Cardiovascular

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21753

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VASOPRESSIN INSTEAD OF EPINEPHRINE ENHANCES EFFICACY OF CPR WITHOUT CAUSING TACHYCARDIA
(2014-03) Cherry, Brandon H.; Nguyen, Ahn Q.; Williams, Arthur G. Jr.; Scott, Gary F.; Hollrah, Roger A.; Ryou, Myoung-Gwi; Hoxha, Besim; Olivencia-Yurvati, Albert H.; Mallet, Robert T.
Survival from cardiac arrest is highly dependent on the arterial pressure generated by cardiopulmonary resuscitation (CPR). To increase efficacy of precordial compressions, the potent vasoconstrictor epinephrine (EPI) is administered. However, EPI also elicits a robust, β1-adrenoceptor-mediated tachycardia following defibrillation, depleting the myocardium of the energy reserves it requires for recovery. We proposed that the adrenoceptor-independent vasoconstrictor vasopressin (AVP) increases arterial pressure as effectively as EPI without producing tachycardia. After 6 min pacing-induced cardiac arrest, domestic swine (25-35 kg; 10 boars, 11 sows) received precordial compressions (100/min) for 4 min, and either EPI (0.1 mg; n=5) or AVP (10 U; n=24) was injected iv at 1 min CPR. EPI and AVP similarly increased mean arterial pressure from 31±3 to 66±4 mmHg vs. 34±3 to 59±3 mmHg after 4 min CPR. The AVP-treated pigs required less countershock energy (12±2 J) to achieve defibrillation vs EPI-treated pigs (16±4 J). Post-arrest tachycardia was less intense in AVP- (133±11 bpm) than EPI-treated (174±14 bpm) pigs. Thus, AVP is as effective as EPI at enhancing CPR, but avoids EPI-induced tachycardia. Purpose (a): The purpose of this study was to test the hypothesis that the adrenoceptor-independent vasoconstrictor vasopressin increases arterial pressure as effectively as epinephrine without producing tachycardia during cardiopulmonary resuscitation (CPR). Methods (b): After 6 min pacing-induced cardiac arrest, domestic swine (25-35 kg; 10 boars, 11 sows) received precordial compressions (100/min) for 4 min, and either epinephrine (0.1 mg; n=5) or vasopressin (10 U; n=24) was injected iv at 1 min CPR. Results (c): Epinephrine and Vasopressin similarly increased mean arterial pressure from 31±3 to 66±4 mmHg vs. 34±3 to 59±3 mmHg after 4 min CPR. The vasopressin-treated pigs required less countershock energy (12±2 J) to achieve defibrillation vs epinephrine-treated pigs (16±4 J). Post-arrest tachycardia was less intense in vasopressin- (133±11 bpm) than epinephrine-treated (174±14 bpm) pigs. Conclusions (d): Vasopressin is as effective as epinephrine at enhancing CPR, but avoids epinephrine-induced tachycardia.
TIME COURSE OF CHANGES IN GLUTAMATERGIC TRANSMISSION WITHIN NTS DURING CIH EXPOSURE AND THE ROLE OF ΔFOSB
(2014-03) Wu, Qiong; Mifflin, Steve
ΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement. ΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs from second order NTS neurons by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both 1 day and 7 days CIH groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement.
THE EFFECT OF CONTINUOUS POSITIVE AIRWAY PRESSURE TREATMENT ON CARDIOVASCULAR REACTIVITY IN OBSTRUCTIVE SLEEP APNEA.
(2014-03) Jouett, Noah; Smith, Michael L.; Sleep Consultants, Inc
Obstructive sleep apnea (OSA) is a disorder in which patients intermittently stop breathing while sleeping. OSA has been associated with an increased risk for cardiovascular disease. This study investigates the role of the blood pressure (BP) response to voluntary breath-holding as an indicator of treatment success. We have found that OSA patients have elevated BP responses to voluntary breath-holding compared to people of a similar age and body weight. This study has found that with adequate treatment, this BP response is effectively attenuated. We propose a voluntary 20 second breath-hold as an effective and objective measure of treatment success in OSA. Purpose (a): To investigate whether or not well-treated obstructive sleep apnea (OSA) subjects will have a decreased Δ systolic blood pressure (SBP) response to voluntary apnea than untreated subjects. Methods (b): 21 OSA patients were stratified into treated (n=15) and untreated (n=6) groups based on their Treatment Success Index (TSI). The TSI takes into account a patient’s continuous positive airway pressure (CPAP) compliance and reduction in apnea-hypopnea index (AHI). Patients with TSIs of less than 85 (out of 100) were considered “untreated” while those over 85 were considered “treated.” This study took place at Sleep Consultants, Inc (Fort Worth, TX). Patients were instrumented with 3-lead ECG, pulse oximeter and a Finometer, which recorded beat-to-beat blood pressure. After respiring normally 3 times, the patient was asked to initiate a voluntary apnea for 20 seconds and the SBP response was recorded. An unpaired t-test was performed on group averages, where a p2values were calculated where indicated with ANOVAs to determine significance. Results (c): The untreated mean ∆ SBP was 24.04 ± 7.271 mm Hg and the treated mean was 12.23 ± 3.57 mm Hg, which was significantly different (p=0.00165). TSI and ∆SBP were inversely and significantly correlated (P= -0.69, p=0.00119). The different treatment groups did not desaturate differently (p>0.05), and greater desaturations did not produce greater ∆ SBP responses (R2=0.003, p> 0.05). Conclusions (d): The SBP response to voluntary breath-holds decreases with adequate CPAP treatment independently from SaO2. Therefore, the underlying increase in sympathetic nervous activity (SNA) that drives the ∆ SBP response is likely attenuated with adequate CPAP treatment. This study proves the utility of this maneuver in evaluating treatment efficacy (i.e. reduction in SNA reactivity) in OSA patients in a clinical setting.
