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Background: Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality. About 80% of SHS-related deaths are due to cardiovascular causes. Reducing SHS exposure with smoking bans is associated with improved cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a great candidate for combating SHS exposure-induced cardiovascular outcomes. The goals of this study were to determine 1) the time course of environmentally relevant SHS exposure effects on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of a soluble epoxide hydrolase inhibitor (TPPU) can protect against SHS-induced autonomic and cardiovascular dysregulation. Methods: Male C57BL/6J mice (11 wk) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to 12 wks of either filtered air (FA, n=19) or 3 mg/m3 of SHS (6 hr/d, 5 d/wk, n=18), followed by 4 weeks recovery window (SHS substituted with FA). Separate mice were randomly assigned to one of the 3 groups: FA (n=12), FA+TPPU (n=12), and SHS+TPPU (n=22). TPPU (15 mg/l) was given by drinking water during the 12 weeks of exposure period (estimated dose = 3.75 ug/g/day). BP and ECG were recorded continuously for 36 hours every month when mice were not exposed. Data were stratified by 12-hour circadian window: The dark cycle (dark 1) immediately after the 5th day of exposure and the following light and dark (dark 2) cycles. Baroreflex sensitivity (BRS) was determined with the sequence method. Standard time domain heart rate variability (HRV) parameters were obtained from normal-to-normal R-R intervals. Results: SHS-exposed mice had significantly higher physical activity levels (~ 50% higher, p R01 ES025229, T32 HL086350, T32 ES007059, R35ES030443 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vascular complications are major causes of morbidity and mortality in diabetic patients. Recently, our group identified a novel purinergic signaling pathway involving the AKAP5/P2Y11/AC5/PKA/CaV1.2 protein network in arterial myocytes that is activated upon diabetic hyperglycemia leading to vasoconstriction. This purinergic complex is activated by the release of nucleotides (e.g. ATP) to the extracellular space in response to hyperglycemic conditions. Pannexin 1 (Panx1) is a channel that mediates ATP efflux, thus inducing purinergic signaling activation. In this study, we hypothesize that Panx1 is a critical regulator mediating ATP release and increasing cAMP synthesis, CaV1.2 potentiation, and vasoconstriction upon diabetic hyperglycemia. Consistent with this hypothesis, we found that elevated glucose (HG) induced an elevation in extracellular ATP concentrations, and this was reduced in the presence of the Panx1 inhibitor spironolactone (spiro). Panx1 was found in complex with AKAP5, P2Y11, AC5, PKA, and CaV1.2 in arterial myocytes. This protein complex was strengthened upon HG treatment, and spiro prevented this effect. Spiro and genetic ablation of smooth muscle Panx1 (iPanx1sm -/-) blocked cAMP production, CaV1.2 potentiation, sustained vasoconstriction, and in vivo elevations in cerebral artery myogenic tone and reduced blood flow in response to HG. In a mouse model of type 1 diabetes (e.g. STZ), the increased CaV1.2 potentiation and enhanced myogenic tone were prevented in arterial myocytes and arteries from iPanx1sm -/- mice. Altogether, these data suggest that Panx1 is part of the AKAP5/P2Y11/AC5/PKA/CaV1.2 signaling module in arterial myocytes. This Panx1-led complex modulates CaV1.2 activity and vascular reactivity in response to diabetic hyperglycemia. Thus, Panx1 could be a new therapeutic target to treat vascular complications during diabetes. This work was supported by NIH grants R01HL149127, R01HL121059, and R01HL161872 (to MFN). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alzheimer’s disease (AD), the most common form of dementia, affects over 6 million Americans and has devastating emotional and physical consequences for both the patient and surrounding loved ones. Current drug therapies are principally targeted at the main disease-causing peptide in AD, amyloid-beta (Aβ). These drugs reduce symptoms at the onset of the disease but have negligible positive impacts once AD has progressed. Thus, new avenues for therapeutic intervention are imperative. It is currently understood that reduced cerebral blood flow (CBF), due to an increased constriction of cerebral blood vessels, contributes to the pathogenesis of AD. Calcium (Ca2+) influx through voltage-gated L-type calcium CaV1.2 channels is essential for vascular smooth muscle (VSM) contractility, arterial diameter, and CBF. Yet, how Aβ affects CaV1.2 channel activity and vascular function is unclear. Here, we test the hypothesis that Aβ promotes vascular CaV1.2 clustering leading to increased CaV1.2 channel activity and enhanced vascular reactivity. The hypothesis was tested using a multi-scale approach that includes super-resolution microscopy, patch-clamp electrophysiology, and pressure myography using freshly isolated cerebral VSM and arteries from WT mice. Initial experiments found that exposing VSM to 100 nM Aβ for 10 minutes stimulates the redistribution of CaV1.2 into superclusters. This observation was correlated with an increase in CaV1.2 channel activity and the frequency and strength of cooperative events, which are expected to amplify Ca2+ influx and alter vascular contractility. Consistent with this premise, pressurized cerebral arteries exposed to Aβ showed an elevation in myogenic tone. These results suggest that Aβ impacts vascular CaV1.2 spatiotemporal properties to control vascular contractility. Moreover, results may provide new mechanistic insight into a link between impaired CaV1.2 function, VSM dysfunction and AD. NIH R01HL149127 and R01HL149127S1(to MFN). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The L-type calcium channel CaV1.2 (LTCC) is essential for vascular smooth muscle (VSM) contraction and blood pressure control. Increased vascular contractility, blood pressure, and enhanced angiotensin II (angII) signaling are hallmarks of hypertension (HTN). It has been proposed that spatial redistribution of CaV1.2 channels at the sarcolemma of VSM contributes to the potentiation of LTCC function and vascular dysfunction during HTN. However, the mechanisms underlying the spatiotemporal regulation of the CaV1.2 channels during angII signaling and HTN are poorly understood. Here, we hypothesize that CaV1.2 phosphorylation at the S1928 site is a key event mediating increased CaV1.2 channel clustering, which in turn leads to an increase in LTCC activity and vascular reactivity during angII signaling and HTN. We first demonstrated that angII-treated wild-type (WT) mesenteric arteries display increased phosphorylation at the S1928 site. Molecularly, angII redistributed CaV1.2 into larger clusters. This was correlated with elevated VSM whole-cell L-type channel currents, global intracellular Ca2+ and contraction, increased arterial tone ex vivo and in vivo in mesenteric arteries, and altered mesenteric blood flow in arteries from WT mice. These changes were prevented or significantly ameliorated in mesenteric VSM cells and arteries from a knock-in mouse expressing a mutant CaV1.2 in which serine was replaced with alanine at position 1928 (S1928A mouse) and by PKC inhibition. In angII-induced hypertensive mice, increased CaV1.2 cluster size and channel activity, enhanced arterial tone, and mean arterial pressure in WT mice were prevented or significantly reduced in S1928A mice. Altogether, these results suggest a key role for the phosphorylation of vascular CaV1.2 at S1928 in regulating LTCC spatial distribution, activity and vascular function during angII signaling and HTN. Phosphorylation of this single amino acid could be an essential regulatory mechanism that could be exploited for therapeutic intervention. This work was supported by NIH grants R01HL149127, R01HL121059, and R01HL161872 (to MFN). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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 (50-100nM) 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.

