Xuefei Sun

The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart

The present study examined the binding of the individual N‐ and C‐lobes of calmodulin (CaM) to Cav1.2 at different Ca2+ concentration ([Ca2+]) from ≈ free to 2 mM, and found that they may bind to Cav1.2 Ca2+‐dependently. In particular, using the patch‐clamp technique, we confirmed that the N‐ or C‐lobes can rescue the basal activity of Cav1.2 from run‐down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca2+], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca2+‐dependent regulation of Cav1.2.

Nonylphenol affects myocardial contractility and L-type Ca2+ channel currents in a non-monotonic manner via G protein-coupled receptor 30

Nonylphenol (NP) is one of the widely spread xenoestrogens (XEs) and is used in the production of industrial and consumer surfactants. In the present study, we examined the acute effects of NP on myocardial contractility to determine its rapid-response cardiac effects in the isolated heart. We also investigated the mechanism of action of NP by determining its effects on the L-type Ca(2+) channel (LTCC) currents (ICa-L) in ventricular myocytes. Lower concentrations (10(-12)-10(-10)M) of NP increased cardiac contractility while higher concentrations (10(-8)-10(-6)M) exhibited opposite effects. These apparently opposing effects suggest that NP has biphasic concentration-dependent rapid effects on cardiac contractility. These non-monotonic changes in contractility correlated with the effects of NP on ICa-L, indicating that ion channels, as rapidly responding membrane proteins, play a very important role in mediating the rapid-response effects of XEs. Further studies revealed that the G protein coupled receptor 30 (GPR30) mediates the effects of NP on LTCC at lower but not higher concentrations, implying that the differential involvement of GPR30 might be responsible for the non-monotonic effects of NP.

The Ca2+-dependent interaction of calpastatin domain L with the C-terminal tail of the Cav1.2 channel

To demonstrate the interaction of calpastatin (CS) domain L (CSL) with Cav1.2 channel, we investigated the binding of CSL with various C‐terminus‐derived peptides at ≈ free, 100 nM, 10 μM, and 1 mM Ca2+ by using the GST pull‐down assay method. Besides binding with the IQ motif, CSL was also found to bind with the PreIQ motif. With increasing [Ca2+], the affinity of the CSL–IQ interaction gradually decreased, and the affinity of the CSL–PreIQ binding gradually increased. The results suggest that CSL may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca2+‐dependent manners.

Reduced DHPRα1S and RyR1 expression levels are associated with diaphragm contractile dysfunction during sepsis

Sepsis often causes diaphragm contractile dysfunction. Dihydropyridine receptors (DHPRα1s and DHPRα1c) and ryanodine receptors (RyR1, RyR2, and RyR3) are essential for excitation–contraction coupling in striated muscles. However, their expression in diaphragm during sepsis have not been explored. Methods: Eight rats received endotoxin, and 8 more rats received placebo. After 24 hours, 3) diaphragm isometric contractile force was measured. The mRNA and protein levels of DHPRs and RyRs in diaphragm muscles were determined.Results: Sepsis weakened diaphragm contractile function. The expression levels of DHPRα1s and RyR1 were significantly lower in septic rats than in control rats. The expression levels of DHPRα1c and RyR3 were unaffected by sepsis. RyR2 was undetectable at both mRNA and protein levels in the control and sepsis groups. Conclusions: Weakened diaphragm contraction in the septic rats was associated with reduced mRNA and protein expression of DHPRα1s and RyR1, the isoforms of skeletal muscles. Muscle Nerve 48:745–751, 2013

Lobe-related concentration- and Ca2+-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca2+ channel CaV1.2 at different CaM and Ca2+ concentrations ([Ca2+]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca2+-dependencies. Our data show that more than one CaM molecule could bind to the CaV1.2 C-terminal tail at high [Ca2+]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca2+] in its binding to the C-terminal tail of CaV1.2, and the binding between CaM and the N-terminal tail of CaV1.2 requires high [Ca2+]. Our data provide new details on the interactions between CaM and the CaV1.2 channel.

Electrophysiological effect and the gating mechanism of astragaloside IV on l-type Ca2+ channels of guinea-pig ventricular myocytes

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca(2+) channel activity of single cardiomyocytes and single Ca(2+) channels. Whole-cell Ca(2+) currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca(2+) channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of L-type Ca(2+) currents (ICaL) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba(2+) current via L-type Ca(2+) channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba(2+) current at 0.1 or 1 µM AS-IV was accounted for a decrease in the channel activity (NPo). In addition to the decrease in NPo, there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of L-type Ca(2+) channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca(2+) channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of L-type Ca(2+) channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus.