EFFECTS ANTIOXIDANTS ON CEREBROVASCULAR HEMODYNAMICS DURING MODERATE AND HIGH INTENSITY EXERCISE
(2014-03) Moralez, Gilbert; White, Daniel; Raven, Peter
It has been identified that central reactive oxygen species (ROS) scavenge nitric oxide (NO) and increase central sympathetic nerve activity (SNA) outflow. During dynamic exercise increases in ROS have been measured across the brain. PURPOSE: to test the hypothesis that during dynamic leg cycling exercise, antioxidants would increase middle cerebral artery blood velocity (MCAv) and cerebrovascular conductance (CVCi). METHODS: Five healthy subjects performed back supported semi-recumbent dynamic leg cycling with antioxidant cocktail (AxT) and without AxT (placebo – Pl). Arterial pressures (AP) and MCAv were measured continuously using Finometer and Doppler ultrasound technologies, respectively, during moderate intensity dynamic leg exercise at heart rates (HR) of 120 bpm (e120) and heavy intensity at HR of 150 bpm (e150). RESULTS: No differences in AP were observed between Pl and Axt at e120 and e150 (P≥0.50). MCAv during AxT at e120 and e150 was increased above Pl at rest (P=0.03) and during exercise (P<0.05). Additionally, the calculated CVCi was significantly greater at rest, e120 and e150 between CT and Pl (P=0.05). CONCLUSIONS: From these data we conclude that Axt scavenged exercise induced central ROS resulting in increased central/peripheral NO induced cerebrovascular vasodilation during exercise. Purpose (a): To test the hypothesis that during dynamic leg cycling exercise, antioxidants would increase middle cerebral artery blood velocity (MCAv) and cerebrovascular conductance (CVCi). Methods (b): Five healthy subjects performed back supported semi-recumbent dynamic leg cycling with antioxidant cocktail (AxT) and without AxT (placebo – Pl). Arterial pressures (AP) and MCAv were measured continuously during moderate intensity dynamic leg exercise at heart rates (HR) of 120 bpm (e120) and heavy intensity at HR of 150 bpm (e150). Results (c): No differences in AP were observed between Pl and Axt at e120 and e150 (P≥0.50). MCAv during AxT at e120 and e150 was increased above Pl at rest (P=0.03) and during exercise (P<0.05). Additionally, the calculated CVCi was significantly greater at rest, e120 and e150 between CT and Pl (P=0.05). Conclusions (d): From these data we conclude that Axt scavenged exercise induced central ROS resulting in increased central/peripheral NO induced cerebrovascular vasodilation during exercise.
HYPERCOAGULATION IN THE POST-ATRIAL FIBRILLATION CATHETER ABLATION PATIENT
(2014-03) Khan, Shamyal H.; Hussain, Omar Z.; Chiapa-Scifres, Ana
Purpose (a): A 72 year old Caucasian female with a past medical history of stroke and treated atrial fibrillation presented to the emergency room with swelling and discomfort in her left leg. The patient denied chest pain, shortness of breath, or hypoxemia. Physical examination was remarkable for edema in the left lower extremity with palpable pulses and no ischemic changes. Results (c): Laboratory findings demonstrated a D-dimer of 4230, prompting a CT angiogram which showed a nonocclusive embolus in the posterior basilar right lower lobe of the pulmonary artery. Ultrasound of the left leg showed thrombus throughout the venous system. She was admitted for deep vein thrombosis and treated with Lovenox 80 mg twice a day as well as Coumadin 5mg daily. After her diagnosis of stroke and non-valvular atrial fibrillation in 2003, the patient had been taking anti-coagulants until her ablation six months ago. At that time the patient had received an AF catheter ablation and her anticoagulation medication was discontinued after she was stable in sinus rhythm. Conclusions (d): While being treated in the hospital, the patient offered a family history of Factor V Leiden mutation in a cousin. Due to present symptoms, testing for clotting factor mutations was not practical given necessary treatment with anticoagulation medication. Hematology recommended treatment with Coumadin for six months, holding medication for two weeks, and testing for protein C, protein S, antithrombin III, and Factor V Leiden. With completion of blood draw, the patient would resume Coumadin and results discussed for long term therapy versus discontinuation of medication.
