescent sorting. First, the effect of collagen I was investigated. Addition of soluble collagen I markedly reduced binucleation as a result of an increase in cytokinesis. Laden with a collagen gel layer, myocyte mobility and cell shape change were impeded. Second, the effect of cell alignment by microcontact printing and nanopattern topography was investigated. Both patterning techniques induced cell alignment and elongation. Microcontact printing of 20 μm line pattern accelerated binucleation and nanotopography with 700 nm ridges and 3.5 μm grooves negatively regulated binucleation. This study highlights the importance of biophysical cues in the morphological changes of differentiated cardiomyocytes and may have important implications on how these cells incorporate into the native myocardium. [ABSTRACT FROM AUTHOR]

The left atrial (LA) posterior wall has been demonstrated to have regional electrophysiological differences with a higher arrhythmogenic potential leading to atrial fibrillation (AF). However, the ionic characteristics and calcium regulation in the LA anterior and posterior myocytes have not been fully elucidated. The purpose of this study was to investigate the electrical characteristics of the LA anterior and posterior myocytes. Whole-cell patch-clamp techniques and the indo-1 fluorimetric ratio technique were used to investigate the characteristics of the ionic currents, action potentials, and intracellular calcium in single isolated rabbit myocytes in the LA anterior and posterior walls. The expression of the Na-Ca exchanger (NCX) and ryanodine receptor (RyR) were evaluated by a Western blot. The LA posterior myocytes ( n = 15) had a higher incidence (53 vs. 19%, P < 0.05) of delayed afterdepolarizations than the LA anterior myocytes ( n = 16). The LA posterior myocytes had larger sodium currents and late sodium currents, but smaller inward rectifier potassium currents than the LA anterior myocytes. The LA posterior myocytes had larger intracellular Ca transient and sarcoplasmic reticulum Ca contents as compared with the LA anterior myocytes. However, the NCX currents in the LA posterior myocytes were smaller than those in the LA anterior myocytes. The LA posterior myocytes had a smaller protein expression of NCX, but a larger protein expression of RyR than the LA anterior myocytes. In conclusion, LA posterior myocytes contain a high arrhythmogenic potential and distinctive electrophysiological characteristics, which may contribute to the pathophysiology of AF. [ABSTRACT FROM AUTHOR]

Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (β-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed ≥50% by the PDE5A inhibitor sildenafil (SIL, 1 μM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of β3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in β3-AR knockout (β3-AR−/−) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not β3-AR−/− myocytes. These data support a cascade involving β3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial β-adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Tissue-engineered heart muscle may provide an alternative treatment modality for end-stage congestive heart failure. We have previously described a method to engineer contractile heart muscle in vitro (termed cardioids). This study describes a method to improve the contractile properties of cardioids utilizing thyroid hormone (T3) stimulation. Cardioids were engineered by promoting the self-organization of primary neonatal cardiac cells into a contractile tissue construct. Cardioids were maintained in standard cell culture media supplemented with varying concentrations of T3 in the range 1–5ng/ml. The contractile properties of the cardioids were evaluated 48h after formation. Stimulation with T3 resulted in an increase in the specific force of cardioids from an average value of 0.52 ± 0.16kPa ( N = 6) for control cardioids to 2.42 ± 0.29kPa ( N = 6) for cardioids stimulated with 3ng/ml T3. In addition, there was also an increase in the rate of contraction and relaxation in response to T3 stimulation. Cardioids that were stimulation with T3 exhibited improved pacing characteristics in response to electrical pacing at 1–5Hz and an increase in the degree of spontaneous contractility. Changes in the gene expression of SERCA2, phospholamban, α-myosin heavy chain, and β-myosin heavy chain correlated with the changes in contractile properties. This study demonstrates the modulation of the contractile properties of tissue-engineered heart muscle using T3 stimulation. [ABSTRACT FROM AUTHOR]

