Frequency, Etiology, and Impact of Unplanned Repeat Coronary Angiography After ST-Elevation Myocardial Infarction.

Unplanned repeat coronary angiography (CAG) after balloon angioplasty for ST-elevation myocardial infarction (STEMI) was common before the advent of coronary stenting. Limited data are available regarding the role of unplanned repeat CAG in contemporary percutaneous coronary intervention (PCI) for STEMI. Therefore, we analyzed a large, 2-center prospective STEMI registry (January 2011 to June 2020) stratified by the presence or absence of unplanned repeat CAG during index hospitalization. Patients with planned CAG for staged PCI or experimental drug administration were excluded. Among 3,637 patients with STEMI, 130 underwent unplanned repeat CAG (3.6%) during index hospitalization. These patients were more likely to have cardiogenic shock (16% vs 9.8%, p = 0.021), left anterior descending culprit (44% vs 31%, p <0.001), lower left ventricular ejection fraction (45% vs 52%, p <0.001), and higher peak troponin levels (22 vs 8 ng/ml, p <0.001) than those without repeat CAG. At repeat CAG, 80 patients had a patent stent (62%) including 65 requiring no further intervention (50%) and 15 who underwent intervention on a nonculprit lesion (12%). Only 32 patients had stent thrombosis (25%). Repeat CAG was associated with a higher incidence of recurrent MI (19% vs 0%, p <0.001) and major bleeding (12% vs 4.5%, p <0.001), yet similar in-hospital mortality (7% vs 6.4%, p = 0.93) than those without repeat CAG. In conclusion, in the era of contemporary PCI for STEMI, unplanned repeat CAG during index hospitalization was infrequent and more commonly observed in patients with left anterior descending culprit in the presence of significant left ventricular dysfunction or shock and was associated with higher in-hospital recurrent myocardial infarction and major bleeding complications.

Lesiones por fuegos artificiales atendidas por Cirugía Plástica en el Hospital General Dr. Manuel Gea González de México, 2015-2019 / Injuries due to fireworks treated by the Plastic Surgery Service of the General Hospital Dr. Manuel Gea González, 2015 to 2019

Introducción y objetivo: La Comisión de Seguridad de Productos del Consumidor de Estados Unidos realiza de forma anual un reporte de lesiones provocadas por fuegos artificiales que orienta la toma de decisiones para disminuir este tipo de accidentes. Actualmente, México no cuenta con reportes, ni leyes específicas que ayuden a disminuir las lesiones por fuegos artificiales.El objetivo del presenta trabajo es recoger las características de las lesiones provocadas por fuegos artificiales atendidas en el Servicio de Cirugía Plástica del Hospital General Dr. Manuel Gea González de la Ciudad de México, con la intención de incentivar la creación de normas y regulaciones de artículos pirotécnicos en México y otros países.Material y método.Estudio retrospectivo, descriptivo, de lesiones provocadas por fuegos artificiales entre 2015 y 2019 atendidas en el Servicio de Cirugía Plástica del Hospital General Dr. Manuel Gea González (México).Resultados.Recogimos 84 pacientes; la mayor incidencia de lesiones fue en varones (76%) y el rango de edad más afectado fue el de 5 a 14 años (37%).La región corporal más afectada fue la extremidad superior, principalmente la mano (61%), seguida de cabeza y cuello (27%) y miembro pélvico (9%). El diagnóstico más frecuente fue quemaduras (32%), seguido de laceraciones (31%), fracturas (20%), amputación (11%) y contusión (4%).Conclusiones.En nuestro ámbito de estudio, los accidentes por fuegos artificiales afectan en mayor proporción a menores de edad, ocasionando lesiones graves que pueden llegar a ser incapacitantes. Creemos necesario implementar estrategias para conocer la incidencia de estos accidentes a fin de crear leyes que regulen el uso de fuegos artificiales. (AU)