CONTRIBUTION OF THE MNPO ANGIOTENSIN RECEPTORS TO BRAIN STEM ACTIVITY AND HYPERTENSION
(2014-03) Shell, Brent; Cunningham, Tom
Sleep apnea can increase blood pressure. It is not understood what changes in the brain occur while experiences lack of oxygen during sleep apnea that results in high blood pressure both during the day and during the sleeping hours. Our lab uses a model of sleep apnea that exposes rodents to periods of reduced oxygen. We have found that removing a receptor for a specific chemical in the front of the brain prevents the increase of blood pressure during the normal oxygen waking hours. This current study shows that the knockdown of this receptor decreases activity in the rear portions of the brain that directly control blood pressure. Understanding the mechanisms how sleep apnea leads to hypertension is essential for effective treatment of the disease. Purpose (a): The repeated bouts of hypoxia experienced by sufferers of sleep apnea results in persistent blood pressure elevation. This pathophysiological increase in pressure exists in both the hypoxic night phase and the normoxic period. Neurological mechanisms that drive this maladaptive blood pressure increase are not well understood. Our lab has shown that knockdown of the Angiotensin type 1a (At1a) receptor in a forebrain nucleus, the median preoptic nucleus (MnPO), prevents the normoxic blood pressure increase. How the MnPO At1a receptors affect downstream nuclei to maintain normal pressure is not known. In the current study, rats were exposed to chronic intermittent hypoxia (CIH) to simulate the hypoxic effects of sleep apnea. We then examined the activity of downstream nuclei by performing immunohistochemistry for ∆FosB, a marker for neuronal activity. We hypothesize that knockdown of AT1a in the MnPO results in decreased ∆FosB expression in downstream hypertensive nuclei such as the caudal ventrolateral medulla (CVLM), the rostral ventrolateral medulla (RVLM), and the nucleus tractus solitaries (NTS). Methods (b): After exposure to chronic intermittent hypoxia (CIH), rats are sacrificed, perfused with 4% paraformaldehyde, dehydrated with sucrose, and serial sectioned at 40 microns on a cryostat. Sections are split into three groups; one group is used for immunohistochemistry. Sections are processed with primary goat antibody for ∆FosB, a secondary biotinilated anti-goat, and finally visualized using diaminobenzidine. Localization of the NTS, CLVM, and RVLM was performed by double labeling for dopamine-β-hydroxylase (DβH), an enzyme used in the production of catecholamines. DβH was visualized using a CY3 fluorophore. Cell counts utilized at least 3 brain sections per nucleus. Results (c): A significant difference was found between the AT1a knockdown rats and the scramble rats in the subpostremal region of the NTS. This region has neurons that are responsible for processing both baroreceptor and chemoreceptor information. Conclusions (d): The MnPO is connected to this region through the paraventricular nucleus. Decreased MnPO activity could cause a decrease in the quantity of inputs to the hindbrain. These results, coupled with the prevention of the normoxic blood pressure increase, indicate that angiotensin acting through the MnPO is affecting the activity of neurons in the brainstem that are directly controlling blood pressure regulation.
EVALUATION OF HEART RATE VARIABILITY IN THE TREATMENT OF OBSTRUCTIVE SLEEP APNEA
(2014-03) Bentkowski, Olivia; Welch, Lindsey; Jouett, Noah; Smith, Michael L.
