L-type Ca Channels Research Articles

α1-Adrenergic receptor–PKC–Pyk2–Src signaling boosts L-type Ca2+ channel CaV1.2 activity and long-term potentiation in rodents

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq-coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR–Pyk2–Src signaling regulates synaptic strength.

Functional LTCC-β2AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure.

Beta-2 adrenergic receptors (β2ARs) but not beta-2 adrenergic receptors (β1ARs) form a functional complex with L-type Ca2+ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and β adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca2+/calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Global signaling between LTCCs and β adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and β1AR or β2AR in different membrane microdomains in control and failing cardiomyocytes. LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when β2AR was locally stimulated in the proximity of the channel (<350 nm) in the transverse tubule microdomain. In failing cardiomyocytes, from both rodents and humans, this transverse tubule coupling between LTCC and β2AR was lost. Interestingly, local stimulation of β1AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via β1AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the β2AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular "global" level PKA plays a major role downstream β1AR and results in an increase in LTCC current. Regulation of the LTCC activity by proximity coupling mechanisms occurs only via β2AR, but not β1AR. This may explain how β2ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Effects and underlying mechanisms of L-arginine on spontaneous muscle contraction of rat colon.

Arginine (Arg), as a basic amino acid, has been reported to be involved in regulation of gut motility. However, the evidence is limited and the underlying mechanism is not fully understood. Our study was conducted to investigate the effects of L-Arg on spontaneous contraction of the longitudinal muscle strip (LMS) of the rat colon and the relevant mechanisms. An organ bath system was used to detect the contractile force of the LMS. Whole-cell voltage-clamp techniques were applied to observe alterations in the currents of large conductance Ca2+-activated K+ (KCa) channels, voltage-dependent potassium (KV) channels, and L-type Ca2+ channels (LTCCs) in smooth muscle cells (SMCs) of the colon. We found that L-Arg within the physiological concentration had no effect on contraction of LMS, while 1mM L-Arg significantly increased both the amplitude and frequency of LMS contractility. And the increase in force was mucosa-dependent, whereas changes in frequency as well as in amplitude were inhibited by atropine. In addition, L-Arg (1mM) activated the LTTCs and inhibited both KCa channels and KV channels on SMCs. Thus, L-Arg above the physiological concentration exerted an excitatory effect on colonic LM contraction, and stimulation by L-Arg was mediated by ACh. In addition, LTCCs, KCa channels, and KV channels on SMCs were involved in the action of L-Arg.

Cav 1.3-selective inhibitors of voltage-gated L-type Ca2+ channels: Fact or (still) fiction?

Voltage-gated L-type Ca2+ -channels (LTCCs) are the target of Ca2+ -channel blockers (CCBs), which are in clinical use for the evidence-based treatment of hypertension and angina. Their cardiovascular effects are largely mediated by the Cav 1.2-subtype. However, based on our current understanding of their physiological and pathophysiological roles, Cav 1.3 LTCCs also appear as attractive drug targets for the therapy of various diseases, including treatment-resistant hypertension, spasticity after spinal cord injury and neuroprotection in Parkinson's disease. Since CCBs inhibit both Cav 1.2 and Cav 1.3, Cav 1.3-selective inhibitors would be valuable tools to validate the therapeutic potential of Cav 1.3 channel inhibition in preclinical models. Despite a number of publications reporting the discovery of Cav 1.3-selective blockers, their selectivity remains controversial. We conclude that at present no pharmacological tools exist that are suitable to confirm or refute a role of Cav 1.3 channels in cellular responses. We also suggest essential criteria for a small molecule to be considered Cav 1.3-selective.

Evaluation of the bisphenol A-induced vascular toxicity on human umbilical artery

Bisphenol A (BPA) is one of the most widely used synthetic compound in the manufacture of polycarbonate plastics and epoxy resins. Worryingly, BPA is an endocrine disrupting chemical (EDC) with an estrogenic, androgenic, or anti-androgenic activities. However, the vascular implications of BPA exposome in pregnancy is unclear. In this sense, the present work proposed to understand how BPA exposure impair the vasculature of the pregnant women. To elucidate this, ex vivo studies were performed using human umbilical arteries to explore the acute and chronic effects of BPA. The mode of action of BPA was also explored by analysing the activity (by ex vivo studies) and expression (in vitro studies) analysis of Ca2+ and K+-channels and soluble guanyl cyclase. Moreover, in silico docking simulations were performed to unveil the modes of interactions of BPA with the proteins involved in these signalling pathways. Our study showed that the exposure to BPA may modify the vasorelaxant response of HUA, interfering with NO/sGC/cGMP/PKG pathway by modulation of sGC and activation of BKCa channels. Moreover, our findings suggest that BPA can modulate the HUA reactivity, increasing the L-type Ca2+ Channels (LTCC) activity, a common vascular response observed in hypertensive disorders of pregnancy.