Background and objective: The United States Consumer Product Safety Commission makes an annual report of injuries caused by fireworks, which guides decision-making strategies to reduce these types of accidents. Currently, Mexico does not have statistics or specific laws that help reduce the injuries from fireworks.Our aim is to collect the characteristics of injuries caused by fireworks treated in the Plastic Surgery Service of the Hospital General Dr. Manuel Gea González in México City, with the intention of encouraging the creation of norms and regulations of pyrotechnics articles in Mexico and other countries.Methods.Retrospective and descriptive study of injuries caused by fireworks in the period from 2015 to 2019 treated at the Plastic Surgery Service of the General Hospital Dr. Manuel Gea González (Mexico).Results.We collected 84 patients; the highest incidence of injuries was in men (76%) and the most afected aged ranged from 5 to 14 years (37%). The most afected body region was the upper extremity, mainly hand (61%), followed by head and neck (27%) and pelvic limb (9%). The most frequent diagnosis was burns (32%), followed by lacerations (31%), fractures (20%), amputation (11%) and contusion (4%).Conclusions.In our environment, accidents caused by fireworks are afecting minors, causing serious injuries that can become disabling. In our opinion, strategies must be implemented to know the incidence of these accidents in order to create laws that regulate and prohibit the use of fireworks.In the case of minors, their use should be prohibited and authorities and parents should be made aware of the risk that these activities entail. Besides, in most cases in our country the responsible family members do not have a formal job with access to social security health institutions. (AU)

The Midwest ST-Elevation Myocardial Infarction Consortium: Design and Rationale.

BACKGROUND: Over the past 20 years, the development of regional ST-elevation myocardial infarction (STEMI) care systems has led to remarkable progress in achieving timely coronary reperfusion with attendant improvement in clinical outcomes, including survival. Despite this progress, contemporary STEMI care does not consistently meet the national guideline-recommended goals, which offers an opportunity for further improvement in STEMI outcomes. The lack of single, comprehensive, national STEMI registry complicates our ability to improve STEMI outcomes in particular for high-risk STEMI subsets such as cardiac arrest (CA) and/or cardiogenic shock (CS). OBJECTIVES: To address this need, the Midwest STEMI Consortium (MSC) was created as a collaboration of 4 large, regional STEMI care systems to provide a comprehensive, multicenter, and prospective STEMI registry without any exclusionary criteria. METHODS: The MSC is a collaboration of 4 large, regional STEMI care systems: Iowa Heart Center in Des Moines, IA; Minneapolis Heart Institute Foundation in Minneapolis, MN; Prairie Heart Institute in Springfield, IL; and The Christ Hospital in Cincinnati, OH. Each has similar standardized STEMI protocol and together include 6 percutaneous coronary intervention (PCI)-capable hospitals and over 100 non-PCI-capable hospitals. Each center had a prospective database that was transferred to a data coordinating center to create the multicenter database. The comprehensive database includes traditional risk factors, cardiovascular history, medications, time to treatment data, detailed angiographic characteristics, and short- and long-term clinical outcomes up to 5-year for myocardial infarction, stroke, and cardiovascular and all-cause mortality. Ten-year mortality rates were assessed by using national death index. RESULTS: Currently, the comprehensive database (03/2003-01/2020) includes 14,911 consecutive STEMI patients with mean age of 62.3 ± 13.6 years, female gender (29%), and left anterior descending artery as the culprit vessel (34%). High risk features included: Age >75 years (19%), left ventricular ejection fraction <35% (15%), CA (10%), and CS (8%). CONCLUSION: This collaboration of 4 large, regional STEMI care systems with broad entry criteria including high-risk STEMI subsets such as CA and/or CS provides a unique platform to conduct clinical research studies to optimize STEMI care.

Características morfológicas y morfométricas del músculo aductor del hállux y sus ramos motores / Morphological and morphometric characteristics of the hallux adductor muscle and its motor branches