Heart Rate Variability (HRV) is a non-invasive assessment of the activity of the autonomic nervous system (Xhyheri et al). Previous studies have shown a correlation between higher low/high frequency (LF/HF) values and poor cardiovascular health; however, there is question on how to effectively incorporate these findings into clinical practice and treatment. The aim of this study is to determine a relationship between HRV values and the success of using Positive airway pressure (PAP) (measured by Treatment Success Index) for the treatment of Obstructive Sleep Apnea (OSA). Purpose (a): Obstructive Sleep Apnea (OSA) patients treated with Positive Airway Pressure (PAP) (classified by a Treatment Success Index) will have a lower LF/HF ratio as compared to the untreated group. Methods (b): Subjects were instrumented for 30-45 minutes with a 3-lead ECG, which exported raw data into our data acquisition software. Using WinCPRS, we performed spectral analysis on the calculated R-R intervals, which were derived from raw data. LF/HF Heart Rate Variability (HRV) ratios were then calculated and reported for each subject group. Subjects were stratified into treated and untreated groups depending on their Treatment Success Index (TSI) scores. Subjects with TSI's greater than or equal to 85 were considered treated, while subjects with TSIs less than 85 were considered untreated. Results (c): There was no statistical difference in the low frequency to high frequency ratio (LF/HF) between the treated and untreated subjects (Mann Whitney U, p = 0.175). SDNN was determined to show no statistical difference between the treated and untreated subjects (Student’s t-test, p = 0.273). There was no statistical difference in pNN50 between the treated and untreated subjects (Mann Whitney U, p = 0.254). RMSSD demonstrated no statistical difference between the treated and untreated subjects (Mann Whitney U, p = 0.205). Conclusions (d): RMSSD and pNN50 were not found to be significantly higher in the treated subjects. LF/HF ratio and SDNN were not significantly lower in the treated subjects. However, this does not suggest ineffectiveness of PAP treatment in OSA, but rather inadequate sample size.
SLEEP APNEA AND ITS ROLE IN OXIDATIVE STRESS AND INFLAMMATION
(2014-03) Snyder, Brina; Cunningham, J. Thomas; Cunningham, Rebecca L.
Inflammation has been linked with sleep apnea. Sleep apnea is a common comorbidity associated with Parkinson’s disease. Furthermore, both sleep apnea and Parkinson’s disease have been linked with inflammation. A possible mechanism underlying increased inflammation in these disorders is oxidative stress, a hallmark of many neurodegenerative disorders. To examine the role of oxidative stress on inflammation, we used chronic intermittent hypoxia (CIH), an established model for the hypoxemia associated with sleep apnea. CIH consists of recurring events of low oxygen followed by reoxygenation. We hypothesize that CIH causes oxidative stress, which induces inflammation. To test this hypothesis, plasma from adult male rats subjected to 7 days of CIH (3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day) or normoxia (room air) was tested for AOPP, an indicator of oxidative stress, and circulating inflammatory markers (IL-10, IL-4, IL-6). Our results showed that CIH significantly increased circulating oxidative stress. These results were then correlated with inflammatory markers in the plasma and statistically analyzed for positive associations. IL-6 was found to be significantly increased in CIH, although not associated with oxidative stress. However, CIH did increase IL-4 and IL-10, and these effects were positively associated with circulating oxidative stress. Inflammatory markers IL-4 and IL-6 are generally associated with macrophage-mediated inflammation. Therefore it is possible that CIH-induced oxidative stress underlies macrophage mediated inflammation. These findings suggest that sleep apnea increases oxidative stress and consequently inflammation.
THE ROLE OF CEREBRAL OXYGENATION ON TOLERANCE TO CENTRAL HYPOVOLEMIA
(2014-03) Kay, Victoria; Rickards, Caroline
This study will assess how the human body responds to a decrease in blood volume entering the heart (central hypovolemia). Central hypovolemia leads to a decrease in blood flow to the brain, which can occur under various clinical conditions including hemorrhage and stroke. People have different tolerance to a decrease in central blood volume, and we want to determine the physiological mechanisms responsible for making some people more tolerant than others, with a particular focus on the role of blood flow and oxygen supply to the brain. We will also determine the potential mechanisms responsible for improved tolerance to central hypovolemia with a special breathing technique called inspiratory resistance breathing. Our goal is to determine how the body responds during decreased central blood volume, and if these mechanisms could be augmented to save lives during hemorrhage. These experiments may provide the clinical community with data to support the implementation of a diagnostic tool for assessing the level of bleeding by measuring brain oxygen levels. Moreover, by providing data to support the use of inspiratory resistance as a therapeutic intervention in the field, this technique could potentially save many lives by giving patients more time to reach a hospital where more advanced medical treatment can take place. Purpose (a): Tolerance to central hypovolemia varies between individuals, and recent studies have shown that protection of absolute cerebral blood flow is not an underlying mechanism. We hypothesized that subjects with high tolerance (HT) to central hypovolemia maintain cerebral oxygenation (ScO2) at higher levels of lower body negative pressure (LBNP) compared to their low tolerant (LT) counterparts, despite similar reductions in absolute flow. Methods (b): 15 healthy human subjects (10 male; 5 female) were instrumented for assessment of ScO2 (via near-infrared spectroscopy, NIRS) and mean middle cerebral artery velocity (MCAv; via transcranial Doppler, TCD). All subjects completed a presyncopal-limited lower body negative pressure (LBNP) protocol with an onset rate of 3 mmHg/min. Subjects who made it to ≥80mmHg LBNP were classified as HT, and subjects who made it to ≤70 mmHg LBNP were classified as LT. Results (c): The minimum difference in LBNP tolerance between the HT (N=6) and LT (N=9) group was 206 s (LT=1400±104 s vs. HT=2080±65 s; P=0.0003). Up to -45 mmHg LBNP, ScO2 was maintained in HT subjects (P≥0.538), while the LT (N=9) subjects had a progressive decrease in ScO2 (P≤0.016) from baseline. MCAv decreased from baseline in both HT and LT subjects (P≤0.022). There was a strong linear relationship between %∆ MCAv and %∆ ScO2 within the LT group (R2=0.98; P=0.013), whereas a weaker association between perfusion and oxygenation (R2=0.53; P=0.271) was observed in the HT group. Conclusions (d): In support of our hypothesis, higher tolerance to progressive central hypovolemia was associated with the protection of ScO2, despite an early and significant reduction in cerebral blood flow. This may have important clinical implications for the monitoring of cerebral perfusion and oxygenation in trauma patients.
