Pervious biochemical and hemodymanic studies have highlighted the important role of εPKC in cardioprotection during ischemic preconditioning. However, little is known about the electrophysiological consequences of εPKC modulation in ischemic hearts. Membrane permeable peptide εPKC selective activator and inhibitor were used to investigate the role of εPKC modulation in reperfusion arrhythmias.
Protein transduction domain from HIV- TAT was used as a carrier for peptide delivery into intact Langendorff perfused guinea pig hearts. Action potentials were imaged and mapped (124 sites) using optical techniques and surface ECG was continuously recorded. Hearts were exposed to 30 min stabilization period, 15 min of no-flow ischemia, followed by 20 min reperfusion. Peptides (0.5 μM) were infused as follows: a) control (vehicle-TAT peptide; TAT-scrambled ψεRACK peptide); b) εPKC agonist (TAT-ψεRACK); c) εPKC antagonist (TAT-εV1).
Hearts treated with εPKC agonist ψεRACK had reduced incidence of ventricular tachycardia (VT, 64%) and fibrillation (VF, 50%) compared to control (VT, 80%, p<0.05) and (VF, 70%, P<0.05). However, the highest incidence of VT (100%, P<0.05) and VF (80%) occurred in hearts treated with εPKC antagonist peptide εV1 compared to control and to εPKC agonist ψεRACK. Interestingly, at 20 min reperfusion, 100% of hearts treated with εPKC agonist ψεRACK exhibited complete recovery of action potentials compared to 40% (p<0.05) of hearts treated with εPKC antagonist peptide, εV1 and 65% (P<0.5) of hearts in control. At 20 min reperfusion, maps of action potential duration from εPKC agonist ψεRACK showed minimal dispersion (48.2±9 ms) compared to exacerbated dispersion (115.4±42 ms, P<0.05) in εPKC antagonist and control (67±20 ms, P<0.05). VT/VF and dispersion from hearts treated with scrambled agonist or antagonist peptides were similar to control.
the results demonstrate that εPKC activation by ψεRACK peptide protects intact hearts from reperfusion arrhythmias and affords better recovery. On the other hand, inhibition of εPKC increased the incidence of arrhythmias and worsened recovery compared to controls. The results carry significant therapeutic implications for the treatment of acute ischemic heart disease by preconditioning-mimicking agents.
cardiac electrophysiology; Protein Kinase C; reperfusion arrhythmia; optical mapping
The balance between endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) and reactive oxygen species (ROS) production determines endothelial-mediated vascular homeostasis. Activation of protein kinase C (PKC) has been linked to imbalance of the eNOS/ROS system, which leads to endothelial dysfunction. We previously found that selective inhibition of delta PKC (δPKC) or selective activation of epsilon PKC (εPKC) reduces oxidative damage in the heart following myocardial infarction. In this study we determined the effect of these PKC isozymes in the survival of coronary endothelial cells (CVEC). We demonstrate here that serum deprivation of CVEC increased eNOS-mediated ROS levels, activated caspase-3, reduced Akt phosphorylation and cell number. Treatment with either the δPKC inhibitor, δV1-1, or the εPKC activator, ψεRACK, inhibited these effects, restoring cell survival through inhibition of eNOS activity. The decrease in eNOS activity coincided with specific de-phosphorylation of eNOS at Ser1179, and eNOS phosphorylation at Thr497 and Ser116. Furthermore, δV1-1 or ψεRACK induced physical association of eNOS with caveolin-1, an additional marker of eNOS inhibition, and restored Akt activation by inhibiting its nitration. Together our data demonstrate that 1) in endothelial dysfunction, ROS and reactive nitrogen species (RNS) formation result from uncontrolled eNOS activity mediated by activation of δPKC or inhibition of εPKC 2) inhibition of δPKC or activation of εePKC correct the perturbed phosphorylation state of eNOS, thus increasing cell survival. Since endothelial health ensures better tissue perfusion and oxygenation, treatment with a δPKC inhibitor and/or an εPKC activator in diseases of endothelial dysfunction should be considered.
The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, δ and εPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.
Methods and results
Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished δPKC translocation by 3.8-fold and increased εPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of δPKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of εPKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of εPKC during IPC restored δPKC levels at the mitochondria while decreasing εPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.
Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, εPKC.
Cardioprotection; Ischaemia/reperfusion; Apoptosis; Proteasome; PKC; Ischaemic preconditioning
Activation of ε protein kinase C (εPKC) protects hearts from ischemic injury. However, some of the mechanism(s) of εPKC mediated cardioprotection are still unclear. Identification of εPKC targets may aid to elucidate εPKC–mediated cardioprotective mechanisms. Previous studies, using a combination of εPKC transgenic mice and difference in gel electrophoresis (DIGE), identified a number of proteins involved in glucose metabolism, whose expression was modified by εPKC. These studies, were accompanied by metabolomic analysis, and suggested that increased glucose oxidation may be responsible for the cardioprotective effect of εPKC. However, whether these εPKC-mediated alterations were due to differences in protein expression or phosphorylation was not determined.
Methods and Results
Here, we used an εPKC-specific activator peptide, ψεRACK, in combination with phosphoproteomics to identify εPKC targets, and identified proteins whose phosphorylation was altered by selective activation of εPKC most of the identified proteins were mitochondrial proteins and analysis of the mitochondrial phosphoproteome, led to the identification of 55 spots, corresponding to 37 individual proteins, which were exclusively phosphorylated, in the presence of ψεRACK. The majority of the proteins identified were proteins involved in glucose and lipid metabolism, components of the respiratory chain as well as mitochondrial heat shock proteins.
In summary the protective effect of εPKC during ischemia involves phosphorylation of several mitochondrial proteins involved in glucose, lipid metabolism and oxidative phosphorylation. Regulation of these metabolic pathways by εPKC phosphorylation may lead to εPKC-mediated cardioprotection induced by ψεRACK.
εPKC; ischemia; phosphorylation; mitochondria
Protein Kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that εPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of εPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of εPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that εPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of εPKC activation during ischemia on cerebral blood flow (CBF). We found that ψε-Receptors for activated C kinase (RACK), a εPKC-selective peptide activator, injected intravenously 30 minutes before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψεRACK compared to Tat peptide (vehicle). Our results suggest that εPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, εPKC may be one of the treatment modalities against ischemic injury.
Ischemia; epsilon Protein Kinase C; Cerebral Blood Flow; Neuroprotection
Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of δPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using δV1-1 — a selective peptide inhibitor of δPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or δV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the δV1-1–treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with δV1-1 was also associated with decreased δPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with δV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that δPKC may specifically act to dysregulate tight junction components. Together, these results suggest that δPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.
In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of ε protein kinase C (εPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of εPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal εPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of εPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific εPKC activating peptide, tat-ψεRACK (tat-ψε-receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that εPKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal εPKC. Activation of εPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, εPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the εPKC signaling pathway.
cerebral ischemia; phosphorylation; electron transport chain; neuroprotection; cell death; hippocampus
Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (εPKC). A major goal in our laboratory is to define the cellular targets by which εPKC confers protection. We tested the hypothesis that εPKC targets the mitochondrial
KATP+ channel (
mtKATP+) after IPC. Our results demonstrated a rapid translocation of εPKC to rat hippocampal mitochondria after IPC. Because in other tissues εPKC targets
mtKATP+ channels, but its presence in brain mitochondria is controversial, we determined the presence of the
KATP+ channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether
mtKATP+ channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the
KATP+ channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or εPKC agonist-induced preconditioning. Blockade of
mtKATP+ channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or εPKC preconditioning. Both ischemic andεPKC agonist-mediated preconditioning resulted in phosphorylation of the
mtKATP+ channel subunit Kir6.2. After IPC, selective inhibition of εPKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of εPKC or its downstream effectors. Our results support the hypothesis that the brain
mtKATP+ channel is an important target of IPC and the signal transduction pathways initiated by εPKC.
ischemic tolerance; diazoxide; protein kinase C; organotypic slice culture; cell death; signal transduction
Cerebral ischemia causes cerebral blood flow (CBF) derangements resulting in neuronal damage by enhanced protein kinase C delta (δPKC) levels leading to hippocampal and cortical neuronal death after ischemia. Contrarily, activation of εPKC mediates ischemic tolerance by decreasing vascular tone providing neuroprotection. However, whether part of this protection is due to the role of differential isozymes of PKCs on CBF following cerebral ischemia remains poorly understood. Rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after two-vessel occlusion plus hypotension. In an asphyxial cardiac arrest (ACA) model, rats treated with δ V1-1 (pre- and postischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 and cortical neuronal death 7 days after ACA. On the contrary, εPKC-selective peptide activator, conferred neuroprotection in the CA1 region of the rat hippocampus 30 min before induction of global cerebral ischemia and decreased regional CBF during the reperfusion phase. These opposing effects of δ v. εPKC suggest a possible therapeutic potential by modulating CBF preventing neuronal damage after cerebral ischemia.