Genetic deletion of STAC2 adaptor protein alters electrical activity of mouse chromaffin cells.

Voltage-gated Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) regulate action potential (AP) firing and catecholamine release in mouse chromaffin cells (MCCs). Src homology 3 and cysteine rich domain adaptor proteins (Stac1-Stac3) have recently been identified as novel regulators of neuronal LTCCs expression and biophysical properties. Upon overexpression in cultured hippocampal neurons Stac2 abolished LTCC Ca2+ dependent inactivation via an allosteric inhibition of calmodulin binding. Additionally, in Drosophila, deletion of the homologous DStac gene resulted in deficient LTCC Ca2+ transients and reduced neuropeptide release. Here we investigated the effect of Stac2 genetic ablation on neuronal LTCC function, electrical activity, and vesicle exocytosis in MCCs. Constitutive deletion of Stac2 does not affect the MCCs resting membrane potential or spontaneous firing frequency. However, the AP depolarization threshold was significantly reduced in Stac2-/- MCCs compared to WT (WT = −35.6 ± 1.2 mV, Stac2-/- = −40.6 ± 0.6 mV; ∗∗ P < 0.01). Additionally, step current injection elicited an electrical activity with higher initial AP firing frequency in Stac2-/- compared to WT that led to earlier depolarization block (F0 ≅2 pA current injection: WT = 0.28 ± 0.28 Hz, Stac2-/- = 0.89 ± 0.40 Hz; ∗ P < 0.05). Surprisingly, initial characterization shows that Stac2 deletion does not alter whole-cell Ca2+ current amplitude or inactivation kinetics but significantly shifts the voltage-dependence of activation to more hyperpolarized potential (V0.5: WT = −5.95 ± 1.65 mV, Stac2-/- = −15.25 ± 2.05 mV; ∗ P < 0.05). We are currently investigating possible effects of Stac2 deletion on other ionic conductances governing MCC excitability and the role on catecholamine vesicle exocytosis. Support: TWF F.18863, LFU 2021-CHEM-8 to SMG; FWF P31434, P36053, DOC30-B30 to PT; P33776 to MC.

Elucidating the role Cav1.2 dysfunction in the pathogenesis of timothy syndrome using iPSC-derived neurons.

The L-type Ca2+ channel (LTCC) is critical for normal function of the heart and brain, and mutations in this channel have been linked to severe cardiac and neurodevelopmental disorders. Specifically, single de novo point mutations within the CaV1.2 LTCC can cause a multi-system disorder known as Timothy Syndrome (TS). TS patients exhibit severe cardiac arrhythmias, long-QT syndrome, developmental delays, and autism spectrum disorder (ASD). TS is one of the most penetrant genetic forms of ASD, making it an ideal model system for understanding the role of Ca2+ dysfunction in ASD. Prior work has demonstrated that TS mutations can cause marked differences in channel gating, with a possible association between enhanced channel activation and the neurological features of TS. Here, we utilize human induced pluripotent stem cell (iPSC)-derived neurons harboring select CaV1.2 mutations to probe the effects of these gating changes on neuronal function. Voltage clamp recordings demonstrate measurable alterations to the biophysical properties of calcium channels within wildtype vs. TS iPSC- derived neurons. In addition, current clamp recordings reveal a difference in the pattern of electrical activity across the neuron populationsbetween distinct neuronal populations, demonstrating an effect of the TS mutations at the level of a single neuron. Overall, these cells enable evaluation of the role of CaV1.2 in the pathogenesis of ASD. Through the study of rare mutations such as TS we stand to gain further insight into ASD pathogenesis, especially as Ca2+ disruption may be a recurrent feature of ASD.

Compensatory calcium handling may underlie increased arrhythmia susceptibility of sarcospan-deficient mice after ischemia-reperfusion injury.

