Age-related regulation of excitation–contraction coupling in rat heart

Hearts from subjects with different ages have different Ca²⁺ signaling. Release of Ca²⁺ from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca²⁺ releases, Ca²⁺ transients and Ca²⁺ sparks, demonstrate the main events of excitation–contraction coupling (ECC). Global increase in free Ca²⁺ concentration ([Ca²⁺] _i ) consists of summation of Ca²⁺ release events in cardiomyocytes. Since the Ca²⁺ flux induced by Ca²⁺ sparks reports a summation of ryanodine-sensitive Ca²⁺ release channels (RyR2s)’s behavior in a spark cluster, evaluation of the properties of Ca²⁺ sparks and Ca²⁺ transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca²⁺] _i and Ca²⁺ sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca²⁺ sparks and Ca²⁺ transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca²⁺ current density and decreased SR Ca²⁺ load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca²⁺ signaling including hyperphosphorylation of RyR2s and higher basal [Ca²⁺] _i together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes.     

The excitation–contraction coupling mechanism in skeletal muscle

First coined by Alexander Sandow in 1952, the term excitation–contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca²⁺ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca²⁺ from the SR and transient increase of Ca²⁺ concentration in the myoplasm, (6) activation of the myoplasmic Ca²⁺ buffering system and the contractile apparatus, followed by (7) Ca²⁺ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca²⁺ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na⁺/Ca²⁺ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca²⁺ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca²⁺ sparks.     

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation–contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation–contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10–11 months following 2–3 months of treadmill exercise training. Shortening, [Ca²⁺]_i and L-type Ca²⁺ current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca²⁺ transients and L-type Ca²⁺ current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca²⁺ transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.     

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is 10-fold below the EC₅₀ values for most purinergic receptors expressed in the heart (1.5–20 µM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP. cardiomyocytes; luciferase     

Muscle fibers from senescent mice retain excitation–contraction coupling properties in culture

In the present study, we test the hypothesis that mouse skeletal muscle in culture retains the fundamental properties of excitation-sarcoplasmic reticulum Ca²⁺ release coupling reported for young-adult (3–4 mo) and senescent (22–23) mice. Dissociated flexor digitorum brevis (FDB) muscles from young-adult and senescent mice were cultured for 7 d in a serum-free medium. During this period, the overall morphology of cultured fibers resembled that exhibited by acutely dissociated cells. In addition, survival analysis revealed that more than 70% of the fibers from both young and old mice remained suitable for electrophysiological studies during this same culture period. Charge movement and intracellular Ca²⁺ recordings in FDB fibers, voltage clamped in the whole cell configuration of the patch-clamp technique, reproduced the maximal values, and voltage dependence similarly displayed by acutely dissociated cells for both parameters in young-adult and senescent mice. The analysis of the dihydropyridine receptor by immunoblots confirmed, in the culture system, the age-dependent decrease in the expression of this protein. In conclusion, FDB fibers from young-adult and old mice retain the excitation–contraction coupling phenotype during the course of a week in serum-free medium culture.     

Effects of inserting fluorescent proteins into the α1S II–III loop: insights into excitation–contraction coupling

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca²⁺ release via RYR1, and retrograde coupling is manifested by increased L-type Ca²⁺ current via DHPR. A critical domain (residues 720–765) of the DHPR α_1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the α_1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca²⁺ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the α_1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the α_1S II–III loop. Significantly, our results indicate that the conserved portion of the α_1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1.     

The phenomenological model of muscle contraction with a controller to simulate the excitation–contraction (E–C) coupling

We previously proposed a systematic motor model for muscle with two parallel Maxwell elements and a force generator P. The motor model showed the non-linear behavior of a muscle, such as the force–velocity relation and the force depression and enhancement, by using weight functions. Our newly proposed muscle model is based on the molecular mechanism of myosin cross-bridges. We assume that each parallel Maxwell element represents the mechanical properties of weak and strong binding of the myosin head to actin. Furthermore, we introduce a controller to simulate the excitation–contraction coupling of the muscle. The new muscle model satisfies all the properties obtained in our previous model and reduces the wasted energy of the viscous component to less than 5% of the total energy. The controller enables us to simulate contractions of slow and fast twitch muscles, which are driven by an artificial action potential or a processing electromyography signal despite their same mechanical components. The maximum velocities are calculated to be 3.4L₀ m/s for the fast twitch muscle model and 2.5L₀ m/s for the slow twitch muscle model, where L₀ is the initial length of the muscle model.     

PKCε inhibits the hyperglycemia-induced apoptosis signal in adult rat ventricular myocytes

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC _I/_II, and ) or activate (PKC). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC _I/_II and blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKC (V1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKC translocation activator (RACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKC in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKC protects ARVM from the hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)     

Effects of micromolar concentrations of manganese, copper, and zinc on α1-adrenoceptor-mediating contraction in rat aorta

To determine the influences of the Mn, Cu, and Zn on α₁-adrenoceptor (AR)-mediated vasoconstriction, we investigated their effects on vasoconstriction produced by the α₁-AR agonist phenylephrine in isolated rings of rat thoracic aorta. The cumulative concentration-contraction curves for phenylephrine were obtained in the absence and presence of Mn (0.3, 1, 3 μM), Cu (1, 10, 16 μM), and Zn (0.3, 1, 10 μM). Mn, Cu, and Zn each inhibited phenylephrine-mediated contraction in a dose-dependent manner. The maximal phenylephrine-induced contraction was significantly reduced by the pretreatment of the arterial rings with 10 and 16 μM Cu (p<0.05). The results suggest that variations in the plasma concentrations of metal might lead to changes in α₁-AR-mediated constrictive response.     