I K,slow1 and I K,slow2 are two important voltage-gated potassium (K+) currents expressed in mouse ventricular myocytes. However, their properties at the single-channel level have not been characterized. In this paper, we report two new single K+ channels, mK1 and mK2, in myocytes isolated from mouse ventricles and their possible correlation with the macroscopic currents I K,slow1 and I K,slow2. The conductance of mK1 and mK2 was 24 and 17 pS, respectively. Ensemble-averaged current demonstrated an inactivation time constant of 400 to 500 ms for mK1 compared with 1,300 to 2,000 ms for mK2. The mK1 channel was more sensitive than the MK2 channel to the K channel blocker 4-AP. In myocytes isolated from Kv1DN mice with functional knock out of the Kv1.5 channel, mK1 was not detectable but mK2 was present. Our data suggest that the newly characterized K+ channels, mK1 and mK2, likely correspond to the macroscopic currents of I K,slow1 and I K,slow2, respectively. [ABSTRACT FROM AUTHOR]

Inhibition of endothelin-A (ETA) receptors has been shown to reduce ventricular electrical abnormalities associated with cardiac failure. In this study, we investigate the effect of ETA-receptor inhibition on the development of regional alterations of the transient outward K+ current ( I to) in the setting of pressure-induced left ventricular (LV) hypertrophy. Cardiac hypertrophy was induced in female Sprague–Dawley rats by stenosis of the ascending aorta (AS) for 7 days. Treatment with the selective ETA-receptor antagonist darusentan (LU135252, 35 mg [kg body weight]−1 day−1) was started 1 day before the surgery. AS induced a 46% increase in the relative LV weight ( p < 0.001) and caused a significant reduction in I to (at +40 mV) in epicardial myocytes (19.5 ± 1.2 pA pF−1, n = 32 vs 23.2 ± 1.2 pA pF−1, n = 35, p < 0.05). Darusentan further reduced I to in AS (15.4 ± 1.3 pA pF−1, n = 37, p < 0.05) and sham-operated animals (19.8 ± 1.6 pA pF−1, n = 48, ns.). The effects of AS and darusentan on I to were significant and independent as tested by two-way analysis of variance. I to was not affected in endocardial myocytes. These results indicate that endothelin-1 may exert a tonic effect on the magnitude of I to in the epicardial region of the left ventricle but that ETA-receptor activation is not necessary for the development of electrical alterations associated with pressure-induced hypertrophy. [ABSTRACT FROM AUTHOR]

The aim of conducting this study was to assess the clinical relevance of matrix metalloproteinase (MMP) inhibition by doxycycline, an effective MMP inhibitor, in a rat model of extensive myocardial infarction (MI) and left ventricular (LV) dysfunction. Rats ( n = 22) were subjected to extensive anterior MI. Doxycycline (25 mg SC, daily) or saline (control) injections were started for nine days thereafter. The effect of doxycycline on MMP activity in the infarcted and remote myocardium was measured by zymography, in another subgroup ( n = 8), nine days after MI. Echocardiography and magnetic resonance imaging (MRI) studies were performed at one and thirty days after MI to assess LV remodeling and function. After 4 weeks, hearts were fixed, and subjected to morphometric and histological analysis. Compared with control, doxycycline treatment attenuated MMP-9 and -2 activity in both infarcted and remote myocardium. Serial echocardiography studies showed that doxycycline failed to attenuate scar thinning, LV dilatation and dysfunction. MRI study showed that doxycycline impaired LV compensatory hypertrophy. Furthermore, compared with control, doxycycline reduced vessel density (/mm2 ± SEM) in the infarcted myocardium (84 ± 16 vs. 46 ± 9/mm2, respectively; p < 0.05). Our work suggest that effective MMPs’ inhibition in the infarcted and remote myocardium by doxycycline does not prevent LV remodeling and dysfunction but impairs angiogenesis and compensatory LV hypertrophy. Our findings caution against aggressive, non-selective inhibition of MMPs in the early healing phase after MI. [ABSTRACT FROM AUTHOR]

This work proposes a method to construct a state-shared model for multiple-input multiple-output (MIMO) systems. A state-shared model is defined as a linear time invariant state-space structure that is driven by measurement signals-the plant outputs and the manipulated variables, but shared by different multiple input/output models. The genesis of the state-shared model is based on a particular reduced non-minimal realization. Any such realization necessarily fulfills the requirement that the output of the state-shared model is an asymptotically correct estimate of the output of the plant, if the process model is selected appropriately. The approach is demonstrated on a nonlinear MIMO system — a physiological model of calcium fluxes that controls muscle contraction and relaxation in human cardiac myocytes. [ABSTRACT FROM AUTHOR]

