G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts.

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by ß-agonist stimulation in contrast to normal controls. ß-adrenergic receptor (ß-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following ß-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater ß-agonist-mediated inhibition of basal and TGFß-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored ß-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFß stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

G alpha(q)-mediated activation of GRK2 by mechanical stretch in cardiac myocytes: the role of protein kinase C.

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of beta-adrenergic receptor (beta-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and beta-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by "fetal" gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating beta-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the G alpha(q)-coupled angiotensin II AT(1) receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-G alpha(q) coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKC alpha also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKC alpha in transgenic mice led to impaired beta-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired beta-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and beta-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function.

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3ß, and ERK1/2. There was also maintenance of cardiac myocyte ß-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.

Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells.

BACKGROUND: Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions. METHODS: Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1alpha) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1alpha), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1alpha. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax. RESULTS: Overexpression of pcDNA3-DN-Hif-1alpha led to a significant reduction in hypoxia -induced apoptosis (17 +/- 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1alpha transfected cells. Moreover, selective ablation of HIF-1alpha protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1alpha exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1alpha led to a two-fold increase in Hif-1alpha levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1alpha also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1alpha constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression. CONCLUSION: These data demonstrate that HIF-1alpha is an important component of the apoptotic signaling machinery in the two cell types.

A device for rapid and quantitative measurement of cardiac myocyte contractility.

Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca(2+) activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure cardiac cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure cardiac cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the ß-adrenergic agonist isoproterenol (100 nmol l(-1)) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac cell contractions.

A method to measure cellular adhesion utilizing a polymer micro-cantilever.

In the present study we engineered a micro-machined polyimide cantilever with an embedded sensing element to investigate cellular adhesion, in terms of its relative ability to stick to a cross-linker, 3,3'-dithiobis[sulfosuccinimidylpropionate], coated on the cantilever surface. To achieve this objective, we investigated adhesive properties of three human prostate cancer cell lines, namely, a bone metastasis derived human prostate cancer cell line (PC3), a brain metastasis derived human prostate cancer cell line (DU145), and a subclone of PC3 (PC3-EMT14). We found that PC3-EMT14, which displays a mesenchymal phenotype, has the least adhesion compared to PC3 and DU145, which exhibit an epithelial phenotype.

Activation of JAK-STAT and nitric oxide signaling as a mechanism for donor heart dysfunction.

BACKGROUND: Donor heart dysfunction (DHD) precluding procurement for transplantation occurs in up to 25% of brain-dead (BD) donors. The molecular mechanisms of DHD remain unclear. We investigated the potential role of myocardial interleukin (IL)-6 signaling through the JAK2-STAT3 pathway, which can lead to the generation of nitric oxide (NO) and decreased cardiac myocyte contractility. METHODS: Hearts were procured using standard technique with University of Wisconsin (UW) solution from 14 donors with a left ventricular (LV) ejection fraction of <35% (DHD). Ten hearts with normal function (NF) after BD served as controls. LV IL-6 was quantitated by enzyme-linked immunoassay (ELISA) and JAK2-STAT3 signaling was assessed by expression of phosphorylated STAT3. Inducible NO synthase (iNOS) and caspase-3 were measured by activity assays. RESULTS: Myocardial IL-6 expression was 8-fold greater in the DHD group vs NF controls. Phosphorylated STAT3 expression was 5-fold higher in DHD than in NF, indicating increased JAK2-STAT3 signaling. LV activity of iNOS was 2.5-fold greater in DHD than in NF. LV expression of the pro-apoptotic gene Bnip3 and caspase-3 activity were 3-fold greater in the DHD group than in the NF group. CONCLUSIONS: Myocardial IL-6 expression is significantly higher in the setting of DHD compared with hearts procured with normal function. This may lead to increased JAK2-STAT3 signaling and upregulation of iNOS, which has been shown to decrease cardiac myocyte contractility. Increased NO production may also lead to increased apoptosis through upregulation of Bnip3 gene expression. Increased iNOS signaling may be an important mechanism of DHD and represents a novel therapeutic target to improve cardiac function after BD.

