Karen M. D'Souza

GRK2-Mediated Inhibition of Adrenergic and Dopaminergic Signaling in Right Ventricular Hypertrophy Therapeutic Implications in Pulmonary Hypertension

Background—The cause and consequences of impaired adrenergic signaling in right ventricular failure/hypertrophy (RVH) are poorly understood. We hypothesized that G protein–coupled receptor kinase-2 (GRK2)–mediated uncoupling of &bgr;-adrenergic receptor signaling impairs inotropic reserve. The implications of right ventricular (RV) adrenergic remodeling for inotrope selection and the therapeutic benefit of interrupting G&bgr;&ggr;–GRK2 interaction, using gallein, were tested. Methods and Results—Chamber-specificity and cellular localization of adrenergic remodeling were compared in rodent RVH associated with pulmonary arterial hypertension (PAH-RVH; SU5416+chronic-hypoxia or Monocrotaline) versus pulmonary artery banding–induced RVH (PAB-RVH). Results were corroborated in RV arrays from 10 PAH patients versus controls. Inotropic reserve was assessed in RV- and left ventricular–Langendorff models and in vivo. Gallein therapy (1.8 mg/kg/day ×2-weeks) was assessed. Despite similar RVH, cardiac output (58.3±4.9 versus 82.9±4.8 mL/min; P<0.001) and treadmill distance (41.5±11.6 versus 244.1±12.4 m; P<0.001) were lower in PAH-RVH versus PAB-RVH. In PAH-RVH versus PAB-RVH there was greater downregulation of &bgr;1-, &agr;1- and dopamine-1 receptors, more left ventricular involvement, and greater impairment of RV contractile reserve. RV GRK2 activity increased in parallel with a reduction in both adrenergic receptor expression and inotrope-stimulated cAMP levels (P<0.01). &bgr;1-receptor downregulation also occurred in human PAH-RVH. Dobutamine was superior to dopamine as an RV inotrope, both ex vivo and in vivo. Conclusions—GRK2-mediated desensitization-downregulation of adrenergic and dopaminergic receptors impairs inotropic reserve in PAH-RVH. Acute inotropic support in RVH is best accomplished by dobutamine, reflecting its better coupling to adenylyl cyclase and the reliance of dopamine on dopamine-1–receptor signaling, which is impaired in RVH. Inhibiting G&bgr;&ggr;–GRK2 interactions has therapeutic benefit in RVH.

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Background— Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1&agr; (also called &agr;-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. Methods and Results— In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1&kgr;. By developing a TPM1&kgr;-specific antibody, we found that the TPM1&kgr; protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1&kgr; in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1&kgr;. Incorporation of increased levels of TPM1&kgr; protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1&kgr; compared with TPM1&agr;. Conclusions— This functional analysis of TPM1&kgr; provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Increased Expression of the Auxiliary β2-subunit of Ventricular L-type Ca2+ Channels Leads to Single-Channel Activity Characteristic of Heart Failure

Background Increased activity of single ventricular L-type Ca2+-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation. Methods and Results By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β1 or β3 isoforms, β2a and β2b induce a high-activity channel behavior typical of failing myocytes. In accordance, β2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac CaV1.2 also reveal increased single-channel activity and sarcolemmal β2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β2 expression. Additional evidence for the cause-effect relationship between β2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive CaV1.2 and inducible β2 cardiac overexpression. Here in non-failing hearts induction of β2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. Conclusions Our study presents evidence of the pathobiochemical relevance of β2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Gαq-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 β-adrenergic receptor (β-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and β-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 β-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the Gαq-coupled angiotensin II AT1 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-Gαq coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKCα 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 PKCα in transgenic mice led to impaired β-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 β-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and β-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts. Karen M. D’Souza, Ricky Malhotra, Jennifer L. Philip, Michelle L. Staron, Tiju Theccanat, Valluvan Jeevanandam, and Shahab A. Akhter This article has been withdrawn by the authors. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, p. 25849, October 23, 2015

Reversal of impaired myocardial β-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.

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-3β, and ERK1/2. There was also maintenance of cardiac myocyte β-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.

Dilated Cardiomyopathy with Increased SR Ca2+ Loading Preceded by a Hypercontractile State and Diastolic Failure in the α1CTG Mouse

Mice over-expressing the α1−subunit (pore) of the L-type Ca2+ channel (α1CTG) by 4months (mo) of age exhibit an enlarged heart, hypertrophied myocytes, increased Ca2+ current and Ca2+ transient amplitude, but a normal SR Ca2+ load. With advancing age (8–11 mo), some mice demonstrate advanced hypertrophy but are not in congestive heart failure (NFTG), while others evolve to frank dilated congestive heart failure (FTG). We demonstrate that older NFTG myocytes exhibit a hypercontractile state over a wide range of stimulation frequencies, but maintain a normal SR Ca2+ load compared to age matched non-transgenic (NTG) myocytes. However, at high stimulation rates (2–4 Hz) signs of diastolic contractile failure appear in NFTG cells. The evolution of frank congestive failure in FTG is accompanied by a further increase in heart mass and myocyte size, and phospholamban and ryanodine receptor protein levels and phosphorylation become reduced. In FTG, the SR Ca2+ load increases and Ca2+ release following excitation, increases further. An enhanced NCX function in FTG, as reflected by an accelerated relaxation of the caffeine-induced Ca2+ transient, is insufficient to maintain a normal diastolic Ca2+ during high rates of stimulation. Although a high SR Ca2+ release following excitation is maintained, the hypercontractile state is not maintained at high rates of stimulation, and signs of both systolic and diastolic contractile failure appear. Thus, the dilated cardiomyopathy that evolves in this mouse model exhibits signs of both systolic and diastolic failure, but not a deficient SR Ca2+ loading or release, as occurs in some other cardiomyopathic models.

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.

Uncoupling of Myocardial β-Adrenergic Receptor Signaling During Coronary Artery Bypass Grafting: The Role of GRK2

BACKGROUND Cardiopulmonary bypass (CPB) and cardioplegic arrest during cardiac surgery leads to desensitization of myocardial beta-adrenergic receptors (beta-ARs). Impaired signaling through this pathway can have a detrimental effect on ventricular function and increased need for inotropic support. The mechanism of myocardial beta-AR desensitization during cardiac surgery has not been defined. This study investigates the role of G protein-coupled receptor kinase-2 (GRK2), a serine-threonine kinase which phosphorylates and desensitizes agonist-occupied beta-ARs, as a primary mechanism of beta-AR uncoupling during coronary artery bypass grafting (CABG) with CPB and cardioplegic arrest. METHODS Forty-eight patients undergoing elective CABG were enrolled in this study. Myocardial beta-AR signaling was assessed by measuring total beta-AR density and adenylyl cyclase activity in right atrial biopsies obtained before CPB and just before weaning from CPB. Myocardial GRK2 expression and activity were also measured before CPB and just before weaning from CPB. RESULTS Myocardial beta-AR signaling was significantly impaired after CPB and cardioplegic arrest during CABG. Cardiac GRK2 expression was not altered; however, there was a twofold increase in GRK2 activity during CABG. There was an even greater elevation in cardiac GRK2 activity in patients with severely depressed ventricular function. CONCLUSIONS Increased myocardial GRK2 activity appears to be the primary mechanism of impaired beta-AR signaling during CABG with CPB and cardioplegic arrest. This may contribute to the greater need for inotropic support in patients with severe ventricular dysfunction. Strategies to inhibit activation of GRK2 during CABG may decrease morbidity in this patient population.