MYOSIN REGULATORY LIGHT CHAIN A13T MUTATION ASSOCIATED WITH CARDIAC HYPERTROPHY IMPOSES DIFFERENCES ON KINETICS AND SPATIAL DISTRIBUTIONS OF CROSS-BRIDGES IN HEALTHY AND DISEASED VENTRICLES
(2014-03) Nagwekar, Janhavi; Duggal, Divya; Midde, Krishna; Kazmierczak, Katarzyna; Huang, Weiwen; Fudala, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Szczesna-Cordary, Danuta; Borejdo, Julian
The study is performed at protein and myofibrilar level to identify key sub-steps of ATP induced XB cycle deregulation in FHC. Humans being heterozygous (50%) for FHC, our experiments with 10% penetrance in the mice will analyze subtle changes in the XB mechanisms to which humans have 40% more chances to develop those defects. This project may also provide insights into other RLC mutations (e.g. R58Q, E22K, D166V, P95A) that may involve the same alterations. The project aims to identify drugs to alter specific rate constants, affect ordering of XBs or reverse A13T effect and thus treating patients with personalized therapy. Purpose (a): Muscle is organized into regular periodic thick myosin and thin actin filaments. Myosin tails interact with each other to form a tight coiled coil rod, and the heads protrude out to interact with actin. Myosin head referred to as cross-bridge has ATPase activity and actin binding domain. The tail has a site (Regulatory Light Chain (RLC) domain) which when mutated at A13T site cause myosin heads to bind slowly to the actin molecules affecting the overall ATPase cycle and power strokes necessary for a muscle to contract in the process. Methods (b): Rabbit ventricle muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were labeled with 0.1 nM rhodamine-phalloidin (RP) + 10 μM unlabeled-phalloidin (UP) in Ca2+-rigor solution in the the ratio of 1:100,000 fluorescent to non-fluorescent phalloidin to ensure 1 in ~105 actin monomers carry a fluorophore. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence to determine kinetic rate constants in the ATPase cycle and distribution of orientations emanating from myosin cross-bridges. Results (c): Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. The mean polarization of a contracting WT myofibril was -0.176±0.018 and that of contracting A13T Mutated myofibril was -0.247±0.017. Significant differences in rate constants k1, k2 and k3 of the ATPase cycle were observed with WT values being 325±34, 0.16±0.03, 0.32±0.08 and A13T mutated values being 54±80*, 0.25±0.04, 0.57±0.12 respectively. On comparing the peaks of the fit of the data, peak 1 assumed to be the pre power stroke was lost in the A13T mutated myofibrils while peak 2 (post power stroke) almost remained constant in both muscle types. Conclusions (d): The study suggest that the functional differences between ventricles containing WT myosin and myosin in which the RLC contains the A13T mutation are caused by a change in the rate of binding of myosin cross-bridges to the thin filaments. Differences in the polarization, FWHM and peaks indicate that pre-power strokes are necessary for myosin cross-bridges and that any alterations in its functions may lead to cardiomyopathy.
ANGIOTENSIN CONVERTING ENZYME 1 (ACE1) KNOCKDOWN IN THE MEDIAN PREOPTIC NUCLEUS (MNPO) ATTENUATES SUSTAINED DIURNAL HYPERTENSION FOLLOWING CHRONIC INTERMITTENT HYPOXIA
(2014-03) Faulk, Katelynn; Cunningham, J. Thomas; Nedungadi, Thekkethil P.
