Jorge A. Sánchez

Mitochondria regulate inactivation of L‐type Ca2+ channels in rat heart

1 L‐type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage‐dependent and a Ca2+‐dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague‐Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m‐chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2 RR and CCCP also caused a significant delay in the recovery from inactivation of L‐type Ca2+ currents (ICa). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L‐type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+‐dependent inactivation of L‐type Ca2+ channels. 3 We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L‐type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L‐type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro‐domain near L‐type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.

Characterization of the interaction of domain III of the envelope protein of dengue virus with putative receptors from CHO cells

Domain III (DIII) of the envelope protein of dengue virus (DENV) contains structural determinants for the interaction with cellular receptors. In the present study a solid phase assay and recombinant fusion proteins containing DENV-DIII of serotypes 1 and 2 were used to study structural features of the interaction of the envelope protein with putative receptors present in the microsomal fraction of CHO cells. Recombinant fusion proteins showed specific interaction with proteins present in the microsomal fraction. Binding of the fusion proteins across the pH range of 5.5-8.0 resembled that of virus particles, peaking at pH 6.0. This suggests that the interaction of DIII with cell receptor(s) is strengthened at endosomal pH. The effect of reduction and carbamidomethylation of cysteine residues on the binding to the microsomal fraction and in their recognition by antibodies suggests that the region of DIII that is interacting with putative receptor(s) overlaps only partially with a dominant epitope of the antibody response. The analysis of the residue conservation profile indicates that the surface of DIII is composed typically of specific sub-complex residues with an increased representation of specific type/subtype residues found at the surface that closely correlates with the dominant neutralizing epitope.

Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP‐sensitive K+ (mitoKATP) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L‐type Ca2+ channels.

Muscle and motor-skill dysfunction in a K+ channel-deficient mouse are not due to altered muscle excitability or fiber type but depend on the genetic background.

Abstract. The voltage-gated K+ channel Kv3.1 is expressed in skeletal muscle and in GABAergic interneurons in the central nervous system. Hence, the absence of Kv3.1 K+ channels may lead to a phenotype of myogenic or neurogenic origin, or both. Kv3.1-deficient (Kv3.1–/–) 129/Sv mice display altered contractile properties of their skeletal muscles and show poor performance on a rotating rod. In contrast, Kv3.1–/– mice on the (129/Sv×C57BL/6)F1 background display normal muscle properties and perform like wild-type mice. The correlation of poor performance on the rotating rod with altered muscle properties supports the notion that the skeletal muscle dysfunction in Kv3.1–/– 129/Sv mice may be responsible for the impaired motor skills on the rotating rod. Surprisingly, we did not find major differences between wild-type and Kv3.1–/– 129/Sv skeletal muscles in either the resting or action potential, the delayed-rectifier potassium conductance (gK) or the distribution of fast and slow muscle fibers. These findings suggest that the Kv3.1 K+ channel may not play a major role in the intrinsic excitability of skeletal muscle fibers although its absence leads to slower contraction and relaxation and to smaller forces in muscles of 129/Sv Kv3.1–/– mice.

Regulation of Muscle Cav1.1 Channels by Long-term Depolarization Involves Proteolysis of the α1s Subunit

The effects of long-term depolarization on frog skeletal muscle Cav1.1 channels were assessed. Voltage-clamp and Western-blot experiments revealed that long-term depolarization brings about a drastic reduction in the amplitude of currents flowing through Cav1.1 channels and in the levels of the α1s subunit, the main subunit of muscle L-type channels. The decline of both phenomena was prevented by the action of the protease inhibitors E64 (50 μM) and leupeptin (50 μM). In contrast, long-term depolarization had no effect on β1, the auxiliary subunit of α1s. The levels of mRNAs coding the α1s and the β1 subunits were measured by RNase protection assays. Neither the content of the α1s nor the β1 subunit mRNAs were affected by long-term depolarization, indicating that the synthesis of Cav1.1 channels remained unaffected. Taken together, our experiments suggest that the reduction in the amplitude of membrane currents and in the α1s subunit levels is caused by increased degradation of this subunit by a Ca2+-dependent protease.

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle

BACKGROUND delta-Sarcoglycan (delta-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca(2+) permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease. METHODS Isolated skeletal muscle fibers of wild-type and delta-SG KO mice were used to investigate whether the absence of delta-SG alters the increase in intracellular Ca(2+) during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca(2+) transient recording techniques with fluorescent indicators were used. RESULTS Ca(2+) transients during single twitches and tetani generated by muscle fibers of delta-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca(2+) and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a delta-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles. CONCLUSIONS delta-SG has a role in calcium homeostasis in skeletal muscle fibers. GENERAL SIGNIFICANCE These results support a possible role of delta-SG on calcium homeostasis. The alterations caused by the absence of delta-SG may be related to the pathogenesis of muscular dystrophy.