Previously we found that neural responses to ethanol and the dopamine D2 receptor (D2) agonist NPA involve both epsilon protein kinase C (εPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of εPKC and the relationship to PKA activation. In the present study, we used a new εPKC antibody, 14E6, that selectively recognizes active εPKC when not bound to its anchoring protein εRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators, to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated εPKC and also induced translocation of both εPKC and its anchoring protein, εRACK to a new cytosolic site. The selective εPKC agonist, pseudo-εRACK, activated εPKC but did not cause translocation of the εPKC/εRACK complex to the cytosol. These data suggest a step-wise activation and translocation of εPKC following NPA or ethanol treatment where εPKC first translocates and binds to its RACK and subsequently the εPKC/εRACK complex translocates to a new subcellular site. Direct activation of PKA by Sp-cAMPS, PGE1 or the adenosine A2A receptor is sufficient to cause εPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between εPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.
The cardioprotective effects of moderate alcohol consumption have been well documented in animal models and in humans. Protection afforded against ischemia and reperfusion injury (I/R) proceeds through an ischemic preconditioning-like mechanism involving the activation of epsilon protein kinase C (εPKC) and is dependent on the time and duration of ethanol treatment. However, the substrates of εPKC and the molecular mechanisms by which the enzyme protects the heart from oxidative damage induced by I/R are not fully described. Using an open-chest model of acute myocardial infarction in vivo, we find that intraperitoneal injection of ethanol (0.5 g/kg) 60 minutes prior to (but not 15 minutes prior to) a 30-minute transient ligation of the left anterior descending coronary artery reduced I/R-mediated injury by 57% (measured as a decrease of creatine phosphokinase release into the blood). Only under cardioprotective conditions, ethanol treatment resulted in the translocation of εPKC to cardiac mitochondria, where the enzyme bound aldehyde dehydrogenase-2 (ALDH2). ALDH2 is an intra-mitochondrial enzyme involved in the detoxification of toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE) and 4-HNE mediates oxidative damage, at least in part, by covalently modifying and inactivating proteins (by forming 4-HNE adducts). In hearts subjected to I/R after ethanol treatment, the levels of 4-HNE protein adducts were lower and JNK1/2 and ERK1/2 activities were diminished relative to the hearts from rats subjected to I/R in the absence of ethanol. Together, this work provides an insight into the mitochondrial-dependent basis of ethanol-induced and εPKC-mediated protection from cardiac ischemia, in vivo.
During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 minutes of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel, prophylactic agents to induce ischemic tolerance in patients at risk of stroke or cardiac arrest. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared to rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na+/K+-ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1–2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1–2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS.
brain ischemia; heart arrest; tolerance; neuroprotection
A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
infarct size; remote; incision; protein kinase C; gamma; epsilon
To investigate the mechanism responsible for the increased cardiac stiffness associated with hypertensive heart failure in Dahl salt-sensitive (DS) rats and the effects of treatment with the combination of a calcium channel blocker [azelnidipine (AZE)] and angiotensin II type 1 receptor blocker [olmesartan (OLM)].
DS rats fed a high-salt diet from 7 weeks of age were treated (or not) from 12 to 19 weeks of age with the vasodilator hydralazine, OLM plus AZE, or the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. Rats fed a low-salt diet served as controls.
Treatment with OLM plus AZE attenuated changes in the expression of collagen isoforms and a decrease in the ratio of elastin to collagen in the left ventricle and prevented the increase in myocardial stiffness and diastolic dysfunction in DS rats in a manner independent of the hypotensive effect of these drugs. Such treatment also inhibited the expression and activation of elastolytic proteases (including cathepsins S and K and metalloproteinases-2, -9, and -12), NADPH oxidase–dependent superoxide production, and inflammatory changes in the failing myocardium. All these effects were mimicked by treatment with apocynin.
