Component Of Excitation-contraction Coupling Research Articles

Unraveling the pathogenesis of novel ANKRD1 missense mutation causing familial dilated cardiomyopathy using human-induced pluripotent stem cells derived cardiomyocytes

Abstract Introduction Familial dilated cardiomyopathy(f-DCM) can be caused by mutations in 30 different genes encoding sarcomeric, cytoskeletal, mechanotransducers and transcription factors. One of the genes ANKRD1 encode Cardiac ankyrin repeat protein (CARP) which is involved in the mechanosensory unit located on the I-band of the sarcomere. Here we demonstrate a novel mutation in ANKRD1 gene causing f-DCM, and the usage of human-induced pluripotent(hiPSC) technology unraveling the pathogenesis of the disease. Purpose of the study: 1) Establishing patient specific hiPSC-derived cardiomyocytes from a family harboring a mutation in ANKRD1 gene causing f-DCM; 2) Phenotypical assessment of the morphological and ultrastructural properties of the diseased cells upon CARP activation; 3) Functional assessment of the 3 component of excitation-contraction coupling at the single-cell and tissue level. Methods and Results Patient-specific hiPSC were generated from a family harboring missense mutation in the ANKRD1 gene, with a substitution of the amino acid arginine to cysteine at position 182 of the Cardiac ankyrin repeat protein (CARP). ANKRD1-mut hiPSC were differentiated into cardiomyocytes and later matured using hormonal treatment. Morphological assessment using immunofluorescence(IF) techniques revealed that ANKRD1-mut hiPSC-CMs demonstrate an attenuated response to hypertrophy (upon Angiotensin-2 and Phenylephrine treatment). In addition ANKRD1-mut hiPSC-CMs displayed a higher cellular death rate upon increasing substrate stiffness and starvation. Electrophysiological assessment of the ANKRD1-mut cardiac tissues exhibit no alternations in action potential durations and conduction velocities while assessment of calcium transients demonstrated slower kinetics. In addition forces were quantified at the single-cell and tissue level , and demonstrated reduced active tension values and abnormal post-extrasystolic potentiation, while only single-cell experiments have demonstrated decreased passive tensions. These phenotypical alternations might be attributed to decreased nuclear translocation of CARP upon phenylephrine treatment as detected in IF experiments. Conclusion Novel mutation in ANKRD1 were found and validated to be causing f-DCM, by revealing phenotypical alternation using hiPSC-CM technology. Increased cellular death rate and impaired nuclear translocation of CARP might be the underlying causes of the morphological and contractile abnormalities detected in-vitro, leading to systolic dysfunction in the affected family.

Impact of aging and oxidative stress on specific components of excitation contraction coupling in regulating force generation

Muscle weakness associated with sarcopenia is a major contributor to reduced health span and quality of life in the elderly. However, the underlying mechanisms of muscle weakness in aging are not fully defined. We investigated the effect of oxidative stress and aging on specific molecular mechanisms involved in muscle force production in mice and skinned permeabilized single fibers in mice lacking the antioxidant enzyme CuZnSod (Sod1KO) and in aging (24-month-old) wild-type mice. Loss of muscle strength occurs in both models, potentially because of reduced membrane excitability with altered NKA signaling and RyR stability, decreased fiber Ca2+ sensitivity and suppressed SERCA activity via modification of the Cys674 residue, dysregulated SR and cytosolic Ca2+ homeostasis, and impaired mitochondrial Ca2+ buffering and respiration. Our results provide a better understanding of the specific impacts of aging and oxidative stress on mechanisms related to muscle weakness that may point to future interventions for countering muscle weakness.

Mass spectrometric identification of dystrophin, the protein product of the Duchenne muscular dystrophygene, in distinct muscle surface membranes.

