Matthew A. Nystoriak

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca(2+)](i) were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca(2+)](i), and constriction in SAH arterioles. This SAH-induced increase in [Ca(2+)](i) and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca(2+)](i) and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC Ca(V)1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca(2+) influx.

AKAP150 Contributes to Enhanced Vascular Tone by Facilitating Large-Conductance Ca2+-Activated K+ Channel Remodeling in Hyperglycemia and Diabetes Mellitus

Rationale: Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes mellitus may arise from impaired large-conductance Ca2+-activated K+ (BKCa) channel function. The scaffolding protein A-kinase anchoring protein 150 (AKAP150) is a key regulator of calcineurin (CaN), a phosphatase known to modulate the expression of the regulatory BKCa &bgr;1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes mellitus is unknown. Objective: To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa &bgr;1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes mellitus. Methods and Results: We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/nuclear factor of activated T-cells c3 (NFATc3) activation, and resistance artery constriction in hyperglycemic animals on high-fat diet. Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. D-glucose–dependent suppression of BKCa channel &bgr;1 subunits required Ca2+ influx via voltage-gated L-type Ca2+ channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, high-fat diet mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa &bgr;1 subunits and attenuated high-fat diet–induced elevation in arterial blood pressure. Conclusions: Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes mellitus.

Reduced Ca2+ Spark Activity after Subarachnoid Hemorrhage Disables BK Channel Control of Cerebral Artery Tone

Intracellular Ca2+ release events (‘Ca2+ sparks’) and transient activation of large-conductance Ca2+-activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca2+ spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed ∼50% reduction in Ca2+ spark activity, reflecting a decrease in the number of functional Ca2+ spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca2+ spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca2+ spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca2+ sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca2+ signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH.

Cav1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter

L-type voltage-dependent Ca(2+) channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Ca(v)1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Ca(v)1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Ca(v)1.2 splice variants both containing (alpha(1)C(9/9*/10)) and lacking (alpha(1)C(9/10)) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both alpha(1)C(9/9*/10) and alpha(1)C(9/10) was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater alpha(1)C(9/9*/10) expression relative to alpha(1)C(9/10) in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for alpha(1)C(9/9*/10), smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting alpha(1)C(9/9*/10) (alpha(1)C(9/9*/10)-AS) or exon 9 (alpha(1)C-AS), expressed in all Ca(v)1.2 splice variants, by reversible permeabilization and organ cultured for 1-4 days. Treatment with alpha(1)C(9/9*/10)-AS reduced maximal constriction induced by elevated extracellular K(+) ([K(+)](o)) by approximately 75% compared with alpha(1)C(9/9*/10-)sense-treated arteries. Maximal constriction in response to the Ca(2+) ionophore ionomycin and [K(+)](o) EC(50) values were not altered by antisense treatment. Decreases in maximal [K(+)](o)-induced constriction were similar between alpha(1)C(9/9*/10)-AS and alpha(1)C-AS groups (22.7 + or - 9% and 25.6 + or - 4% constriction, respectively). We conclude that although cerebral artery myocytes express both alpha(1)C(9/9*/10) and alpha(1)C(9/10) VDCC splice variants, alpha(1)C(9/9*/10) is functionally dominant in the control of cerebral artery diameter.

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons

β-Adrenergic regulation of the L-type calcium channel Cav1.2 exhibits surprising differences in the heart and brain. How adrenaline activates Cav1.2 The L-type Ca2+ channel Cav1.2 controls heart rate and neuronal excitability. Qian et al. found that enhancement of Cav1.2 channel activity in the brain by β-adrenergic receptor (βAR) signaling required phosphorylation of Ser1928, whereas in the heart, this site was dispensable for βAR-mediated regulation. In contrast to those from wild-type mice, hippocampal neurons from mice, in which Ser1928 of Cav1.2 was mutated to alanine, did not exhibit increased L-type calcium channel activity in response to β-adrenergic stimulation. Phosphorylation of Ser1928 involved signaling through the β2AR, but not through the β1AR, and this phosphorylation event enabled a particular form of long-term potentiation, a process linked to learning and memory. These results were in marked contrast to βAR-mediated regulation of Cav1.2 activity in cardiomyocytes, which involved β1AR and was independent of Ser1928. This differential regulation in the heart and brain implies that tissue-specific therapeutics could be identified. The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)–cAMP–PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.

Relationship between Ca2+ sparklets and sarcoplasmic reticulum Ca2+ load and release in rat cerebral arterial smooth muscle

Ca(+) sparklets are subcellular Ca(2+) signals produced by the opening of sarcolemmal L-type Ca(2+) channels. Ca(2+) sparklet activity varies within the sarcolemma of arterial myocytes. In this study, we examined the relationship between Ca(2+) sparklet activity and sarcoplasmic reticulum (SR) Ca(2+) accumulation and release in cerebral arterial myocytes. Our data indicate that the SR is a vast organelle with multiple regions near the sarcolemma of these cells. Ca(2+) sparklet sites were located at or <0.2 μm from SR-sarcolemmal junctions. We found that while Ca(2+) sparklets increase the rate of SR Ca(2+) refilling in arterial myocytes, their activity did not induce regional variations in SR Ca(2+) content or Ca(2+) spark activity. In arterial myocytes, L-type Ca(2+) channel activity was independent of SR Ca(2+) load. This ruled out a potential feedback mechanism whereby SR Ca(2+) load regulates the activity of these channels. Together, our data suggest a model in which Ca(2+) sparklets contribute Ca(2+) influx into a cytosolic Ca(2+) pool from which sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps Ca(2+) into the SR, indirectly regulating SR function.

