Cardiac Protein Kinase Research Articles

Abstract 5290: Cypher Long but not Short Isoform Specific Knockout Mice Result in Cardiac Signaling Defects and Dilated Cardiomyopathy

Our previous studies have demonstrated that Cypher, a PDZ-LIM protein localized at the Z line, plays a pivotal role in heart function. We recently identified long and short splice isoforms of Cypher, which are characterized by the presence and absence of LIM domains, respectively. The LIM domain of Cypher is thought to be involved in signaling, based on its ability to directly interact with signaling proteins. In human patients with dilated cardiomyopathy (DCM) we discovered Cypher gene mutations, which affect either long or short isoform or both isoforms. However, the precise molecular mechanisms underlying the role of Cypher isoforms in DCM remain unclear. To determine the role of Cypher isoforms in cardiac signaling and disease in vivo , we generated two Cypher isoform specific knockout mice. Selective ablation of Cypher long isoforms in mice resulted in partial neonatal lethality. However, hearts from viable Cypher long isoform deficient mice displayed Z line abnormalities and decreased cardiomyocyte widths, which resulted in a progressive form of DCM, characterized by fibrosis, calcification and lethality. The effects on cardiac function and disease observed in long-isoform specific Cypher knockout mice were preceded by significant decreases in cardiac protein kinase C and extracellular signal-regulated kinase signaling. These results are in contrast to Cypher short isoform deficient mice, which were viable with no overt cardiac morphology and signaling abnormalities. These results reveal distinct functional roles for Cypher isoforms in the heart as well as shed light into the molecular mechanisms underlying dilated cardiomyopathy.

Mechanism of protection of moderately diet restricted rats against doxorubicin-induced acute cardiotoxicity

Clinical use of doxorubicin (Adriamycin®), an antitumor agent, is limited by its oxyradical-mediated cardiotoxicity. We tested the hypothesis that moderate diet restriction protects against doxorubicin-induced cardiotoxicity by decreasing oxidative stress and inducing cardioprotective mechanisms. Male Sprague–Dawley rats (250–275 g) were maintained on diet restriction [35% less food than ad libitum]. Cardiotoxicity was estimated by measuring biomarkers of cardiotoxicity, cardiac function, lipid peroxidation, and histopathology. A LD 100 dose of doxorubicin (12 mg/kg, ip) administered on day 43 led to 100% mortality in ad libitum rats between 7 and 13 days due to higher cardiotoxicity and cardiac dysfunction, whereas all the diet restricted rats exhibited normal cardiac function and survived. Toxicokinetic analysis revealed equal accumulation of doxorubicin and doxorubicinol (toxic metabolite) in the ad libitum and diet restricted hearts. Mechanistic studies revealed that diet restricted rats were protected due to (1) lower oxyradical stress from increased cardiac antioxidants leading to downregulation of uncoupling proteins 2 and 3, (2) induction of cardiac peroxisome proliferators activated receptor-α and plasma adiponectin increased cardiac fatty acid oxidation (666.9 ±14.0 nmol/min/g heart in ad libitum versus 1035.6 ± 32.3 nmol/min/g heart in diet restriction) and mitochondrial AMPα2 protein kinase. The changes led to 51% higher cardiac ATP levels (17.7 ± 2.1 μmol/g heart in ad libitum versus 26.7 ± 1.9 μmol/g heart in diet restriction), higher ATP/ADP ratio, and (3) increased cardiac erythropoietin and decreased suppressor of cytokine signaling 3, which upregulates cardioprotective JAK/STAT3 pathway. These findings collectively show that moderate diet restriction renders resiliency against doxorubicin cardiotoxicity by lowering oxidative stress, enhancing ATP synthesis, and inducing the JAK/STAT3 pathway.

Quercetin-Supplemented Diets Lower Blood Pressure and Attenuate Cardiac Hypertrophy in Rats With Aortic Constriction