In order to study hypertension associated with Sleep Apnea, our lab uses a hypoxia model on rats. Rats are routinely used to model cardiovascular diseases of humans. We try to discover how the brain controls blood pressure in certain cardiovascular risk groups such as sleep apnea patients. This will help the scientific community better understand how sustained hypertension develops and progresses in sleep apnea patients and may lead to other discoveries about cardiovascular diseases. Purpose (a): Chronic Intermittent Hypoxia (CIH) is a model for the arterial hypoxemia seen in sleep apnea and is associated with a sustained increase in blood pressure throughout the diurnal cycle. Studies indicate that the MnPO contributes to this sustained component of CIH hypertension that persists during normoxia. MnPO neurons from rats show increased expression of the transcription factor FosB following CIH. Dominant-negative inhibition of a FosB splice variant in MnPO attenuates the sustained hypertension in CIH. We identified the pro-hypertensive ACE1 as a possible FosB target gene that may contribute to the sustained hypertension seen in CIH. Methods (b): We tested this hypothesis using a viral vector to knockdown ACE1 in the MnPO. Isoflurane anesthetized adult male rats were microinjected in the MnPO with 500nl of an adeno-associated virus containing GFP and either shRNA against ACE1 (shACE1) or a scrambled shRNA (shSCM). Changes in Mean arterial blood pressure (MAP) were recorded using radio telemetry. Rats were then exposed to CIH for 7 days through 3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day (0800-1600 h). Normoxic controls were exposed to room air. Laser capture microdissection followed by qRT-PCR showed that shACE1 significantly decreased ACE1 message in MnPO. Results (c): During CIH exposure, MAP significantly increased in both shACE1 and shSCM treated rats. During the normoxic dark phase, knockdown of ACE1 in the MnPO statistically decreased the sustained MAP component of CIH as compared to shSCM controls (P<0.001). Conclusions (d): These results show that ACE1 in the MnPO contributes to the sustained hypertension seen in our CIH model.
ANGIOTENSIN II RECEPTOR TYPE-1A KNOCKDOWN IN SUBFORNICAL ORGAN PREVENTS SUSTAINED INCREASE IN MEAN ARTERIAL PRESSURE ASSOCIATED WITH CHRONIC INTERMITTENT HYPOXIA
(2014-03) Saxena, Ashwini; Little, Joel T.; Nedungadi, Thekkethil P.; Cunningham, J. Thomas
Purpose (a): Sleep apnea (SA) is associated with a sustained increase in mean arterial pressure (MAP) even during waking hours. Chronic intermittent hypoxia (CIH) models the hypoxemia associated with SA and produces elevated MAP during CIH and normoxia. Angiotensin II (Ang II) is implicated in the CIH associated increase in MAP. Subfornical organ (SFO), a forebrain circumventricular organ, lacks blood brain barrier and is a major site for the central effects of circulating Ang II. We investigated the effects of Ang II type 1a receptor knockdown (AT1aRKD) in the SFO on CIH hypertension in adult male rats. Methods (b): Adeno-associated viral vectors carrying GFP and either AT1aR shRNA or scrambled shRNA (SCM) were injected in SFO. Continuous measurements of mean arterial pressure, heart rate, respiratory rate, and activity were measured using radio-telemetry device implanted in abdominal aorta. Rats were exposed to cyclic hypoxia (3 min 21% O2 - 3 min 10% O2) for 8 hours/day for 7 days. Rats were sacrificed on Day 8. Results (c): Using laser-capture microdissection and qRT-PCR of amino-allyl RNA, AT1aRKD rats showed decreased SFO AT1aRmRNA in comparison with SCM rats. During intermittently-hypoxic light phase, the AT1aRKD rats exposed to CIH (3 min 10% O2 and 3 min room air cycles for 8 h during light phase for 7 d) exhibited significant increases in MAP vs. AT1aRKD-Normoxia group (p(p<0.05). During the normoxic dark phase, there was no difference in MAP between CIH and normoxic AT1aRKD rats (p=0.69). SCM-CIH group showed significant increase in MAP from SCM-Normoxia group during light phase CIH (p<0.001) and the normoxic dark phase (p<0.001). Conclusions (d): Our data indicate that AT1aRs in SFO may play a role in the sustained increases in MAP during normoxia associated with CIH.
