Differential regulation of C-type natriuretic peptide-induced cGMP and functional responses by PDE2 and PDE3 in failing myocardium

Recently, we showed C-type natriuretic peptide (CNP)-induced negative inotropic (NIR) and positive lusitropic response (LR) in failing rat heart. We wanted to study whether, and if so, how phosphodiesterases (PDEs) regulate CNP-induced cyclic 3′,5′-guanosine monophosphate (cGMP) elevation and functional responses. Inotropic and lusitropic responses were measured in left ventricular muscle strips and cyclic nucleotide levels, PDE activity and phospholamban (PLB) and troponin I (TnI) phosphorylation were measured in ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. CNP-mediated increase in global cGMP was mainly regulated by PDE2, as reflected by a marked amplification of the cGMP increase during PDE2 inhibition and by a high PDE2 activity in cardiomyocytes. PDE3 inhibition, on the other hand, caused no significant cGMP increase by CNP. The functional consequences did not correspond to the changes of cGMP. PDE3 inhibition increased the potency of the CNP-induced NIR and LR, while PDE2 inhibition desensitized the CNP-induced NIR, but not LR. A role for PDE2 on the maximal LR and PDE5 on the maximal NIR to CNP was revealed in the presence of PDE3 inhibition. CNP increased PLB phosphorylation about 25- to 30-fold and tended to increase TnI phosphorylation about twofold. As a whole, CNP-induced functional responses were only modestly regulated by PDEs compared to the cAMP-mediated functional responses to β₁-adrenoceptor stimulation, which are highly regulated by PDEs. There is a mismatch between the CNP-induced cGMP increase and functional responses. Global cGMP levels are mainly regulated by PDE2 after CNP stimulation, whereas the functional responses are modestly regulated by both PDE2 and PDE3, indicating cGMP compartmentation by PDEs affecting CNP-induced responses in failing hearts.     

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Background and Purpose

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca²⁺ cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity.

Experimental Approach

To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells.

Key Results

We showed that istaroxime enhances SERCA2a activity, Ca²⁺ uptake and the Ca²⁺-dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca²⁺ cycling.

Conclusions and Implications

Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca²⁺ uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.     

Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

BACKGROUND AND PURPOSE

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents.

EXPERIMENTAL APPROACH

Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture.

KEY RESULTS

Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose.

CONCLUSIONS AND IMPLICATIONS

NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.     

Differential regulation of β2 -adrenoceptor-mediated inotropic and lusitropic response by PDE3 and PDE4 in failing and non-failing rat cardiac ventricle

BACKGROUND AND PURPOSE

β-Adrenoceptors play a major role in regulating myocardial function through cAMP-dependent pathways. Different phosphodiesterases (PDEs) regulate intracellular cAMP-pools and thereby contribute to the compartmentalization of cAMP-dependent effects. We explored the involvement of PDEs in limiting the β₂ adrenoceptor-mediated positive inotropic (PIR) and lusitropic (LR) responses in sham-operated (Sham) and failing rat hearts.

EXPERIMENTAL APPROACH

Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Rats developing heart failure were studied 6 weeks after surgery. Contractility was measured in left ventricular strips from failing and Sham hearts. cAMP was quantified by RIA.

KEY RESULTS

In ventricular strips, stimulation of β₂-adrenoceptors with (-)-adrenaline (300 nM CGP20712A present) exerted a small PIR and LR. In Sham hearts, β₂-adrenoceptor-mediated as well as β₁-adrenoceptor-mediated PIR and LR were increased by selective inhibition of either PDE3 (1 µM cilostamide) or PDE4 (10 µM rolipram). In failing rat hearts, PDE3 inhibition enhanced PIR and LR to both β₁- and β₂-adrenoceptor stimulation while PDE4 inhibition had no effect on these responses despite a significant increase in cAMP levels. Combined PDE3/4 inhibition further enhanced the PIR and LR of β₂- and β₁-adrenoceptor activation both in Sham and failing hearts, compared with PDE3 inhibition alone. PDE4 enzyme activity was reduced in failing hearts.

CONCLUSIONS AND IMPLICATIONS

Both PDE3 and PDE4 attenuated β₂- and β₁-adrenoceptor-mediated contractile responses in Sham hearts. In failing hearts, these responses are attenuated solely by PDE3 and thus even selective PDE3 inhibitors may provide a profound enhancement of β-adrenoceptor-mediated responses in heart failure.     

