Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's 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).

Discovered in the cardiac sarcolemma, ATP-sensitive K+(KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATPchannel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of −180 ± 15 mV, K+ channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome cand adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.

. Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPK␣2 but not AMPK␣1. Am J Physiol Endocrinol Metab 290: E780 -E788, 2006. First published December 6, 2005 doi:10.1152/ajpendo.00443.2005.-Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPK␣1/␣2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPK␣2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPK␣2 at the site of LKB1 phosphorylation (Thr 172 ) or phosphorylation of acetylCoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPK␣2, significant basal activity of AMPK␣1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPK␣1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPK␣2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPK␣1 in cardiac muscle. cellular energy metabolism; hypoxia; cardiovascular physiology; AMP-activated protein kinase THE AMP-ACTIVATED PROTEIN KINASE (AMPK) is switched on by increases in levels of AMP, resulting from reduced availability of ATP. AMPK functions to restore ATP concentrations by stimulating energy-producing processes, such as nutrient uptake and oxidation of fatty acids, and inhibiting unnecessary energy-consuming processes, such as protein synthesis and cell proliferation (reviewed in Refs. 8, 11). AMPK is a heterotrimeric complex comprising a catalytic ␣-subunit and regulatory ␤-and ␥-subunits. AMP activates the AMPK complex by binding to the Bateman domains made up of pairs of CBS sequences located on the ␥-subunit and by stimulating the phosphorylation of Thr172 in the T-loop of both mammalian AMPK␣ catalytic subunits, termed AMPK␣1 and AMPK␣2.

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 de®ciency of the PDK1 pathway might contribute to development of cardiac disease in humans. Keywords: cardiac muscle/heart failure/hypoxia/PDK1/ PI 3-kinase/PKB/Akt IntroductionHormones and growth factors trigger the activation of members of a group of protein kinases including protein kinase B (PKB) and p70 ribosomal S6K (S6K), which belong to the AGC family of protein kinases (Brazil and Hemmings, 2001;Lawlor and Alessi, 2001; Newton, 2002). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating these AGC kinase members by phosphorylating these enzymes at their activation loop (Toker and Newton, 2000;Alessi, 2001). Much research has shown that the PDK1/AGC kinasesignalling pathway regulates diverse cellular processes, such as those relevant to cell survival, proliferation and metabolic responses to insulin. Misregulation of AGC kinase members is thought to contribute to many diseases. For example, hyperactivation of this pathway is implicated in inducing cardiac hypertrophy (Sugden, 2001) and promoting the survival and proliferation of a signi®cant number of cancers (Simpson and Parsons, 2001). A de®ciency in the activation of AGC kinases may be a primary cause of the insulin-resistant form of diabetes (Saltiel and Kahn, 2001), as well as neuronal cell death following a stroke (Wick et al., 2002).The activation of PKB and S6K isoforms by insulin and growth factors, as well as being dependent on PDK1, requires the prior activation of the phosphoinositide 3-kinase (PI 3-kinase) (Vanhaesebroeck et al., 2001). This produces the second messenger, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ], which binds to the pleckstrin homology domains of PKB and PDK1, recruiting these enzymes to the plasma membrane where PKB is activated by phosphorylation of its activation-loop residue (Thr308 in PKBa) by PDK1 (Brazil and Hemmings, 2001;Scheid and Woodgett, 2001). PtdIns(3,4,5)P 3 also stimulates the phosphorylation of PKB at its hydrophobic motif res...