The changes in collagen isoform expression and the decrease in the elastin to collagen ratio in the failing myocardium likely account for the increase in diastolic stiffness in this model of hypertensive heart failure. Administration of angiotensin receptor and calcium channel blockers prevented these changes in a manner independent of the hypotensive effect of these drugs by inhibiting the increase in elastolytic activity induced by activation of NADPH oxidase.
cardiac stiffness; heart failure; collagen; elastin; oxidative stress; elastase
We previously reported that the angiotensin II type 1 (AT1) receptor blocker (ARB) olmesartan has two important interactions to evoke inverse agonism (IA). We refer to these interactions as the “double-chain domain (DCD).” Since the clinical pharmacotherapeutic relevance of olmesartan is still unclear, we examined these effects in rats and humans. We analyzed the effects at an advanced stage of renal insufficiency in Dahl salt-sensitive hypertensive rats (Study 1). Rats were fed a high-salt diet from age 9 weeks and arbitrarily assigned to three treatment regimens at age 16 to 21 weeks: olmesartan (2 mg/kg/day) with DCD, a compound related to olmesartan without DCD (6 mg/kg/day, R-239470) or placebo. We also compared the depressor effects of olmesartan to those of other ARBs in patients with essential hypertension (Study 2). Thirty essential hypertensive outpatients who had been receiving ARBs other than olmesartan were recruited for this study. Our protocol was approved by the hospital ethics committee and informed consent was obtained from all patients 12 weeks prior to switching from ARBs other than olmesartan to olmesartan. In Study 1, olmesartan induced a more prominent suppression of the ratio of urinary protein excretion to creatinine at age 21 weeks without lowering blood pressure among the three groups. In Study 2, the depressor effect of olmesartan was significantly stronger than those of other ARBs, which do not contain the DCD. These additive effects by olmesartan may be due to DCD.
double-chain domain; angiotensin II type 1 receptor blocker; olmesartan; renal dysfunction; Dahl salt-sensitive rat
One major precursor of carbonyl stress, methylglyoxal (MG), is elevated in the plasma of chronic kidney disease (CKD) patients, and this precursor contributes to the progression of vascular injury, hypertension and renal injury in diabetic nephropathy patients. This molecule induces salt-sensitive hypertension via a reactive oxygen species-mediated pathway. We examined the role of MG in the pathogenesis of hypertension and cardio–renal injury in Dahl salt-sensitive (Dahl S) rats, which is a rat model of CKD. Nine-week-old Dahl S rats were fed a 1% NaCl diet, and 1% MG was added to their drinking water for up to 12 weeks. Blood pressure and cardio–renal injuries were compared with rats treated with tap water alone. The angiotensin II receptor blocker (ARB), candesartan (10 mg kg−1 day−1), was administered to MG Dahl S rats to determine the impact of this drug on the pathogenesis of MG-induced CKD. A progressive increase in systolic blood pressure was observed (123±1–148±5 mm Hg) after 12 weeks of MG administration. MG administration significantly increased urinary albumin excretion, glomerular sclerosis, tubular injury, myocardial collagen content and cardiac perivascular fibrosis. MG also enhanced the renal expression of Nɛ-carboxyethyl-lysine (an advanced glycation end product), 8-hydroxydeoxyguanosine (a marker of oxidative stress), macrophage (ED-1) positive cells (a marker of inflammation) and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity. Candesartan treatment for 4 weeks significantly reduced these parameters. These results suggest that MG-induced hypertension and cardio–renal injury and increased inflammation and carbonyl and oxidative stress, which were partially preventable by an ARB.
carbonyl stress; chronic kidney disease; methylglyoxal; salt sensitivity
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted over-expression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of heart failure in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of heart failure over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT47-57 alone) (3mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT47-57 alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, hematoxylin-eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodeling mediated by the TGF-SMAD signaling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodeling.