Virus-induced activation of nuclear factor-kappa B (NF-?B) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-?B is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-?B activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-?B regulation, where NF-?B activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-?B activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-?B is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-?B also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-?B-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-?B activation in reovirus-infected cells. Further, inhibition of NF-?B activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-?B and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis. [ABSTRACT FROM AUTHOR]

Hypertension leads to impaired contractile function. This study examined the impact of inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin or cyclopiazonic acid (CPA) on cardiac contractile function in ventricular myocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Mechanical properties were examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt). Intracellular Ca2+ transients were evaluated as fura-2 fluorescent intensity (FFI), excitation-induced change in FFI (ΔFFI=peak-basal), and fluorescence decay rate (τ). Expression of Ca2+ regulatory proteins SERCA2a, Na+−Ca2+ exchanger (NCX), and phospholamban (PLB) were assessed by reverse transcriptase polymerase chain reaction and Western blot. SHR rats exhibited elevated blood pressure. SHR myocytes displayed decreased PS±dL/dt, peak FFI, and ΔFFI; shortened TPS; prolonged τ with normal TR90; and basal FFI compared with WKY myocytes. Inhibition of SERCA with thapsigargin (5μ M) or CPA (10 μ M) significantly depressed PS±dL/dt, baseline FFI, and ΔFFI, and prolonged TPS, TR90, and τ in WKY myocytes. However, SHR myocytes were relatively insensitive to thapsigargin or CPA with only TPS and TR90 prolonged. Both mRNA and protein expressions of NCX and PLB were significantly enhanced, whereas SERCA2a protein abundance was reduced in SHR rats compared with the WKY group. Our data suggest that inhibition of SERCA function differentially affected cardiac contractile function in ventricular myocytes from normotensive and hypertensive rats possibly through reduced SERCA2a, elevated PLB, and NCX expression under hypertension. [ABSTRACT FROM AUTHOR]

The anti-anginal drug nicorandil has been demonstrated to protect the myocardium against ischemic injury in both experimental and clinical studies. Although nicorandil seems to protect the myocardium via activation of mitochondrial ATP-sensitive K+ (mitoKATP) channels, the mechanisms underlying its cardioprotection have remained elusive. We therefore examined whether nicorandil depolarizes the mitochondrial membrane and attenuates the mitochondrial Ca2+ overload. With the use of a Nipkow confocal system, the mitochondrial Ca2+ concentration ([Ca2+]m) and the mitochondrial membrane potential (ΔΨm) in rat ventricular myocytes were measured by loading cells with rhod-2 and JC-1 respectively. The number of cell hypercontractures resulting from mitochondrial Ca2+ overload was counted. Exposing cells to ouabain (1 mM) evoked mitochondrial Ca2+ overload and increased the intensity of rhod-2 fluorescence to 180±15% of baseline (p<0.001). Nicorandil (100 μM) significantly attenuated the ouabain-induced mitochondrial Ca2+ overload (129±4% of baseline; p<0.001 vs. ouabain). Nicorandil decreased the ΔΨm during application of ouabain, thereby reducing the intensity of JC-1 fluorescence to 89±2% of baseline (p<0.05). Exposure of myocytes to ouabain eventually resulted in cell hypercontracture (51±2%). This ouabain-induced cell hypercontracture was blunted by application of nicorandil (37±2%, p<0.05 vs. ouabain). Moreover, these effects of nicorandil were abolished by 5-hydroxydecanoate (500 μM), a putative mitoKATP channel blocker, and by glibenclamide (10 μM), a nonselective KATP channel blocker. Our results suggest that nicorandil attenuates the matrix Ca2+ overload with accompanying depolarization of the mitochondrial membrane. Such effect might potentially be attributed to the mechanism of cardioprotection afforded by nicorandil. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of protein tyrosine kinase inhibitors on the fast sodium current (INa) in rabbit ventricular myocytes. Single rabbit ventricular myocytes were isolated enzymatically using Langendorff perfusion. INa was recorded using the whole-cell patch-clamp technique at room temperature. The protein tyrosine kinase inhibitors genistein, AG957, ST638, and PP2 reversibly inhibited INa in a concentration-dependent manner. At a test pulse potential of -30 mV, genistein (n=7) inhibited INa by 37.7±3.2%, 53.4±2.5%, and 71.8±2.7% at concentrations of 15, 50, and 100 µM, respectively, without changing the voltage dependence of activation, while 100 µM AG957, 100 µM ST638, and 30 µM PP2 inhibited INa by 38.7±2.4, 35.8±3.4, and 21.1±3.9%, respectively. Genistein (100 µM) and AG957 (100 µM) shifted the voltage for half-maximal inactivation of INa from -76.7±2.0 mV (n=10) in control to -88.37±2.6 mV (n=6, P<0.05), and -82.9±1.7 (n=4, P<0.05), respectively, without changing the slope factor. Genistein and AG957 also significantly prolonged the time course of INa recovery from inactivation. Daidzein and PP3, inactive analogs of genistein and PP2, respectively, did not inhibit INa significantly. We conclude that protein tyrosine kinase signaling pathways may play an important role in regulation of INa in cardiac myocytes. [ABSTRACT FROM AUTHOR]

