SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data.

BACKGROUND: Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis. METHODS: We developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting. RESULTS: SM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions. CONCLUSIONS: SparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

Effects of chronic secondhand smoke exposure on cardiovascular regulation and the role of soluble epoxide hydrolase in mice.

Background: Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality with an estimated 80% of SHS-related deaths attributed to cardiovascular causes. Public health measures and smoking bans have been successful both in reducing SHS exposure and improving cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a promising target for mitigating SHS exposure-induced cardiovascular outcomes. Objectives: The objectives of this study were to determine 1) effects of environmentally relevant SHS exposure on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of an sEH inhibitor (TPPU) can reduce the adverse cardiovascular effects of SHS exposure. Methods: Male C57BL/6J mice (11 weeks old) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to filtered air or 3 mg/m3 of SHS (6 hr/d, 5 d/wk) for 12 weeks, followed by 4 weeks of recovery in filtered air. Some mice received TPPU in drinking water (15 mg/L) throughout SHS exposure. BP, heart rate (HR), HR variability (HRV), baroreflex sensitivity (BRS), and BP variability were determined monthly. Results: SHS exposure significantly decreased 1) short-term HRV by ∼20% (p < 0.05) within 4 weeks; 2) overall HRV with maximum effect at 12 weeks (-15%, p < 0.05); 3) pulse pressure (-8%, p < 0.05) as early as week 4; and 4) BRS with maximum effect at 12 weeks (-11%, p < 0.05). Four weeks of recovery following 12 weeks of SHS ameliorated all SHS-induced cardiovascular detriments. Importantly, mice exposed to TPPU in drinking water during SHS-related exposure were protected from SHS cardiovascular consequences. Discussion: The data suggest that 1) environmental relevant SHS exposure significantly alters cardiac autonomic function and BP regulation; 2) cardiovascular consequences from SHS can be reversed by discontinuing SHS exposure; and 3) inhibiting sEH can prevent SHS-induced cardiovascular consequences.

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.

Mechanisms of Vascular CaV1.2 Channel Regulation During Diabetic Hyperglycemia.

Diabetes is a leading cause of disability and mortality worldwide. A major underlying factor in diabetes is the excessive glucose levels in the bloodstream (e.g., hyperglycemia). Vascular complications directly result from this metabolic abnormality, leading to disabling and life-threatening conditions. Dysfunction of vascular smooth muscle cells is a well-recognized factor mediating vascular complications during diabetic hyperglycemia. The function of vascular smooth muscle cells is exquisitely controlled by different ion channels. Among the ion channels, the L-type CaV1.2 channel plays a key role as it is the main Ca2+ entry pathway regulating vascular smooth muscle contractile state. The activity of CaV1.2 channels in vascular smooth muscle is altered by diabetic hyperglycemia, which may contribute to vascular complications. In this chapter, we summarize the current understanding of the regulation of CaV1.2 channels in vascular smooth muscle by different signaling pathways. We place special attention on the regulation of CaV1.2 channel activity in vascular smooth muscle by a newly uncovered AKAP5/P2Y11/AC5/PKA/CaV1.2 axis that is engaged during diabetic hyperglycemia. We further describe the pathophysiological implications of activation of this axis as it relates to myogenic tone and vascular reactivity and propose that this complex may be targeted for developing therapies to treat diabetic vascular complications.

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2.

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100 nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.

BACKGROUND: L-type CaV1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the CaV1.2 pore-forming subunit α1C at S1928 mediates vascular CaV1.2 cooperativity during diabetic hyperglycemia. METHODS: A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaV1.2 cooperativity, α1C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. RESULTS: CaV1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. CaV1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. CONCLUSIONS: These results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1C into "superclusters" upon hyperglycemia and diabetes. This triggers CaV1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct CaV1.2 and arterial function during diabetic hyperglycemia.

Vascular CaV1.2 channels in diabetes.

Diabetic vasculopathy is a significant cause of morbidity and mortality in the diabetic population. Hyperglycemia, one of the central metabolic abnormalities in diabetes, has been associated with vascular dysfunction due to endothelial cell damage. However, studies also point toward vascular smooth muscle as a locus for hyperglycemia-induced vascular dysfunction. Emerging evidence implicates hyperglycemia-induced regulation of vascular L-type Ca2+ channels CaV1.2 as a potential mechanism for vascular dysfunction during diabetes. This chapter summarizes our current understanding of vascular CaV1.2 channels and their regulation during physiological and hyperglycemia/diabetes conditions. We will emphasize the role of CaV1.2 in vascular smooth muscle, the effects of elevated glucose on CaV1.2 function, and the mechanisms underlying its dysregulation in hyperglycemia and diabetes. We conclude by examining future directions and gaps in knowledge regarding CaV1.2 regulation in health and during diabetes.

Ion channel molecular complexes in vascular smooth muscle.

