Regulation of Focal Adhesion Kinase through a Direct Interaction with an Endogenous Inhibitor.

Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK-related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis. FRNK has been proposed to compete for FAK anchoring sites in focal adhesions, but we did not detect displacement of FAK by WT-FRNK or superinhibitory S217A-FRNK. Instead, we found FRNK interacted directly with FAK, and this interaction is markedly strengthened for the superinhibitory S217A-FRNK. The results suggest that FRNK limits growth and survival signaling pathways by binding directly to FAK in an inhibitory complex, and this inhibition is relieved by phosphorylation of FRNK at S217.

Myocardial infarction sensitizes medial prefrontal cortex to inhibitory effect of locus coeruleus stimulation in rats.

RATIONALE: Anxiety is a common comorbidity that develops after myocardial infarction and is now an established independent risk factor for cardiovascular mortality. OBJECTIVE: Here, we assessed anxiety and mapped neural activity of forebrain regions that regulate anxiety in a rat model of myocardial infarction in order to identify sites of dysregulation. METHODS: Anxiety responses to novel (open field) or aversive stimuli (discriminative auditory fear conditioning) were assessed in rats subjected to coronary artery ligation (CAL) or sham ligation. Forebrain metabolic activity was measured by cytochrome oxidase (CO) histochemistry. Changes in CO activity and the incidence of ventricular arrhythmias were also assessed during modulation of fear circuitry induced by electrical stimulation of the locus coeruleus. RESULTS: Coronary artery ligation had negligible effects on open-field behavior, but increased expression of learned fear and impaired fear cue discrimination. Cytochrome oxidase activity was increased in the medial prefrontal cortex and in the lateral amygdala after CAL. Locus coeruleus stimulation reduced CO activity in the infralimbic medial prefrontal cortex only in rats subjected to CAL. Stimulation of the LC also elicited new ventricular arrhythmias in rats subjected to CAL. CONCLUSION: Coronary artery ligation sensitizes the infralimbic medial prefrontal cortex to the inhibitory effects of locus coeruleus stimulation. Suppression of infralimbic medial prefrontal cortical activity may impair the ability of rats subjected to CAL to discriminate between cues that signal aversive and neutral events which, in turn, may promote excessive sympathetic activation of the cardiovascular system in response to innocuous stimuli.

Connective tissue growth factor regulates cardiac function and tissue remodeling in a mouse model of dilated cardiomyopathy.

Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective tissue growth factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3 months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3 months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3 months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic functions in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling was elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.

PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis.

AIMS: Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε. METHODS AND RESULTS: Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1((Ser133)) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. CONCLUSIONS: PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury.

Cardiomyocyte-specific expression of CRNK, the C-terminal domain of PYK2, maintains ventricular function and slows ventricular remodeling in a mouse model of dilated cardiomyopathy.

Up-regulation and activation of PYK2, a member of the FAK family of protein tyrosine kinases, is involved in the pathogenesis of left ventricular (LV) remodeling and heart failure (HF). PYK2 activation can be prevented by CRNK, the C-terminal domain of PYK2. We previously demonstrated that adenoviral-mediated CRNK gene transfer improved survival and LV function, and slowed LV remodeling in a rat model of coronary artery ligation-induced HF. We now interrogate whether cardiomyocyte-specific, transgenic CRNK expression prevents LV remodeling and HF in a mouse model of dilated cardiomyopathy (DCM) caused by constitutively active Protein Kinase Cε (caPKCε). Transgenic (TG; FVB/N background) mice were engineered to express rat CRNK under control of the α-myosin heavy chain promoter, and crossed with FVB/N mice with cardiomyocyte-specific expression of caPKCε to create double TG mice. LV structure, function, and gene expression were evaluated in all 4 groups (nonTG FVB/N; caPKCε(+/-); CRNK(+/-); and caPKCε×CRNK (PXC) double TG mice) at 1, 3, 6, 9 and 12mo of age. CRNK expression followed a Mendelian distribution, and CRNK mice developed and survived normally through 12mo. Cardiac structure, function and selected gene expression of CRNK mice were similar to nonTG littermates. CRNK had no effect on caPKCε expression and vice versa. PYK2 was up-regulated ~6-fold in caPKCε mice, who developed a non-hypertrophic, progressive DCM with reduced systolic (Contractility Index=151±5 vs. 90±4s(-1)) and diastolic (Tau=7.5±0.5 vs. 14.7±1.3ms) function, and LV dilatation (LV Remodeling Index (LVRI)=4.2±0.1 vs. 6.0±0.3 for FVB/N vs. caPKCε mice, respectively; P<0.05 for each at 12mo). In double TG PXC mice, CRNK expression significantly prolonged survival, improved contractile function (Contractile Index=115±8s(-1); Tau=9.5±1.0ms), and reduced LV remodeling (LVRI=4.9±0.1). Cardiomyocyte-specific expression of CRNK improves contractile function and slows LV remodeling in a mouse model of DCM.

Focal adhesion signaling in heart failure.