Supramolecular membrane complexes of low abundance are difficult to study by routine bioanalytical techniques. The plasmalemmal complex consisting of sarcoglycans, dystroglycans, dystrobrevins and syntrophins, which is closely associated with the membrane cytoskeletal protein dystrophin, represents such a high-molecular-mass protein assembly in skeletal muscles. The almost complete loss of the dystrophin isoform Dp427-M and concomitant reduction in the dystrophin-associated glycoprotein complex is the underlying cause of the highly progressive neuromuscular disorder named Duchenne muscular dystrophy. This gives the detailed characterization of the dystrophin complex considerable pathophysiological importance. In order to carry out a comprehensive mass spectrometric identification of the dystrophin-glycoprotein complex, in this study, we used extensive subcellular fractionation and enrichment procedures prior to subproteomic analysis. Mass spectrometry identified high levels of full-length dystrophin isoform Dp427-M, α/β-dystroglycans, α/β/γ/δ-sarcoglycans, α1/β1/β2-syntrophins and α/β-dystrobrevins in highly purified sarcolemma vesicles. By contrast, lower levels were detected in transverse tubules and no components of the dystrophin complex were identified in triads. For comparative purposes, the presence of organellar marker proteins was studied in crude surface membrane preparations vs. enriched fractions from the sarcolemma, transverse tubules and triad junctions using gradient gel electrophoresis and on-membrane digestion. This involved the subproteomic assessment of various ion-regulatory proteins and excitation-contraction coupling components. The comparative profiling of skeletal muscle fractions established a relatively restricted subcellular localization of the dystrophin-glycoprotein complex in the muscle fibre periphery by proteomic means and clearly demonstrated the absence of dystrophin from triad junctions by sensitive mass spectrometric analysis.

Gut Motility Alterations in Neonates and Young Infants

C olic usually affects babies in the first few weeks of their lives and persists for about 4 months. Crying can be intense and furious and it may last for several hours per day for several weeks. Although crying can occur at any time, it is usually worse in the late afternoon and evening, and occasionally affects the baby’s sleep. Infantile colic is not considered a disease, fulfilling instead criteria for a functional disorder. Research shows that babies with colic continue to eat and gain weight appropriately, despite the crying (1). The main risk for these babies relates to the stress and anxiety that the condition creates at home, especially when it affects the first child. The cause of colic is not known. Painful flatus may contribute to colic, but there is little evidence to prove that it is linked to digestive problems (2). Another theory is that while the digestive system is maturing, some babies are more sensitive to substances such as lactose ingested through breast-feeding and formula milk; however, evidence to support this hypothesis also is limited. Other possible causes relate to the baby’s behavior and temperament. Among the gastrointestinal (GI) factors, we focus on non-nutritive pathophysiology such as the relation of colic to gastroesophageal reflux (GER), GI motility disorders, the role of gut hormones, and intestinal microflora.

Heat acclimation and exercise training interact when combined in an overriding and trade-off manner: physiologic-genomic linkage

Combined heat acclimation (AC) and exercise training (EX) enhance exercise performance in the heat while meeting thermoregulatory demands. We tested the hypothesis that different stress-specific adaptations evoked by each stressor individually trigger similar cardiac alterations, but when combined, overriding/trade-off interactions take place. We used echocardiography, isolated cardiomyocyte imaging and cDNA microarray techniques to assay in situ cardiac performance, excitation-contraction (EC) coupling features, and transcriptional programs associated with cardiac contractility. Rat groups studied were controls (sedentary 24°C); AC (sedentary, 34°C, 1 mo); normothermic EX (treadmill at 24°C, 1 mo); and heat-acclimated, exercise-trained (EXAC; treadmill at 34°C, 1 mo). Prolonged heat exposure decreased heart rate and contractile velocity and increased end ventricular diastolic diameter. Compared with controls, AC/EXAC cardiomyocytes demonstrated lower l-type Ca(2+) current (I(CaL)) amplitude, higher Ca(2+) transient (Ca(2+)T), and a greater Ca(2+)T-to-I(CaL) ratio; EX alone enhanced I(CaL) and Ca(2+)T, whereas aerobic training in general induced cardiac hypertrophy and action potential elongation in EX/EXAC animals. At the genomic level, the transcriptome profile indicated that the interaction between AC and EX yields an EXAC-specific molecular program. Genes affected by chronic heat were linked with the EC coupling cascade, whereas aerobic training upregulated genes involved with Ca(2+) turnover via an adrenergic/metabolic-driven positive inotropic response. In the EXAC cardiac phenotype, the impact of chronic heat overrides that of EX on EC coupling components and heart rate, whereas EX regulates cardiac morphometry. We suggest that concerted adjustments induced by AC and EX lead to enhanced metabolic and mechanical performance of the EXAC heart.