Glyburide, also known as glibenclamide, was shown to have positive inotropic effect in human and animal hearts. The objectives of the present study was to investigate the effects of glyburide on developed left ventricular pressure (DLVP), coronary flow (CF), and heart rate (HR), in isolated rabbit heart as well as its effects on myocardial contractility and L-type calcium current, iCa, in guinea pig myocytes. Rabbit hearts were mounted on Langendorff apparatus and perfused with an oxygenated Krebs for 30 min until reaching steady state to be followed by 20 min of experimental perfusion divided into 5 min of control perfusion and 15 min of perfusion with Glyburide (10 μM). Ventricular myocytes were isolated by enzymatic dispersion technique and superfused in an oxygenated Tyrode solution. Cells were voltage-clamped at holding potential −40 mV to inactivate Na+ current and a step depolarizations, 200 msec duration, to 0 mV was applied to elicit iCa. The contractions of the myocytes were measured by optical methods. Glyburide significantly increased DLVP by 30% and CF by 36% but had no effect on HR. Glyburide increased cell contractility by 7 ± 6, 18 ± 7, 28 ± 9 and 54 ± 15% for 0.1, 1, 10 and 100 μM respectively, p < 0.001. Meanwhile it depressed iCa by 9 ± 6 and 19 ± 8% for 1 and 10 μM respectively. In conclusion, glyburide increased contractility of guinea pig single myocytes and of isolated rabbit heart, as indicated by increased developed left ventricular pressure while it depressed iCa. It is hypothesized that an elevation in intracellular calcium, which caused increased myocardial contractility, could be attributed to an increase in intracellular Na+ that could increase intracellular calcium via Na+/Ca2+ exchange. [ABSTRACT FROM AUTHOR]

Na+/Ca2+ exchange (NCX) is a major Ca2+ extrusion system in cardiac myocytes, but can also mediate Ca2+ influx and trigger sarcoplasmic reticulum Ca2+ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na+]i may lead to a rise in [Ca2+]i through NCX, causing Ca2+ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca2+ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca2+ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 μM) did not alter control steady-state twitch contractions or Ca2+ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na+-loaded by perfusion of strophanthidin (50 μM), the addition of KBR reduced diastolic [Ca2+]i and abolished spontaneous Ca2+ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 μM applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca2+ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca2+ influx via NCX may be critical in causing myocardial Ca2+ overload and triggered activities induced by cardiac glycoside or reoxygenation. [ABSTRACT FROM AUTHOR]

The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes. In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed. Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min. The cell fraction obtained at 80 ml/min consisted of 68–90% myocytes. Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60±2% at day 5. A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4–5 days. By this approach a fibroblast-rich cell fraction (87±5%) and a myocyte-rich cell fraction (82±6%) were obtained. In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing. Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately. [ABSTRACT FROM AUTHOR]

This study was designed to elucidate the relationship between enhanced cytoplasmic calcium levels (Ca2+ i) and membrane phospholipid degradation, a key step in the loss of cellular integrity during cardiac ischemia/reperfusion-induced damage. Isolated rat hearts were subjected to 15 min ischemia followed by 30 min reperfusion. Ca2+ i was estimated by the Indo-1 fluorescence ratio technique. Degradation of membrane phospholipids as indicated by the increase of tissue arachidonic acid content was assessed in tissue samples taken from the myocardium at various points of the ischemia/reperfusion period. The hemodynamic parameters showed almost complete recovery during reperfusion. Fluorescence ratio increased significantly during ischemia, but showed a considerable heart-to-heart variation during reperfusion. Based upon the type of change of fluorescence ratio during reperfusion, the hearts were allotted to two separate subgroups. Normalization of fluorescence ratio was associated with low post-ischemic arachidonic acid levels. In contrast, elevated fluorescence ratio coincided with enhanced arachidonic acid levels. This observation is suggestive for a relationship between the Ca2+-related fluorescence ratio and arachidonic acid accumulation probably due to a calcium-mediated stimulation of phospholipase A2. [ABSTRACT FROM AUTHOR]

Release of bacterial endotoxin and cytokines induce cardiac failure during sepsis. We investigated the direct effects of E. coli endotoxin (lipopolysaccharide, LPS) and cytokines induced by LPS on the cardiac myocyte gene program. For in vivo-experiments adult Wistar rats were given 600 µg/day LPS i.v. for 24 h or 7 days. In addition, cultured adult rat cardiac myocytes were treated with LPS, interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα), interferon-γ (IFNγ) or IL-6 for 24 h. mRNA expression was evaluated for cardiac-α-actin (cAct), skeletal-α-actin (skAct), β- and α-myosin heavy chain (MHC). LPS induced βMHC-mRNA 3.6-fold and repressed αMHC 2.7-fold and cAct 2.5-fold after 24 h in vivo. Up-regulation of βMHC (3-fold) and repression of cAct (2.5-fold) were still observed after 7 days LPS infusion, whereas αMHC-mRNA levels had returned to normal. At the protein level, increased expression of βMHC by LPS treatment occurred already after 24 h and was maintained thereafter. LPS had no influence on skAct-mRNA. Similar changes in contractile protein mRNA expression were observed in LPS-treated cardiomyocytes in culture, whereas the tested cytokines either activated (IL-1β, IFNγ) or repressed (TNFα, IL-6) both MHC-isoforms and cAct. In conclusion, LPS and pro-inflammatory cytokines induce changes in contractile protein expression that may contribute to the acute heart failure observed during endotoxaemia. [ABSTRACT FROM AUTHOR]

The cardiac subtype of Na+/H+ exchanger (NHE-1) plays an important role in the regulation of intracellular pH (pHi) and also can be a major route for Na+ influx. Although intracellular ATP is required for the optimal function of NHE-1, the regulation of the exchanger by ATP is less well characterized. This study was designed to investigate which intracellular ATP generated by oxidative phosphorylation or by glycolysis is dominant for the activation of NHE-1 in intact cardiac myocytes. Isolated guinea pig ventricular myocytes were loaded with the pHi-sensitive fluorescent indicator, 2′-7′-bis(carboxyl)-5′,6′-carboxy fluorescein (BCECF), and exposed to 20 mM 2-deoxyglucose (2-DG) or 2 mM sodium cyanide (CN) to inhibit glycolysis or oxidative phosphorylation, respectively. The activity of NHE-1 was estimated with pHi recovery following transient application of 15 mM NH4Cl (NH4Cl prepulse). After the NH4Cl prepulse, pHi decreased from 7.00 ± 0.03 (mean ± S.E.) to 6.60 ± 0.06 and recovered to 6.94 ± 0.13 at 10 min (n = 7). The pHi recovery was suppressed in the presence of 2-DG (6.67 ± 0.05, p < 0.01, n = 7), but was not changed in the presence of CN (6.88 ± 0.18, n = 6). Since there was no difference in the intrinsic H+ buffering power, the estimation of the net acid efflux demonstrated that the activity of NHE-1 was significantly depressed in 2-DG. The inhibitory effect of 2-DG was not due to more extensive depletion of global intracellular ATP or secondary to the change in either intracellular Na+ or Ca2+ concentration. We concluded that ATP generated by glycolysis rather than by oxidative phosphorylation is essential to activate NHE-1 in ventricular myocytes. [ABSTRACT FROM AUTHOR]