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Effects of β-phenylethylamine on polyphosphoinositide turnover in rat cerebral cortex

The effects of the trace amine, -phenylethylamine, on the hydrolysis of inositol phospholipids in rat cerebral cortical slices was studied using a direct assay involving prelabeling with [³H]inositol and then examining the production of [³H]inositol phosphates in the presence of lithium. Phenylethylamine exhibited two different effects. Millimolar concentrations of phenylethylamine stimulated the production of [³H]inositol phosphates to about 200% of control, while much smaller concentrations (micromolar) inhibited noradrenaline(NE)-stimulated [³H]inositol phosphate formation dose-dependently. The ₁-antagonist, prazosin, inhibited the increases in [³H]polyphosphoinositide turnover stimulated by phenylethylamine and by NE, though it inhibited phenylethylamine to a lesser extent than NE. It appears, therefore, that phenylethylamine affects [³H]inositol phosphate formation by acting as a partial ₁-agonist.     

Effects of endogenous serum neuregulin-1β on morbidity and mortality in patients with heart failure and left ventricular systolic dysfunction

Abstract

Context: Improved left ventricular ejection fraction (LVEF) following administration of recombinant human Neuregulin-1β (NRG), epidermal growth factor (EGF) involved in cardiomyocyte repair/survival, has been observed in patients with systolic heart failure (HF).

Methods: Serum NRG was measured by ELISA in 248 patients with NYHA class I–IV HF.

Results: NRG exhibited a marginally significant effect on LVEF trajectory over 11?months (p?=?0.07). There is no apparent level of NRG that predicts improved survival.

Conclusions: There is a potential relationship between serum NRG and improved LVEF, indicating the need to investigate the utility of NRG in predicting HF outcomes, including LVEF maintenance.     

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of β-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or α-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [β-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ from 29 to 16 to 10 mM, entry $\text{V}$ being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external β-hydroxybutyrate enhanced efflux slightly. At high external pH, external β-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal β-hydroxybutyrate (βHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled βHB^?/OH^? exchange, βHB^? and OH^? competing for influx, C : OH^? moving faster than C : βHB^?, empty C being immobile. They are also compatible with coupled βHB^?/H⁺ copermeation, empty C moving inward faster than H⁺ : C : βHB^?, H⁺ : C being immobile, and C : βHB^? (without H⁺) being so unstable as not to be formed in significant amounts (relative to C, H⁺ : C, and H⁺ : C : βHB^?).     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Effects of Pyrazinamide on Tryptophan–Niacin Conversion in Rats

In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC₅₀ values of 9.1 μM and 23.4 μM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. α-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.     

Influence of pH on Ca²⁺ current and its control of electrical and Ca²⁺ signaling in ventricular myocytes

Modulation of L-type Ca(2+) current (I(Ca,L)) by H(+) ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of I(Ca,L) was investigated (whole cell voltage clamp) while measuring intracellular Ca(2+) (Ca(2+)(i)) or pH(i) (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pH(o) 6.5 and pH(i) 6.7) had opposite effects on I(Ca,L) gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pH(o), this decreased I(Ca,L), whereas at low pH(i), it increased I(Ca,L) at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca(2+)(i) was buffered with BAPTA, the stimulatory effect of low pH(i) was even more marked, with essentially no inhibition. We conclude that extracellular H(+) ions inhibit whereas intracellular H(+) ions can stimulate I(Ca,L). Low pH(i) and pH(o) effects on I(Ca,L) were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H(+) ion screening of fixed negative charge at the sarcolemma, with additional channel block by H(+)(o) and Ca(2+)(i). Action potential duration (APD) was also strongly H(+) sensitive, being shortened by low pH(o), but lengthened by low pH(i), caused mainly by H(+)-induced changes in late Ca(2+) entry through the L-type Ca(2+) channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca(2+) signaling. We conclude that the pH(i)-versus-pH(o) control of I(Ca,L) will exert a major influence on electrical and Ca(2+)-dependent signaling during acid-base disturbances in the heart.     

Effects of ring contraction on the conformational preferences of α-substituted proline analogs

The structural consequences derived from the incorporation of either a methyl or a phenyl group at the α carbon of proline were recently investigated by quantum mechanical calculations (J Org Chem 2008, 73, 3418). In this work, the effect produced by contraction of the pyrrolidine ring on such α-substituted proline analogs has been explored using the same computational methods. Specifically, the intrinsic conformational preferences of the N-acetyl-N'-methylamide derivatives of the lower proline homolog L-azetidine-2-carboxylic acid (Aze), characterized by a four- instead of a five-membered ring, and its α-methyl (αMeAze) and α-phenyl (αPhAze) derivatives have been determined using quantum mechanical calculations and compared to those observed before for the proline counterparts. Replacement of the pyrrolidine ring by an azetidine cycle leads to a reduction of the conformational flexibility, especially for the Aze and αMeAze derivatives, which should be attributed to the quasi-planar geometry of the four-membered ring. Furthermore, the azetidine nitrogen shows pyramidalization, which depending on the peptide backbone conformation favors the formation of an attractive N-H···N interaction or alleviates a severe steric hindrance. Calculations on different environments predict that the tendency of αMeAze to adopt γ-turns is higher than that of unsubstituted Aze and α-methylproline, this feature being in full agreement with the experimental observations available.