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 TPM1alpha (also called alpha-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 TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa 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 TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa 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 TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

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.

Traumatic diaphragmatic injury: experience from a level I trauma center.

OBJECTIVE: Traumatic diaphragmatic injuries (TDI) are uncommon but associated with substantial morbidity and mortality. We sought to analyze patients with TDI at a large trauma center and associated county coroner to identify characteristics predictive of increased mortality. METHODS: We queried a level I university trauma center and associated county coroner databases containing >20,000 patients to identify patients with ICD-9 diagnoses pertaining to TDI from January 1992 through May 2005. Once identified, hospital records, operative details, and autopsy reports were reviewed to determine injury characteristics, treatment provided, and outcome. Statistical analyses were performed using the Student t-test, chi-square analysis, analysis of variance, and multiple logistic regression. RESULTS: TDI were identified in 254 individuals. Two hundred (79%) survived to undergo operation. Of the 81 (32%) deaths, 33 (41%) occurred before arrival at the trauma center. Survivors were younger, had lesser injury severity scores (ISS), were more likely to be female, and had more bilateral injuries (P < or = .002 all) than nonsurvivors. By multiple logistic regression analyses, increased age (odds ratio [OR], 1.044; 95% confidence interval [CI], 1.015-1.074; P = .0029) and greater ISS (OR, 1.145; 95% CI, 1.103-1.188; P < .0001) were predictors of the probability of death in all patients. CONCLUSION: Although TDI may indicate substantive trauma burden in any patient, those with greater ISS and advanced age are at the greatest risk of death.

Uncoupling of myocardial beta-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.

Acute beta-blockade prevents myocardial beta-adrenergic receptor desensitization and preserves early ventricular function after brain death.

OBJECTIVE: Beta-adrenergic receptor desensitization through activation of the G protein-coupled receptor kinase 2 is an important mechanism of early cardiac dysfunction after brain death. We hypothesized that acute beta-blockade can prevent myocardial beta-adrenergic receptor desensitization after brain death through attenuation of G protein-coupled receptor kinase 2 activity, resulting in improved cardiac function. METHODS: Adult pigs underwent either sham operation, induction of brain death, or treatment with esmolol (beta-blockade) for 30 minutes before and 45 minutes after brain death (n = 8 per group). Cardiac function was assessed at baseline and for 6 hours after the operation. Myocardial beta-adrenergic receptor signaling was assessed 6 hours after operation by measuring sarcolemmal membrane adenylate cyclase activity, beta-adrenergic receptor density, and G protein-coupled receptor kinase 2 expression and activity. RESULTS: Baseline left ventricular preload recruitable stroke work was similar among sham, brain death, and beta-blockade groups. Preload recruitable stroke work was significantly decreased 6 hours after brain death versus sham, and beta-blockade resulted in maintenance of baseline preload recruitable stroke work relative to brain death and not different from sham. Basal and isoproterenol-stimulated adenylate cyclase activities were preserved in the beta-blockade group relative to the brain death group and were not different from the sham group. Left ventricular G protein-coupled receptor kinase 2 expression and activity in the beta-blockade group were markedly decreased relative to the brain death group and similar to the sham group. Beta-adrenergic receptor density was not different among groups. CONCLUSION: Acute beta-blockade before brain death attenuates beta-adrenergic receptor desensitization mediated by G protein-coupled receptor kinase 2 and preserves early cardiac function after brain death. These data support the hypothesis that acute beta-adrenergic receptor desensitization is an important mechanism in early ventricular dysfunction after brain death. Future studies with beta-blocker therapy immediately after brain death appear warranted.

Beta-adrenergic receptor antagonism preserves myocardial function after brain death in a porcine model.

BACKGROUND: Cardiac dysfunction after brain death decreases the already limited number of potential donors for cardiac transplantation. Acute beta-adrenergic receptor (betaAR) desensitization after the brain death-associated catecholamine surge is an important mechanism. We hypothesized that acute betaAR antagonism could improve myocardial function after brain death by preserving betaAR signaling. METHODS: Pigs were randomly assigned to three study groups (n = 5): sham; brain death; and brain death with betaAR antagonist (200 microg/kg/min esmolol), 30 minutes before brain death until 45 minutes after brain death. Functional data were collected for 6 hours after brain death and tissues procured. RESULTS: Compared with baseline, pre-load recruitable stroke work (PRSW), a pre-load-independent measure of systolic function (21.4 +/- 7.5 vs 43.3 +/- 6.8, slope of regression line during vena caval occlusion, p < 0.001), diastolic function (Tau, 101 +/- 54.7 vs 36.4 +/- 5.4 ms, p = 0.03) and systemic oxygen delivery (151 +/- 79.7 vs 298 +/- 78.7 ml/min, p < 0.001) deteriorated in untreated animals at 6 hours after brain death. In contrast, betaAR antagonist maintained baseline systolic function (PRSW, 37.8 +/- 5.6 vs 38.2 +/- 4.7, slope of regression line during vena caval occlusion, p = 0.92), diastolic function (Tau, 32.6 +/- 5.1 vs 48.5 +/- 28.3 ms, p = 0.57) and oxygen delivery (427 +/- 116 vs 397 +/- 98.8 ml/min, p = 0.36) at 6 hours after brain death. betaAR antagonist preserved betaAR signaling, as demonstrated by similar left ventricular (LV) basal (55.4 +/- 32.8 vs 58.8 +/- 10.9 pmol/mg/min, p = 0.40) and isoproterenol-stimulated (125 +/- 70.5 vs 124 +/- 52.0 pmol/mg/min, p = 0.49) adenylate cyclase activity at 6 hours after brain death, upon comparing betaAR antagonist and sham treatment groups. Both LV basal and isoproterenol-stimulated adenyl cyclase activity were higher with betaAR antagonist (25.9 +/- 4.8 pmol/mg/min, p = 0.03) than with untreated brain death (55.6 +/- 17.3 pmol/mg/min, p = 0.02). CONCLUSIONS: Beta-adrenergic receptor antagonism before brain death preserves cardiac function by preventing betaAR desensitization. This therapy in potential donors might increase the number of organs available for transplantation.