Activation of alpha-1 adrenergic receptors in the heart has been shown to result in increased contractile activity, cardiac fetal gene re-expression, and myocyte hypertrophy. Three alpha-1 adrenergic receptors have been identified through molecular cloning. Due to the limited selectivities of the currently available alpha-1 adrenergic receptor antagonists, the signaling pathways activated by specific subtypes in the heart remain unresolved. To resolve this dilemma, we have used a molecular approach to identify the signaling pathways and downstream genes that are engaged in response to activation of individual alpha-1 adrenergic subtypes in cardiac cells. We have transfected constitutively active alpha-1 adrenergic receptors (α1a-S290/293-AR [1] or the α1b-S288/294-AR [2]) subtypes into the cardiac murine myocyte cell line (HL-1) and studied the signal transduction pathway(s) and cardiac gene(s) activated by them. In this study, we demonstrate that the α1a-S290/293-AR [1] subtype preferentially couples to cardiac-specific atrial natriuretic factor (ANF) gene expression, while the α1b-S288/294-AR preferentially couples to activation of mitogen-activated protein kinase (MAPK), Ets-like transcription factor-1 (Elk1) and serum response element (SRE) signaling pathways. Endogenous alpha-1 adrenergic receptors are expressed, and stimulate phosphatidylinositol-hydrolysis upon activation with the alpha-1 agonist, phenylephrine. [ABSTRACT FROM AUTHOR]

Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% ( P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% ( P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain. [ABSTRACT FROM AUTHOR]

The mechanisms that regulate cardiac myocyte apoptosis are not well understood. To study the role of protein phosphatase 1 (PP1) and 2A (PP2A) in apoptosis, we exposed cultured neonatal rat cardiac myocytes to the phosphatase inhibitor okadaic acid (OA). Exposure (18 h) to 100 nM OA (a concentration which inhibits both PP1 and PP2A) decreased the number of adherent cells, caused genomic DNA fragmentation, and increased the percentage of apoptotic cells. These effects did not occur at a lower concentration of OA (1 nM) which is relatively specific for PP2A. Stimulation of α1- or β-adrenergic receptors with norepinephrine (NE) in the presence of propranolol or prazosin partially blocked OA-induced apoptosis as measured by flow cytometry. Likewise, stimulation of adenylyl cyclase with forskolin reduced OA-induced apoptosis. Conversely, inhibition of protein kinase A with H89 or protein kinase C with chelerethrine potentiated OA-induced apoptosis. OA increased caspase-3 activity, and this effect was reduced by NE. Thus, inhibition of PP1 stimulates apoptosis in NRVM and stimulation of adrenergic receptors protects against OA-induced apoptosis. [ABSTRACT FROM AUTHOR]

Our purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 μ N. When stimulated electrically the myooids contracted to produce force. The myooids (n=14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 μ N, maximum isometric force of 440 μ N, resting baseline force of 181 μ N, and specific force of 2.9k N/m2. The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms. [ABSTRACT FROM AUTHOR]

Functional adaptation of cardiac cells in response to haemodynamic load requires dynamic alteration of gene expression. In this study, we examined early changes in gene expression following stretch in myocytes and fibroblasts isolated from neonatal rat hearts. In the first hour of biaxially applied static stretch, the changes in expression of immediate-early genes, such as c-fos, c-jun and fra-1, were quantified. The expression of the atrial natriuretic peptide (ANP) gene in myocytes was measured as an indication of the hypertrophic response. In stretched myocytes, expression of c-fos and ANP increased transiently to 227% and 176% respectively after 30 min stretch, whereas c-jun and fra-1 expression decreased in the 1st hour of stretch. In stretched fibroblasts the expression of c-fos and fra-1 increased transiently to maxima of 145% and 146% respectively after 30 min stretch, whereas c-jun expression did not change significantly. To study the indirect effects of stretch, as an indication of cross-talk between cardiac cells, stationary myocytes and fibroblasts were incubated with stretch-conditioned medium (CM) from stretched (0–60 min) myocytes and fibroblasts. CM from stretched myocytes reduced c-fos and induced c-jun expression in myocytes and fibroblasts, reduced fra-1 expression in myocytes but induced fra-1 expression in fibroblasts. CM from stretched fibroblasts induced c-fos expression and had little effect on c-jun expression in myocytes and fibroblasts, induced the fra-1 expression in myocytes but had little effect on fra-1 expression in fibroblasts. CM from myocytes and CM from fibroblasts induced ANP expression in myocytes to 206% and 120% respectively after 45 min stretch. Static stretch of myocytes and fibroblasts appears to stimulate, within 1 h, secretion of cell type-specific factors that participate in the regulation of proto-oncogene and ANP expression of stationary myocytes and fibroblasts. These early changes in gene transcription suggest that stretch of the myocardium initiates intracellular gene expression as well as cross-talk between the cell types. [ABSTRACT FROM AUTHOR]

