Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum

Sarcolemmal membrane-associated proteins (SLMAPs) are components of cardiac membranes involved in excitation-contraction (E-C) coupling. Here, we assessed the role of SLMAP in cardiac structure and function. We generated transgenic (Tg) mice with cardiac-restricted overexpression of SLMAP1 bearing the transmembrane domain 2 (TM2) to potentially interfere with endogenous SLMAP through homodimerization and subcellular targeting. Histological examination revealed vacuolated myocardium; the severity of which correlated with the expression level of SLMAP1-TM2. High resolution microscopy showed dilation of the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and confocal imaging combined with biochemical analysis indicated targeting of SLMAP1-TM2 to the SR/ER membranes and inappropriate homodimerization. Older (28 wk of age) Tg mice exhibited reduced contractility with impaired relaxation as assessed by left ventricle pressure monitoring. The ventricular dysfunction was associated with electrophysiological abnormalities (elongated QT interval). Younger (5 wk of age) Tg mice also exhibited an elongated QT interval with minimal functional disturbances associated with the activation of the fetal gene program. They were less responsive to isoproterenol challenge (ΔdP/dt(max)) and developed electrical and left ventricular pressure alternans. The altered electrophysiological and functional disturbances in Tg mice were associated with diminished expression level of calcium cycling proteins of the sarcoplasmic reticulum such as the ryanodine receptor, Ca(2+)-ATPase, calsequestrin, and triadin (but not phospholamban), as well as significantly reduced calcium uptake in microsomal fractions. These data demonstrate that SLMAP is a regulator of E-C coupling at the level of the SR and its perturbation results in progressive deterioration of cardiac electrophysiology and function.     

Hypertension-induced remodeling of cardiac excitation-contraction coupling in ventricular myocytes occurs prior to hypertrophy development

Hypertension is a major risk factor for developing cardiac hypertrophy and heart failure. Previous studies show that hypertrophied and failing hearts display alterations in excitation-contraction (E-C) coupling. However, it is unclear whether remodeling of the E-C coupling system occurs before or after heart disease development. We hypothesized that hypertension might cause changes in the E-C coupling system that, in turn, induce hypertrophy. Here we tested this hypothesis by utilizing the progressive development of hypertensive heart disease in the spontaneously hypertensive rat (SHR) to identify a window period when SHR had just developed hypertension but had not yet developed hypertrophy. We found the following major changes in cardiac E-C coupling during this window period. 1) Using echocardiography and hemodynamics measurements, we found a decrease of left ventricular ejection fraction and cardiac output after the onset of hypertension. 2) Studies in isolated ventricular myocytes showed that myocardial contraction was also enhanced at the same time. 3) The action potential became prolonged. 4) The E-C coupling gain was increased. 5) The systolic Ca(2+) transient was augmented. These data show that profound changes in E-C coupling already occur at the onset of hypertension and precede hypertrophy development. Prolonged action potential and increased E-C coupling gain synergistically increase the Ca(2+) transient. Functionally, augmented Ca(2+) transient causes enhancement of myocardial contraction that can partially compensate for the greater workload to maintain cardiac output. The increased Ca(2+) signaling cascade as a molecular mechanism linking hypertension to cardiac hypertrophy development is also discussed.     

Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad.

Increased interest in the mechanism of excitation-contraction (E-C) coupling over the last few years has been accompanied by numerous investigations into the development of the underlying cellular structures. Areas of particular interest include: (1) the compartmentalization and specialization of an external and an internal membrane system, the T-tubules, and the sarcoplasmic reticulum, respectively; (2) interactions between the membrane proteins of both systems upon the formation of a junction, the triad; and (3) membrane-cytoskeletal interactions leading to the orderly arrangement of the triads with respect to the myofibrils. Structural studies using newly available specific molecular probes and a variety of in vivo and in vitro model systems have provided new insights into the cellular and molecular mechanisms involved in the development of the E-C coupling apparatus in skeletal muscle.     