APPLICATION OF TREATMENT SUCCESS INDEX ON MULTIPLE VARIABLES IMPLICATED IN SLEEP APNEA
(2014-03) Welch, Lindsey; Bentkowski, Olivia; Jouett, Noah; Raven, Ph.D., Peter
The NIH states that Positive Airway Pressure (PAP) is the optimal treatment for obstructive sleep apnea (OSA), with successful treatment benefits ranging from an increased quality of sleep to decreasing or preventing high blood pressure. Previous research has explored the positive effects of PAP use on various health indicators, however this data is based on subjective PAP use data from patients. The goal of this study was to evaluate the relationship between various health indicators and the success of using PAP with a Treatment Success Index (TSI), a measured value of treatment success based on PAP compliance and treatment efficacy. The TSI assumes that usage and effectiveness are equally important, and that decrements in either impose multiplicative impact on treatment quality. Purpose (a): The goal of this study was to evaluate the relationship between various health indicators and the success of using PAP with a Treatment Success Index (TSI), a measured value of treatment success based on PAP compliance and treatment efficacy. The literature demonstrates quite clearly that untreated OSA issues in a wide array of physical malady, ranging from obesity to cardiovascular disease. Therefore, we hypothesize that Obstructive Sleep Apnea (OSA) patients treated with Positive Airway Pressure (PAP) will demonstrate more favorable values of health indicators as compared to untreated patients (classified by Treatment Success Index). Methods (b): Data collection was performed at an office visit for each subject. PAP machines were brought from home to provide at-home data. Health indicators collected included Epworth score, heart rate, systolic and diastolic blood pressure and BMI. Subjects were assigned a Treatment Success Index (TSI) percentage based on compliance and at-home effectiveness data. Analysis was completed to determine the relationship between TSI score and the health indicators collected. Subjects with a TSI greater than or equal to 85 were considered treated, while subjects with a TSI less than 85 were considered untreated. Analysis was run on these groups to compare health indicators to their treated or untreated status. Results (c): The Epworth score was found to be decreased in the treated group, determined by the Mann-Whitney U Test results of a median value of 5 in the treated group and 7 in the untreated group (p<0.001). Systolic blood pressure was increased in the treated group, with a Mann-Whitney U Test demonstrating a median of 130 for the treated group and 124 for the untreated group (p <0.001). BMI was also increased in the treated group at 33.6, compared to 31.7 in the untreated group (p=0.002, Mann-Whitney U). There were no significant findings related to changes in heart rate of diastolic blood pressure. Conclusions (d): While Epworth score showed a favorable response to PAP treatment as determined by TSI values, systolic blood pressure and BMI were found to be higher in the treated groups. This does not indicate a negative effect of PAP use on these variables, but rather suggests other underlying mechanisms.
PYRUVATE'S ANTI-INFLAMMATORY EFFECTS IN LUNG TISSUE POST CARDIAC ARREST
(2014-03) Baker, Charla L.; Nquyen, Anh Q.; Cherry, Brandon H.; Olivencia-Yurvati, Albert H.; Mallet, Robert T.
Ischemia-reperfusion injury sustained following cardiac arrest initiates an injury cascade that leads to inflammatory changes in multiple organs. The inflammatory response is characterized by an increase in cytokines and infiltrative neutrophils. In a study focused on the myocardial and systemic effects of an induced hemorrhagic shock in goats, pyruvate administration blunted inflammation in cardiac tissue. Further, pyruvate-fortified Ringer’s has been shown to suppress inflammation during reperfusion by decreasing the amount of reactive oxygen species and improving the glucose metabolism, providing multi-organ protection. These findings suggest a pyruvate-fortified resuscitation fluid infusion following cardiac arrest may attenuate the inflammatory response to ischemia-reperfusion. Of particular interest, pulmonary tissue has been shown to be affected by ischemia-reperfusion. Therefore, we hypothesized that intravenous administration of pyruvate during cardiac arrest and early recovery would exert anti-inflammatory effects to protect the lungs from ischemia-reperfusion injury. To test this hypothesis, Yorkshire swine were subjected to cardiac arrest and infused with either NaCl (n=3) or Na-pyruvate (n=3) during cardiopulmonary resuscitation (CPR). Lung biopsies collected after 4 hours recovery were embedded in paraffin, sectioned, and stained for haematoxylin and eosin (H&E), and compared to biopsies from sham (non-arrest; n=3) animals using brightfield microscopy. Sections were examined for the histological marker of acute lung injury—i.e., neutrophils in the alveolar or interstitial space. The CPR NaCl showed appreciable neutrophil infiltration in the alveoli in contrast to the CPR Na-pyruvate. These early results suggest that the Na-pyruvate may suppress pulmonary inflammation following cardiac arrest. Purpose (a): To identify a new pharmacological strategy to protect the lungs from ischemia-reperfusion injury due to cardiac arrest. Methods (b): Yorkshire swine (30-40 kg) were subjected to cardiac arrest-resuscitation or non-arrest sham protocols. Ventricular fibrillation was induced by a train of electric impulses transmitted to the right ventricle via a pacing wire. Precordial compressions (100/min) were given from 6-10 min arrest, and then sinus rhythm was restored with defibrillatory transthoracic countershocks. NaCl or Na-pyruvate was infused iv at 0.1 mmol/kg/min during chest compressions and the first 60 min post-defibrillation. After 4 h recovery, lung tissue was excised, fixed in 4% paraformaldehyde and embedded in paraffin wax. Sections were cut and stained with H&E. Twenty random high power fields independently were scored in a blinded fashion. Scoring: No neutrophils in the alveolar space= 0; 1-5 neutrophils= 1; >5 neutrophils= 2. Interstitial neutrophils were counted, averaged, and compared among the three experimental groups. Results (c): H&E staining showed no statistically significant difference of interstitial (P=0.49) and alveolar (P=0.65) neutrophil infiltrate among the sham, CPR and CPR + Pyruvate. Conclusions (d): There was no statically significant difference in pulmonary neutrophil infiltration at 4 hour post cardiac arrest and resuscitation in CPR and pyruvate treated CPR compared to sham. Further analysis of longer post-arrest recoveries are necessary to better understand the anti-inflammatory effect of pyruvate in lung tissue in this experiment model.