Cardiac Excitation-Contraction Coupling Proteins: A 3D Spatial Analysis

The vital heart beat that pumps blood around the body is caused by millions of cardiac muscle cells exciting and contracting in concert. Cellular contraction is inextricably linked to the sarcoplasmic reticulum (SR) release event and subsequent binding of Ca2+ to myofibril binding sites. An integral protein in the release event is the SR membrane bound Ryanodine Receptor (RyR). Ca2+ enters the narrow spaces between transverse tubules and the termini of the SR via voltage-gated L-type Ca2+ channels. It then binds to RyR's to evoke the release of more Ca2+ from the SR, into the cytosol. Mathematical models examining this mechanism of excitation-contraction coupling (ECC) have thus far lacked anatomically realistic parameters for representing the ultrastructure and the distributions of key proteins in these myocytes. Our work has focused on producing an anatomically correct 3D model of a healthy cardiomyocyte that represents the spatial organization of the different cellular components involved in ECC. To this end, this study seeks to develop and perform statistical analyses on the 3D spatial distribution of RyR's and other ECC proteins in the healthy cell. Using immunofluorescence confocal microscopy images of rat ventricular muscle, we have carried out 3D spatial analyses which have enabled us to investigate the anisotropy, clustering, randomness and scatter of RyR's. We have also examined aspects of the spatial relationship between RyR's and myofibrils at Z-disks via the implementation of computational models. We have thus developed a novel anatomically based model of cardiomyocyte RyR distribution. This combined with models of other ECC component proteins, will enable us to gain new insights into the role of cardiomyocyte ultrastructure in determining the spatiotemporal changes in intracellular Ca2+ which is central in ECC.

Ultrastructural differences and histochemical characteristics in swimming muscles between wild and reared Atlantic salmon

The swimming capacity of wild and reared fish differs. Whether the differences are associated with metabolic, contractile or structural variation in swimming musculature is unknown. In the present study, some aspects of contractile machinery in swimming muscles of wild and reared salmon are compared. Several morphological parameters and key enzyme activities were measured using electron microscopy and histochemical methods. The density and size of the mitochondria was significantly higher in the muscle samples from wild fish when compared with the reared ones. Similar variability was also seen in the density of triads. Conversely, the size and density of lipid droplets was significantly lower in the red muscle of wild vs. reared salmon. The densities of two excitation contraction coupling components, dihydropyridine and ryanodine receptor, were considerably higher in swimming muscles of wild salmon than in reared fish. A similar difference was observed in the activities of aerobic enzymes. Moreover, oxygen consumption followed the same pattern, being significantly higher in the samples of wild salmon. Phosphorylase activity was, on the other hand, significantly lower in the muscles of wild fish. There are significant differences in morphology, Ca(2+)-regulating capacity and enzyme activities in swimming muscles between wild and reared salmon. These results provide evidence that the prerequisites for efficient contraction of the swimming muscles are better met in wild than in reared salmon. Importantly, the results also suggest that the observed variation is a major contributing factor to the difference in the swimming capacity between wild and hatchery-reared salmon.