Critical role of cardiac t-tubule system for the maintenance of contractile function revealed by a 3D integrated model of cardiomyocytes

T-tubules in mammalian ventricular myocytes constitute an elaborate system for coupling membrane depolarization with intracellular Ca(2+) signaling to control cardiac contraction. Deletion of t-tubules (detubulation) has been reported in heart diseases, although the complex nature of the cardiac excitation-contraction (E-C) coupling process makes it difficult to experimentally establish causal relationships between detubulation and cardiac dysfunction. Alternatively, numerical simulations incorporating the t-tubule system have been proposed to elucidate its functional role. However, the majority of models treat the subcellular spaces as lumped compartments, and are thus unable to dissect the impact of morphological changes in t-tubules. We developed a 3D finite element model of cardiomyocytes in which subcellular components including t-tubules, myofibrils, sarcoplasmic reticulum, and mitochondria were modeled and realistically arranged. Based on this framework, physiological E-C coupling was simulated by simultaneously solving the reaction-diffusion equation and the mechanical equilibrium for the mathematical models of electrophysiology and contraction distributed among these subcellular components. We then examined the effect of detubulation in this model by comparing with and without the t-tubule system. This model reproduced the Ca(2+) transients and contraction observed in experimental studies, including the response to beta-adrenergic stimulation, and provided detailed information beyond the limits of experimental approaches. In particular, the analysis of sarcomere dynamics revealed that the asynchronous contraction caused by a large detubulated region can lead to impairment of myocyte contractile efficiency. These data clearly demonstrate the importance of the t-tubule system for the maintenance of contractile function.     

Endothelial dysfunction in Type 2 diabetes correlates with deregulated expression of the tail-anchored membrane protein SLMAP

The Type 2 diabetic db/db mouse experiences vascular dysfunction typified by changes in the contraction and relaxation profiles of small mesenteric arteries (SMAs). Contractions of SMAs from the db/db mouse to the alpha1-adrenoceptor agonist phenylephrine (PE) were significantly enhanced, and acetylcholine (ACh)-induced relaxations were significantly depressed. Drug treatment of db/db mice with a nonthiazolidinedione peroxisome prolifetor-activated receptor-gamma agonist and insulin sensitizing agent 2-[2-(4-phenoxy-2-propylphenoxy)ethyl]indole-5-acetic acid (COOH) completely prevented the changes in endothelium-dependent relaxation, but, with the discontinuation of therapy, endothelial dysfunction returned. Dysfunctional SMAs were found to specifically upregulate the expression of a 35-kDa isoform of sarcolemmal membrane-associated protein (SLMAP), which is a component of the excitation-contraction coupling apparatus and implicated in the regulation of membrane function in muscle cells. Real-time PCR revealed high SLMAP mRNA levels in the db/db microvasculature, which were markedly downregulated during COOH treatment but elevated again when drug therapy was discontinued. These data reveal that the microvasculature in db/db mice undergoes significant changes in vascular function with the endothelial component of vascular dysfunction specifically correlating with the overexpression of SLMAP. Thus changes in SLMAP expression may be an important indicator for microvascular disease associated with Type 2 diabetes.     

Ca2+ signaling in microdomains: Homer1 mediates the interaction between RyR2 and Cav1.2 to regulate excitation-contraction coupling

Excitation-contraction (E-C) coupling and Ca(2+)-induced Ca(2+) release in smooth and cardiac muscles is mediated by the L-type Ca(2+) channel isoform Ca(v)1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca(v)1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca(v)1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1(-/-) mice and by demonstrating a Homer1-mediated dynamic interaction between Ca(v)1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca(v)1.2. Most notably, expression of Ca(v)1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca(v)1.2 and RyR2 to regulate the intensity of Ca(2+)-induced Ca(2+) release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca(v)1.2 and RyR2. In one state, Ca(v)1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca(v)1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca(v)1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.     

