Store-operated calcium entry is present in HL-1 cardiomyocytes and contributes to resting calcium.

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) potentiates cardiac contractility via activation of the ryanodine receptor.

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.

Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency.

OBJECTIVE: The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD+/+ and MnSOD-deficient (MnSOD+/-) mice. METHODS AND RESULTS: Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD+/+ (n=9) and young MnSOD+/- (n=6) mice. This response was impaired in old MnSOD+/+ (n=8) mice and old MnSOD+/- mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 micromol/L ACh produced 77+/-3% [mean+/-SE], 77+/-3%, 70+/-4%, and 57+/-4% relaxation in young MnSOD+/+, young MnSOD+/-, old MnSOD+/+, and old MnSOD+/- mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD+/+ and MnSOD+/- mice compared with both groups of young mice (P<0.05). Superoxide levels, as measured using lucigenin-enhanced chemiluminescence, were increased more than 2-fold in old MnSOD+/- mice compared with old MnSOD+/+ and young mice (P<0.05). CONCLUSIONS: These data provide the first direct evidence that MnSOD haploinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.

Evidence for two-pore domain potassium channels in rat cerebral arteries.

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.

Effects of carbon monoxide and heme oxygenase inhibitors in cerebral vessels of rats and mice.

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10(-6) to 10(-4) M). Mouse MCAs, however, dilated by 21 +/- 10% at 10(-4) M CO. Authentic nitric oxide (NO., 10(-10) to 10(-7) M) dilated both rat and mouse MCAs. At 10(-8) M NO., rat vessels dilated by 84 +/- 4%, and at 10(-7) M NO., mouse vessels dilated by 59 +/- 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor delta-aminolevulinic acid (10(-10) to 10(-4) M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 +/- 2% at 3 x 10(-5) M). Importantly, this constriction was unaltered by exogenous CO (10(-4) M) or CO plus 10(-5) M biliverdine (both HO products). In contrast, exogenous CO (10(-4) M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 +/- 1% in response to 10(-5) M CrMP. Magnesium protoporphyrin IX (10(-5) M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.

Vasomotor responses in MnSOD-deficient mice.

MnSOD is the only mammalian isoform of SOD that is necessary for life. MnSOD(-/-) mice die soon after birth, and MnSOD(+/-) mice are more susceptible to oxidative stress than wild-type (WT) mice. In this study, we examined vasomotor function responses in aortas of MnSOD(+/-) mice under normal conditions and during oxidative stress. Under normal conditions, contractions to serotonin (5-HT) and prostaglandin F2alpha (PGF2alpha), relaxation to ACh, and superoxide levels were similar in aortas of WT and MnSOD(+/-) mice. The mitochondrial inhibitor antimycin A reduced contraction to PGF2alpha and impaired relaxation to ACh to a similar extent in aortas of WT and MnSOD(+/-) mice. The Cu/ZnSOD and extracellular SOD inhibitor diethyldithiocarbamate (DDC) paradoxically enhanced contraction to 5-HT and superoxide more in aortas of WT mice than in MnSOD(+/-) mice. DDC impaired relaxation to ACh and reduced total SOD activity similarly in aortas of both genotypes. Tiron, a scavenger of superoxide, normalized contraction to 5-HT, relaxation to ACh, and superoxide levels in DDC-treated aortas of WT and MnSOD(+/-) mice. Hypoxia, which reportedly increases superoxide, reduced contractions to 5-HT and PGF2alpha similarly in aortas of WT and MnSOD(+/-) mice. The vasomotor response to acute hypoxia was similar in both genotypes. In summary, under normal conditions and during acute oxidative stress, vasomotor function is similar in WT and MnSOD(+/-) mice. We speculate that decreased mitochondrial superoxide production may preserve nitric oxide bioavailability during oxidative stress.

Gene transfer of extracellular superoxide dismutase reduces cerebral vasospasm after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Superoxide may play an important role in cerebral vasospasm after subarachnoid hemorrhage (SAH). Our first goal was to determine the effect of gene transfer of extracellular superoxide dismutase (ECSOD) on vasospasm after experimental SAH. Our second goal was to determine whether tissue binding of ECSOD via the heparin-binding domain (HBD) is important for the effect of the enzyme. Thus, we examined effects of gene transfer of ECSOD with the HBD deleted (ECSODDeltaHBD). METHODS: Adenovirus expressing human ECSOD (AdECSOD), ECSODDeltaHBD (AdECSODDeltaHBD), or no transgene (AdBglII) was injected into the cisterna magna of anesthetized rabbits 30 minutes after induction of experimental SAH. Cerebral angiography, an assay for ECSOD activity in cerebrospinal fluid (CSF), and Western blotting for human ECSOD in the basilar artery were performed. RESULTS: Baseline diameter of the basilar artery averaged 0.77+/-0.01 mm (mean+/-SEM) and was similar in all treatment groups. Decreases in diameter of the basilar artery 2 days after SAH were smaller after AdECSOD (11+/-3%; n=10) than after AdBglII (25+/-4%; n=7; P<0.05). ECSOD activity was not detected in CSF before SAH and gene transfer. Of 8 rabbits treated with AdECSOD, in which ECSOD activity in CSF was measured after SAH, 5 animals had detectable ECSOD activity in CSF (46+/-13 U/mL). In these 5 rabbits, the diameter decreased by only 6+/-3%, and ECSOD protein was detected in the basilar artery. After AdECSODDeltaHBD (n=4), despite high levels of ECSOD activity in CSF (91+/-19 U/mL), vessel diameter decreased by 20+/-2%, and no ECSODDeltaHBD protein was detected in the basilar artery. CONCLUSIONS: Gene transfer of ECSOD reduces cerebral vasospasm after experimental SAH. Tissue binding of the enzyme is essential for cerebral vascular effects of ECSOD.