Differential Downregulation of ß1-Adrenergic Receptor Signaling in the Heart.

BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of ß1 adrenergic receptor (ß1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ß1AR signaling in the heart. METHODS AND RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine ß1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular ß1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced ß1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the ß1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the ß1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced ß1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular ß1AR signaling in the heart under chronic adrenergic stimulation.

Identifying Key Binding Interactions Between the Cardiac L-Type Calcium Channel and Calmodulin Using Molecular Dynamics Simulations.

Defects in the binding of the calcium sensing protein calmodulin (CaM) to the L-type calcium channel (CaV1.2) or to the ryanodine receptor type 2 (RyR2) can lead to dangerous cardiac arrhythmias with distinct phenotypes, such as long-QT syndrome (LQTS) and catecholaminergic ventricular tachycardia (CPVT). Certain CaM mutations lead to LQTS while other mutations lead to CPVT, but the mechanisms by which a specific mutation can lead to each disease phenotype are not well-understood. In this study, we use long, 2 µs molecular dynamics simulations and a multitrajectory approach to identify the key binding interactions between the IQ domain of CaV1.2 and CaM. Five key interactions are found between CaV1.2 and CaM in the C-lobe, 1 in the central linker, and 2 in the N-lobe. In addition, while 5 key interactions appear between residues 120-149 in the C-lobe of CaM when it interacts with CaV1.2, only 1 key interaction is found within this region of CaM when it interacts with the RyR2. We show that this difference in the distribution of key interactions correlates with the known distribution of CaM mutations that lead to LQTS or CPVT. This correlation suggests that a disruption of key binding interactions is a plausible mechanism that can lead to these two different disease phenotypes.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons.

Pacemaking activity in substantia nigra dopaminergic neurons is generated by the coordinated activity of a variety of distinct somatodendritic voltage- and calcium-gated ion channels. We investigated whether these functional interactions could arise from a common localization in macromolecular complexes where physical proximity would allow for efficient interaction and co-regulations. For that purpose, we immunopurified six ion channel proteins involved in substantia nigra neuron autonomous firing to identify their molecular interactions. The ion channels chosen as bait were Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 channel proteins, and the methods chosen to determine interactions were co-immunoprecipitation analyzed through immunoblot and mass spectrometry as well as proximity ligation assay. A macromolecular complex composed of Cav1.3, HCN, and SK3 channels was unraveled. In addition, novel potential interactions between SK3 channels and sclerosis tuberous complex (Tsc) proteins, inhibitors of mTOR, and between HCN4 channels and the pro-degenerative protein Sarm1 were uncovered. In order to demonstrate the presence of these molecular interactions in situ, we used proximity ligation assay (PLA) imaging on midbrain slices containing the substantia nigra, and we could ascertain the presence of these protein complexes specifically in substantia nigra dopaminergic neurons. Based on the complementary functional role of the ion channels in the macromolecular complex identified, these results suggest that such tight interactions could partly underly the robustness of pacemaking in dopaminergic neurons.

CardioDPi: An explainable deep-learning model for identifying cardiotoxic chemicals targeting hERG, Cav1.2, and Nav1.5 channels.

The cardiotoxic effects of various pollutants have been a growing concern in environmental and material science. These effects encompass arrhythmias, myocardial injury, cardiac insufficiency, and pericardial inflammation. Compounds such as organic solvents and air pollutants disrupt the potassium, sodium, and calcium ion channels cardiac cell membranes, leading to the dysregulation of cardiac function. However, current cardiotoxicity models have disadvantages of incomplete data, ion channels, interpretability issues, and inability of toxic structure visualization. Herein, an interpretable deep-learning model known as CardioDPi was developed, which is capable of discriminating cardiotoxicity induced by the human Ether-à-go-go-related gene (hERG) channel, sodium channel (Na_v1.5), and calcium channel (Ca_v1.5) blockade. External validation yielded promising area under the ROC curve (AUC) values of 0.89, 0.89, and 0.94 for the hERG, Na_v1.5, and Ca_v1.5 channels, respectively. The CardioDPi can be freely accessed on the web server CardioDPipredictor (http://cardiodpi.sapredictor.cn/). Furthermore, the structural characteristics of cardiotoxic compounds were analyzed and structural alerts (SAs) can be extracted using the user-friendly CardioDPi-SAdetector web service (http://cardiosa.sapredictor.cn/). CardioDPi is a valuable tool for identifying cardiotoxic chemicals that are environmental and health risks. Moreover, the SA system provides essential insights for mode-of-action studies concerning cardiotoxic compounds.

Chamaecyparis lawsoniana and Its Active Compound Quercetin as Ca2+ Inhibitors in the Contraction of Airway Smooth Muscle.

The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.

AMPK inhibits voltage-gated calcium channel-current in rat chromaffin cells.

Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.

Impact of quetiapine on ion channels and contractile dynamics in rat ventricular myocyte.