Ca2+handling and oxidative capacity are greatly impaired in swimming muscles of hatchery-reared versus wild Atlantic salmon (Salmo salar)

The swimming capacity of fish is strongly associated with muscle performance, although the prerequisites for effective movements have not been fully described at the molecular level. To compare the condition of swimming musculature of hatchery-reared Atlantic salmon (Salmo salar) with that of wild fish, we analyzed the relative level of two excitation–contraction coupling components (i.e., dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)) and the oxidative capacity of muscles with histochemical and Western blot methods. The density of DHPR and RyR was considerably higher in swimming muscles of wild fingerlings (age 0+) (109.8% and 123.3% in red muscle; 128.6% and 186.0% in white muscle, respectively) and yearlings (age 1+) (153.5% and 459.1% in red muscle; 131.2% and 858.4% in white muscle) as compared with those in reared fish. Similar difference was also observed in the oxidative capacity of muscles. Moreover, the oxidative activity correlated positively with the level of DHPR and RyR. Our data indicate that calcium handling, as well as oxidative capacity of swimming muscles of reared salmon, is clearly separable from the corresponding capacities of wild fish. We suggest that the observed alteration is a major contributing factor to the well-documented differences in swimming ability between wild and hatchery-reared salmon.

Myocardial calcium signalling and arrhythmia pathogenesis

Myocardial calcium signalling is a vital component of the normal physiological function of the heart. Key amongst the many roles calcium plays is its use as the primary signalling component of excitation–contraction coupling, the intracellular process that links cardiomyocyte depolarisation to contraction. Defective cellular calcium handling, due to abnormalities of the various components which mediate and control excitation–contraction coupling, is widely recognised as a significant patho-physiological event in the contractile dysfunction of the failing heart. In addition, similar defects also appear to be increasingly recognised as mediators of certain forms of cardiac arrhythmias. Such defects include single gene defects in excitation–contraction coupling components that lead to inherited sudden death arrhythmia syndromes. Alternatively, arrhythmogenesis occurring within the context of acquired cardiac disease, in particular heart failure, also appears to be highly dependent on abnormal calcium homeostasis. In this article we review the defects in cardiomyocyte calcium homeostasis that lead to particular pro-arrhythmogenic phenomena and discuss recent insights gained into a variety of inherited and acquired arrhythmia syndromes that appear to involve defective calcium signalling as a central component of their patho-physiology. Potential opportunities for new anti arrhythmic therapeutic strategies based on these recent insights are also discussed.

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes.

We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca(2+) current (I(Ca-L)) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca(2+) transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f(340)/f(380)) decreased by 12% after 3 min in 0.6T, whereas shortening and I(Ca-L) increased at the same time. After 8 min, shortening, I(Ca-L), and the f(340)/f(380) ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I(Ca-L) that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca(2+) transient initiated by a given I(Ca-L), and later, both I(Ca-L) and the Ca(2+) transient are inhibited.

Cardiac excitation-contraction coupling in the portal hypertensive rat.

Basal contractility and responses to beta-adrenoceptor activation are compromised in hearts from rats with chronic portal vein stenosis. Here we report the effect of partial ligation of the portal vein on myocardial G protein expression, beta-adrenoceptor-G protein coupling, and excitation-contraction coupling (ECC). Contractility (dT/dt) was reduced 30-50% in right and left ventricles, but the rate of relaxation (-dT/dt) was unaffected. Isoproterenol-induced positive inotropism was diminished, but there was no difference in ED(50). The concentration-dependent increase in -dT/dt was unaffected. G(s)alpha and G(i)alpha expression, cholera toxin- and pertussis toxin-induced ADP-ribosylation, and formation of the agonist-receptor-G(s) complex were unaffected by portal vein stenosis. Of the components of ECC examined, the caffeine-sensitive sarcoplasmic reticulum Ca(2+) pool was reduced 35%, although the Ca(2+) uptake and release processes were unchanged; the apparent density of L-type Ca(2+) channels decreased 60% with no change in affinity; the dihydropyridine Ca(2+) channel agonist BAY K 8644 produced relative changes in dT/dt that were similar in both groups, suggesting normal function in the remaining Ca(2+) channels; and Na(+)/Ca(2+) exchange was reduced 50% in the portal vein stenosis group. These data suggest that the effect of portal vein stenosis on the myocardium is the result of alterations to ECC.

