Ronen Beeri

Troponin elevation in severe sepsis and septic shock: The role of left ventricular diastolic dysfunction and right ventricular dilatation

Objective:Serum troponin concentrations predict mortality in almost every clinical setting they have been examined, including sepsis. However, the causes for troponin elevations in sepsis are poorly understood. We hypothesized that detailed investigation of myocardial dysfunction by echocardiography can provide insight into the possible causes of troponin elevation and its association with mortality in sepsis. Design:Prospective, analytic cohort study. Setting:Tertiary academic institute. Patients:A cohort of ICU patients with severe sepsis or septic shock. Interventions:Advanced echocardiography using global strain, strain-rate imaging and 3D left and right ventricular volume analyses in addition to the standard echocardiography, and concomitant high-sensitivity troponin-T measurement in patients with severe sepsis or septic shock. Measurements and Main Results:Two hundred twenty-five echocardiograms and concomitant high-sensitivity troponin-T measurements were performed in a cohort of 106 patients within the first days of severe sepsis or septic shock (2.1 ± 1.4 measurements/patient). Combining echocardiographic and clinical variables, left ventricular diastolic dysfunction defined as increased mitral E-to-strain-rate e′-wave ratio, right ventricular dilatation (increased right ventricular end-systolic volume index), high Acute Physiology and Chronic Health Evaluation-II score, and low glomerular filtration rate best correlated with elevated log-transformed concomitant high-sensitivity troponin-T concentrations (mixed linear model: t = 3.8, 3.3, 2.8, and –2.1 and p = 0.001, 0.0002, 0.006, and 0.007, respectively). Left ventricular systolic dysfunction determined by reduced strain-rate s′-wave or low ejection fraction did not significantly correlate with log(concomitant high-sensitivity troponin-T). Forty-one patients (39%) died in-hospital. Right ventricular end-systolic volume index and left ventricular strain-rate e′-wave predicted in-hospital mortality, independent of Acute Physiology and Chronic Health Evaluation-II score (logistic regression: Wald = 8.4, 6.6, and 9.8 and p = 0.004, 0.010, and 0.001, respectively). Concomitant high-sensitivity troponin-T predicted mortality in univariate analysis (Wald = 8.4; p = 0.004), but not when combined with right ventricular end-systolic volume index and strain-rate e′-wave in the multivariate analysis (Wald = 2.3, 4.6, and 6.2 and p = 0.13, 0.032, and 0.012, respectively). Conclusions:Left ventricular diastolic dysfunction and right ventricular dilatation are the echocardiographic variables correlating best with concomitant high-sensitivity troponin-T concentrations. Left ventricular diastolic and right ventricular systolic dysfunction seem to explain the association of troponin with mortality in severe sepsis and septic shock.

Volatile anesthetic preconditioning attenuates myocardial apoptosis in rabbits after regional ischemia and reperfusion via Akt signaling and modulation of Bcl-2 family proteins

We tested whether isoflurane preconditioning inhibits cardiomyocyte apoptosis and evaluated the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic preconditioning and determined whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in anesthetic preconditioning. Six-month-old New Zealand rabbits subjected to 40 min of myocardial ischemia followed by 180 min of reperfusion were assigned to the following groups: ischemia-reperfusion (I/R), isoflurane preconditioning and isoflurane plus PI3K inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002) (0.6 and 0.3 mg/kg i.v., respectively). Sham-operated, wortmannin + I/R, wortmannin + sham, LY294002 + I/R, and LY294002 + sham groups were also included. Infarct size was assessed by triphenyltetrazolium chloride staining. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and activated caspase-3 assays. Akt phosphorylation, Bax, Bcl-2, Bad, and phosphorylated Bad (phospho-Bad) expression was assessed by immunoblotting. Isoflurane preconditioning reduced infarct size compared with the I/R group: 22 ± 4 versus 41 ± 5% (p < 0.05). The percentage of apoptotic cells decreased in the isoflurane group (3.8 ± 1.2%) compared with the I/R group (12.4 ± 1.6%; p < 0.05). These results were also confirmed by the activated caspase-3 assay. Wortmannin and LY294002 inhibited the effects of isoflurane. Myocardial infarction increased to 44 ± 3 and 45 ± 2% and the percentage of apoptotic cells was 11.9 ± 2.1 and 11.7 ± 3.3%, respectively. Akt phosphorylation and Bcl-2 and phospho-Bad expression increased after isoflurane preconditioning, whereas Bax expression decreased. These effects were inhibited by wortmannin and LY294002. The data indicate that isoflurane preconditioning reduces infarct size and myocardial apoptosis after I/R. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play a role in isoflurane-induced myocardial protection.