Extracellular heparin inhibits Ca2+ transients and contraction in mammalian cardiac myocytes.

The effect of heparin on Ca2+ transients and cell shortening was studied in isolated cardiac myocytes from rat and guinea-pig ventricles. Ca2+ signals were measured with the fluorescent indicator fura-2. Heparin reversibly decreased Ca2+ transients and cell shortening in a dose-dependent manner. Half and complete blockade were obtained with 50 μg/ml and 200 μg/ml heparin, respectively. The dihydropyridine agonist BAY K 8644 (50 nM) antagonized the effects of heparin. However, Ca2+ release elicited by caffeine (10 mM) was not affected by heparin. The actions of heparin were also studied in multicellular preparations. In papillary muscle, heparin (5 mg/ml) reversibly reduced the amplitude of the plateau of the action potential and the associated peak tension. BAY K 8644 (500 nM) also antagonized these effects. It is proposed that heparin interacts with dihydropyridine-sensitive Ca2+ channels to cause a decrease of Ca2+ transients and contractility in heart.

An embryonic heart cell line is susceptible to dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

The gamma subunit runs in the family

Ion channels are ubiquitous membrane proteins that play fundamental roles in cell physiology such as muscle contraction, secretion, excitability and gene expression, among others. Ion channels that are permeant to Ca2+ are especially relevant to cells where this cation acts as a second messenger. In skeletal muscle, the Cav1.1 channel, a key element in excitation–contraction coupling, is composed of four subunits, α1, β, α2δ and γ (Arikkath & Campbell, 2003). The γ subunit was initially described as a major component of the muscle channel complex (Curtis & Catterall, 1984) and while heart muscle and other excitable cells express their α1, β and α2δ subunit counterparts, the γ subunit appeared for many years to be uniquely expressed in skeletal muscle (Jay et al. 1990) and its role on channel function remained largely unknown. This view changed dramatically when it was found that the stargazer mouse, a mutant mouse with epileptic seizures, is deficient in stargazin, a brain protein that is similar to the muscle γ subunit and whose absence leads to alterations in neuronal Ca2+ channel function. When stargazin is expressed in model systems, Ca2+ channel inactivation is accentuated and channel availability is significantly reduced (Letts et al. 1998). In skeletal muscle, inactivation of Cav1.1 channels is shifted to more positive potentials when the γ subunit is absent (Freise et al. 2000). In both cases, the role of the skeletal muscle γ1 subunit and the role of the neuronal γ2 subunit are to avoid an inappropriate Ca2+ entry. To these two family members of γ subunits, other members, γ3, γ4 and γ5, were added using conserved structural criteria (Burgess et al. 1999) and more recently, a cluster of three new γ genes (γ6, γ7 and γ8) was described and it was found that the profile of gene expression of these eight γ subunits is wider than previously thought (Burgess et al. 2001). Soon, new roles for members of the γ family emerged such as trafficking of ligand-gated ion channels to postsynaptic sites (Chu et al. 2001). More recently, a role of γ6 subunit as a potential regulator of the low-threshold cardiac Ca2+ channel Cav3.1 was characterized. It was found that γ6 has the unique ability to decrease current density by more than half without changing the voltage dependence of channel inactivation or the amount of total Cav3.1 protein (Hansen et al. 2004). It is unclear what makes γ6 so unique in the sense that it is the only γ subunit known to modulate the low-voltage-activated Ca2+ channel in atria, as is whether or not this subunit actually forms a physical association with the Cav3.1 channel. If it does, what is the nature of this interaction? In this issue of The Journal of Physiology, Lin et al. (2008) identify a domain in γ6 that is responsible for the inhibition of Cav3.1 currents. Furthermore, they describe a unique GxxxA motif within this domain that is required for the function of the γ6 subunit, pointing to helix–helix interactions and demonstrate physical interactions of this subunit with the channels. The γ6 subunit is highly selective on its inhibitory action, with no effects on the high-voltage-activated Ca2+ channel that is also expressed in heart muscle. This implies a fine modulation of Ca2+ entry in atria. Is this also the case in other tissues? Are there other unsuspected functions for the γ6 subunit? For example, the expression of low-voltage-activated Ca2+ channels in adult skeletal muscle is practically non-existent; it is the high-voltage-activated Cav1.1 channel that is the predominant one, and yet γ6 is expressed in this tissue.