Quercetin (Q), a flavonoid found in berries and onions, can reduce blood pressure in hypertensive animals and inhibit signal transduction pathways in vitro that regulate cardiac hypertrophy. We hypothesized that quercetin could prevent cardiovascular complications in rats with abdominal aortic constriction (AAC). Rats consumed standard or Q-supplemented chow (1.5 g Q/kg chow) for 7 days before AAC or sham surgery (SHAM, n = 15; AAC, n = 15; SHAMQ, n = 15; AACQ, n = 14). Fourteen days after surgery, plasma and liver Q concentrations were elevated (P < 0.05) and hepatic lipid oxidation was reduced (P < 0.05) in Q-treated versus untreated rats. Carotid arterial blood pressure and cardiac hypertrophy were attenuated (P < 0.05), and cardiac protein kinase C betaII translocation was normalized (P < 0.05) in AACQ versus AAC. Expression of cardiac beta-myosin heavy-chain mRNA was also reduced in AACQ versus AAC (P < 0.05). However, extracellular regulated kinase 1/2 phosphorylation was similar in AAC versus AACQ. The level of aortic endothelial dysfunction (wire myography) was also similar between AAC and AACQ, in spite of reduced aortic thickening in AACQ. Importantly, Q-treated rats did not show any deleterious changes in myocardial function (echocardiography). Our data supports an antihypertensive and antihypertrophic effect of Q in vivo in the absence of changes concerning vascular and myocardial function.

Combined therapy with PPARα agonist and l-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPARalpha by fenofibrate, a PPARalpha agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD. Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine + 0.2% fenofibrate containing chow (CA + FE) from 4 weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis. At 8 weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA + FE than by CA or FE alone. CA + FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n-7,9) and 18:2(n-6) fatty acids compared with that in WT mice. CA + FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C beta2 in JVS mice. Furthermore, CA + FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%, P < 0.0001). PPARalpha activation, in addition to l-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.

Acute PKCδ inhibition limits ischaemia–reperfusion injury in the aged rat heart: Role of GSK-3β

Age is a leading risk factor for the development of ischaemic heart disease and failure. However, the efficacy of cardioprotective strategies designed to rescue the aged myocardium remains controversial. We have previously demonstrated increased levels of basal cardiac protein kinase Cdelta (PKCdelta) with ageing, a well-known mediator of apoptotic cell death following ischaemia and reperfusion (I/R) in adult hearts. Our objective was to determine the contribution of PKCdelta signaling mechanisms to reperfusion injury in the aged heart using local delivery of a novel PKCdelta inhibitory peptide (KID1-1). Contractile responses were assessed in hearts isolated from adult (4 months, n=38) and aged (24 months, n=45) male Fisher 344 rats treated with either KID1-1 (500 nM) or Tat vehicle peptide (500 nM) upon reperfusion for 10 min following 31-min global ischaemia. Recovery of left ventricular (LV) developed pressure was significantly improved by KID1-1 and associated with smaller infarct size in 24 months vs. age-matched controls (p<0.005). We also observed significant reductions in DNA laddering and cytochrome c and caspase 3 levels in aged hearts treated with KID1-1. Interestingly, KID1-1 attenuated mitochondrial and nuclear PKCdelta levels during reperfusion in aged vs. age-matched controls (p<0.01). Further, increases in mitochondrial phosphorylated glycogen synthase kinase-3beta (pGSK-3beta) levels were hastened in aged and adult hearts following KID1-1 (p<0.05), increasing the pGSK-3beta/GSK-3beta ratio. These results provide novel evidence for cardioprotection through acute PKCdelta inhibition in aged rat heart following I/R. Our results also suggest, for the first time, a key role for mitochondrial GSK-3beta as a cellular basis for the protection associated with PKCdelta inhibition with ageing.

Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).

Impaired α1‐adrenergic responses in aged rat hearts

To determine age-related changes in the cardiac effect of alpha1-adrenergic stimulation, both cardiomyocyte Ca2+-transient and cardiac protein kinase C (PKC) activity were measured in 3-month- (3MO) and 24-month- (24MO) old Wistar rats. Ca2+ transients obtained under 1 Hz pacing by microfluorimetry of cardiomyocyte loaded with indo-1 (405/480 nm fluorescence ratio) were compared in control conditions (Kreb's solution alone) and after alpha1-adrenergic stimulation (phenylephrine or cirazoline, an alpha1-specific agonist). PKC activity and PKC translocation index (particulate/total activity) were also assayed before and after alpha1-adrenergic stimulation. In 3MO, cirazoline induced a significant increase in Ca2+ transient for a 10(-9) M concentration which returned to control values for larger concentrations. In contrast, in 24MO, we observed a constant negative effect of cirazoline on the Ca2+ transient with a significant decrease at 10(-6) M compared with both baseline and Kreb's solution. Preliminary experiments showed that, in a dose-response curve to phenylephrine, the response of Ca2+ transient was maximal at 10(-7) M. This concentration induced a significant increase in Ca2+ transient in 3MO and a significant decrease in 24MO. The same concentration was chosen to perform PKC activity measurements under alpha1-adrenergic stimulation. In the basal state, PKC particulate activity was higher in 24MO than that in 3MO but was not different in cytosolic fractions; so that the translocation index was higher in 24MO (P < 0.01). After phenylephrine, a translocation of PKC toward the particulate fraction was observed in 3MO but not in 24MO. In conclusion, cardiac alpha1-adrenoceptor response was found to be impaired in aged hearts. The negative effect of alpha1-adrenergic stimulation on Ca2+ transient in cardiomyocytes obtained from old rats can be related to an absence of alpha1-adrenergic-induced PKC translocation.