SERCA2a stimulation by istaroxime: a novel mechanism of action with translational implications

Sarcoplasmic reticular (SR) Ca²⁺-ATPase (SERCA2a) is central to cardiac electrophysiological and mechanical function. It ensures full diastolic relaxation minimizing delayed after-potentials that would otherwise compromise membrane electrophysiological stability, and optimizes SR Ca²⁺ refilling and systolic contraction. Previous studies demonstrated that the small molecule agent istaroxime stimulates SERCA2a-ATPase activity, restoring its function in failing hearts, and enhancing indices of mechanical, and SR Ca²⁺ release and re-uptake, activity. Ferrandi et al (2013) now elegantly demonstrate its ability to dissociate the phospholamdan (PB) bound to cardiac SERCA2a, thereby removing the inhibitory effect of PB on SERCA2a. This effect was independent of the cAMP/PKA system and modified a specific SERCA2a reaction step. They used SERCA-enriched SR preparations from a rigorously validated and realistic physiological, canine model of cardiac failure with established Na⁺-K⁺-ATPase sensitivity to cardiac glycosides and SR Ca²⁺ handling features. These findings potentially translate into a novel management of the major and increasingly important public health challenge of chronic cardiac failure.

Linked Article

This article is a commentary on Ferrandi et al., pp. 1849–1861 of volume 169 issue 8. To view this paper visit http://dx.doi.org/10.1111/bph.12278.     

Amplified NO/cGMP-mediated relaxation and ryanodine receptor-to-BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice

BACKGROUND AND PURPOSE

Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK_Ca) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK_Ca activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ release from the SR via ryanodine receptors (RyRs) is important for BK_Ca channel activation in response to NO/cGMP.

EXPERIMENTAL APPROACH

In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB^−/−) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS).

KEY RESULTS

In CCSM strips from PLB^−/− mice, a model of increased SR Ca²⁺ load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB^−/− strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB^−/−, as demonstrated by a shift in the concentration–response curve towards lower concentrations. Blocking RyRs and BK_Ca channels diminished the induced relaxations and eliminated the difference between wild-type and PLB^−/−.

CONCLUSIONS AND IMPLICATIONS

NO/cGMP activates BK_Ca channels through RyR-mediated Ca²⁺ release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca²⁺ concentrations.     

Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation

Background and purpose:

Muscarinic stimulation increases myofilament Ca²⁺ sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca²⁺ sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC).

Experimental approach:

Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified.

Key results:

Carbachol (10 µmol·L⁻¹) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 µmol·L⁻¹ isoproterenol (20 ± 1.5% and 56 ± 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M₂ receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment.

Conclusion and implications:

In failing ventricular muscle, muscarinic receptor activation, most likely via M₂ receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca²⁺ sensitivity. Enhancement of myofilament Ca²⁺ sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

cAMP- and Ca²(+) /calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

BACKGROUND AND PURPOSE

Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms.

EXPERIMENTAL APPROACH

Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca²⁺]_i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16.

KEY RESULTS

Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC₅₀: 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF₂ receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca²⁺]_i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca²⁺]_i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca²⁺]_i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93.

CONCLUSIONS AND IMPLICATIONS

Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF₂ receptors in a cAMP/PKA- and Ca²⁺/CaMKII-dependent manner. This enhancement was accompanied by Ca²⁺-dependent arrhythmogenic effects mediated by PKA and CaMKII.     

Characterization of the mechanisms of the increase in PPARδ expression induced by digoxin in the heart using the H9c2 cell line

BACKGROUND AND PURPOSE

Digoxin has been used as an inotropic agent in heart failure for a long time. Troponin I (TnI) phosphorylation is related to cardiac contractility, and the genes are regulated by peroxisome proliferator-activated receptors (PPARs). Our previous studies indicated that cardiac abnormality related to the depressed expression of PPARδ in the hearts of STZ rats is reversed by digoxin. However, the cellular mechanisms for this effect of digoxin have not been elucidated. The aim of the present study was to investigate possible mechanisms for this effect of digoxin using the H9c2 cell line cultured in high glucose (HG) conditions.

METHODS

The effects of digoxin on PPARδ expression, intracellular calcium and TnI phosphorylation were investigated in cultured H9c2 cells, maintained in a HG medium, by using Western blot analysis.

RESULTS

Digoxin increased PPARδ expression in H9c2 cells subjected to HG conditions, and increase the intracellular calcium concentration. This effect of digoxin was blocked by BAPTA-AM at concentrations sufficient to chelate calcium ions. In addition, the calcineurin inhibitor cyclosporine A and KN93, an inhibitor of calcium/calmodulin-dependent protein kinase, inhibited this action. Digoxin also increased TnI phosphorylation and this was inhibited when PPARδ was silenced by the addition of RNAi to the cells. Similar changes were observed on the contraction of H9c2 cells.

CONCLUSION

The results suggest that digoxin appears, through calcium-triggered signals, to reverse the reduced expression of PPARδ in H9c2 cells caused by HG treatment.     

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.     

Protective action of doxycycline against diabetic cardiomyopathy in rats

Background and purpose:

Reactive oxygen and nitrogen species play an important role in the development of diabetic cardiomyopathy. They can activate matrix metalloproteinases (MMPs), and MMP-2 in particular is known to mediate early consequences of oxidative stress injury in the heart. Therefore, we investigated the role of MMP-2 and the effect of the MMP inhibitor doxycycline on the changes of heart function caused by diabetes.