The protein sarcospan (SSPN) is an integral member of the dystrophin glycoprotein complex (DGC) and has been shown important in the heart during development and periods of acute stress. In this study, ex vivo hearts obtained from WT and SSPN-deficient (SSPN-/-) mice were used to investigate the role of SSPN in protecting against arrhythmia after acute ischemia-reperfusion (IR) injury. Measurements of sinus heart rates in ex vivo hearts showed that SSPN-/- hearts trended toward slower cardiac conduction post-IR injury than WT hearts. Premature ventricular contractions (PVCs) were increased in SSPN-/- hearts post-IR injury during reperfusion. The regulation of intracellular Ca2+ was examined in Fura-2AM loaded WT and SSPN-/- cardiomyocytes. Acute beta-adrenergic stimulation of cardiomyocytes with 1 μM isoproterenol resulted in reduced sarcomere length, increased diastolic Ca2+ levels of WT but not SSPN-/- cardiomyocytes. After isoproterenol exposure changes in systolic Ca2+ levels similar between WT and SSPN-/- cardiomyocytes. Cardiomyocytes were treated with caffeine (10 mM) to deplete the sarcoplasmic reticulum (SR) Ca2+ store. It was found that WT cardiomyocytes had greater Ca2+ SR store than SSPN-/-. However, the total Ca2+ release from WT and SSPN-/- cardiomyocytes was similar. To better understand these results in context of IR injury, qRT-PCR was utilized to examine changes in Ca2+ handling genes. Overall, SSPN-/- hearts exhibited significantly increased expression of major Ca2+ handling genes suggesting compensation for perturbed Ca2+ handling dynamics. Interestingly, after acute IR injury the expression of L-type Ca2+-channel (LTCC) and phospholamban (PLN) were reduced in SSPN-/- hearts but not WT hearts. These results suggest that increased arrhythmia susceptibility in SSPN-/- hearts post-IR injury may arise from disruption of compensatory Ca2+ handling mechanisms in the SSPN-/- heart.

Sinoatrial node cells and tissue operate via stochastic resonance.

Sinoatrial node (SAN), the primary heart pacemaker, is a highly heterogeneous, complex cellular network. Recent imaging data indicate that synchronized action potentials (APs) that exit the SAN emerge from heterogeneous signals, including subthreshold, irregular signals. We tested the hypothesis that SAN cells and tissue operate via stochastic resonance, i.e. biological noise within and among SAN cells plays an important role in generation of cardiac impulses. We performed patch-clamp experiments in current-clamp mode in isolated single rabbit SAN cells. When uniformly random current noise of a few pA amplitude with a zero average was applied via the patch pipette, cells that had been firing APs rarely and irregularly began firing more frequently and rhythmically. Further, similar noise application to non-firing cells (dormant cells or artificially hyperpolarized cells) initiated AP firing. These results were supported by simulations of Maltsev-Lakatta numerical model of SAN cells. Further, simulations using a numerical model of heterogeneous SAN cellular network, in which the rate of Ca pumping and ICaL conductance varied, revealed that random current noise applied within cells also improved the tissue function. In scenarios at the edge of criticality, in which the cell network could not operate (99.84% of non-firing cells), almost all cells (97.12%) fired APs when noise was applied to cells within the network. Although artificial noise was applied in our experiments and simulations, similar noise is likely to be generated under physiological conditions by stochastic openings of ion channels (RyRs and L-type Ca-channels) within cells and by subthreshold signals from neighboring cells in SAN tissue. Our results support the idea that the SAN operates via stochastic resonance, thus shifting the paradigm of SAN operation from dictatorship and democracy theories, to a complex neuronal-type modus operandi, i.e. the SAN operates as “the heart's brain”.

CACNA1D-Related Channelopathies: From Hypertension to Autism.

Tightly controlled Ca2+ influx through voltage-gated Ca2+ channels (Cavs) is indispensable for proper physiological function. Thus, it is not surprising that Cav loss and/or gain of function have been implicated in human pathology. Deficiency of Cav1.3 L-type Ca2+ channels (LTCCs) causes deafness and bradycardia, whereas several genetic variants of CACNA1D, the gene encoding the pore-forming α1 subunit of Cav1.3, have been linked to various disease phenotypes, such as hypertension, congenital hypoglycemia, or autism. These variants include not only common polymorphisms associated with an increased disease risk, but also rare de novo missense variants conferring high risk. This review provides a concise summary of disease-associated CACNA1D variants, whereas the main focus lies on de novo germline variants found in individuals with a neurodevelopmental disorder of variable severity. Electrophysiological recordings revealed activity-enhancing gating changes induced by these de novo variants, and tools to predict their pathogenicity and to study the resulting pathophysiological consequences will be discussed. Despite the low number of affected patients, potential phenotype-genotype correlations and factors that could impact the severity of symptoms will be covered. Since increased channel activity is assumed as the disease-underlying mechanism, pharmacological inhibition could be a treatment option. In the absence of Cav1.3-selective blockers, dihydropyridine LTCC inhibitors clinically approved for the treatment of hypertension may be used for personalized off-label trials. Findings from in vitro studies and treatment attempts in some of the patients seem promising as outlined. Taken together, due to advances in diagnostic sequencing techniques the number of reported CACNA1D variants in human diseases is constantly rising. Evidence from in silico, in vitro, and in vivo disease models can help to predict the pathogenic potential of such variants and to guide diagnosis and treatment in the clinical practice when confronted with patients harboring CACNA1D variants.