Antipsychotic drugs often lead to adverse effects, including those related to the cardiovascular system. Of these, quetiapine is known to cause significant changes in the QT interval although the underlying mechanism remains mysterious, prompting us to examine its effects on cardiac electrophysiological properties. Therefore, we investigated the effect of quetiapine on contraction, action potential (AP), and the associated membrane currents such as L-type Ca2+ and K+ using the whole-cell patch clamp method to examine its impacts on isolated rat ventricular myocytes. Our results showed that (1) quetiapine reduces cell contractility in a concentration-dependent manner and (2) leads to a significant prolongation in the duration of AP in isolated ventricular myocytes. This effect was both concentration and frequency-dependent; (3) quetiapine significantly decreased the Ca2+, transient outward K+, and steady-state K+ currents. However, only high concentration of quetiapine (100 µM) could significantly change the activation and reactivation kinetics of L-type Ca2+ channels. This study demonstrates that QT extension induced by quetiapine is mainly associated with the prolongation of AP. Moreover, quetiapine caused a significant decrease in contractile force and excitability of ventricular myocytes by suppressing Ca2+ and K+ currents.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

Cardiac L-type calcium channel regulation by Leucine-Rich Repeat-Containing Protein 10.

L-type calcium channels (LTCCs), the major portal for Ca2+ entry into cardiomyocytes, are essential for excitation-contraction coupling and thus play a central role in regulating overall cardiac function. LTCC function is finely tuned by multiple signaling pathways and accessory proteins. Leucine-rich repeat-containing protein 10 (LRRC10) is a little studied cardiomyocyte-specific protein recently identified as a modulator of LTCCs. LRRC10 exerts a remarkable effect on LTCC function, more than doubling L-type Ca2+ current (ICa,L) amplitude in a heterologous expression system by altering the gating of the channels without changing their surface membrane expression. Genetic ablation of LRRC10 expression in mouse and zebrafish hearts leads to a significant reduction in ICa,L density and a slowly progressive dilated cardiomyopathy in mice. Rare sequence variants of LRRC10 have been identified in dilated cardiomyopathy and sudden unexplained nocturnal cardiac death syndrome, but these variants have not been clearly linked to disease. Nevertheless, the DCM-associated variant, I195T, converted LRRC10 from a ICa,L potentiator to a ICa,L suppressor, thus illustrating the wide dynamic range of LRRC10-mediated ICa,L regulation. This review focuses on the contemporary knowledge of LTCC modulation by LRRC10 and discusses potential directions for future investigations.

Role of the CaV1.2 distal carboxy terminus in the regulation of L-type current.

L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVß2b. The CaV1.2 channel's C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels' basal activity in cardiomyocytes.

CaV1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy.

A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3. In the heart, it is required for voltage-dependent Ca2+-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of CaV1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify CaV1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify CaV1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. CaV1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.

Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia.

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague-Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current-voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications.

Novel protocol for multiple-dose oral administration of the L-type Ca2+ channel blocker isradipine in mice: A dose-finding pharmacokinetic study.

Studies in genetically modified animals and human genetics have recently provided new insight into the role of voltage-gated L-type Ca2+ channels in human disease. Therefore, the inhibition of L-type Ca2+ channels in vivo in wildtype and mutant mice by potent dihydropyridine (DHP) Ca2+ channel blockers serves as an important pharmacological tool. These drugs have a short plasma half-life in humans and especially in rodents and show high first-pass metabolism upon oral application. In the vast majority of in vivo studies, they have therefore been delivered through parenteral routes, mostly subcutaneously or intraperitoneally. High peak plasma concentrations of DHPs cause side effects, evident as DHP-induced aversive behaviors confounding the interpretation of behavioral readouts. Nevertheless, pharmacokinetic data measuring the exposure achieved with these applications are sparse. Moreover, parenteral injections require animal handling and can be associated with pain, discomfort and stress which could influence a variety of physiological processes, behavioral and other functional readouts. Here, we describe a noninvasive oral application of the DHP isradipine by training mice to quickly consume small volumes of flavored yogurt that can serve as drug vehicle. This procedure does not require animal handling, allows repeated drug application over several days and reproducibly achieves peak plasma concentrations over a wide range previously shown to be well-tolerated in humans. This protocol should facilitate ongoing nonclinical studies in mice exploring new indications for DHP Ca2+ channel blockers.

Arterial Smooth Muscle Cell AKAP150 Mediates Exercise-Induced Repression of CaV1.2 Channel Function in Cerebral Arteries of Hypertensive Rats.