Astragaloside IV Inhibits Membrane Ca2+ Current but Enhances Sarcoplasmic Reticulum Ca2+ Release

Astragaloside IV (AS-IV) is one of the active ingredients in Astragalus membrananceus (Huangqi), a traditional Chinese medicine. The present study investigated the effects of AS-IV on Ca[Formula: see text] handling in cardiac myocytes to elucidate its possible mechanism in the treatment of cardiac disease. The results showed that AS-IV at 1 and 10[Formula: see text][Formula: see text]M reduced KCl-induced [Ca[Formula: see text]]i increase ([Formula: see text] from 1.33[Formula: see text][Formula: see text][Formula: see text]0.04 (control, [Formula: see text] 28) to 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text], [Formula: see text] 29) and 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text] 0.01, [Formula: see text]), but it enhanced Ca[Formula: see text] release from SR ([Formula: see text] from 1.04[Formula: see text][Formula: see text][Formula: see text]0.01 (control, [Formula: see text]) to 1.44[Formula: see text][Formula: see text][Formula: see text]0.03 ([Formula: see text], [Formula: see text]) and 1.60[Formula: see text][Formula: see text][Formula: see text]0.04 ([Formula: see text] 0.01, [Formula: see text]0), in H9c2 cells. Similar results were obtained in native cardiomyocytes. AS-IV at 1 and 10[Formula: see text][Formula: see text]M inhibited L-type Ca[Formula: see text] current ([Formula: see text] from [Formula: see text]4.42[Formula: see text][Formula: see text][Formula: see text]0.58 pA/pF of control to [Formula: see text]2.25[Formula: see text][Formula: see text][Formula: see text]0.12 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) and [Formula: see text]1.78[Formula: see text][Formula: see text][Formula: see text]0.28 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) respectively, when the interference of [Ca[Formula: see text]]i was eliminated due to the depletion of SR Ca[Formula: see text] store by thapsigargin, an inhibitor of Ca[Formula: see text] ATPase. Moreover, when BAPTA, a rapid Ca[Formula: see text] chelator, was used, CDI (Ca[Formula: see text]-dependent inactivation) of [Formula: see text] was eliminated, and the inhibitory effects of AS-IV on ICaL were significantly reduced at the same time. These results suggest that AS-IV affects Ca[Formula: see text] homeostasis through two opposite pathways: inhibition of Ca[Formula: see text] influx through L-type Ca[Formula: see text] channel, and promotion of Ca[Formula: see text] release from SR.

Dynamic Alterations in the CaV1.2/CaM/CaMKII Signaling Pathway in the Left Ventricular Myocardium of Ischemic Rat Hearts

Cardiac L-type calcium channel (CaV1.2), calmodulin (CaM), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) form the CaV1.2/CaM/CaMKII signaling pathway, which plays an important role in maintaining intracellular Ca(2+) homeostasis. The roles of CaM and CaMKII in the regulation of CaV1.2 in Ca(2+)-dependent inactivation and facilitation have been reported; however, alterations in this signaling pathway in the heart after myocardial ischemia (MI) had not been well characterized. In this study, we investigated the dynamic changes in CaV1.2, CaM, and CaMKII mRNA and protein expression levels in the left ventricles of the heart following MI in rats. The MI model was induced by ligating the left anterior descending coronary artery; the rats were divided into the following five groups: the 6 h post-MI group (MI-6h), 24 h post-MI group (MI-24h), 1 week post-MI group (MI-1w), 2 weeks post-MI group (MI-2w), and the sham group. The mRNA levels were measured by quantitative real-time polymerase chain reaction and the protein expression was determined by western blotting and immunohistochemistry. There were no observable differences in the CaV1.2 mRNA and protein levels at the early stages of MI, but these levels decreased at MI-2w. Both the mRNA and protein levels of CaM increased at MI-6h, peaked at MI-24h, and then reduced to normal levels at MI-2w. CaMKII mRNA and protein levels decreased at MI-6h and reached their lowest level at MI-24h. Taken together, these data demonstrate that there are dynamic changes in the CaV1.2/CaM/CaMKII signaling pathway following MI injuries, which suggests that different therapeutic regimens should be used at different time points after MI injuries.

Evaluation of cardiac function at different time points after myocardial infarction of rats.

Results Compared with sham group, cardiac function did not change in 24h group after ligation, but the interventricular septum thickness at end-systole and interventricular septum thickness at end-diastole in groups of 1, 2, 4 weeks after operation were significantly decreased (P<0.05); the left ventricular internal dimension at end-systole in groups of 1, 4 weeks after MI and left ventricular internal dimension at end-diastole in 4w group were dramastically increased (P<0.05); the ejection fraction and left ventricular fraction shortening in groups of 1, 2, 4 weeks after MI declined significantly (P<0.05); in addition, the mitral valve E peak in 2w group was greatly reduced (P<0.05). These data suggested that both left ventricular systolic and diastolic function were affected 1 week after MI, with changes of cardiac sturcture. Moreover, TEM illustration showed that nuclear pyknosis, myofilament disruption, mitochondrial swelling were present in all MI groups and severe reconstruction and fibrosis were observed especially in 4w-MI group.

Decreased intracellular [Ca2+] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis

Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca2+]) and the distribution and co‐localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca2+] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis.