Sofija Jovanović

Role of the WNK-activated SPAK kinase in regulating blood pressure

Mutations within the with‐no‐K(Lys) (WNK) kinases cause Gordons syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na+:Cl− cotransporter (NCC) and Na+:K+:2Cl− cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock‐in mice in which SPAK cannot be activated by WNKs. The SPAK knock‐in animals are viable, but display significantly reduced blood pressure that was salt‐dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na+‐diet, the SPAK knock‐in mice were normokalaemic, but developed mild hypokalaemia when the renin–angiotensin system was activated by a low Na+‐diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda‐Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059).

Deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia.

We employed Cre/loxP technology to generate mPDK1−/− mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6‐phosphofructo‐2‐kinase and production of fructose 2,6‐bisphosphate, in the hearts of mPDK1−/− mice, consistent with PDK1 mediating these processes. All mPDK1−/− mice died suddenly between 5 and 11 weeks of age. The mPDK1−/− animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1−/− muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1−/− mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single‐cell assay we found that cardiomyocytes from mPDK1−/− mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a deficiency of the PDK1 pathway might contribute to development of cardiac disease in humans.

AMP-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K+ channels

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP‐activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the α2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC‐1, a mitochondrial membrane potential‐sensitive dye, and laser confocal microscopy. In contrast, experiments with di‐8‐ANEPPS, a sarcolemmal‐potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning‐induced shortening of the action membrane potential. The preconditioning‐induced activation of sarcolemmal KATP channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal KATP channels opening, inhibited preconditioning‐induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning‐induced recruitment of sarcolemmal KATP channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal KATP channels without being a part of signaling pathway that regulates mitochondrial membrane potential. J. Cell. Physiol. 210: 224–236, 2007.

M-LDH serves as a sarcolemmal KATP channel subunit essential for cell protection against ischemia

ATP‐sensitive K+ (KATP) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K+ channel core, and SUR2A, a regulatory subunit implicated in ligand‐dependent regulation of channel gating. Here, we have shown that the muscle form (M‐LDH), but not heart form (H‐LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal KATP channel by interacting with the Kir6.2 subunit via its N‐terminus and with the SUR2A subunit via its C‐terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M‐LDH in the channel protein complex was required for opening of KATP channels during ischemia and ischemia‐resistant cellular phenotype. We conclude that M‐LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the KATP channels activity that is essential for cell protection against ischemia.

Hypoxia-induced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal KATP channels.

The opening of sarcolemmal and mitochondrial ATP‐sensitive K+ (KATP) channels in the heart is believed to mediate ischemic preconditioning, a phenomenon whereby brief periods of ischemia/reperfusion protect the heart against myocardial infarction. Here, we have applied digital epifluorescent microscopy, immunoprecipitation and Western blotting, perforated patch clamp electrophysiology, and immunofluorescence/laser confocal microscopy to examine the involvement of KATP channels in cardioprotection afforded by preconditioning. We have shown that adult, stimulated‐to‐beat, guinea‐pig cardiomyocytes survived in sustained hypoxia for ~17 min. An episode of 5‐min‐long hypoxia/5‐min‐long reoxygenation before sustained hypoxia dramatically increased the duration of cellular survival. Experiments with different antagonists of KATP channels, applied at different times during the experimental protocol, suggested that the opening of sarcolemmal KATP channels at the beginning of sustained hypoxia mediate preconditioning. This conclusion was supported by perforated patch clamp experiments that revealed activation of sarcolemmal KATP channels by preconditioning. Immunoprecipitation and Western blotting as well as immunofluorescence and laser confocal microscopy showed that the preconditioning is associated with the increase in KATP channel proteins in sarcolemma. Inhibition of trafficking of KATP channel subunits prevented preconditioning without affecting sensitivity of cardiomyocytes to hypoxia in the absence of preconditioning. We conclude that the preconditioning is mediated by the activation and trafficking of sarcolemmal KATP channels.

Overexpression of SUR2A generates a cardiac phenotype resistant to ischemia

ATP‐sensitive K+ (KATP) channels are present in the sarcolemma of cardiac myocytes where they link membrane excitability with the cellular bioen‐ergetic state. These channels are in vivo composed of Kir6.2, a pore‐forming subunit, SUR2A, a regulatory subunit, and at least four accessory proteins. In the present study, real‐time RT‐PCR has demonstrated that of all six sarcolemmal KATP channel‐forming proteins, SUR2A was probably the least expressed protein. We have generated mice where the SUR2A was under the control of a cytomegalovirus promoter, a promoter that is more efficient than the native promoter. These mice had an increase in SUR2A mRNA/protein levels in the heart whereas levels of mRNAs of other channel‐forming proteins were not affected at all. Imunopre‐cipitation/Western blot and patch clamp electrophysi‐ology has shown an increase in KATP channel numbers in the sarcolemma of transgenic mice. Cardiomyocytes from transgenic mice responded to hypoxia with shortening of action membrane potential and were significantly more resistant to this insult than cardiomyocytes from the wild‐type. The size of myocardial infarction in response to ischemia‐reperfusion was much smaller in hearts from transgenic mice compared to those in wild‐type. We conclude that overexpression of SUR2A generates cardiac phenotype resistant to hypoxia/isch‐emia/reperfusion injury due at least in part to increase in levels of sarcolemmal KATP channels.—Du, Q., Jovanovic, S., Clelland, A., Sukhodub, A., Budas, G., Phelan, K., Murray‐Tait, V., Malone, L., Jovanovic, A. Overexpression of sur2a generates cardiac phenotype resistant to ischemia. FASEBJ. 20, 1131–1141 (2006)

Glyceraldehyde 3‐phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal KATP channel

Cardiac sarcolemmal ATP‐sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal KATP‐channel protein complex. A polypeptide of ∼42 kDa was immunoprecipitated with an anti‐SUR2A antibody from guinea‐pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti‐Kir6.2, anti‐SUR2A and anti‐GAPDH antibodies showed that GAPDH is a part of the sarcolemmal KATP‐channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two‐hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch‐clamp electrophysiology showed that GAPDH regulates KATP‐channel activity irrespective of high intracellular ATP, by producing 1,3‐bisphosphoglycerate, a KATP‐channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal KATP‐channel protein complex, where it couples glycolysis with the KATP‐channel activity.

M-LDH physically associated with sarcolemmal KATP channels mediates cytoprotection in heart embryonic H9C2 cells

Muscle form of lactate dehydrogenase (M-LDH) physically associate with KATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K+ (KATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the KATP channel protein complex. The K+ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes KATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal KATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.

Effect of the vascular endothelium on noradrenaline-induced contractions in non-pregnant and pregnant guinea-pig uterine arteries

1 The effect of pregnancy on noradrenaline‐mediated contraction of guinea‐pig uterine artery rings with both intact and denuded endothelium was investigated. 2 Noradrenaline (25 nm − 100 μm) induced concentration‐dependent contraction of non‐pregnant and pregnant guinea‐pig uterine arterial rings with intact endothelium with similar pD2 and maximal response values (non‐pregnant: pD2 = 5.85 ± 0.02, maximal response = 121 ± 8.2%; pregnant: pD2 = 5.81 ± 0.04, maximal response = 122 ± 9.1%). Removal of endothelium did not affect noradrenaline‐induced contractions in non‐pregnant guinea‐pig uterine artery (pD2 = 5.97 ± 0.02, maximal response = 119 ± 8.6%). In contrast, in arteries from pregnant guinea‐pigs, removal of endothelium shifted concentration‐response curve for noradrenaline to the left, without affecting maximal response value (pD2 = 6.36 ± 0.03, maximal response = 120 ± 9.0%). 3 The pKA values for noradrenaline were: 5.76 ± 0.09 and 5.82 ± 0.10 for non‐pregnant guinea‐pig uterine artery with intact and denuded endothelium, respectively and 5.74 ± 0.09 and 5.72 ± 0.07 for pregnant guinea‐pig uterine artery with intact and denuded endothelium, respectively. 4 The receptor occupancy‐response relationship for noradrenaline was linear for all types of vessels, except for pregnant guinea‐pig uterine artery with denuded endothelium, since half‐maximal response to noradrenaline was obtained with 44.8 ± 6.9% (non‐pregnant guinea‐pig uterine artery with intact endothelium), 43.3 ± 6.1% (non‐pregnant guinea‐pig uterine artery with denuded endothelium) and 44.3 ± 6.3% (pregnant guinea‐pig uterine artery with intact endothelium) receptor occupancy. In pregnant guinea‐pig uterine artery with denuded endothelium, occupancy‐response relationship for noradrenaline was non‐linear since half‐maximal response to noradrenaline was obtained with 19.7 ± 3.3% receptor occupancy. 5 NG‐monomethyl‐l‐arginine (100 μm) and indomethacin (10 μm) did not affect concentration‐response curve for noradrenaline in guinea‐pig uterine arteries, regardless of pregnancy status or endothelial condition. 6 In quiescent preparations, the α‐adrenoceptor antagonists, prazosin (5–50 nm) and yohimbine (1–10 μm) produced parallel rightward shifts of the curves for noradrenaline and the slopes of the Schild plots were not significantly different from unity. The plots constrained to a slope of unity gave the following—log KB values: prazosin vs. yohimbine 8.78 ± 0.03 vs. 6.41 ± 0.02 for non‐pregnant guinea‐pig uterine artery with intact endothelium, 8.95 ± 0.03 vs. 6.34 ± 0.02 for non‐pregnant guinea‐pig uterine artery with denuded endothelium, 8.91 ± 0.01 vs. 6.44 ± 0.03 for pregnant guinea‐pig uterine artery with intact endothelium and 9.07 ± 0.01 vs. 6.52 ± 0.03 for pregnant guinea‐pig uterine artery with denuded endothelium. 7 It is concluded that initially there is no difference in noradrenaline action between uterine arteries from non‐pregnant and pregnant guinea‐pigs, but after removal of the endothelium the pregnant guinea‐pig uterine artery is more sensitive to noradrenaline, which is related to increased receptor reserve for noradrenaline in this tissue. It is probable that relaxing factor derived from the endothelium mediates this effect, but it is unlikely to be nitric oxide or prostacyclin. Antagonist affinities and affinity of noradrenaline itself suggests that an identical subtype of α‐adrenoceptor, probably the α1 subtype, is involved in the noradrenaline‐induced contraction of non‐pregnant and pregnant guinea‐pig uterine artery with or without endothelium.

Nicotinamide-rich diet protects the heart against ischaemia―reperfusion in mice: A crucial role for cardiac SUR2A

Graphical abstract