BACKGROUND: Hypertension is a major, prevalent risk factor for the development and progression of cerebrovascular disease. Regular exercise has been recommended as an excellent choice for the large population of individuals with mild-to-moderate elevations in blood pressure, but the mechanisms that underlie its vascular-protective and antihypertensive effects remain unknown. Here, we describe a mechanism by which myocyte AKAP150 (A-kinase anchoring protein 150) inhibition induced by exercise training alleviates voltage-dependent L-type Ca2+ channel (CaV1.2) activity and restores cerebral arterial function in hypertension. METHODS: Spontaneously hypertensive rats and newly generated smooth muscle-specific AKAP150 knockin mice were used to assess the role of myocyte AKAP150/CaV1.2 channel in regulating cerebral artery function after exercise intervention. RESULTS: Activation of the AKAP150/PKCα (protein kinase Cα) signaling increased CaV1.2 activity and Ca2+ influx of cerebral arterial myocyte, thus enhancing vascular tone in spontaneously hypertensive rats. Smooth muscle-specific AKAP150 knockin mice were hypertensive with higher CaV1.2 channel activity and increased vascular tone. Furthermore, treatment of Ang II (angiotensin II) resulted in a more pronounced increase in blood pressure in smooth muscle-specific AKAP150 knockin mice. Exercise training significantly reduced arterial myocyte AKAP150 expression and alleviated CaV1.2 channel activity, thus restoring cerebral arterial function in spontaneously hypertensive rats and smooth muscle-specific AKAP150 knockin mice. AT1R (AT1 receptor) and AKAP150 were interacted closely in arterial myocytes. Exercise decreased the circulating Ang II and Ang II-involved AT1R-AKAP150 association in myocytes of hypertension. CONCLUSIONS: The current study demonstrates that aerobic exercise ameliorates CaV1.2 channel function via inhibiting myocyte AKAP150, which contributes to reduced cerebral arterial tone in hypertension.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Non-ionotropic voltage-gated calcium channel signaling.

Voltage-gated calcium channels (VGCCs) are the major conduits for calcium ions (Ca2+) within excitable cells. Recent studies have highlighted the non-ionotropic functionality of VGCCs, revealing their capacity to activate intracellular pathways independently of ion flow. This non-ionotropic signaling mode plays a pivotal role in excitation-coupling processes, including gene transcription through excitation-transcription (ET), synaptic transmission via excitation-secretion (ES), and cardiac contraction through excitation-contraction (EC). However, it is noteworthy that these excitation-coupling processes require extracellular calcium (Ca2+) and Ca2+ occupancy of the channel ion pore. Analogous to the "non-canonical" characterization of the non-ionotropic signaling exhibited by the N-methyl-D-aspartate receptor (NMDA), which requires extracellular Ca2+ without the influx of ions, VGCC activation requires depolarization-triggered conformational change(s) concomitant with Ca2+ binding to the open channel. Here, we discuss the contributions of VGCCs to ES, ET, and EC coupling as Ca2+ binding macromolecules that transduces external stimuli to intracellular input prior to elevating intracellular Ca2+. We emphasize the recognition of calcium ion occupancy within the open ion-pore and its contribution to the excitation coupling processes that precede the influx of calcium. The non-ionotropic activation of VGCCs, triggered by the upstroke of an action potential, provides a conceptual framework to elucidate the mechanistic aspects underlying the microseconds nature of synaptic transmission, cardiac contractility, and the rapid induction of first-wave genes.

BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

Abnormal phosphorylation / dephosphorylation and Ca2+ dysfunction in heart failure.

Heart failure (HF) can be caused by a variety of causes characterized by abnormal myocardial systole and diastole. Ca2+ current through the L-type calcium channel (LTCC) on the membrane is the initial trigger signal for a cardiac cycle. Declined systole and diastole in HF are associated with dysfunction of myocardial Ca2+ function. This disorder can be correlated with unbalanced levels of phosphorylation / dephosphorylation of LTCC, endoplasmic reticulum (ER), and myofilament. Kinase and phosphatase activity changes along with HF progress, resulting in phased changes in the degree of phosphorylation / dephosphorylation. It is important to realize the phosphorylation / dephosphorylation differences between a normal and a failing heart. This review focuses on phosphorylation / dephosphorylation changes in the progression of HF and summarizes the effects of phosphorylation / dephosphorylation of LTCC, ER function, and myofilament function in normal conditions and HF based on previous experiments and clinical research. Also, we summarize current therapeutic methods based on abnormal phosphorylation / dephosphorylation and clarify potential therapeutic directions.

Glial KCNQ K+ channels control neuronal output by regulating GABA release from glia in C. elegans.

KCNQs are voltage-gated K+ channels that control neuronal excitability and are mutated in epilepsy and autism spectrum disorder (ASD). KCNQs have been extensively studied in neurons, but their function in glia is unknown. Using voltage, calcium, and GABA imaging, optogenetics, and behavioral assays, we show here for the first time in Caenorhabditis elegans (C. elegans) that glial KCNQ channels control neuronal excitability by mediating GABA release from glia via regulation of the function of L-type voltage-gated Ca2+ channels. Further, we show that human KCNQ channels have the same role when expressed in nematode glia, underscoring conservation of function across species. Finally, we show that pathogenic loss-of-function and gain-of-function human KCNQ2 mutations alter glia-to-neuron GABA signaling in distinct ways and that the KCNQ channel opener retigabine exerts rescuing effects. This work identifies glial KCNQ channels as key regulators of neuronal excitability via control of GABA release from glia.

Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart.

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.