Nitric Oxide and Postconditioning: Cardioprotective Methods for Acute Care of Ischemia Reperfusion Injury

Timely coronary artery reperfusion is essential to prevent myocyte death following myocardial infarction. The act of restoring blood flow however, paradoxically reduces the beneficial effects of reperfusion. This phenomenon, termed myocardial reperfusion injury, refers to the injury of cardiac myocytes that were viable immediately before reperfusion. Recent studies have shown that the timing and hemodynamic sequence of events which govern reperfusion can help to minimize the severity of reperfusion injury. The term postconditioning describes a modified form of reperfusion that involves a series of flow interruptions which confer significant cardioprotection to the heart. This thesis investigates ischemic postconditioning and endothelial nitric oxide synthase (eNOS) phosphorylation as cardioprotective therapies against reperfusion injury. In the first half of this thesis, we test the hypothesis that phosphorylation of eNOS serves as a cardioprotection nodal point for ischemic postconditioning. We show that phosphorylation of eNOS increases enzyme activity and that its product, nitric oxide, plays a critical role in cardioprotection. A number of cardiac dysfunctions arise after reperfusion and we address the effects of postconditioning on infarct size and myocardial blood flow. The second half of this thesis introduces the use of magnetic relaxometry sensors to detect cardiac biomarkers. The ability to non-invasively measure infarct size in small animals would be helpful in studying models of myocardial ischemia-reperfusion injury. We investigate the use of implantable biosensors in vivo and show that the cumulative detection of cardiac biomarkers correlates with infarct severity.Engineering and Applied Science

Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice

Objective: Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection. Methods and Results: We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation. Conclusions and Significance: Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.American Heart Association (Predoctoral Fellowship)National Institutes of Health (U.S.) (R01 NS33335)National Institutes of Health (U.S.) (R01 HL57818

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Talent management in a New Zealand multi-national corporation (Fonterra co-operative group limited).

This study focuses on understanding the talent management programme of a New Zealand multi-national cooperation, Fonterra Co-operative Group Limited. Specifically, it examines the key components of this programme against an established framework adopted in the human resource consultancy fraternity and its implementation challenges.Master of Business Administratio

Postconditioning activates Akt and eNOS.

<p><b>A.</b> Western blot of total eNOS and GADPH in WT (C57BL6/J), S1176D mice (S1176ki), and eNOS ko mice. <b>B.</b> Western blot demonstrating phosphorylation (Ser1176) and total protein levels of eNOS in wild-type mice under conditions of control (CTL), MIR, and MIPc. <b>C.</b> Representative Western blot demonstrating phosphorylated (Ser473) and total protein levels of Akt. Densities (arbitrary units, AU) show that MIPc phosphorylates Akt in WT and eNOS ko mice. n = 5 per group. Data are expressed as the mean±SD. *P<0.05.</p

Temporal myocardial contrast echocardiography.

<p>Myocardial blood flow (Aβ) profiles in the apical region for <b>A.</b> C57BL/6J mice. <b>B.</b> eNOS knockout mice, and <b>C.</b> S1176D knockin mice. Levels of myocardial myocardial blood flow were normalized to baseline values and measured at 2, 10 and 30 minutes after reperfusion. <b>D.</b> Apical myocardial blood flow 30 minutes after reperfusion. MIR: Traditional myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.</p

eNOS S1176 phosphorylation protects against I/R injury in vivo.

<p>Wild-type, S1176D and eNOS knockout mice were subjected to 45 minutes of myocardial ischemia (LAD ligation) followed by traditional reperfusion (MIR) or postconditioned reperfusion (MIPc: 6 cycles of 10sec reperfusion, 10 sec ischemia). <b>A.</b> Percentage of left ventricle area at risk (AAR), (P = NS). <b>B.</b> Quantitative analysis of infarct size over AAR, *P<0.05 compared to wild-type control. <b>C.</b> Representative heart sections perfused with 1% Evans blue and stained with 2% TTC; infarct areas are outlined in black. MIR: Myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning, AAR: Area at Risk, LV: Left Ventricle. n = 6–9 mice per group. Data are expressed as the mean±SD.</p

Effect of postconditioning and S1176D mutation on no-reflow zones.

<p><b>A.</b> Representative images 30 minutes after reperfusion. Superimposed areas (blue) indicate regions with ≤20% residual blood flow. <b>B.</b> Composite graph showing areas of the myocardium with ≤20% (black) and ≤30% (white) residual blood flow compared to preischemic baseline. MBF: myocardial blood flow. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.</p

Hierarchical architecture influences calcium dynamics in engineered cardiac muscle.

Changes in myocyte cell shape and tissue structure are concurrent with changes in electromechanical function in both the developing and diseased heart. While the anisotropic architecture of cardiac tissue is known to influence the propagation of the action potential, the influence of tissue architecture and its potential role in regulating excitation-contraction coupling (ECC) are less well defined. We hypothesized that changes in the shape and the orientation of cardiac myocytes induced by spatial arrangement of the extracellular matrix (ECM) affects ECC. To test this hypothesis, we isolated and cultured neonatal rat ventricular cardiac myocytes on various micropatterns of fibronectin where they self-organized into tissues with varying degrees of anisotropy. We then measured the morphological features of these engineered myocardial tissues across several hierarchical dimensions by measuring cellular aspect ratio, myocyte area, nuclear density and the degree of cytoskeletal F-actin alignment. We found that when compared with isotropic tissues, anisotropic tissues have increased cellular aspect ratios, increased nuclear densities, decreased myocyte cell areas and smaller variances in actin alignment. To understand how tissue architecture influences cardiac function, we studied the role of anisotropy on intracellular calcium ([Ca(2+)](i)) dynamics by characterizing the [Ca(2+)](i)-frequency relationship of electrically paced tissues. When compared with isotropic tissues, anisotropic tissues displayed significant differences in [Ca(2+)](i) transients, decreased diastolic baseline [Ca(2+)](i) levels and greater [Ca(2+)](i) influx per cardiac cycle. These results suggest that ECM cues influence tissue structure at cellular and subcellular levels and regulate ECC.</p