Ion channels that influence membrane potential and intracellular calcium concentration control vascular smooth muscle excitability. Voltage-gated calcium channels (VGCC), transient receptor potential (TRP) channels, voltage (KV), and Ca2+-activated K+ (BK) channels are key regulators of vascular smooth muscle excitability and contractility. These channels are regulated by various signaling cues, including protein kinases and phosphatases. The effects of these ubiquitous signaling molecules often depend on the formation of macromolecular complexes that provide a platform for targeting and compartmentalizing signaling events to specific substrates. This manuscript summarizes our current understanding of specific molecular complexes involving VGCC, TRP, and KV and BK channels and their contribution to regulating vascular physiology.

Deciphering cellular signals in adult mouse sinoatrial node cells.

Sinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct ß-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.

Secondhand Smoke Exposure Impairs Ion Channel Function and Contractility of Mesenteric Arteries.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

Cellular and molecular effects of hyperglycemia on ion channels in vascular smooth muscle.

Diabetes affects millions of people worldwide. This devastating disease dramatically increases the risk of developing cardiovascular disorders. A hallmark metabolic abnormality in diabetes is hyperglycemia, which contributes to the pathogenesis of cardiovascular complications. These cardiovascular complications are, at least in part, related to hyperglycemia-induced molecular and cellular changes in the cells making up blood vessels. Whereas the mechanisms mediating endothelial dysfunction during hyperglycemia have been extensively examined, much less is known about how hyperglycemia impacts vascular smooth muscle function. Vascular smooth muscle function is exquisitely regulated by many ion channels, including several members of the potassium (K+) channel superfamily and voltage-gated L-type Ca2+ channels. Modulation of vascular smooth muscle ion channels function by hyperglycemia is emerging as a key contributor to vascular dysfunction in diabetes. In this review, we summarize the current understanding of how diabetic hyperglycemia modulates the activity of these ion channels in vascular smooth muscle. We examine underlying mechanisms, general properties, and physiological relevance in the context of myogenic tone and vascular reactivity.

AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Purinergic Signaling During Hyperglycemia in Vascular Smooth Muscle Cells.

The activation of purinergic receptors by nucleotides and/or nucleosides plays an important role in the control of vascular function, including modulation of vascular smooth muscle excitability, and vascular reactivity. Accordingly, purinergic receptor actions, acting as either ion channels (P2X) or G protein-coupled receptors (GCPRs) (P1, P2Y), target diverse downstream effectors, and substrates to regulate vascular smooth muscle function and vascular reactivity. Both vasorelaxant and vasoconstrictive effects have been shown to be mediated by different purinergic receptors in a vascular bed- and species-specific manner. Purinergic signaling has been shown to play a key role in altering vascular smooth muscle excitability and vascular reactivity following acute and short-term elevations in extracellular glucose (e.g., hyperglycemia). Moreover, there is evidence that vascular smooth muscle excitability and vascular reactivity is severely impaired during diabetes and that this is mediated, at least in part, by activation of purinergic receptors. Thus, purinergic receptors present themselves as important candidates mediating vascular reactivity in hyperglycemia, with potentially important clinical and therapeutic potential. In this review, we provide a narrative summarizing our current understanding of the expression, function, and signaling of purinergic receptors specifically in vascular smooth muscle cells and discuss their role in vascular complications following hyperglycemia and diabetes.

TRPML1ng on sparks.

In this issue of Science Signaling, Thakore et al. report that the Ca2+-permeable channel TRPML1 closely associates with ryanodine receptors to induce Ca2+ sparks in native arterial myocytes. Functional studies revealed a key role for TRPML1 channels in regulation of arterial myocyte contractility and blood pressure.

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

ß-blockers augment L-type Ca2+ channel activity by targeting spatially restricted ß2AR signaling in neurons.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report neuronal excitability of ß-blockers carvedilol and alprenolol at clinically relevant nanomolar concentrations. Carvedilol and alprenolol activate ß2AR, which promote G protein signaling and cAMP/PKA activities without action of G protein receptor kinases (GRKs). The cAMP/PKA activities are restricted within the immediate vicinity of activated ß2AR, leading to selectively enhance PKA-dependent phosphorylation and stimulation of endogenous L-type calcium channel (LTCC) but not AMPA receptor in rat hippocampal neurons. Moreover, we have engineered a mutant ß2AR that lacks the catecholamine binding pocket. This mutant is preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant ß2AR in mouse hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which ß-blocker-dependent activation of GPCRs promotes spatially restricted cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Adenylyl cyclase 5-generated cAMP controls cerebral vascular reactivity during diabetic hyperglycemia.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

A Gs-coupled purinergic receptor boosts Ca2+ influx and vascular contractility during diabetic hyperglycemia.

Elevated glucose increases vascular reactivity by promoting L-type CaV1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a Gs-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y11, the only Gs-coupled P2Y receptor, was detected in nanometer proximity to CaV1.2 and PKA. FRET-based experiments revealed that the selective P2Y11 agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y11 inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y11-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y11 in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.