Protein kinase; PKCβII inhibitor peptide; cardiac remodeling; heart failure; myocardial infarction; mast cells, myocardial fibrosis; inflammation
Enhanced production of angiotensin II and excessive release of norepinephrine in the ischemic heart are major causes of arrhythmias and sudden cardiac death. Mast cell-dependent mechanisms are pivotal in the local formation of angiotensin II and modulation of norepinephrine release in cardiac pathophysiology. Cardiac mast cells increase in number in myocardial ischemia and are located in close proximity to sympathetic neurons expressing angiotensin AT1- and histamine H3-receptors. Once activated, cardiac mast cells release a host of potent pro-inflammatory and pro-fibrotic cytokines, chemokines, preformed mediators (e.g., histamine) and proteases (e.g., renin). In myocardial ischemia, angiotensin II (formed locally from mast cell-derived renin) and histamine (also released from local mast cells) respectively activate AT1- and H3-receptors on sympathetic nerve endings. Stimulation of angiotensin AT1-receptors is arrhythmogenic whereas H3-receptor activation is cardioprotective. It is likely that in ischemia/reperfusion the balance may be tipped toward the deleterious effects of mast cell renin, as demonstrated in mast cell-deficient mice, lacking mast cell renin and histamine in the heart. In these mice, no ventricular fibrillation occurs at reperfusion following ischemia, as opposed to wild-type hearts which all fibrillate. Preventing mast cell degranulation in the heart and inhibiting the activation of a local reninangiotensin system, hence abolishing its detrimental effects on cardiac rhythmicity, appears to be more significant than the loss of histamine-induced cardioprotection. This suggests that therapeutic targets in the treatment of myocardial ischemia, and potentially congestive heart failure and hypertension, should include prevention of mast cell degranulation, mast cell renin inhibition, local ACE inhibition, ANG II antagonism and H3-receptor activation.
arrhythmias; cardiac renin-angiotensin system; histamine H3-receptors; mast-cell renin; myocardial ischemia-reperfusion; norepinephrine; sensory and sympathetic nerve endings; sodium-proton exchanger
Extracellular matrix (ECM) remodeling is the hallmark of hypertensive nephropathy. Uncontrolled proteolytic activity due to an imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases (MMPs/TIMPs) has been implicated in renovascular fibrosis. We hypothesized that inhibition of MMPs will reduce excess ECM deposition and modulate autophagy to attenuate hypertension. Dahl salt-sensitive (Dahl/SS) and Lewis rats were fed on high salt diet and treated without or with 1.2 mg/kg b.w. of GM6001 (MMP inhibitor) by intraperitoneal injection on alternate days for 4 weeks. Blood pressure (BP), renal cortical blood flow, vascular density, collagen, elastin, and MMPs/TIMPs were measured. GM6001 treatment significantly reduced mean BP in hypertensive Dahl/SS rats. Renal resistive index (RI) was increased in hypertensive Dahl/SS rats and Doppler flowmetry showed reduced cortical perfusion. Barium angiography demonstrated a reduction in terminal branches of renal vasculature. Inhibition of MMPs by GM6001 resulted in a significant improvement in all the parameters including renal function. In hypertensive Dahl/SS rats, protein levels of MMP-9, -2, and -13 were increased including the activity of MMP-9 and -2; TIMP-1 and -2 levels were increased whereas TIMP-3 levels were similar to Lewis controls. Administration of GM6001 reduced the activity of MMPs and increased the levels of TIMP-1, -2, and -3. MMP inhibition reduced type 1 collagen deposition and increased elastin in the intrarenal vessels indicating reduced fibrosis. Autophagy markers were decreased in hypertensive Dahl/SS rats and GM6001 treatment enhanced their levels. We conclude that MMP inhibition (GM6001) reduces adverse renovascular remodeling in hypertension by modulating ECM turnover and stimulating autophagy.