El hállux se encuentra en aducción en relación al eje del pie y para mantener esta posición requiere de una adecuada alineación ósea, la que está determinada principalmente por la actividad muscular. Una de las estructuras involucradas en esta función es el músculo aductor del hállux, el cual puede producir hállux valgus o hállux rígido cuando ocurre un desbalance en su actividad normal. A pesar de la importancia de este músculo, existen pocos estudios de su complejo neuromuscular. El objetivo de esta investigación fue describir las características morfológicas y morfométricas del músculo aductor del hállux y sus ramos motores en 30 miembros inferiores. Se disecó la planta del pie hasta alcanzar el plano del músculo aductor del hállux y sus ramos motores. La longitud media de la cabeza oblicua del músculo aductor del hállux fue de 78,16 mm (±13,35) con un ancho máximo promedio de 20,55 mm (±2,59) y un tendón de 25,87 mm (±7,97) de longitud. Respecto a las mismas medidas en la cabeza transversa, estas fueron 39,55 (±8,26), 15,04 (±3,52) y 18,51 (±10,04), respectivamente. La inervación de ambas cabezas del músculo aductor del hállux provenía del ramo profundo del nervio plantar lateral. En la mayoría de las muestras dicho nervio emitió un ramo para la cabeza oblicua y uno para la cabeza transversa. La cabeza oblicua presentaba uno o dos puntos motores, localizados generalmente en su tercio medio. La cabeza transversa presentaba sólo un punto motor localizado frecuentemente en su tercio lateral. El conocimiento de las características morfológicas y morfométricas del músculo aductor del hállux y de sus ramos motores son clínicamente significativos, puesto que permiten realizar una aproximación de la localización del punto motor en los procedimientos electromiográficos.

The hallux is adducted in relation to the axis of the foot and to maintain this position requires adequate bone alignment, which is determined mainly by muscle activity. One of the structures that is involved in this function is the adductor muscle of the hallux, which can produce hallux valgus or rigid hallux when an imbalance occurs in its normal activity. Despite the importance of this muscle, there are few studies of its neuromuscular complex. The objective of this study was to describe the morphological and morphometric characteristics of the adductor muscle of the hallux and its motor branches in 30 lower limbs. The sole of the foot was dissected until it reached the plane of the muscle and its motor branches. The average length of the oblique head of the adductor muscle of the hallux was 78.16 mm (± 13.35), with an average maximum width of 20.55 mm (± 2.59) and a tendon of 25.87 mm (± 7, 97) in length. Regarding the same measurements of the transverse head were 39.55 (± 8.26), 15.04 (± 3.52) and 18.51 (± 10.04), respectively. The innervation of both heads came from the deep branch of the lateral plantar nerve. In most of the samples, said nerve emitted a bouquet for the oblique head and one for the transverse head. The oblique head had one or two motor points, generally located in its middle third. The transverse head had only one motor point that was usually in its lateral third. The knowledge of the morphological and morphometric characteristics of the adductor muscle of the hallux and its motor branches are clinically significant, since they allow an approximation of the location of the motor point in electromyographic procedures.

Maturity Onset Diabetes of the Young: case report / Diabetes del adulto de inicio juvenil: reporte de un caso

Background: Maturity Onset Diabetes of the Young (MODY) is a type of diabetes that results from mutations in 13 known genes that play a role in the development and maturation of pancreatic beta cells. It represents 5% of all individuals diagnosed with diabetes before the age of 45 years and it is misdiagnosed as type 1 or type 2 diabetes in 80% of the cases. Clinical case: We present the case of a 21-year old female, initially diagnosed with type 1 diabetes when she was 19 years, and glycemic dyscontrol despite adequate use of long-acting insulin. She had family history of diabetes and persisting glycosuria. Due to high suspicions of MODY diabetes we requested an oral glucose tolerance test (basal 130 mg/dL, 2 hours post-glucose load 240 mg/dL) and serum concentrations of C-peptide (1.83 ng/mL) that confirmed our diagnosis. The patient improved after treatment with sulfonylurea with discontinuation of insulin treatment. Conclusion: Prompt identification of MODY allows patients to have effective and safe treatments and prevent the development of premature complications; in addition, its identification allows genetic counseling and can guide the management of other first-degree relatives who also suffer from it.