New Efficient Catheter-Based System for Myocardial Gene Delivery

Background—Manipulating gene expression in the failing heart has therapeutic promise, but until now efficient and homogeneous cardiac gene delivery has required an open-chest approach. This study examines the hypothesis that vector delivery promoted by echo contrast microbubbles will be maximized by injection of the vectors into the aortic root with brief balloon occlusion above the sinuses, while at the same time prolonging diastole and vasodilating with acetylcholine (ACh) to maximize coronary exposure. Methods and Results—After incubation with albumin-coated perfluorocarbon microbubbles, an adenovirus encoding a reporter gene was infused into the aortic root of rats. To maximize delivery, the aortic root was transiently occluded with a balloon catheter during a brief ACh-induced asystole. Ultrasound was used to image the delivery and disrupt the microbubbles. Aortic occlusion with concomitant ACh increased myocardial gene expression for virus + microbubbles by >2.5-fold, from 925±165 to 2358±376 relative units (RU;P <0.01). This delivery system also produced substantial expression with vector alone (1473±549 RU). All uptakes were significant compared with 433±332 RU without virus. Conclusions—An adenoviral delivery system combining echo contrast with a catheter-based technique to maximize coronary perfusion increases gene delivery compared with echo contrast alone. This novel method permits efficient percutaneous gene delivery in closed-chest animals.

Mitral Regurgitation Augments Post-Myocardial Infarction Remodeling: Failure of Hypertrophic Compensation

OBJECTIVES We examined whether mitral regurgitation (MR) augments post-myocardial infarction (MI) remodeling. BACKGROUND MR doubles mortality after MI, but its additive contribution to left ventricular (LV) remodeling is debated and has not been addressed in a controlled fashion. METHODS Apical MIs were created in 12 sheep, and 6 had an LV-to-left atrial shunt implanted, consistently producing regurgitant fractions of approximately 30%. The groups were compared at baseline, 1, and 3 months. RESULTS Left ventricular end-systolic volume progressively increased by 190% with MR versus 90% without MR (p < 0.02). Pre-load-recruitable stroke work declined by 82 +/- 13% versus 25 +/- 16% (p < 0.01) with MR, with decreased remote-zone sarcoplasmic reticulum Ca(2+)-ATPase levels (0.56 +/- 0.03 vs. 0.76 +/- 0.02, p < 0.001), and decreased isolated myocyte contractility. In remote zones, pro-hypertrophic Akt and gp130 were upregulated in both groups at 1 month, but significantly lower and below baseline in the MR group at 3 months. Pro-apoptotic caspase 3 remained high in both groups. Matrix metalloproteinase (MMP)-13 and membrane-type MMP-1 were increased in remote zones of MR versus infarct-only animals at 1 month, then fell below baseline. The MMP tissue inhibitors rose from baseline to 3 months in all animals, rising higher in the MI + MR-group border zone. CONCLUSIONS In this controlled model, moderate MR worsens post-MI remodeling, with reduced contractility. Pro-hypertrophic pathways are initially upregulated but subsequently fall below infarct-only levels and baseline; with sustained caspase 3 elevation, transformation to a failure phenotype occurs. Extracellular matrix turnover increases in MR animals. Therefore, MR can precipitate an earlier onset of dilated heart failure.

Gene Delivery of Sarcoplasmic Reticulum Calcium ATPase Inhibits Ventricular Remodeling in Ischemic Mitral Regurgitation

Background—Mitral regurgitation (MR) doubles mortality after myocardial infarction (MI). We have demonstrated that MR worsens remodeling after MI and that early correction reverses remodeling. Sarcoplasmic reticulum Ca+2-ATPase (SERCA2a) is downregulated in this process. We hypothesized that upregulating SERCA2a might inhibit remodeling in a surgical model of apical MI (no intrinsic MR) with independent MR-type flow. Methods and Results—In 12 sheep, percutaneous gene delivery was performed by using a validated protocol to perfuse both the left anterior descending and circumflex coronary arteries with occlusion of venous drainage. We administered adeno-associated virus 6 (AAV6) carrying SERCA2a under a Cytomegalovirus promoter control in 6 sheep and a reporter gene in 6 controls. After 2 weeks, a standardized apical MI was created, and a shunt was implanted between the left ventricle and left atrium, producing regurgitant fractions of ≈30%. Animals were compared at baseline and 1 and 3 months by 3D echocardiography, Millar hemodynamics, and biopsies. The SERCA2a group had a well-maintained preload-recruitable stroke work at 3 months (decrease by 8±10% vs 42±12% with reporter gene controls; P<0.001). Left ventricular dP/dt followed the same pattern (no change vs 55% decrease; P<0.001). Left ventricular end-systolic volume was lower with SERCA2a (82.6±9.6 vs 99.4±9.7 mL; P=0.03); left ventricular end-diastolic volume, reflecting volume overload, was not significantly different (127.8±6.2 vs 134.3±9.4 mL). SERCA2a sheep showed a 15% rise in antiapoptotic pAkt versus a 30% reduction with the reporter gene (P<0.001). Prohypertrophic activated STAT3 was also 41% higher with SERCA2a than in controls (P<0.001). Proapoptotic activated caspase-3 rose >5-fold during 1 month in both SERCA2a and control animals (P=NS) and decreased by 19% at 3 months, remaining elevated in both groups. Conclusions—In this controlled model, upregulating SERCA2a induced better function and lesser remodeling, with improved contractility, smaller volume, and activation of prohypertrophic/antiapoptotic pathways. Although caspase-3 remained activated in both groups, SERCA2a sheep had increased molecular antiremodeling “tone.” We therefore conclude that upregulating SERCA2a inhibits MR-induced post-MI remodeling in this model and thus may constitute a useful approach to reduce the vicious circle of remodeling in ischemic MR.