Protein kinase C δ and ε mediate positive inotropy in adult ventricular myocytes

To examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the δ and ε isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture. PKC-δ-GFP and PKC-ε-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced. PKC-δ-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker, whereas PKC-ε-GFP redistributed preferentially to the surface sarcolemma. Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5 Hz, 22 °C. In myocytes expressing PKC-δ-GFP, PDBu caused a transient negative inotropic response followed by a robust and sustained positive inotropic response that paralleled perinuclear PKC-δ accumulation. In PKC-ε-GFP myocytes, PDBu caused a sustained negative inotropic response that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone. At higher expression levels, PKC-ε-GFP myocytes responded more like PKC-δ-GFP myocytes including perinuclear accumulation and a sustained positive inotropic response. Positive inotropic responses were markedly attenuated if PKC translocation was biased toward the surface sarcolemma by use of a more hydrophobic PKC activator, and were completely and selectively blocked by the PKC antagonist bis-indoylmaleimide. In contrast, transient and sustained negative inotropic responses were selectively blocked by the Ca 2+-dependent PKC isoform antagonist Go6976. The data indicate that the novel PKC isoforms δ and ε have little effect on contractility when accumulating at the cell surface, but produce a strong positive inotropic response upon accumulation at the Golgi or other intracellular sites.

Calcineurin A-α But Not A-β Is Required for Normal Kidney Development and Function

Calcineurin is an important signaling molecule in the kidney and may be involved in a variety of processes. The phosphatase subunit of calcineurin (CnA) has three isoforms, alpha, beta, and gamma. In this study, we investigated the effect of loss of calcineurin A-alpha (CnA-alpha) or calcineurin A-beta (CnA-beta) on the development and function of the kidney. Total calcineurin expression and activity was significantly reduced in whole kidney homogenates from both CnA-alpha -/- and CnA-beta -/- mice. Kidneys of CnA-beta -/- mice appear normal and the mice develop with no phenotypic abnormalities. In contrast, kidneys of CnA-alpha -/- animals fail to fully develop. In particular, postnatal maturation of the nephrogenic zone (NZ) is defective. Within the NZ, glomeruli also fail to mature and lack mesangial cells. In addition to alterations in development, there is an absence of proliferation and an increase of cell death in the NZ with loss of CnA-alpha. Finally, increased collagen deposition is observed and serum creatinine levels are significantly increased in CnA-alpha -/- animals compared to wild-type littermates, indicating that kidney function is impaired. In summary, absence of CnA-alpha but not CnA-beta leads to a defect in normal maturation of the NZ and glomeruli, alterations in the cell cycle, and impaired kidney function.

Regulations of thyroid hormone on cardiac protein kinase C signal pathwayin vitro

The experiments were conducted to assess the influences of thyroid hormone on cardiac protein kinase C(PKC) signal pathway with cultured cardiac myocytes and fibroblasts as the models. Cells were pretreated with 1% newborn calf serum (NCS) or angiotensin II (Ang II), and then following by a triiodothyronine (T3) treatment. The PKC activity, PKCα and PKCe expressions were analyzed and compared. In 1% NCS pretreatment, T3 could inhibit PKC activity and PKCe expression in cardiac myocytes. The AngII pretreatment led to an increase of PKC activity and PKCe expression in cardiac myocytes, and an increase of PKC activity in cardiac fibroblasts. Following by T3 treatment, the increased PKC activity and PKCe expression in cardiac myocytes were markedly decreased. In conclusion, whether in 1% NCS or in Ang II pretreatment, T3 could inhibit PKC activity and PKCe expression in cardiac myocytes.

Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction.