In this brief review, recent evidence is presented to indicate a role for specific components of the cardiomyocyte costamere (and its related structure the focal adhesion complex of cultured cardiomyocytes) in initiating and sustaining the aberrant signal transduction that contributes to myocardial remodeling and the progression to heart failure (HF). Special attention is devoted to the focal adhesion kinase family of nonreceptor protein tyrosine kinases in bidirectional signal transduction during cardiac remodeling and HF progression. Finally, some speculations and directions for future study are provided for this rapidly developing field of research.

Detection of differentially methylated gene promoters in failing and nonfailing human left ventricle myocardium using computation analysis.

Human dilated cardiomyopathy (DCM) is characterized by congestive heart failure and altered myocardial gene expression. Epigenetic changes, including DNA methylation, are implicated in the development of DCM but have not been studied extensively. Clinical human DCM and nonfailing control left ventricle samples were individually analyzed for DNA methylation and expressional changes. Expression microarrays were used to identify 393 overexpressed and 349 underexpressed genes in DCM (GEO accession number: GSE43435). Gene promoter microarrays were utilized for DNA methylation analysis, and the resulting data were analyzed by two different computational methods. In the first method, we utilized subtractive analysis of DNA methylation peak data to identify 158 gene promoters exhibiting DNA methylation changes that correlated with expression changes. In the second method, a two-stage approach combined a particle swarm optimization feature selection algorithm and a discriminant analysis via mixed integer programming classifier to identify differentially methylated gene promoters. This analysis identified 51 hypermethylated promoters and six hypomethylated promoters in DCM with 100% cross-validation accuracy in the group assignment. Generation of a composite list of genes identified by subtractive analysis and two-stage computation analysis revealed four genes that exhibited differential DNA methylation by both methods in addition to altered gene expression. Computationally identified genes (AURKB, BTNL9, CLDN5, and TK1) define a central set of differentially methylated gene promoters that are important in classifying DCM. These genes have no previously reported role in DCM. This study documents that rigorous computational analysis applied to microarray analysis of healthy and diseased human heart samples helps to define clinically relevant DNA methylation and expressional changes in DCM.

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation.

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

Regulation of connective tissue growth factor gene expression and fibrosis in human heart failure.

BACKGROUND: Heart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective tissue growth factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited. METHODS AND RESULTS: Left ventricle (LV) tissue from patients undergoing cardiac transplantation for ischemic (ICM; n = 20) and dilated (DCM; n = 20) cardiomyopathies and from nonfailing (NF; n = 20) donor hearts were examined. Paired samples (n = 15) from patients undergoing LV assist device (LVAD) implantation as "bridge to transplant" (34-1,145 days) also were analyzed. There was more interstitial fibrosis in both ICM and DCM compared with NF hearts. Hydroxyproline concentration was also significantly increased in DCM compared with NF samples. The expression of CTGF, transforming growth factor (TGF) ß1, collagen (COL) 1-α1, COL3-α1, matrix metalloproteinase (MMP) 2, and MMP9 mRNA in ICM and DCM were also significantly elevated compared with NF samples. Although TGF-ß1, CTGF, COL1-α1, and COL3-α1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading. CONCLUSIONS: CTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.

Talin1 has unique expression versus talin 2 in the heart and modifies the hypertrophic response to pressure overload.

Integrins are adhesive, signaling, and mechanotransduction proteins. Talin (Tln) activates integrins and links it to the actin cytoskeleton. Vertebrates contain two talin genes, tln1 and tln2. How Tln1 and Tln2 function in cardiac myocytes (CMs) is unknown. Tln1 and Tln2 expression were evaluated in the normal embryonic and adult mouse heart as well as in control and failing human adult myocardium. Tln1 function was then tested in the basal and mechanically stressed myocardium after cardiomyocyte-specific excision of the Tln1 gene. During embryogenesis, both Tln forms are highly expressed in CMs, but in the mature heart Tln2 becomes the main Tln isoform, localizing to the costameres. Tln1 expression is minimal in the adult CM. With pharmacological and mechanical stress causing hypertrophy, Tln1 is up-regulated in CMs and is specifically detected at costameres, suggesting its importance in the compensatory response to CM stress. In human failing heart, CM Tln1 also increases compared with control samples from normal functioning myocardium. To directly test Tln1 function in CMs, we generated CM-specific Tln1 knock-out mice (Tln1cKO). Tln1cKO mice showed normal basal cardiac structure and function but when subjected to pressure overload showed blunted hypertrophy, less fibrosis, and improved cardiac function versus controls. Acute responses of ERK1/2, p38, Akt, and glycogen synthase kinase 3 after mechanical stress were strongly blunted in Tln1cKO mice. Given these results, we conclude that Tln1 and Tln2 have distinct functions in the myocardium. Our data show that reduction of CM Tln1 expression can lead to improved cardiac remodeling following pressure overload.

Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100-µm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions.