Experimental approach:

Using streptozotocin-induced diabetic rats, we evaluated the effect of doxycycline on both mechanical and electrical function of isolated hearts, papillary muscle and cardiomyocytes.

Key results:

Doxycycline abolished the diabetes-induced depression in left ventricular developed pressure and the rates of changes in developed pressure in isolated hearts and normalized the prolongation of the action potential in papillary muscles. In cardiomyocytes isolated from doxycycline-treated diabetic rats, the altered kinetic parameters of Ca²⁺ transients, depressed Ca²⁺ loading of sarcoplasmic reticulum and basal intracellular Ca²⁺ level, and the spatio-temporal properties of Ca²⁺ sparks were significantly restored. Gelatin zymography and western blot data indicated that the diabetes-induced alterations in MMP-2 activity and protein level, level of tissue inhibitor of matrix metalloproteinase-4 and loss of troponin I were restored to control levels with doxycycline.

Conclusions and implications:

Our data suggest that these beneficial effects of doxycycline on the mechanical, electrical and biochemical properties of the diabetic rat heart appear, at least in part, to be related to inhibition of MMP activity, implying a role for MMPs in the development of diabetic cardiomyopathy.     

Ca2+ signalling by P2Y receptors in cultured rat aortic smooth muscle cells

Background and purpose:

P2Y receptors evoke Ca²⁺ signals in vascular smooth muscle cells and regulate contraction and proliferation, but the roles of the different P2Y receptor subtypes are incompletely resolved.

Experimental approach:

Quantitative PCR was used to define expression of mRNA encoding P2Y receptor subtypes in freshly isolated and cultured rat aortic smooth muscle cells (ASMC). Fluorescent indicators in combination with selective ligands were used to measure the changes in cytosolic free [Ca²⁺] in cultured ASMC evoked by each P2Y receptor subtype.

Key results:

The mRNA for all rat P2Y receptor subtypes are expressed at various levels in cultured ASMC. Four P2Y receptor subtypes (P2Y1, P2Y2, P2Y4 and P2Y6) evoke Ca²⁺ signals that require activation of phospholipase C and comprise both release of Ca²⁺ from stores and Ca²⁺ entry across the plasma membrane.

Conclusions and implications:

Combining analysis of P2Y receptor expression with functional analyses using selective agonists and antagonists, we isolated the Ca²⁺ signals evoked in ASMC by activation of P2Y1, P2Y2, P2Y4 and P2Y6 receptors.     

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity

Background and purpose:

Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na⁺ channel. However, long-term effects of bepridil on the Na⁺ channel remain unclear. We explored the long-term effect of bepridil on the Na⁺ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na_v1.5 channel.

Experimental approach:

Na⁺ currents were recorded by whole-cell voltage-clamp technique. Na⁺ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis.

Key results:

Treatment of cardiomyocytes with 10 µmol·L⁻¹ bepridil for 24 h augmented Na⁺ channel current (I_Na) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca²⁺/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I_Na increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na_v1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na_v1.5 were not modified. Bepridil and W-7 did not increase I_Na any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner.

Conclusions and implications:

We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na_v1.5 α-subunit, which in turn increased Na⁺ current.     

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

BACKGROUND AND PURPOSE

Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K⁺ (mitoK_ATP) 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.

EXPERIMENTAL APPROACH

PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca²⁺ signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the α_1c subunit of L-type channels in the cellular membrane were measured by Western blot.

KEY RESULTS

PPC was accompanied by a 50% reduction in α_1c subunit levels, and by a reversible fall in L-type current amplitude and Ca²⁺ transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD). PPC signficantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the α_1c subunit and Ca²⁺ channel function were prevented in part by the protease inhibitor leupeptin.

CONCLUSIONS AND IMPLICATIONS

PPC downregulated the α_1c subunit, possibly through ROS. Downregulation involved increased degradation of the Ca²⁺ channel, which in turn reduced Ca²⁺ influx, which may attenuate Ca²⁺ overload during reperfusion.     

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose:

Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery.

Experimental approach:

The cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery.

Key results:

Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR₁) antagonist, while it was mimicked by PAR₁-activating peptide (PAR₁AP), but not PAR₄AP. The thrombin-induced contraction was associated with a small elevation of [Ca²⁺]_i and an increase in MLC20 phosphorylation. Thrombin and PAR₁AP induced a greater increase in tension for a given [Ca²⁺]_i elevation than that obtained with high K⁺-depolarization. They also induced a contraction at a fixed Ca²⁺ concentration in α-toxin-permeabilized preparations.

Conclusions and implications:

The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR₁ and thereby increasing the sensitivity of the myofilament to Ca²⁺. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.