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to highconcentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c)and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca2+]c and [Ca2+]m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca2+]m but not[Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator,the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover,inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused anincrease in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released,or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity. [ABSTRACT FROM AUTHOR]

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N′-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 μM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome c concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c. [ABSTRACT FROM AUTHOR]

This study addressed the question of whether the sarcolemmal fragility of cardiomyocytes after anoxia and subsequent reoxygenation can be altered by modulation of the cellular glutathione state. Isolated ventricular cardiomyocytes (from adult rats) were exposed to 120 min anoxia and subsequently to 30 min reoxygenation. Osmotic stress was generated by reduction of medium osmolarity from 270 to 80 mosmol/l and sarcolemmal fragility assessed by the leakage of lactate dehydrogenase (LDH). Under normoxic conditions 6.7±1.0 % of total LDH activity was found extracellularly. Hyposmolar reoxygenation, but not hypoosmolar anoxia, increased LDH release (17.9±2.7% of total, P<0.05). Increasing cellular glutathione content by pretreatment with N-acetylcysteine (1 mM) reduced LDH release following hyposmolar reoxygenation (12.3±1.9% vs. 18.2±2.9% of LDH in medium, P<0.05). Depletion of glutathione content by pretreatment with buthionine sulphoximine (BSO, 200 µM), increased LDH release following osmotic stress already in normoxia (10.5±1.8% of LDH in medium; P<0.05 vs. no BSO), and even further after reoxygenation (21.8±3.2%, P<0.05 vs. normoxia). We conclude that the increased sarcolemmal fragility in reoxygenated cardiomyocytes is due to reoxygenation in the presence of reduced antioxidant defence. [ABSTRACT FROM AUTHOR]

Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or by the replating technique. [H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function of the culture's age, of seeding density, and replating. It was shown that (a) replating maintains predominance of myocyte population for at least 2 wk in culture; (b) heavy seeding density allows homogeneous myocyte population for the 1st wk in culture; and (c) the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and its state of differentiation. [ABSTRACT FROM AUTHOR]

The phenotype of perivascular placental cells has previously been studied using tissue sections from the fetal villi. The examination of these cells in culture by scanning electron microscopy gives us the opportunity to observe their three-dimensional phenotypes and associations outside their normal constraints. Human umbilical endothelial cells, which have a phenotype comparable to that observed in other sutdies, seem more flattened in culture than in their usual environment. Microvascular endothelial cells did not attain an epithelioid phenotype with close contacts between cells but formed a network of branched, elongated cells with phagocytotic activity. Some circular associations were observed when using a gelatinized matrix. Microvascular pericytes were large, flattened cells with an irregular border that pushed up nodular associations on a gelatin matrix. Chorioplacental myocytes adopted a network template comparable to that developed by microvascular endothelial cells. However, these elongated cells were thicker, without microvilli, and superficial filaments could be observed. In culture, confluent endothelial cells from the umbilical cord or microvascular pericytes associated as nodules reached a cell phenotype close to their in vivo counterparts. This attainment of an in vivo phenotype remains questionable for chorioplacental myocytes. Microvascular endothelial cells, however, though there was sparse formation of circular associations, remained far from their in vivo phenotype. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury is probably caused by the generation of oxygen free radicals. The final common pathway to cell injury may be mediated by intracellular calcium overloading induced by oxygen free radicals. Volatile anesthetics have been shown to improve myocardial function following reperfusion. To determine whether or not oxygen radicals are involved in the mechanism by which volatile anesthetics improve myocardial function following reperfusion, we investigated the effects of hydrogen peroxide (HO) on the intracellular calcium concentration ([Ca]) in isolated rat ventricular cells. First, the effects of volatile anesthetics, halothane, isoflurane, or sevoflurane, on [Ca] were studied in the absence of HO. Next, myocytes were perfused with volatile anesthetics in the presence of HO. [Ca] was measured using fura-2, a Ca-sensitive fluorescent dye. None of the volatile anesthetics changed [Ca] in the absence of HO. In the presence of HO, [Ca] gradually increased during HO perfusion. Halothane delayed the onset of the increase in [Ca] induced by HO, whereas sevoflurane and isoflurane accelerated the onset. Furthermore, sevoflurane caused more pronounced accumulation of intracellular calcium than did halothane and isoflurane. Therefore, the reduction of excessive intracellular calcium accumulation caused by halothane may have beneficial effects on myocardial function following reperfusion. [ABSTRACT FROM AUTHOR]

We have used site-directed antibodies against various segments of the connexin43 (Cx43) gap-junction protein in an attempt to explore the role of different portions of this molecule in regulating junctional permeability. The antibodies used in the present study were raised against epitopes exposed at the cytoplasmic face of the junctions, specifically the amino (AT-2) and carboxy (CT-360) termini and the cytoplasmic loop (CL-100) of Cx43. Neonatal rat cardiac myocytes, which are known to express Cx43, were microinjected with a series of anti-Cx43 antibodies, followed by Lucifer yellow. The extent of cell coupling was quantified as the percentage of instances of intercellular transfer of the dye. The effectiveness of the AT-2 and CT-360 antibodies varied strongly and differentially with the external calcium concentration. In the absence of antibody, the dye permeability was unaffected by calcium. In medium containing physiological concentrations of calcium, the antibodies inhibited dye transfer to different degrees: AT-2 and CT-360 antibodies inhibited well while the CL-100 antibody had very little effect on dye permeability. Our results indicate that several highly conserved cytoplasmic domains of Cx43 could be involved in regulating junctional permeability, and that calcium modulates the effect of antibodies on junctional permeability. [ABSTRACT FROM AUTHOR]

To measure the mechanical activity of enzymatically isolated mammalian myocytes the principle of laser light diffraction was used. Since the viability of isolated cardiac myocytes showed a marked dependence on the laser power used, an opto-electronic system with improved light sensitivity and low susceptibility to optical noise was developed. The high sensitivity was achieved by a novel approach in the detection of diffraction patterns, that provides a significant reduction of the amount of laser power required. This improvement rendered possible the application of laser diffraction during extended experiments including pharmacological interventions. The static performance of the system, as assessed by means of calibration gratings, showed a resolution in the order of 5 nm for small changes in sarcomere length in the range from 1.2 μm to 2.0 μm. Examples of measurements on resting and contracting cells are presented, and the limitations of the application of the system to biological specimens are discussed. [ABSTRACT FROM AUTHOR]

Objective: Ischemic or hypoxic preconditioning has been shown, in multicellular preparations, to reduce post-ischemic injury. In the present study, we attempted to develop a model of preconditioning in isolated rat myocytes in order to facilitate investigation into the mechanism of preconditioning. Methods: The protective effect of a short period (10 min) of anoxia and reoxygenation against a subsequent longer period of anoxia was studied in single, electrically stimulated (0.2 Hz, 37°C) adult rat cardiac myocytes. The control group received only the long period of anoxia. Three protocols were tested: Protocol 1 in which octanoate was the only substrate; Protocol 2 in which only glucose was present during all normoxic phases and during the preconditioning anoxia and octanoate alone during the prolonged period of anoxia and; Protocol 3 in which protocol 2 was repeated with the addition of adenosine (100 μM) and insulin (15 μU/ml) during the prolonged anoxic period. The end-point of assessment was loss of cell morphology, i.e., hypercontracture (death or relengthening (survival) on reoxygenation following the prolonged anoxic period. Membrane integrity was also examined at the end of each protocol by observing if the cells excluded trypan blue. Results: No protective effect of preconditioning on cell survival was observed in protocols 1 or 2. In contrast, in protocol 3, a significant protection was observed in the preconditioned versus control group (58% vs 27% survival respectively; p<0.001). However, in the absence of preconditioning, adenosine and insulin provided no additional protection in the control group. No significant differences in trypan blue exclusion were observed between the groups in any protocol. Conclusions: These results suggest that preconditioning cannot protect against a subsequent period of anoxia where the accumulation of metabolic products, e.g., adenosine is prevented. However, that protection can be re-instated by the presence of adenosine and insulin during the period of prolonged anoxia. Furthermore, this study suggests that the preconditioning by anoxia may induce a change in the A-receptor or its second messenger system such that adenosine is able to provide protection. [ABSTRACT FROM AUTHOR]