Glucocorticoids alter the balance between pro- and anti-inflammatory mediators in the myocardium in a porcine model of brain death.

BACKGROUND: Cardiac dysfunction after brain death (BD) limits donors for cardiac transplantation. Glucocorticoids ameliorate brain death-induced donor heart dysfunction. We hypothesized that glucocorticoid therapy alleviates myocardial depression through altering the balance between pro- and anti-inflammatory mediators via the nuclear factor-kappaB (NF-kappaB)/inhibitor of kappaB-alpha (IkappaBalpha) pathway and/or by preserving beta-adrenergic receptor (betaAR) signaling in the heart. METHODS: Crossbred pigs (25 to 35 kg) were randomly assigned to the following groups (n = 5/treatment): sham (Group 1); BD (Group 2); and BD with glucocorticoids (30 mg/kg methylprednisolone), either 2 hours before (Group 3) or 1 hour after BD (Group 4). Tumor necrosis factor-alpha (TNF-alpha) levels were measured in plasma at baseline and 1 hour and 6 hours after BD. Protein levels were measured in left ventricular homogenates procured 6 hours after BD. RESULTS: Pro-inflammatory proteins (TNF-alpha) and interleukin-6 were lower in Group 3 and Group 4 compared with Group 2 at 6 hours after BD (p < 0.01). Intracellular adhesion molecule-1 was also lower in Group 4 compared with Group 2 (p = 0.001). Interleukin-10, an anti-inflammatory mediator, was lower in Group 4 than in Group 2 (p < 0.001), but not different between Groups 2 and 3. At 6 hours after BD, neither NF-kappaB activity nor basal adenylate cyclase activity differed between Groups 3 and 4 compared with Group 2. CONCLUSIONS: Glucocorticoids maintained myocardial function and shifted the balance of pro- and anti-inflammatory mediators after BD. The mechanisms by which glucocorticoids preserve myocardial function, however, do not appear to involve the NF-kappaB pathway or betaAR signaling.

Restoration of myocardial beta-adrenergic receptor signaling after left ventricular assist device support.

OBJECTIVE: Left ventricular assist device support for patients with chronic heart failure can significantly improve beta-adrenergic receptor signaling, which is likely critical to myocardial recovery. The mechanism underlying the restoration of beta-adrenergic receptor signaling is unclear. This study investigates our hypothesis that restoration of cardiac beta-adrenergic receptor signaling by left ventricular assist devices results from inhibition of the G protein-coupled receptor kinase-2, a G protein-coupled receptor kinase that specifically phosphorylates and desensitizes agonist-occupied beta-adrenergic receptors. METHODS: Left ventricular beta-adrenergic receptor signaling was assessed in biopsy specimens taken from patients with chronic heart failure (n = 12) at the time of left ventricular assist device implantation (heart failure group) and again at the time of heart transplantation (left ventricular assist device group). Signaling was also studied in left ventricular biopsy specimens from nonfailing control (n = 8) hearts (nonfailing control group). Signaling was assessed by measuring sarcolemmal membrane beta-adrenergic receptor density, adenylyl cyclase activity, G protein expression, and G protein-coupled receptor kinase-2 expression and activity. RESULTS: Left ventricular beta-adrenergic receptor signaling was severely decreased in the heart failure group versus that seen in the nonfailing control group, as demonstrated by adenylyl cyclase activity. G protein-coupled receptor kinase-2 expression and activity was increased 3-fold in the heart failure group versus that seen in the nonfailing control group. After left ventricular assist device support, beta-adrenergic receptor signaling was restored to levels similar to those seen in the nonfailing control group. G protein-coupled receptor kinase-2 expression and activity were markedly diminished after left ventricular assist device support compared with that seen in the heart failure group and were not different from that seen in the nonfailing control group. CONCLUSION: In chronic heart failure left ventricular assist device support leads to restoration of cardiac beta-adrenergic receptor signaling. The primary mechanism appears to be diminished myocardial G protein-coupled receptor kinase-2 activity. This demonstrates the potentially beneficial effects of G protein-coupled receptor kinase-2 inhibition on beta-adrenergic receptor signaling in heart failure and might represent a novel therapeutic strategy for this disease process.