A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling

Recent perspectives on sinoatrial nodal cell (SANC)^? function indicate that spontaneous sarcoplasmic reticulum (SR) Ca²⁺ cycling, i.e. an intracellular “Ca²⁺ clock,” driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules (“surface membrane clock”) to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca²⁺ signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca²⁺ cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca²⁺ releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca²⁺]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca²⁺ cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca²⁺ cycling is disrupted. Thus, AC-cAMP-PKA-Ca²⁺ signaling cascade is a major mechanism of normal automaticity in mouse SANC.     

New drugs vs. old concepts: a fresh look at antiarrhythmics

Common arrhythmias, particularly atrial fibrillation (AF) and ventricular tachycardia/fibrillation (VT/VF) are a major public health concern. Classic antiarrhythmic (AA) drugs for AF are of limited effectiveness, and pose the risk of life-threatening VT/VF. For VT/VF, implantable cardiac defibrillators appear to be the unique, yet unsatisfactory, solution. Very few AA drugs have been successful in the last few decades, due to safety concerns or limited benefits in comparison to existing therapy. The Vaughan-Williams classification (one drug for one molecular target) appears too restrictive in light of current knowledge of molecular and cellular mechanisms. New AA drugs such as atrial-specific and/or multichannel blockers, upstream therapy and anti-remodeling drugs, are emerging. We focus on the cellular mechanisms related to abnormal Na⁺ and Ca²⁺ handling in AF, heart failure, and inherited arrhythmias, and on novel strategies aimed at normalizing ionic homeostasis. Drugs that prevent excessive Na⁺ entry (ranolazine) and aberrant diastolic Ca²⁺ release via the ryanodine receptor RyR2 (rycals, dantrolene, and flecainide) exhibit very interesting antiarrhythmic properties. These drugs act by normalizing, rather than blocking, channel activity. Ranolazine preferentially blocks abnormal persistent (vs. normal peak) Na⁺ currents, with minimal effects on normal channel function (cell excitability, and conduction). A similar “normalization” concept also applies to RyR2 stabilizers, which only prevent aberrant opening and diastolic Ca²⁺ leakage in diseased tissues, with no effect on normal function during systole. The different mechanisms of action of AA drugs may increase the therapeutic options available for the safe treatment of arrhythmias in a wide variety of pathophysiological situations.     

The NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na⁺/Ca²⁺ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na⁺ and Ca²⁺ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke.     

Contribution of Na(+)/Ca(2+) exchange to excessive Ca(2+) loading in dendrites and somata of CA1 neurons in acute slice

Multiple Ca(2+) entry routes have been implicated in excitotoxic Ca(2+) loading in neurons and reverse-operation of sodium-calcium exchangers (NCX) has been shown to contribute under conditions where intracellular Na(+) levels are enhanced. We have investigated effects of KB-R7943, an inhibitor of reverse-operation NCX activity, on Ca(2+) elevations in single CA1 neurons in acute hippocampal slices. KB-R7943 had no significant effect on input resistance, action potential waveform, or action potential frequency adaptation, but reduced L-type Ca(2+) entry in somata. Nimodipine was therefore included in subsequent experiments to prevent complication from effects of L-type influx on evaluation of NCX activity. NMDA produced transient primary Ca(2+) increases, followed by propagating secondary Ca(2+) increases that initiated in apical dendrites. KB-R7943 had no significant effect on primary or secondary Ca(2+) increases generated by NMDA. The Na(+)/K(+) ATPase inhibitor ouabain (30 microM) produced degenerative Ca(2+) overload that was initiated in basal dendrites. KB-R7943 significantly reduced initial Ca(2+) increases and delayed the propagation of degenerative Ca(2+) loads triggered by ouabain, raising the possibility that excessive intracellular Na(+) loading can trigger reverse-operation NCX activity. A combination of NMDA and ouabain produced more rapid Ca(2+) overload, that was contributed to by NCX activity. These results suggest that degenerative Ca(2+) signaling can be triggered by NMDA in dendrites, before intracellular Na(+) levels become sufficient to reverse NCX activity. However, since Na(+)/K(+) ATPase inhibition does appear to produce significant reverse-operation NCX activity, this additional Ca(2+) influx pathway may operate in ATP-deprived CA1 neurons and play a role in ischemic neurodegeneration.     

Effect of Low-Intensity Pulsed Ultrasound on the Expression of Calcium Ion Transport-Related Proteins during Tertiary Dentin Formation

Low-intensity pulsed ultrasound (LIPUS) is known for its positive effect on bone healing and reparative regeneration. This study investigated whether LIPUS affects reparative progression of the tooth and the expression of calcium ion transport-related proteins in odontoblasts and dental pulp cells using a rat dentin–pulp complex injury model. Forty male adult Sprague-Dawley rats underwent cavity preparation in the right maxillary first molar: 20 received LIPUS irradiation on the cavity-prepared tooth; 20 received LIPUS irradiation on the left maxillary first molar. Rats were randomly allocated into four groups: blank control group, LIPUS group, cavity-prepared group, cavity-prepared?+?LIPUS group. LIPUS irradiation (frequency: 1.5?MHz, 200-µs pulse width, 1-kHz pulse repetition frequency, 30 mW/cm² spatial averaged temporal averaged intensity) was administered individually for 20?min daily. Rats were sacrificed 1, 3, 7 and 14 d post-operation. The histopathological and cellular morphologic changes in the dentin–pulp complex were detected with hematoxylin and eosin staining. Expression of calcium ion transport-related proteins (Cav1.2, NCX1 and TRPV1) was determined with immunohistochemical staining and imaging analysis. Histopathological analysis revealed obvious reparative dentin formation at day 14 in the cavity-prepared?+?LIPUS group compared with the other groups. Expression levels of Cav1.2, NCX1 and TRPV1 increased significantly by 22%, 53% and 23%, respectively, at day 1 and increased significantly by 23%, 27% and 22%, respectively, at day 3 in the cavity-prepared?+?LIPUS group (p?<0.05) compared with the cavity-prepared group. LIPUS has a positive effect on the expression of calcium transport-related proteins during early-stage dentin injury and facilitates tertiary dentin formation; the mechanism for this likely relates to the inflammatory reaction and a mechanical effect.     