Introducción: la diabetes del adulto de inicio juvenil (MODY, por sus siglas en inglés: maturity onset diabetes of the young) es un tipo de diabetes que resulta de mutaciones en 13 genes conocidos que desempeñan un papel en el desarrollo y la maduración de las células beta pancreáticas. Representa el 5% de todas las personas diagnosticadas con diabetes antes de los 45 años y se diagnostica erróneamente como diabetes tipo 1 o tipo 2 hasta en el 80% de los casos. Caso clínico: presentamos el caso de una mujer de 21 años, inicialmente diagnosticada con diabetes tipo 1 a los 19 años y con descontrol glucémico a pesar del uso adecuado de insulina de acción prolongada. Tenía antecedentes familiares de diabetes y glucosuria persistente. Debido a la alta sospecha de diabetes MODY, solicitamos una prueba oral de tolerancia a la glucosa (basal 130 mg/dL y dos horas después de la carga de glucosa: 240 mg/dL) y de concentraciones séricas de péptido C (1.83 ng/mL), que confirmaron el diagnóstico. La paciente mejoró después del tratamiento con sulfonilurea y se logró la interrupción del tratamiento con insulina. Conclusiones: la identificación temprana de la diabetes tipo MODY permite a los pacientes tener tratamientos efectivos y seguros, y prevenir el desarrollo de complicaciones prematuras; además, su identificación permite el asesoramiento genético y puede orientar el manejo de otros parientes de primer grado que también la padecen.

Colombia a Source of Cacao Genetic Diversity As Revealed by the Population Structure Analysis of Germplasm Bank of Theobroma cacao L.

Beans of the species Theobroma cacao L., also known as cacao, are the raw material to produce chocolate. Colombian cacao has been classified as a fine flavor cacao that represents the 5% of cacao world's production. Colombian genetic resources from this species are conserved in ex situ and in-field germplasm banks, since T. cacao has recalcitrant seeds to desication and long-term storage. Currently, the collection of T. cacao of the Colombian Corporation of Agricultural Research (CORPOICA) has approximately 700 germplasm accessions. We conducted a molecular analysis of Corpoica's cacao collection and a morphological characterization of some accessions with the goal to study its genetic diversity and population structure and, to select interesting accessions for the cacao's breeding program. Phenotypic evaluation was performed based on 18 morphological traits and 4 biochemical traits. PCA analysis of morphological traits explained 60.6% of the total variation in seven components and 100% of the total variation of biochemical traits in four components, grouping the collection in 4 clusters for both variables. We explored 565 accessions from Corpoica's germplasm and 252 accessions from reference populations using 96 single nucleotide polymorphism (SNP) molecular markers. Molecular patterns of cacao Corpoica's collection were obtained amplifying specific alleles in a Fluidigm platform that used integrated circuits of fluids. Corpoica's collection showed highest genetic diversity [Expected Heterozygosity (HE = 0.314), Observed Heterozygosity (HO = 0.353)] that is reduced when reference populations were included in the dataset (HE = 0.294, HO = 0.261). The collection was divided into four clusters based on population structure analysis. Cacao accessions from distinct groups showed some taxonomic concordance and reflected their geographic origins. For instance, accessions classified as Criollo were clearly differentiated in one group and we identified two new Colombian genetic groups. Using a number of allelic variations based on 87 SNP markers and 22 different morphological/biochemical traits, a core collection with a total of 232 accessions was selected as a primary genetic resource for cacao breeders.

Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling.

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR_ _R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR_ _R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.

Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle.