Reversal of impaired myocardial beta-adrenergic receptor signaling by continuous-flow left ventricular assist device support.

BACKGROUND: Myocardial beta-adrenergic receptor (beta-AR) signaling is severely impaired in chronic heart failure (HF). This study was conducted to determine if left ventricular (LV) beta-AR signaling could be restored after continuous-flow LV assist device (LVAD) support. METHODS: Twelve patients received LVADs as a bridge to transplant. Paired LV biopsy specimens were obtained at the time of LVAD implant (HF group) and transplant (LVAD group). The mean duration of LVAD support was 152 +/- 34 days. Myocardial beta-AR signaling was assessed by measuring adenylyl cyclase (AC) activity, total beta-AR density (B(max)), and G protein-coupled receptor kinase-2 (GRK2) expression and activity. LV specimens from 8 non-failing hearts (NF) were used as controls. RESULTS: Basal and isoproterenol-stimulated AC activity was significantly lower in HF vs NF, indicative of beta-AR uncoupling. Continuous-flow LVAD support restored basal and isoproterenol-stimulated AC activity to levels similar to NF. B(max) was decreased in HF vs NF and increased to nearly normal in the LVAD group. GRK2 expression was increased 2.6-fold in HF vs NF and was similar to NF after LVAD support. GRK2 activity was 3.2-fold greater in HF vs NF and decreased to NF levels in the LVAD group. CONCLUSIONS: Myocardial beta-AR signaling can be restored to nearly normal after continuous-flow LVAD support. This is similar to previous data for volume-displacement pulsatile LVADs. Decreased GRK2 activity is an important mechanism and indicates that normalization of the neurohormonal milieu associated with HF is similar between continuous-flow and pulsatile LVADs. This may have important implications for myocardial recovery.

Glucose uptake and adenoviral mediated GLUT1 infection decrease hypoxia-induced HIF-1alpha levels in cardiac myocytes.

Hypoxia causes a large array of adaptive and physiological responses in all cells including cardiac myocytes. In order to elucidate the molecular effects of increased glucose flux on hypoxic cardiac myocytes we focused on the basic helix-loop-helix transcription factor, hypoxia inducible factor 1 alpha (HIF-1alpha), which is rapidly upregulated in hypoxic cells and elicits a number of responses including augmentation of glucose uptake. Primary cultures of neonatal rat cardiac myocytes as well as embryonic rat heart-derived myogenic H9c2 cells demonstrated a significant upregulation of HIF-1alpha when subjected to hypoxia of 6-8h in the absence of glucose. Re-addition of extracellular glucose to the medium resulted in a decrease of HIF-1alpha levels by almost 50%. This glucose effect was blocked by addition of glycolytic inhibitors. In addition, glucose uptake and glycolysis resulted in substantial decreased levels of p53, which is regulated by HIF-1alpha. Adenoviral infection of cultures of cardiac myocytes with the facilitative glucose transporter, GLUT1 followed by hypoxia of 24h also resulted in a significant reduction in the protein expression of HIF-1alpha compared to control vector-infected cultures. GLUT1 infected cultures also demonstrated fewer apoptotic cells and a reduction in the release of cytochrome c after hypoxia. Inhibition of the ubiquitin-proteasomal pathway by a variety of 26S proteasomal inhibitors increased HIF-1alpha to similar levels under both normoxic and hypoxic conditions and in the presence or absence of glucose. This result suggested that glucose induces HIF-1alpha degradation via a proteasomal pathway. This conclusion was substantiated by immunoprecipitation experiments of total cell extracts, which demonstrated an increase of ubiquitinated HIF-1alpha relative to total HIF-1alpha in the presence of glucose during hypoxia. Thus, glucose as well as GLUT1 overexpression diminishes hypoxia-induced HIF-1alpha protein via an ubiquitin-proteasomal pathway in hypoxic cardiac myocytes. This represents a novel feedback mechanism that may play an important role in adaptation of cardiac myocytes to hypoxia and ischemia.