Isoflurane Preconditioning Decreases Myocardial Infarction in Rabbits via Up-regulation of Hypoxia Inducible Factor 1 That Is Mediated by Mammalian Target of Rapamycin

Background:Volatile anesthetics are known to protect the heart against ischemia-reperfusion injury. The authors tested whether anesthetic preconditioning with isoflurane is mediated via activation of the transcription factor hypoxia inducible factor 1 (HIF-1) and evaluated the role of mammalian target of rapamycin signaling in this process. Methods:New Zealand White rabbits subjected to 40 min of regional myocardial ischemia, followed by 180 min of reperfusion, were assigned to the following groups: ischemia and reperfusion (I/R) only, isoflurane (1 minimal alveolar concentration) preconditioning, and isoflurane preconditioning in the presence of the mammalian target of rapamycin inhibitor rapamycin (0.25 mg/kg). Sham-operated, isoflurane + sham, rapamycin + sham, rapamycin + I/R, and dimethyl sulfoxide + I/R groups were also included. Creatine kinase-MB levels were assessed as an indicator of myocardial damage, and infarct size was evaluated by triphenyl tetrazolium chloride staining. HIF-1&agr; expression and DNA binding were assessed by Western blotting and electrophoretic mobility shift analysis, respectively. Results:Isoflurane preconditioning reduced infarct size compared with the I/R group: 26 ± 4% versus 44 ± 6% (P < 0.05). Creatine kinase-MB concentrations in the preconditioned animals (103 ± 8% above baseline) were lower than in the I/R group (243 ± 12% above baseline; P < 0.05). Rapamycin inhibited the cardioprotective effect of isoflurane: myocardial infarction increased to 44 ± 4% and creatine kinase-MB level increased to 254 ± 9% above baseline. HIF-1&agr; protein expression and DNA binding activity increased after isoflurane preconditioning compared with the ischemia group. These effects were also inhibited by rapamycin. Conclusions:The current results indicate that isoflurane-induced myocardial protection involves activation of the HIF-1 pathway that is mediated by the mammalian target of rapamycin.

Early Repair of Moderate Ischemic Mitral Regurgitation Reverses Left Ventricular Remodeling A Functional and Molecular Study

Background— Mitral regurgitation (MR) doubles postmyocardial infarction (MI) mortality. We have shown that moderate MR augments remodeling in an apical MI model (no intrinsic MR) with independent left ventricle-to-left atrial MR-type flow. We hypothesized that repairing moderate MR 1 month after MI reverses this remodeling. Methods and Results— Anteroapical MIs were created in 18 sheep, and a left ventricle-to-left atrial shunt implanted in 12 (regurgitant fraction, 30%). Six sheep had the shunt closed at 1 month (repair group). Sheep were compared at baseline, and at 1 and 3 months. Sheep in the MI+MR (unrepaired) and repaired groups remodeled during the first month (120% increased left ventricular end-systolic volume [ESV; P<0.01]), but shunt closure reversed remodeling at 3 months, with end-diastolic volume (EDV) and ESV 135% and 128% of baseline versus 220% and 280% without repair (P<0.001). At 3 months, dP/dt and preload-recruitable stroke work were relatively maintained in the repaired and MI-only groups versus nearly 50% decreases without repair. Prohypertrophic gp130 and antiapoptotic pAkt increased followed by exhaustion below baseline without repair, but remained elevated at 3 months with repair or MI only. With repair, matrix metalloproteinase-2 decreased to ≤50% that without repair in remote and border zones at 3 months, and the matrix metalloproteinase inhibitor TIMP-4 increased dramatically. Conclusions— Early repair of moderate MR in the setting of apical MI substantially reverses the otherwise progressive remodeling process, with reduced left ventricular volumes, relatively maintained contractility, persistently activated intracellular signals promoting hypertrophy and opposing apoptosis, and reduced matrix proteolytic activity. These findings are of interest for the current controversy regarding potential benefits of repair of MR after MI.