心脏钠钙交换--心力衰竭治疗的一个新目标

心力衰竭是一个非常复杂的过程,其发生与细胞内钙调节的异常有关,Na /Ca2 交换体(NCX)是心肌细胞内钙稳态的重要调节机制之一,也是心脏收缩功能决定性因素.目前对肥厚衰竭心肌Ca2 交换活性改变及其与心功能障碍以及心律失常产生的关系已有较多研究,本文就其研究现状予以综述.     

Is timing everything? Therapeutic potential of modulators of cardiac Na+ transporters

Sodium ion (Na⁺) transporters have roles in the modulation of cardiomyocyte pH and Na⁺ and Ca²⁺ handling. Activation of the cardiac Na⁺-H⁺ exchanger 1 (NHE1) during ischaemia induces arrhythmias, myocardial stunning and irreversible cell injury. As the benefits of NHE1 inhibitors (e.g., amiloride, cariporide) in models of myocardial infarction are usually much greater when used as pretreatment, rather than during or after ischaemia, it is probably not surprising that clinical trials with cariporide in ischaemia have shown little shortterm benefit. NHE1 inhibitors have been shown to be beneficial in animal models of ventricular fibrillation and resuscitation, cardioplegia, hypertrophy and heart failure, and their therapeutic potential in these conditions should be further developed. The Na⁺-HCO₃^- cotransporter (NBC) is also stimulated by intracellular acidification, and part of the benefit of angiotensin-converting enzyme inhibitors after myocardial infarction may be due to inhibition of the NBC. Selective inhibitors of the NBC are required to determine the therapeutic potential of this mechanism. The Na⁺-Ca²⁺ exchanger (NCX) has a major role in cardiac Na⁺ and Ca²⁺ homeostasis and influences cardiac electrical activity. The NCX also has a role in ischaemia/infarction, arrhythmias, hypertrophy and heart failure. NCX inhibitors may have beneficial effects in animal models of ischaemia and reperfusion injury and the therapeutic benefit of these should be further studied in animal models.     

A dual acting compound with latanoprost amide and nitric oxide releasing properties, shows ocular hypotensive effects in rabbits and dogs

The IOP lowering effects of NCX 139, a new chemical entity comprising latanoprost amide and a NO-donating moiety, were compared to those of the respective des-nitro analog in in vitro assays and in rabbit and dog models of ocular hypertension. The NO donor, molsidomine as well as the prostamide bimatoprost (Lumigan^®) and the prostaglandin agonist, latanoprost (Xalatan^®) were also investigated for comparison.NCX 139 but not its des-nitro analog resulted in NO-mediated vascular relaxant effect in pre-contracted rabbit aortic rings (EC₅₀ = 0.70 ± 0.06 μM; E_max = 80.6 ± 2.9%). Like bimatoprost (IC₅₀ = 3.07 ± 1.3 μM) or latanoprost (IC₅₀ = 0.48 ± 0.15 μM), NCX 139 displaced ³H-PGF2α binding on recombinant human prostaglandin-F (FP) receptors with an estimated potency of 0.77 ± 0.13 μM. In transient ocular hypertensive rabbits, bimatoprost and latanoprost were not effective while molsidomine elicited a dose-dependent reduction of IOP confirming the responsiveness of rabbits to NO but not to FP receptor agonists. NCX 139 tested at a therapeutically relevant dose, significantly lowered IOP while the des-nitro analog was not effective (0.03% NCX 139, Δ_max = −12.8 ± 2.0 mmHg). In glaucomatous dogs, 0.03% NCX 139 decreased IOP to a greater extent compared to an equimolar dose of the respective des-nitro derivative (Δ_max = −4.6 ± 1.0 and −2.7 ± 1.3 mmHg, respectively for NCX 139 and its des-nitro analog). Albeit with low potency, NCX 139 also resulted effective in normotensive dogs while it did not reduce IOP in normotensive rabbits. NCX 139, a compound targeting two different and important mechanisms, is endowed with ocular hypotensive effects more evident in hypertensive conditions which may be of interest in the search of more effective treatments for hypertensive glaucoma.     

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase β1-subunit

Na,K-ATPase is composed of two essential α- and β-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its α₁, α₃ and β₁ isoforms are reduced in the failing human heart. The catalytic α-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase β₁-subunit (Na,K-β₁) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-β₁ gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-β₁ gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13–14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-β₁ KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-β₁ KO in older mice. Further, we show that intact hearts of Na,K-β₁ KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-β₁ plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.     

A comparison of adenovirally delivered molecular methods to inhibit Na+/Ca2+ exchange

The ability to use molecular biology tools to down-regulate Na+/Ca2+ exchanger (NCX) expression will allow us to better understand the regulation of Ca(i)2+ and contractility in heart. Three different techniques to deplete NCX expression were compared: short hairpin RNA (shRNA), antisense RNA and exchanger inhibitory peptide expression via adenoviral transfection. Our results demonstrate that the most efficient method to deplete NCX expression and activity from cardiomyocytes is shRNA. It is also possible to replace the endogenous NCX with alternative isoforms or mutant forms of the NCX. Adenovirally delivered shRNA is an efficient tool for the study of the NCX and could be adapted for many other cardiac proteins.