Calcium-induced calcium release in smooth muscle: the case for loose coupling

This article reviews the key experiments demonstrating calcium-induced calcium release (CICR) in smooth muscle and contrasts the biophysical and molecular features of coupling between the sarcolemmal (L-type Ca²⁺ channel) and sarcoplasmic reticulum (ryanodine receptor) Ca²⁺ channels in smooth and cardiac muscle. Loose coupling refers to the coupling process in smooth muscle in which gating of ryanodine receptors is non-obligate and may occur with a variable delay following opening of the sarcolemmal Ca²⁺ channels. These features have been observed in the earliest studies of CICR in smooth muscle and are in marked contrast to cardiac CICR, where a close coupling between T-tubular and SR membranes results in tight coupling between the gating events. The relationship between this “loose coupling” and distinct subcellular release sites within smooth muscle cells, termed frequent discharge sites, is discussed.     

Functional studies of Ca2+ channels from plasmalemma and sarcoplasmic reticulum membranes in muscle cells

Physiological and biochemical studies (channel characteristics, intracellular Ca2+ determinations and, channel purification, cloning and expression) of the different components involved in the regulation of intercellular Ca2+ have provided new information about their specific role. Recent information favors a major role for plasmalemma Ca2+ channels in E-C coupling of cardiac muscle, while a major role for sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors) is proposed for E-C coupling of skeletal muscle. In smooth muscle, both plasmalemma and sarcoplasmic reticulum (IP3 receptors) Ca2+ channels are involved in E-C coupling. These studies will be comparatively discussed for skeletal, cardiac and smooth muscle cells.     

Simulated de novo assembly of golgi compartments by selective cargo capture during vesicle budding and targeted vesicle fusion

The Golgi apparatus is comprised of stacked cisternal membranes forming subcompartments specialized for posttranslational processing of newly synthesized secretory cargo. Recent experimental evidence indicates that the Golgi apparatus can undergo de novo biogenesis from the endoplasmic reticulum, but the mechanism by which the membranes self assemble into compartmentalized structures remains unknown. We developed a discrete-event computer simulation model to test whether two fundamental mechanisms—vesicle-coat-mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation, and SNARE-mediated selective fusion of vesicles—suffice to generate and maintain compartments. Simulations verified that this minimal model is adequate for homeostasis of preestablished compartments, even in response to small perturbations, and for de novo formation of stable compartments. The model led to a novel prediction that Golgi size is in part dependent on target SNARE expression level. This prediction was supported by a demonstration that exogenous expression of the Golgi target SNARE syntaxin-5 alters Golgi size in living cells.     

To the heart of myofibril assembly

One of the most fascinating examples of cytoskeletal assembly is the myofibril, the contractile structure of striated (i.e. skeletal and cardiac) muscle. Myofibrils are composed of repeating contractile units known as sarcomeres, perhaps the most highly ordered macromolecular structures in eukaryotic cells. When skeletal and cardiac muscle cells differentiate, thousands of structural and regulatory molecules assemble into the semicrystalline sarcomeric contractile units. As a consequence of this precise assembly, many different classes of proteins function together to convert the molecular interactions of actin and myosin efficiently into the macroscopic movements of contractile activity.     

Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres

Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.     

Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle

The plasmalemmal dihydropyridine receptor (DHPR) is the voltage sensor in skeletal muscle excitation-contraction (e-c) coupling. It activates calcium release from the sarcoplasmic reticulum via protein-protein interactions with the ryanodine receptor (RyR). To enable this interaction, DHPRs are arranged in arrays of tetrads opposite RyRs. In the DHPR alpha(1S) subunit, the cytoplasmic loop connecting repeats II and III is a major determinant of skeletal-type e-c coupling. Whether the essential II-III loop sequence (L720-L764) also determines the skeletal-specific arrangement of DHPRs was examined in dysgenic (alpha(1S)-null) myotubes reconstituted with distinct alpha(1) subunit isoforms and II-III loop chimeras. Parallel immunofluorescence and freeze-fracture analysis showed that alpha(1S) and chimeras containing L720-L764, all of which restored skeletal-type e-c coupling, displayed the skeletal arrangement of DHPRs in arrays of tetrads. Conversely, alpha(1C) and those chimeras with a cardiac II-III loop and cardiac e-c coupling properties were targeted into junctional membranes but failed to form tetrads. However, an alpha(1S)-based chimera with the heterologous Musca II-III loop produced tetrads but did not reconstitute skeletal muscle e-c coupling. These findings suggest an inhibitory role in tetrad formation of the cardiac II-III loop and that the organization of DHPRs in tetrads vis-a-vis the RyR is necessary but not sufficient for skeletal-type e-c coupling.     

Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro

Muscular dysgenesis (mdg) in mice causes the failure of excitation-contraction (E-C) coupling in skeletal muscle. Cultured dysgenic muscle fails to contract upon depolarization, lacks typical muscle ultrastructure, including normal triads, and lacks functional voltage-dependent slow calcium channels. We show that normal rodent fibroblasts and 3T3 fibroblasts "rescue" dysgenic myotubes, reestablishing contractions (i.e., E-C coupling), normal ultrastructure, and functional slow calcium channels. These results support the finding that the expression of the slow calcium channel is affected in the mdg mutation and that this protein is essential for E-C coupling. Additionally, fibroblast rescue provides a system for examining the mechanisms of heterotypic cellular influence on cell function.     

Functional Expression of GFP-linked Human Heart Sodium Channel (hH1) and Subcellular Localization of the a Subunit in HEK293 Cells and Dog Cardiac Myocytes

Recent evidence suggests that biosynthesis of the human heart Na+ channel (hH1) protein is rapidly modulated by sympathetic interventions. However, data regarding the intracellular processing of hH1 in vivo are lacking. In this study we sought to establish a model that would allow us to study the subcellular localization of hH1 protein. Such a model could eventually help us to better understand the trafficking of hH1 in vivo and its potential role in cardiac conduction. We labeled the C-terminus of hH1 with the green fluorescent protein (GFP) and compared the expression of this construct (hH1-GFP) and hH1 in transfected HEK293 cells. Fusion of GFP to hH1 did not alter its electrophysiological properties. Confocal microscopy revealed that hH1-GFP was highly expressed in intracellular membrane structures. Immuno-electronmicrographs showed that transfection of hH1-GFP and hH1 induced proliferation of three types of endoplasmic reticulum (ER) membranes to accommodate the heterologously expressed proteins. Labeling with specific markers for the ER and the Golgi apparatus indicated that the intracellular channels are almost exclusively retained within the ER. Immunocytochemical labeling of the Na+ channel in dog cardiomyocytes showed strong fluorescence in the perinuclear region of the cells, a result consistent with our findings in HEK293 cells. We propose that the ER may serve as a reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct enhancers regulate skeletal and cardiac muscle-specific expression programs of the cardiac alpha-actin gene in Xenopus embryos

During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.     

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.     

Molecular simulation of self-assembly of hydrophilic functionalized aromatics in aqueous solutions

This study identifies mechanisms of self-assembly of hydrophilic functionalized aromatic molecules—a distinct class of lyotropic materials. Results from molecular dynamics studies used to understand the moieties of the lyotropic molecule that affect the structure are consistent with experimental observations of these self-assembled structures. Coulombic forces, dominated by π–π interactions drives the self-assembly of this class of materials. Intra-molecular configurations of the aromatic rings—the extent of torsion or bending between the rings—as well as the structure of functional groups connected to the rings affect the self-assembled structures. In addition, the chemistry of the functional groups also affects how the molecules are oriented as they self assemble. Molecular modeling provides insight into design of these molecules.     

Immunocytochemical studies of spectrin in hamster cardiac tissue

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.