The activities of cardiac protein kinase C (PKC) were examined in hemodynamically assessed rats subsequent to myocardial infarction (MI). Both Ca(2+)-dependent and Ca(2+)-independent PKC activities increased significantly in left ventricular (LV) and right ventricular (RV) homogenates at 1, 2, 4, and 8 wk after MI was induced. PKC activities were also increased in both LV and RV cytosolic and particulate fractions from 8-wk infarcted rats. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were significantly increased in LV homogenate, cytosolic (except PKC-alpha), and particulate fractions from the failing rats. On the other hand, the protein contents of PKC-alpha, -beta, and -epsilon isozymes, unlike the PKC-zeta isozyme, were increased in RV homogenate and cytosolic fractions, whereas the RV particulate fraction showed an increase in the PKC-alpha isozyme only. These changes in the LV and RV PKC activities and protein contents in the 8-wk infarcted animals were partially corrected by treatment with the angiotensin-converting enzyme inhibitor imidapril. No changes in protein kinase A activity and its protein content were seen in the 8-wk infarcted hearts. The results suggest that the increased PKC activity in cardiac dysfunction due to MI may be associated with an increase in the expression of PKC-alpha, -beta, and -epsilon isozymes, and the improvement of heart function in the infarcted animals by imidapril may be due to partial prevention of changes in PKC activity and isozyme contents.

Effects of nifedipine-induced pulmonary vasodilatation on cardiac receptors and protein kinase C isoforms in the chronically hypoxic rat.

In chronic hypoxia, pulmonary hypertension induces a right ventricular (RV) hypertrophy (RVH) and the catecholamine-activated adrenergic system modulates cardiovascular responses through alpha- and beta-adrenergic pathways. The alpha(1)-adrenergic receptor (alpha(1)-AR) and protein kinase C (PKC) may play an important role in the signaling pathway leading to RVH. The aim of this study was to examine the relationship between nifedipine-induced pulmonary vasodilatation, the blunting of RVH and the modifications in the density of alpha(1)-AR, PKC activity and expression of PKC isoforms. In rats exposed to 15 days of hypoxia (380 torr, 50.66 kPa), RV pressure increased and RVH developed. Nifedipine, a calcium antagonist, given through gastric administration, partially decreased RV pressure and RVH. In both ventricles, hypoxia decreased alpha(1)-AR and beta-AR density and increased muscarinic acetylcholine receptor density. Nifedipine decreased alpha(1)-AR density only in normoxia. Expression of epsilon, delta and zeta PKC isoforms increased with RVH and normalized with nifedipine treatment. In conclusion, in this in vivo model of hypoxic rat, no relation was found between a RVH decrease and cardiac receptor densities. However, the development and regression of pulmonary hypertension and RVH were related to the expression of some PKC isoforms suggesting that pathways other than alpha(1)-AR might be involved in hypoxia-induced ventricular hypertrophy.

Increased Translocation of Cardiac Protein Kinase C β2 Accompanies Mild Cardiac Hypertrophy in Rats Fed Saturated Fat

Signal transduction through protein kinase C (PKC) beta2 may modulate cardiac hypertrophy in pressure-overloaded rat myocardium. Because PKC beta2 can be activated by fatty acids and diacylglycerol, we hypothesized that altering the level and type of dietary fat might modulate cardiac PKC activation and stimulate hypertrophy in otherwise normal rat myocardium. Male Sprague-Dawley rats (n = 32) were randomly assigned to either a low fat [10% total energy intake (TEI)] or high fat diet (40% TEI) based on corn or coconut oil as a source of saturated or unsaturated fat. After 40 d of isoenergetic diet consumption, the heart/body weight ratio was slightly greater in rats fed saturated fat diets compared with those fed unsaturated fat (P = 0.05). Increased activation of PKC beta2, as evidenced by greater membrane translocation, was also observed in all rats fed saturated fat diets (P < 0.01). PKC alpha, beta1 and epsilon did not change. These results suggest that dietary fat type can alter PKC beta2 activation in the heart, and exert a mild hypertrophic effect on the heart.

Differential regulation of cardiac protein kinase C isozyme expression after aortic banding in rat.