Hesperetin ameliorates ischemia/hypoxia-induced myocardium injury via inhibition of oxidative stress, apoptosis, and regulation of Ca2+ homeostasis.

Ischemia/hypoxia (I/H)-induced myocardial injury has a large burden worldwide. Hesperetin (HSP) has a cardioprotective effect, but the molecular mechanism underlying this is not clearly established. Here, we focused on the protective mechanisms of HSP against I/H-induced myocardium injury. H9c2 cardiomyocytes were challenged with CoCl2 for 22 h to imitate hypoxia after treatment groups received HSP for 4h. The viability of H9c2 cardiomyocytes was evaluated, and cardiac function indices, reactive oxygen species, apoptosis, mitochondrial membrane potential (MMP), and intracellular Ca2+ concentration ([Ca2+ ]i ) were measured. L-type Ca2+ current (ICa-L ), myocardial contraction, and Ca2+ transients in isolated ventricular myocytes were also recorded. We found that HSP significantly increased the cell viability, and MMP while significantly decreasing cardiac impairment, oxidative stress, apoptosis, and [Ca2+ ]i caused by CoCl2 . Furthermore, HSP markedly attenuated ICa-L , myocardial contraction, and Ca2+ transients in a concentration-dependent manner. Our findings suggest a protective mechanism of HSP on I/H-induced myocardium injury by restoring oxidative balance, inhibiting apoptosis, improving mitochondrial function, and reducing Ca2+ influx via L-type Ca2+ channels (LTCCs). These data provide a new direction for HSP applied research as a LTCC inhibitor against I/H-induced myocardium injury.

The CaV1.2 G406R mutation decreases synaptic inhibition and alters L-type Ca2+ channel-dependent LTP at hippocampal synapses in a mouse model of Timothy Syndrome

Genetic alterations in autism spectrum disorders (ASD) frequently disrupt balance between synaptic excitation and inhibition and alter plasticity in the hippocampal CA1 region. Individuals with Timothy Syndrome (TS), a genetic disorder caused by CaV1.2 L-type Ca2+ channel (LTCC) gain-of function mutations, such as G406R, exhibit social deficits, repetitive behaviors, and cognitive impairments characteristic of ASD that are phenocopied in TS2-neo mice expressing G406R. Here, we characterized hippocampal CA1 synaptic function in male TS2-neo mice and found basal excitatory transmission was slightly increased and inhibitory transmission strongly decreased. We also found distinct impacts on two LTCC-dependent forms of long-term potentiation (LTP) synaptic plasticity that were not readily consistent with LTCC gain-of-function. LTP induced by high-frequency stimulation (HFS) was strongly impaired in TS2-neo mice, suggesting decreased LTCC function. Yet, CaV1.2 expression, basal phosphorylation, and current density were similar for WT and TS2-neo. However, this HFS-LTP also required GABAA receptor activity, and thus may be impaired in TS2-neo due to decreased inhibitory transmission. In contrast, LTP induced in WT mice by prolonged theta-train (PTT) stimulation in the presence of a β-adrenergic receptor agonist to increase CaV1.2 phosphorylation was partially induced in TS2-neo mice by PTT stimulation alone, consistent with increased LTCC function. Overall, our findings provide insights regarding how altered CaV1.2 channel function disrupts basal transmission and plasticity that could be relevant for neurobehavioral alterations in ASD.This article is part of the Special Issue on ‘L-type calcium channel mechanisms in neuropsychiatric disorders’.

Disorder in Ca2+ release unit locations confers robustness but cuts flexibility of heart pacemaking.