Autophagy; collagen; Dahl salt sensitive; extracellular matrix; fibrosis; vascular density
Extracellular matrix (ECM) remodeling is the hallmark of hypertensive nephropathy. Uncontrolled proteolytic activity due to an imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases (MMPs/TIMPs) has been implicated in renovascular fibrosis. We hypothesized that inhibition of MMPs will reduce excess ECM deposition and modulate autophagy to attenuate hypertension. Dahl-salt sensitive (Dahl/SS) and Lewis rats were fed on high salt diet and treated without or with 1.2 mg/kg b.w. of GM6001 (MMP inhibitor) by intra-peritoneal injection on alternate days for 4 weeks. Blood pressure, renal cortical blood flow, vascular density, collagen, elastin and MMPs/TIMPs were measured. GM6001 treatment significantly reduced mean blood pressure in hypertensive Dahl/SS rats. Renal resistive index was increased in hypertensive Dahl/SS rats and Doppler flowmetry showed reduced cortical perfusion. Barium angiography demonstrated a reduction in terminal branches of renal vasculature. Inhibition of MMPs by GM6001 resulted in a significant improvement in all the parameters including renal function. In hypertensive Dahl/SS rats, protein levels of MMP-9, -2 and -13 were increased including the activity of MMP-9 and -2; TIMP-1 and -2 levels were increased whereas, TIMP-3 levels were similar to Lewis controls. Administration of GM6001 reduced the activity of MMPs and increased the levels of TIMP-1, -2 and -3. MMP inhibition reduced type -1 collagen deposition and increased elastin in the intra-renal vessels indicating reduced fibrosis. Autophagy markers were decreased in hypertensive Dahl/SS rats and GM6001 treatment enhanced their levels. We conclude that MMP inhibition (GM6001) reduces adverse renovascular remodeling in hypertension by modulating ECM turnover and stimulating autophagy.
Dahl-salt sensitive; fibrosis; autophagy; extracellular matrix; collagen; vascular density
The cellular response to excessive endoplasmic reticulum (ER) stress includes the activation of signaling pathways, which lead to apoptotic cell death. Here we show that treatment of cultured cardiac myocytes with tunicamycin, an agent that induces ER stress, causes the rapid translocation of δPKC to the ER. We further demonstrate that inhibition of δPKC using the δPKC-specific antagonist peptide, δV1-1, reduces tunicamycin-induced apoptotic cell death, and inhibits expression of specific ER stress response markers such as CHOP, GRP78 and phosphorylation of JNK. The physiological importance of δPKC in this event is further supported by our findings that the ER stress response is also induced in hearts subjected to ischemia and reperfusion injury and that this response also involves δPKC translocation to the ER. We found that the levels of the ER chaperone, GRP78, the spliced XBP-1 and the phosphorylation of JNK are all increased following ischemia and reperfusion and that δPKC inhibition by δV1-1 blocks these events. Therefore, ischemia-reperfusion injury induces ER stress in the myocardium in a mechanism that requires δPKC activity. Taken together, our data show for the first time that δPKC activation plays a critical role in the ER stress-mediated response and the resultant cell death.
Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen–glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.
Cerebral ischemia; Ischemic tolerance; Epsilon protein kinase C; Extracellular signal-regulated kinase (ERK1/2); Neuroprotection; Mixed cortical neuron/astrocyte cell cultures
To improve myocardial flow during reperfusion after acute myocardial infarction and to elucidate the molecular and cellular basis that impedes it. According to the AHA/ACC recommendation, an ideal reperfusion treatment in patients with acute myocardial infarction (AMI) should not only focus on restoring flow in the occluded artery, but should aim to reduce microvascular damage to improve blood flow in the infarcted myocardium.
Transgenic mouse hearts expressing the δPKC (protein kinase C) inhibitor, δV1-1, in their myocytes only were treated with or without the δPKC inhibitor after ischemia in an ex vivo AMI model. δV1-1 or vehicle was also delivered at reperfusion in an in vivo porcine model of AMI. Microvascular dysfunction was assessed by physiological and histological measurements.
δPKC inhibition in the endothelial cells improved myocardial perfusion in the transgenic mice. In the porcine in vivo AMI model, coronary flow reserve (CFR), which is impaired for 6 days following infarction, was improved immediately following a one-minute treatment at the end of the ischemic period with the δPKC-selective inhibitor, δV1-1 (∼250 ng/Kg), and was completely corrected by 24 hrs. Myocardial contrast echocardiography, electron microscopy studies, and TUNEL staining demonstrated δPKC-mediated microvascular damage. δPKC-induced preconditioning, which also reduces infarct size by >60%, did not improve microvascular function.
These data suggest that δPKC activation in the microvasculature impairs blood flow in the infarcted tissue after restoring flow in the occluded artery and that AMI patients with no-reflow may therefore benefit from treatment with a δPKC inhibitor given in conjunction with removal of the coronary occlusion.