Protein kinase C (PKC) plays a key role in myocardial hypertrophy. To evaluate whether its isoforms are expressed differentially during gradual development of pressure-overload-induced cardiac hypertrophy, banding of the ascending aorta was used as an experimental model of left ventricular hypertrophy. One, 7 and 30 days after sham operation or aortic banding in male Wistar rats, the PKC activity and the expression of the cardiac PKC isozymes (PKC-alpha, -delta, - epsilon and -zeta), both at the protein and the mRNA level, were determined in the left and right ventricle. Left ventricular hypertrophy developed rapidly as early as 1 day after aortic banding followed by further progression at day 7 and day 30. This was paralleled by an increased total PKC enzyme activity in the cytosol fraction and a selectively enhanced protein expression of PKC-delta (day 7, 267+/-18%; day 30, 289+/-12%) and PKC-alpha (day 7, 212+/-20%; day 30, 193+/-14%). The protein amount of PKC- epsilon was not changed in either group. This differential protein expression was associated with a significant increase of the absolute mRNA levels for PKC-delta and PKC-alpha up to 202+/-20% (day 30) and 177+/-17% (day 30), whereas significant alterations in the PKC- epsilon mRNA levels were not detected. A selective upregulation of PKC-alpha and PKC-delta, both on the protein and on the mRNA level, was also noted in the right ventricle during the development of right ventricular hypertrophy, suggesting an adaptive response following elevated left ventricular enddiastolic pressure after long-term aortic banding for 30 days. This study characterizes in the right and left ventricle a differential regulation of the dominant PKC isozymes in pressure-overload cardiac hypertrophy both at the protein and the mRNA level.

Phosphorylation of cardiac protein kinase B is regulated by palmitate.

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 microU/ml insulin or 100 microU/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor C(2)-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation.

Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I.

Activation of the protein kinase C (PKC) family is a potential signaling mechanism by which high ambient glucose concentration modulates the phenotype and physiological function of cells. Recently, the cardiac renin angiotensin system (RAS) has been reported to promote PKC translocation in the diabetic heart via the angiotensin (ANG) II type 1 receptor (AT-1R). To evaluate the molecular events coupled with high glucose-induced PKC translocation and to examine the role of endogenously released ANG II in myocyte PKC signaling, primary cultures of adult rat ventricular myocytes were exposed to normal (5 mmol/l) or high (25 mmol/l) glucose for 12-24 h. Western blot analysis indicated that adult rat ventricular myocytes coexpress six PKC isozymes (alpha, beta(1,) beta(2,) delta, epsilon, and zeta). Translocation of five PKC isozymes (beta(1), beta(2), delta, epsilon, and zeta) was detected in response to 25 mmol/l glucose. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate blocked glucose-induced translocation of PKC-beta(2), -delta, and -zeta. Inhibition of tyrosine kinase with genistein blocked glucose-induced translocation of PKC-beta(1) and -delta, whereas chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane N,N,N,'N'-tetraacetic acid blocked translocation of PKC-beta(1) and -beta(2). Enzyme-linked immunosorbent assay performed on culture media from myocytes maintained in 25 mmol/l glucose detected a twofold increase in ANG II. Addition of an AT-1R antagonist (losartan; 100 nmol/l) to myocyte cultures blocked translocation of PKC-beta(1), -beta(2), -delta, and -epsilon. Phosphorylation of troponin (Tn) I was increased in myocytes exposed to 25 mmol/l glucose. Losartan selectively inhibited Tn I serine phosphorylation but did not affect phosphorylation at threonine residues. We concluded that 1) 25 mmol/l glucose triggers the release of ANG II by myocytes, resulting in activation of the ANG II autocrine pathway; 2) differential translocation of myocyte PKC isozymes occurs in response to 25 mmol/l glucose and ANG II; and 3) AT-1R-dependent PKC isozymes (beta(1), beta(2), delta, and epsilon) target Tn I serine residues.

Alterations of Load-Induced p38 MAP Kinase Activation in Failing Rat Hearts

Hemodynamic load-induced cardiac p38 mitogen-activated protein kinase (MAPK) activation was studied in normotensive control Dahl rats (n = 10) and hypertensive Dahl rats with heart failure (n = 16). The isolated heart from each animal was stretched on a Langendorff apparatus at an equivalent diastolic wall stress, and the p38-MAPK activity of the left ventricular (LV) myocardium was analyzed by immunoprecipitation-kinase assay. Compared to the control hearts, the stretch-induced p38-MAPK activities were significantly decreased, and inversely correlated with the LV diameter (r = −0.73, P < 0.01). Chronic treatment with an angiotensin II AT1-receptor antagonist, valsartan (10 mg/kg/day), ameliorated cardiac function and remodeling process in the failing hearts, which was associated with an improvement of the p38-MAPK activities. Thus, the mechano-signal transduction of p38-MAPK pathway is downregulated in the failing hearts, along with progressive ventricular remodeling. The data also suggest that the beneficial effects of the AT1-receptor antagonists are potentially mediated by the restoration of cardiac growth-related signal transduction.