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

Targeting a protein complex that phosphorylates the calcium channel CaV1.2 in arteries may prevent vascular pathologies associated with diabetes. How sugar constricts arteries Pathological vasoconstriction compromises blood flow to tissues and contributes to various conditions associated with diabetes, including stroke, hypertension, diabetic neuropathy, and diabetic retinopathy. Nystoriak et al. identified a molecular signaling complex—protein kinase A, a scaffolding protein in the AKAP family, and the L-type calcium channel CaV1.2—in arterial myocytes from mice that mediates the phosphorylation of CaV1.2 and enhances the activity of this channel, leading to vasoconstriction. Exposing isolated arterial myocytes from mice or humans to increased extracellular glucose promoted this modification and increased channel activity. Furthermore, myocytes from diabetic mice or human diabetic subjects had increased amount of phosphorylation of CaV1.2 at Ser1928, which resulted in increased channel activity. Arteries from the diabetic mice exhibited a more pronounced vasoconstriction response to pressure than did arteries from control mice. Knocking in S1928A mutant form of the channel blocked this response. Thus, targeting this CaV1.2 regulatory complex may prevent vascular dysfunction in diabetic patients. Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes.

Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia.

Rationale: Endothelial progenitor cells (EPCs) respond to stromal cell–derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus–induced EPCs dysfunction remains unknown. Objective: To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. Methods and Results: CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3&bgr; phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. Conclusions: Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus–induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3&bgr;/Fyn pathway via increased activity of Nrf2.

Impact of subarachnoid hemorrhage on local and global calcium signaling in cerebral artery myocytes

BACKGROUND Ca2+ signaling mechanisms are crucial for proper regulation of vascular smooth muscle contractility and vessel diameter. In cerebral artery myocytes, a rise in global cytosolic Ca2+ concentration ([Ca2+]i) causes contraction while an increase in local Ca2+ release events from the sarcoplasmic reticulum (Ca2+ sparks) leads to increased activity of large-conductance Ca2+-activated (BK) K+ channels, hyperpolarization and relaxation. Here, we examined the impact of SAH on Ca2+ spark activity and [Ca2+]i in cerebral artery myocytes following SAH. METHODS A rabbit double injection SAH model was used in this study. Five days after the initial intracisternal injection of whole blood, small diameter cerebral arteries were dissected from the brain for study. For simultaneous measurement of arterial wall [Ca2+]i and diameter, vessels were cannulated and loaded with the ratiometric Ca2+ indicator fura-2. For measurement of Ca2+ sparks, individual myocytes were enzymatically isolated from cerebral arteries and loaded with the Ca2+ indicator fluo-4. Sparks were visualized using laser scanning confocal microscopy. RESULTS Arterial wall [Ca2+]i was significantly elevated and greater levels of myogenic tone developed in arteries isolated from SAH animals compared with arteries isolated from healthy animals. The L-type voltage-dependent Ca2+ channel (VDCC) blocker nifedipine attenuated increases in [Ca2+]i and tone in both groups suggesting increased VDCC activity following SAH. Membrane potential measurement using intracellular microelectrodes revealed significant depolarization of vascular smooth muscle following SAH. Further, myocytes from SAH animals exhibited significantly reduced Ca2+ spark frequency (~50%). CONCLUSIONS Our findings suggest decreased Ca2+ spark frequency leads to reduced BK channel activity in cerebral artery myocytes following SAH. This results in membrane potential depolarization, increased VDCC activity, elevated [Ca2+]i and decreased vessel diameter. We propose this mechanism of enhanced cerebral artery myocyte contractility may contribute to decreased cerebral blood flow and development of neurological deficits in SAH patients.

Predominant contribution of L-type Cav1.2 channel stimulation to impaired intracellular calcium and cerebral artery vasoconstriction in diabetic hyperglycemia

ABSTRACT Enhanced L-type Ca2+ channel (LTCC) activity in arterial myocytes contributes to vascular dysfunction during diabetes. Modulation of LTCC activity under hyperglycemic conditions could result from membrane potential-dependent and independent mechanisms. We have demonstrated that elevations in extracellular glucose (HG), similar to hyperglycemic conditions during diabetes, stimulate LTCC activity through phosphorylation of CaV1.2 at serine 1928. Prior studies have also shown that HG can suppress the activity of K+ channels in arterial myocytes, which may contribute to vasoconstriction via membrane depolarization. Here, we used a mathematical model of membrane and Ca2+ dynamics in arterial myocytes to predict the relative roles of LTCC and K+ channel activity in modulating global Ca2+ in response to HG. Our data revealed that abolishing LTCC potentiation normalizes [Ca2+]i, despite the concomitant reduction in K+ currents in response to HG. These results suggest that LTCC stimulation may be the primary mechanism underlying vasoconstriction during hyperglycemia.