Excitation-contraction coupling kinetics is dictated by the action potential rate of sinoatrial-nodal cells. These cells generate local Ca releases (LCRs) that activate Na/Ca exchanger current, which accelerates diastolic depolarization and determines the pace. LCRs are generated by clusters of ryanodine receptors, Ca release units (CRUs), residing in the sarcoplasmic reticulum. While CRU distribution exhibits substantial heterogeneity, its functional importance remains unknown. Using numerical modeling, here we show that with a square lattice distribution of CRUs, Ca-induced-Ca-release propagation during diastolic depolarization is insufficient for pacemaking within a broad range of realistic ICaL densities. Allowing each CRU to deviate randomly from its lattice position allows sparks to propagate, as observed experimentally. As disorder increases, the CRU distribution exhibits larger empty spaces and simultaneously CRU clusters, as in Poisson clumping. Propagating within the clusters, Ca release becomes synchronized, increasing action potential rate and reviving pacemaker function of dormant/nonfiring cells. However, cells with fully disordered CRU positions could not reach low firing rates and their β-adrenergic-receptor stimulation effect was substantially decreased. Inclusion of Cav1.3, a low-voltage activation L-type Ca channel isoform into ICaL, strongly increases recruitment of CRUs to fire during diastolic depolarization, increasing robustness of pacemaking and complementing effects of CRU distribution. Thus, order/disorder in CRU locations along with Cav1.3 expression regulates pacemaker function via synchronization of CRU firing. Excessive CRU disorder and/or overexpression of Cav1.3 boosts pacemaker function in the basal state, but limits the rate range, which may contribute to heart rate range decline with age and disease.

Initiation of ventricular arrhythmia in the acquired long QT syndrome.

Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ -40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with ICaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of ICaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. These data demonstrate that ICaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS.

Protective mechanisms of 10-gingerol against myocardial ischemia may involve activation of JAK2/STAT3 pathway and regulation of Ca2+ homeostasis

10-Gingerol (10-Gin), an active ingredient extracted from ginger, has been reported to have beneficial effects on the cardiovascular system. However, its protective effects on myocardial ischemia (MI) and the underlying cellular mechanisms are still unclear. To investigate the protection conferred by 10-Gin against MI injury and its potential mechanisms in cardiomyocytes via patch-clamp and molecular biology techniques. A rat MI model was established using the subcutaneous injection of isoproterenol (85 mg/kg) administered on two consecutive days. 10-Gin was pre-administered to rats for seven days to assess its cardio-protection. The patch-clamp and IonOptix Myocam detection techniques were used to investigated 10-Gin’s effects on L-type Ca2+ channels (LTCCs), Ca2+ transients and cell contractility in isolated rat cardiomyocytes. 10-Gin administration alleviated MI injury, improved cardiac function and myocardial histopathology, reduced myocardial infarct area, downregulated oxidative stress and Ca2+ levels, and decreased the expression of apoptotic factors. Importantly, 10-Gin led to an increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2 and STAT3, respectively) expressions. Furthermore, 10-Gin inhibited LTCCs in a concentration-dependent manner with a half-maximal inhibitory concentration of 75.96 μM. Moreover, 10-Gin administration inhibited Ca2+ transients and cell contractility. Our results suggest that 10-Gin exerts cardioprotective effects on MI in vivo and in vitro in connection with the inhibition of oxidative stress and apoptosis via activation of the JAK2/STAT3 signalling pathway, and regulation of Ca2+ homeostasis by LTCCs.