Protein kinase Cε activity induces anti-inflammatory and anti-apoptotic genes via an ERK1/2- and NF-κB-dependent pathway to enhance vascular protection.

Vascular endothelial injury predisposes to endothelial dysfunction and atherogenesis. We have investigated the hypothesis that PKCε (protein kinase Cε) is an important upstream regulator of cytoprotective pathways in vascular ECs (endothelial cells). Depletion of PKCε in human ECs reduced expression of the cytoprotective genes A1, A20 and Bcl-2. Conversely, constitutively active PKCε expressed in human ECs increased mRNA and protein levels of these cytoprotective genes, with up-regulation dependent upon ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. Furthermore, inhibition of NF-κB (nuclear factor κB) by the pharmacological antagonist BAY 11-7085 or an IκB (inhibitor of NF-κB) SuperRepressor prevented cytoprotective gene induction. Activation of PKCε enhanced p65 NF-κB DNA binding and elevated NF-κB transcriptional activity. Importantly, although NF-κB activation by PKCε induced cytoprotective genes, it did not up-regulate pro-inflammatory NF-κB targets [E-selectin, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1)]. Indeed, PKCε exhibited cytoprotective and anti-inflammatory actions, including inhibition of TNFα (tumour necrosis factor α)-induced JNK (c-Jun N-terminal kinase) phosphorylation and ICAM-1 up-regulation, a response attenuated by depletion of A20. Thus we conclude that PKCε plays an essential role in endothelial homoeostasis, acting as an upstream co-ordinator of gene expression through activation of ERK1/2, inhibition of JNK and diversion of the NF-κB pathway to cytoprotective gene induction, and propose that PKCε represents a novel therapeutic target for endothelial dysfunction.

Contractile Activity Regulates Inducible Nitric Oxide Synthase Expression and NO(i) Production in Cardiomyocytes via a FAK-Dependent Signaling Pathway.

Intracellular nitric oxide (NO(i)) is a physiological regulator of excitation-contraction coupling, but is also involved in the development of cardiac dysfunction during hypertrophy and heart failure. To determine whether contractile activity regulates nitric oxide synthase (NOS) expression, spontaneously contracting, neonatal rat ventricular myocytes (NRVM) were treat with L-type calcium channel blockers (nifedipine and verapamil) or myosin II ATPase inhibitors (butanedione monoxime (BDM) and blebbistatin) to produce contractile arrest. Both types of inhibitors significantly reduced iNOS but not eNOS expression, and also reduced NO(i) production. Inhibiting contractile activity also reduced focal adhesion kinase (FAK) and AKT phosphorylation. Contraction-induced iNOS expression required FAK and phosphatidylinositol 3-kinase (PI(3)K), as both PF573228 and LY294002 (10 µM, 24 h) eliminated contraction-induced iNOS expression. Similarly, shRNAs specific for FAK (shFAK) caused FAK knockdown, reduced AKT phosphorylation at T308 and S473, and reduced iNOS expression. In contrast, shRNA-mediated knockdown of PYK2, the other member of the FAK-family of protein tyrosine kinases, had much less of an effect. Conversely, overexpression of a constitutively active form of FAK (CD2-FAK) or AKT (Myr-AKT) reversed the inhibitory effect of BDM on iNOS expression and NO(i) production. Thus, contraction-induced iNOS expression and NO(i) production in NRVM are mediated via a FAK-PI(3)K-AKT signaling pathway.

Serine-910 phosphorylation of focal adhesion kinase is critical for sarcomere reorganization in cardiomyocyte hypertrophy.

AIMS: Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM). METHODS AND RESULTS: Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ET(A)R-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK-paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM. CONCLUSION: FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.

Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding.

OBJECTIVE: Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. METHODS AND RESULTS: Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas). CONCLUSION: We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.

Protein kinase C-epsilon activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307-F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F(0)F(1)-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F(0)F(1)-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.

Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes.

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 µM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.

FRNK inhibition of focal adhesion kinase-dependent signaling and migration in vascular smooth muscle cells.

OBJECTIVE: To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation. METHODS AND RESULTS: Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase-related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein-tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II-stimulated focal adhesion kinase, paxillin, and extracellular signal-regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK-expressing VSMCs. Angiotensin II-induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2. CONCLUSIONS: FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.

Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width.

A ventricular myocyte experiences changes in length and load during every beat of the heart and has the ability to remodel cell shape to maintain cardiac performance. Specifically, myocytes elongate in response to increased diastolic strain by adding sarcomeres in series, and they thicken in response to continued systolic stress by adding filaments in parallel. Myocytes do this while still keeping the resting sarcomere length close to its optimal value at the peak of the length-tension curve. This review focuses on the little understood mechanisms by which direction of growth is matched in a physiologically appropriate direction. We propose that the direction of strain is detected by differential phosphorylation of proteins in the costamere, which then transmit signaling to the Z-disc for parallel or series addition of thin filaments regulated via the actin capping processes. In this review, we link mechanotransduction to the molecular mechanisms for regulation of myocyte length and width.