Contractile properties of rat skeletal muscles following storage at 4 degrees C.

The purpose of this study was to assess the potential of preservation solutions for protecting skeletal muscle function during storage at 4 degrees C. The soleus and the cutaneus trunci (CT) from the rat were stored for 2, 8 or 16 h at 4 degrees C in University of Wisconsin solution (UW), HTK-Bretschneider solution (HTK) or Krebs-Henseleit solution (KH). After storage, muscles were stimulated electrically to analyse the isometric contractile properties, such as the maximum tetanic tension (P(0)). Histological analysis was also performed. In separate experiments, the effect of the diffusion distance on muscle preservation was studied by bisecting the soleus. After 8 h of storage in UW or HTK, the contractile properties of the CT were similar to those of the control, whereas those of the soleus were reduced (P(0) values of 16% and 69% of control in UW and HTK respectively). At 16 h, the contractile properties of the CT (P(O) 28%) were again better preserved than those of the soleus (P(0) 9%). Muscle function deteriorated most after storage in KH (P(0) at 16 h: soleus, 3%; CT, 17%). The bisected soleus was equally well preserved as the CT (P(O) of bisected soleus at 8 h in UW and HTK: 86%). The functional data corresponded well with the histological data, which showed increasing muscle fibre derangement with increasing storage time. For both muscles and all solutions, the threshold stimulus current increased with increasing storage time (control, 0.1 mA; 16 h, 1.2 mA) and was strongly correlated with the deterioration in contractile properties. It is concluded that, at 4 degrees C, muscle is preserved better in UW and HTK (intracellular-like solutions) than in KH (extracellular-like solution). The soleus and CT were best protected in HTK. The diffusion distance is a critical factor for successful preservation of muscle function at 4 degrees C. The reduced function after 16 h of storage at 4 degrees C was caused by hypercontraction and necrosis of about 25% of the muscle fibres, and by deterioration of the electrical component of excitation-contraction coupling of the remaining fibres.

Changes in the Expression of the L-Type Voltage-Dependent Calcium Channel during Pregnancy and Parturition in the Rat1

The L-type voltage-dependent calcium channel (L-VDCC) is assumed to be a critical component of excitation-contraction coupling in smooth muscle. Using pregnant rat myometrium, we examined the hypothesis that parturition is associated with significant changes in the expression of the alpha 1 subunit of the L-VDCC at the mRNA or protein level. The binding of radiolabeled dihydropyridine, which correlates with the total number of calcium channels in the membrane, was increased by 14 days' gestation, in comparison to that in nonpregnant controls. The elevation in binding capacity persisted through labor and fell postpartum. Northern and RNA dot-blot analysis demonstrated the highest level of expression on Days 20 and 21, with a 3- to 10-fold decrease during parturition. We believe these studies are most consistent with a one-day lag time between mRNA and protein expression, and generally support a modest increase in L-VDCC expression in pregnancy and labor. Reverse transcriptase polymerase chain reaction was used to examine changes in isoform expression in Motif IV, a region of the alpha 1 subunit known to be alternatively spliced. These studies revealed the presence of multiple isoforms in rat myometrium, with a predominance of IVS3B. Interestingly, a marked increase in the ratio of S3B:S3A was noted at parturition. In summary, these data demonstrate that the number of L-type calcium channels, although increased in pregnancy, do not change prior to, or with the onset of, myometrial contraction. Intriguingly, mRNA expression was markedly decreased at parturition. The change in isoform expression during labor is of unknown, but potential, physiologic significance.