The L-type Ca

The cellular mechanisms mediating norepinephrine 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 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 norepinephrine. Indeed, a form of hippocampal 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.

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. We identified 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 pre-associates with CaV1.2 to promote Po and CDI. We 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. 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 wildtype CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI identifying Ca2/CaM as an important mediator of Ca signaling.

RATIONALE: Cardiotoxic β(1) adrenergic receptor (β(1)AR)-CaMKII signaling is a major and critical feature associated with development of heart failure. Synapse-associated protein 97 (SAP97) is a multi-functional scaffold protein that binds directly to the C-terminus of β(1)AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of β(1)AR-SAP97 signalosome and its potential role in chronic cardiotoxic β(1)AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac β(1)AR-SAP97 complex was examined in heart failure. Cardiac specific deletion of SAP97 was developed to examine β(1)AR signaling in ageing mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β(1)AR-SAP97 signaling complex is reduced in heart failure. Cardiac specific deletion of SAP97 yields an ageing-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-dependent association of β(1)AR with arrestin2 and CaMKII and turns on an Epac-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 is necessary to promote agonist-induced dissociation of SAP97 from β(1)AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β(1)AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β(1)AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy.

The L-type Ca2+channel CaV1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1646-1664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV1.2 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 CaV1.2 to defined surface regions and simultaneously boosts its open probability so that CaV1.2 is mostly active when appropriately localized.