Reliability of Visual Assessment of Global and Segmental Left Ventricular Function: A Multicenter Study by the Israeli Echocardiography Research Group

BACKGROUND The purpose of this multicenter study was to determine the reliability of visual assessments of segmental wall motion (WM) abnormalities and global left ventricular function among highly experienced echocardiographers using contemporary echocardiographic technology in patients with a variety of cardiac conditions. METHODS The reliability of visual determinations of left ventricular WM and global function was calculated from assessments made by 12 experienced echocardiographers on 105 echocardiograms recorded using contemporary echocardiographic equipment. Ten studies were reread independently to determine intraobserver reliability. RESULTS Interobserver reliability for visual differentiation between normal, hypokinetic, and akinetic segments had an intraclass correlation coefficient of 0.70. The intraclass correlation coefficient for dichotomizing segments into normal versus other abnormal was 0.63, for hypokinetic versus other scores was 0.26, and for akinetic versus other scores was 0.58. Similar results were found for intraobserver reliability. Interobserver reliability for WM score index was 0.84 and for left ventricular ejection fraction was 0.78. Similar values were obtained for the intraobserver reliability of WM score index and ejection fraction. Compared to angiographic data, the accuracy of segmental WM assessments was 85%, and correct determination of the culprit artery was achieved in 59% of patients with myocardial infarctions. CONCLUSION Among experienced readers using contemporary echocardiographic equipment, interobserver and intraobserver reliability was reasonable for the visual quantification of normal and akinetic segments but poor for hypokinetic segments. Reliability was good for the visual assessment of global left ventricular function by WM score index and ejection fraction.

Transcription factor MITF regulates cardiac growth and hypertrophy

High levels of microphthalmia transcription factor (MITF) expression have been described in several cell types, including melanocytes, mast cells, and osteoclasts. MITF plays a pivotal role in the regulation of specific genes in these cells. Although its mRNA has been found to be present in relatively high levels in the heart, its cardiac role has never been explored. Here we show that a specific heart isoform of MITF is expressed in cardiomyocytes and can be induced by beta-adrenergic stimulation but not by paired box gene 3 (PAX3), the regulator of the melanocyte MITF isoform. In 2 mouse strains with different MITF mutations, heart weight/body weight ratio was decreased as was the hypertrophic response to beta-adrenergic stimulation. These mice also demonstrated a tendency to sudden death following beta-adrenergic stimulation. Most impressively, 15-month-old MITF-mutated mice had greatly decreased heart weight/body weight ratio, systolic function, and cardiac output. In contrast with normal mice, in the MITF-mutated mice, beta-adrenergic stimulation failed to induce B-type natriuretic peptide (BNP), an important modulator of cardiac hypertrophy, while atrial natriuretic peptide levels and phosphorylated Akt were increased, suggesting a cardiac stress response. In addition, cardiomyocytes cultured with siRNA against MITF showed a substantial decrease in BNP promoter activity. Thus, for what we believe is the first time, we have demonstrated that MITF plays an essential role in beta-adrenergic-induced cardiac hypertrophy.

Layer-specific strain analysis by speckle tracking echocardiography reveals differences in left ventricular function between rats and humans

The rat heart is commonly used as an experimental model of the human heart in both health and disease states, assuming that heart function of rats and humans is alike. When studying a rat model, echocardiography is usually performed on sedated rats, whereas standard echocardiography on adult humans does not require any sedation. Since echocardiography results of sedated rats are usually inferred to alert humans, in the present study, we tested the hypothesis that differences in left ventricular (LV) function may be present between rats sedated by a low dose of ketamine-xylazine and alert humans. Echocardiography was applied to 110 healthy sedated rats and 120 healthy alert humans. Strain parameters were calculated from the scans using a layer-specific speckle tracking echocardiography program. The results showed that layer longitudinal strain is equal in rats and humans, whereas segmental strain is heterogeneous (P < 0.05) in a different way in rats and humans (P < 0.05). Furthermore, layer circumferential strain is larger in humans (P < 0.001), and the segmental results showed different segmental heterogeneity in rats and humans (P < 0.05). Radial strain was found to be homogeneous at the apex and papillary muscle levels in humans and heterogeneous in rats (P < 0.001). Additionally, whereas LV twist was equal in rats and humans, in rats the rotation was larger at the apex (P < 0.01) and smaller at the base (P < 0.001). The torsion-to-shortening ratio parameter, which indicates the transmural distribution of contractile myofibers, was found to be equal in rats and humans. Thus, when evaluating LV function of sedated rats under ketamine-xylazine, it is recommended to measure the global longitudinal strain, LV twist, and torsion-to-shortening ratio, since no scaling is required when converting these parameters and inferring them to humans.