Protein dephosphorylation rates in myocytes after isoproterenol withdrawal

Dephosphorylation of substrates for cyclic AMP-dependent protein kinase is essential for reversing the effects of this enzyme. It has been proposed that the relevant phosphatase(s) is stimulated by muscarinic cholinergic agonists, thereby accentuating cholinergic antagonism of β-adrenergic agonists in the heart. To test this hypothesis, dephosphorylation of the three major substrates of cardiac cyclic AMP-dependent protein kinase (phospholamban, troponin-I, and C-protein) was examined. In isolated myocytes, isoproterenol caused concentration-dependent phosphorylation of these three proteins. Simultaneous exposure to acetylcholine with the isoproterenol caused a rightward shift in the concentration–response curve that was similar for protein phosphorylation in myocytes and for the inotropic response of the intact heart. The addition of propranolol after exposure to isoproterenol resulted in protein dephosphorylation, the onset of which was accelerated by acetylcholine. However, acetylcholine did not affect the rate of dephosphorylation for any of the proteins, indicating that phosphatase activity in cardiac muscle is not enhanced by acetylcholine.

Altered Cardiac Endothelin Receptors and Protein Kinase C in Deoxycorticosterone-Salt Hypertensive Rats

J. Fareh, R. M. Touyz, E. L. Schiffrin and G. Thibault. Altered Cardiac Endothelin Receptors and Protein Kinase C in Deoxycorticosterone-Salt Hypertensive Rats. Journal of Molecular and Cellular Cardiology (2000) 32, 665–676. The aim of the present study was to assess the status of ET-1 receptor subtypes (ETAand ETB) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ETAsubtype, whereas in fibroblasts ETAand ETBreceptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ETAreceptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ETBreceptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC α, γ and ϵ was significantly increased, whereas PKC δ was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKCδ was significantly increased and PKC ϵ was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ETAreceptors and fibroblast ETBreceptors; (2) cell-specific enhanced expression of some PKC isoenzymes (α, γ,δ and ϵ); and (3) unresponsiveness of PKC ϵ to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca2+handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats.

Signaling from β‐adrenoceptor to L‐type calcium channel: identification of a novel cardiac protein kinase A target possessing similarities to AHNAK

A novel calcium channel-associated protein of approximately 700 kDa has been identified in mammalian cardiomyocytes that undergoes substantial cAMP-dependent protein kinase (PKA) phosphorylation. It was therefore designated as phosphoprotein 700 (pp700). The pp700 interacts specifically with the beta(2) subunit of cardiac L-type calcium channels as revealed by coprecipitation experiments using affinity-purified antibodies against different calcium channel subunits. It is surprising that amino acid sequence analysis of pig pp700 revealed homology to AHNAK-encoded protein, which was originally identified in human cell lines of neural crest origin as 700-kDa phosphoprotein. Cardiac AHNAK expression was assessed on mRNA level by reverse transcriptase-polymerase chain reaction. Sequence-directed antibodies raised against human AHNAK recognized pp700 in immunoblotting and immunoprecipitation experiments, confirming the homology between both proteins. Anti-AHNAK antibodies labeled preferentially the plasma membrane of cardiomyocytes in cryosections of rat cardiac tissue and isolated cardiomyocytes. Sarcolemmal pp700/AHNAK localization was not influenced by stimulation of either the PKA or the protein kinase C pathway. In back-phosphorylation studies with cardiac biopsies, we identified distinct pp700 pools. The membrane-associated fraction of pp700 underwent substantial in vivo phosphorylation on beta-adrenergic receptor stimulation by isoproterenol, whereas the cytoplasmic fraction of pp700 was not accessible to endogenous PKA. It is important that in vivo phosphorylation occurred in that pp700 fraction which coprecipitated with the calcium channel beta subunit. We hypothesize that both phosphorylation of pp700 and its coupling to the beta subunit play a physiological role in cardiac beta-adrenergic signal transduction. Haase, H., Podzuweit, T., Lutsch, G., Hohaus, A., Kostka, S., Lindschau, C., Kott, M., Kraft, R., Morano, I. Signaling from beta-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target that has similarities to AHNAK.