Understanding which cytosolic domains of the dihydropyridine receptor participate in excitation-contraction (EC) coupling is critical to validate current structural models. Here we quantified the contribution to skeletal-type EC coupling of the alpha1S (CaV1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of alpha1S. Chimeras consisting of alpha1C (CaV1.2) with alpha1S substitutions at each of the interrepeat loops (I-II, II-III, and III-IV loops) and N- and C-terminal domains were evaluated in dysgenic (alpha1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the alpha1C/alpha1S II-III loop chimera, but none of the other single-loop chimeras, recovered a sigmoidal fluorescence-voltage curve indicative of skeletal-type EC coupling. To quantify Ca2+ transients in the absence of inward Ca2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of alpha1C. The Ca2+ transients expressed by the alpha1C(E736K)/alpha1S II-III loop chimera were approximately 70% smaller than those expressed by the Ca2+-conducting alpha1C/alpha1S II-III variant. The low skeletal-type EC coupling expressed by the alpha1C/alpha1S II-III loop chimera was confirmed in the Ca2+-conducting alpha1C/alpha1S II-III loop variant using Cd2+ (10(-4) M) as the Ca2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca2+ transients expressed by an alpha1C/alpha1S chimera carrying all tested skeletal alpha1S domains (all alpha1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type alpha1S, and did not change in the presence of the E736K mutation or in the presence of 10(-4) M Cd2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca2+ permeant alpha1S(E1014K) variant, the alpha1C(E736K)/alpha1S II-III loop chimera, and the alpha1C(E736K)/alpha1S chimera carrying all tested alpha1S domains. The data indicate that the functional recovery produced by the alpha1S II-III loop is incomplete and that multiple cytosolic domains of alpha1S are necessary for a quantitative recovery of the EC-coupling phenotype of skeletal myotubes. Thus, despite the importance of the II-III loop there may be other critical determinants in alpha1S that influence the efficiency of EC coupling.

Maurocalcine and domain A of the II-III loop of the dihydropyridine receptor Cav 1.1 subunit share common binding sites on the skeletal ryanodine receptor.

Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Cav1.1) subunit. This domain belongs to the II-III loop of Cav1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. Proof that peptide A and maurocalcine bind onto the same site is provided by competition experiments in which binding of fragment 7.2 to peptide A is inhibited by preincubation with maurocalcine. Moreover, when expressed in COS-7 cells, RyR1 carrying a deletion of fragment 7 shows a loss of interaction with both peptide A and maurocalcine. At the functional level, this deletion abolishes the maurocalcine induced stimulation of [3H]ryanodine binding onto microsomes of transfected COS-7 cells without affecting the caffeine and ATP responses.

Myocardial infarction after taking zolmitriptan.

We report the case of a patient with mild non-obstructive coronary artery disease who sustained an inferior wall myocardial infarction shortly after taking zolmitriptan as abortive therapy for migraine headaches. A Medline search was performed to review all reported cases of myocardial infarction related to migraine therapy with zolmitriptan and related medications. Zolmitriptan may cause myocardial infarction (MI) even in the absence of significant coronary artery disease.

Involvement of a heptad repeat in the carboxyl terminus of the dihydropyridine receptor beta1a subunit in the mechanism of excitation-contraction coupling in skeletal muscle.

Chimeras consisting of the homologous skeletal dihydropyridine receptor (DHPR) beta1a subunit and the heterologous cardiac/brain beta2a subunit were used to determine which regions of beta1a were responsible for the skeletal-type excitation-contraction (EC) coupling phenotype. Chimeras were transiently transfected in beta1 knockout myotubes and then voltage-clamped with simultaneous measurement of confocal fluo-4 fluorescence. All chimeras expressed a similar density of DHPR charge movements, indicating that the membrane density of DHPR voltage sensors was not a confounding factor in these studies. The data indicates that a beta1a-specific domain present in the carboxyl terminus, namely the D5 region comprising the last 47 residues (beta1a 478-524), is essential for expression of skeletal-type EC coupling. Furthermore, the location of beta1aD5 immediately downstream from conserved domain D4 is also critical. In contrast, chimeras in which beta1aD5 was swapped by the D5 region of beta2a expressed Ca(2+) transients triggered by the Ca(2+) current, or none at all. A hydrophobic heptad repeat is present in domain D5 of beta1a (L478, V485, V492). To determine the role of this motif, residues in the heptad repeat were mutated to alanines. The triple mutant beta1a(L478A/V485A/V492A) recovered weak skeletal-type EC coupling (DeltaF/F(max) = 0.4 +/- 0.1 vs. 2.7 +/- 0.5 for wild-type beta1a). However, a triple mutant with alanine substitutions at positions out of phase with the heptad repeat, beta1a(S481A/L488A/S495A), was normal (DeltaF/F(max) = 2.1 +/- 0.4). In summary, the presence of the beta1a-specific D5 domain, in its correct position after conserved domain D4, is essential for skeletal-type EC coupling. Furthermore, a heptad repeat in beta1aD5 controls the EC coupling activity. The carboxyl terminal heptad repeat of beta1a might be involved in protein-protein interactions with ryanodine receptor type 1 required for DHPR to ryanodine receptor type 1 signal transmission.

Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells.

Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mM MbetaCD for 1 h at 37 degrees C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca(2+) current amplitude was significantly reduced, and its voltage dependence was shifted approximately 15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca(2+) transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca(2+) current was not affected by MbetaCD treatment. Most of the L-type Ca(2+) conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca(2+) transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Functional equivalence of dihydropyridine receptor alpha1S and beta1a subunits in triggering excitation-contraction coupling in skeletal muscle.

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.

Functional equivalence of dihydropyridine receptor a1S and b1a subunits in triggering excitation-contraction coupling in skeletal muscle

Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in a1S and b1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the a1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the a1S/b1a pair.

Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes.

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.

Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes.

We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.

Ca2+ current and charge movements in skeletal myotubes promoted by the beta-subunit of the dihydropyridine receptor in the absence of ryanodine receptor type 1.

The beta-subunit of the dihydropyridine receptor (DHPR) enhances the Ca(2+) channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the beta-subunit in the expression of L-type Ca(2+) current and charge movements in RyR1 knock-out (KO), beta1 KO, and double beta1/RyR1 KO myotubes generated by mating heterozygous beta1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various beta-isoforms and then whole-cell voltage-clamped for measurements of Ca(2+) and gating currents. Overexpression of the endogenous skeletal beta1a isoform resulted in a low-density Ca(2+) current either in RyR1 KO (36 +/- 9 pS/pF) or in beta1/RyR1 KO (34 +/- 7 pS/pF) myotubes. However, the heterologous beta2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca(2+) current that was entirely wild-type in density in RyR1 KO (195 +/- 16 pS/pF) and was significantly enhanced in double beta1/RyR1 KO (115 +/- 18 pS/pF) myotubes. Other variants tested from the four beta gene families (beta1a, beta1b, beta1c, beta3, and beta4) were unable to enhance Ca(2+) current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in beta2a-expressing beta1a/RyR1 KO myotubes were significantly lower than in beta1a-expressing beta1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, beta2a had a preferential ability to recover Ca(2+) current, whereas beta1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (beta2a C3,4S) eliminated the RyR1-independent Ca(2+) current expression. Furthermore, Ca(2+) current enhancement was observed with a beta2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the beta2a variant to the myotube surface activated the DHPR Ca(2+) current and bypassed the requirement for RyR1. The data suggest that the Ca(2+) current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the beta1a-subunit is critically involved in process.

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels.

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.

Molecular characterization of a two-domain form of the neuronal voltage-gated P/Q-type calcium channel alpha(1)2.1 subunit.

We characterized the neuronal two-domain (95kD-alpha(1)2.1) form of the alpha(1)2.1 subunit of the voltage-gated calcium channels using genetic and molecular analysis. The 95kD-alpha(1)2.1 is absent in neuronal preparations from CACNA1A null mouse demonstrating that alpha(1)2.1 and 95kD-alpha(1)2.1 arise from the same gene. A recombinant two-domain form (alpha(1AI-II)) of alpha(1)2.1 associates with the beta subunit and is trafficked to the plasma membrane. Translocation of the alpha(1AI-II) to the plasma membrane requires association with the beta subunit, since a mutation in the alpha(1AI-II) that inhibits beta subunit association reduces membrane trafficking. Though the alpha(1AI-II) protein does not conduct any voltage-gated currents, we have previously shown that it generates a high density of non-linear charge movements [Ahern et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6935-6940]. In this study, we demonstrate that co-expression of the alpha(1AI-II) significantly reduces the current amplitude of alpha(1)2.1/beta(1a)/alpha(2)delta channels, via competition for the beta subunit. Taken together, our results demonstrate a dual functional role for the alpha(1AI-II) protein, both as a voltage sensor and modulator of P/Q-type currents in recombinant systems. These studies suggest an in vivo role for the 95kD-alpha(1)2.1 in altering synaptic activity via protein-protein interactions and/or regulation of P/Q-type currents.