Elucidating the Mechanisms of CaMKII‐CaMKAP Interactions

Dynamic changes of intracellular calcium ion (Ca2+) concentrations are critical signaling mechanisms in all cells and organisms, and Ca2+ oscillations must be precisely decoded by intracellular signaling molecules, such as the ubiquitous Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Four mammalian CaMKII genes encode diverse alternative mRNA splice variants, which are thought to be targeted to different subcellular compartments to regulate numerous cell responses (exocytosis, gene expression, cell cycle). Our lab hypothesized that the localization, activity, and biological functions of CaMKII are precisely modulated by CaMKII‐associated proteins (CaMKAPs), and we have detected over 100 CaMKAPs associated with brain CaMKII holoenzymes. Some CaMKII‐CaMKAP interactions are critical for normal physiological function. CaMKIIδ binding to the L‐type Ca2+ channel (LTCC) b2a subunit is essential for cardiac function1, and binding of neuronal CaMKII to the GluN2B subunit of the NMDA‐type glutamate receptor is necessary for synaptic plasticity2. While b2a, GluN2B and most other CaMKAPs preferentially interact with activated CaMKII, the exact molecular mechanisms for these interactions, and their physiological roles, have yet to be determined. Several residues in the catalytic domain of CaMKII have been implicated in CaMKAP binding. Previous mutagenesis studies from our lab indicated that mutation of valine 102 (V102) to glutamate disrupts CaMKII binding to LTCC α1 subunits, but not to b2a, GluN2B, or densin, and also disrupts neuronal excitation‐transcription coupling3. Additionally, a naturally‐occurring CaMKIIα Glu183Val mutation linked to autism spectrum disorder and intellectual disability disrupted interactions with all CaMKAPs tested4. To further probe the molecular basis of CaMKII‐CaMKAP interactions, we are exploiting available structural information to design additional CaMKII catalytic domain mutations, and then test their effect on CaMKII‐CaMKAP interactions, as well as CaMKII activity toward multiple substrates. We are also examining the impact of additional human CaMKII mutations. Initial studies indicate that some CaMKII mutations differentially affect binding to various CaMKAPs, CaMKII autophosphorylation, and phosphorylation of a representative substrate, the GluA1 subunit of AMPA‐type glutamate receptors. Our ongoing studies are further exploring the impact of these CaMKII mutations. Expanding our knowledge of mechanisms underlying CaMKII‐CaMKAP interactions is required to better understand CaMKII biology, the role of CaMKAPs, and the molecular basis of Ca2+signaling. Koval et al., 2010. PNAS 107:4996. Halt et al., 2012. EMBO J. 31:1203. Wang et al., 2017. J Biol Chem 292:17324. Stephenson et al., 2017. J Neurosci 37:2216.

A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts

Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.

L-type Ca2+ channel recovery from inactivation in rabbit atrial myocytes.

Adaptation of the myocardium to varying workloads critically depends on the recovery from inactivation (RFI) of L‐type Ca2+ channels (LCCs) which provide the trigger for cardiac contraction. The goal of the present study was a comprehensive investigation of LCC RFI in atrial myocytes. The study was performed on voltage‐clamped rabbit atrial myocytes using a double pulse protocol with variable diastolic intervals in cells held at physiological holding potentials, with intact intracellular Ca2+ release, and preserved Na+ current and Na+/Ca2+ exchanger (NCX) activity. We demonstrate that the kinetics of RFI of LCCs are co‐regulated by several factors including resting membrane potential, [Ca2+]i, Na+ influx, and activity of CaMKII. In addition, activation of CaMKII resulted in increased I Ca amplitude at higher pacing rates. Pharmacological inhibition of NCX failed to have any significant effect on RFI, indicating that impaired removal of Ca2+ by NCX has little effect on LCC recovery. Finally, RFI of intracellular Ca2+ release was substantially slower than LCC RFI, suggesting that inactivation kinetics of LCC do not significantly contribute to the beat‐to‐beat refractoriness of SR Ca2+ release. The study demonstrates that CaMKII and intracellular Ca2+ dynamics play a central role in modulation of LCC activity in atrial myocytes during increased workloads that could have important consequences under pathological conditions such as atrial fibrillations, where Ca2+ cycling and CaMKII activity are altered.

Abstract 11: Should all subjects with Type 2 diabetes mellitus receive magnesium supplementation

Background: Deficiency of Mg may increase the incidence of diabetes mellitus and occurrence of diabetic complications.Aim: To evaluate an association between serum magnesium level, glycaemic status and diabetic complications in T2DM subjects in Punjab.Methods: Participants were 650 T2DM subjects who underwent clinical evaluation, relevant investigations including serum Mg levels. Hypomagnesemia was a Mg level below 1.8 mg/dL.Results: Mean age of the participants was 51.2 years, mean duration of diabetes 9.5 years and 53.6% were males. The mean HbA1c was 9.13 ±2.4%. 51.3% of the participants had hypomagnesemia. Hypomagnesemia was significantly associated with retinopathy, microalbuminuria, neuropathy and HbA1c but not with hypertension, BMI, CAD, Age, Sex and duration of diabetes.Conclusions: Our findings suggest that Mg deficiency is common in subjects with T2DM in Punjab. Intracellular Mg regulates glucokinase, KATP channels and L-type Ca channels in pancreatic b-cells. Insulin receptor autophosphorylation is also dependent on intracellular Mg concentration. Hypomagnesemia in T2DM leads to reduced pancreatic b-cell activity and increases insulin resistance thereby resulting in poor glycaemic control and increased risk of complications. Dietary Mg supplementation for all patients with T2DM can be a simple and cost effective strategy to improve glucose metabolism and insulin sensitivity and prevent long term complications.