THE K104E MUTATION OF THE MYOSIN REGULATORY LIGHT CHAIN ALTERS KINETICS AND DISTRIBUTION OF ORIENTATIONS OF CROSS-BRIDGES IN TRANSGENIC CARDIAC MYOFIBRILS
(2014-03) Duggal, Divya; Nagwekar, Janhavi; Rich, Ryan; Huang, W.; Midde, Krishna; Fudala, Rafal; Gryczynski, Ignacy; Szczesna-Cordary, Danuta; Borejdo, Julian
My project aims at finding out the differences in a healthy and diseased heart. The disease I am focusing on is Familial Hypertrophic Cardiomyopathy(FHC), in particular, the K104E mutation, which is a mutation in the Regulatory Light Chain(RLC) of Myosin. The differences are established in terms of the rate of association and dissociation of myosin from actin, as well as the order of cross bridges. This study will help us to investigate the role of such mutations in causing FHC as well as offer us the opportunity to investigate other mutations causing this disease. Purpose (a): The purpose of my study is to examine the cross-bridge (XB) kinetics and the degree of order in contracting myofibrils from the ex-vivo left ventricles of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) Regulatory Light Chain (RLC) mutation K104E. Methods (b): 1. Myofibrils were prepared from the frozen hearts of Tg-WT mice and newly generated Tg-K104E mice. 2. Since the kinetics and degree of order are best studied when an individual cross bridge (XB) makes a significant contribution to the overall signal, the number of observed XBs was minimized to ~20 by sparsely labeling the Essential Light Chain(ELC) of myosin. Autofluorescence and photobleaching were minimized by labeling ELC with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule, SeTau 647. 3. Myofibrils were crosslinked with a cross linker prior to labeling. 4. Following labeling, fluorescence was measured by PicoQuant MT 200 inverse time-resolved fluorescence instrument coupled to Olympus IX 71 microscope. Results (c): We show that the K104E mutation, when compared with Wild Type (WT) ventricles, had significant effect on both the kinetics of the interaction between actin and myosin and on the degree of order of the myosin lever arm. In particular, the K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke, while decreasing the rate of XB dissociation from actin. Mutated XBs were significantly better ordered during steady-state contraction and during rigor but mutation had no effect on the degree of order in relaxed myofibrils. Conclusions (d): This implies that the mutated ventricle may be prone to decreased maximal tension and increased muscle relaxation time suggesting a potential for diastolic dysfunction in patients.
DELAYED NEURONAL DEATH IN SWINE FOLLOWING CARDIAC ARREST AND RESUSCITATION
(2014-03) Nguyen, Anh Q.; Cherry, Brandon H.; Myoung-Gwi, Ryou; Williams, Arthur G.; Hollrah, Roger; Baker, Charla L.; Choudhury, Gourav; Olivencia-Yurvati, Albert H.; Mallet, Robert T.
Purpose (a): Cardiac arrest, a leading cause of death in the U.S., kills >90% of its victims, and survivors often are disabled by permanent brain injury inflicted by ischemia-reperfusion. Purkinje cells of the cerebellum and CA1 neurons of the hippocampus are especially vulnerable to post-ischemic neuronal death. We tested the hypothesis that cardiac arrest in a swine model caused delayed neuronal death. Methods (b): Yorkshire swine (25-35 kg) were subjected to cardiac arrest-resuscitation (n = 9) or non-arrest sham (n = 5) protocols. Ventricular fibrillation was induced by electrical pacing. Precordial compressions (100/min) were given at 6-10 min arrest, and then sinus rhythm was restored with transthoracic countershocks. NaCl was infused iv at 0.1 mmol/kg/min during CPR and the first 60 min after return of spontaneous circulation (ROSC). At 7 d ROSC, brain regions were fixed in 4% paraformaldehyde and H&E stained. Results (c): More than 70% of the Purkinje cells were shrunken, lacked dendrites and displayed condensed cytoplasm at 7 d ROSC; in contrast, in shams the majority of Purkinje cells retained the characteristic thick dendrites and well-defined nuclei. Conclusions (d): Thus, cardiac arrest-resuscitation produced marked changes in cerebellar neurons evident 7d after acute insult.
INTERMITTENT HYPOXIA CONDITIONING REDUCES INOS AND ENOS EXPRESSION IN RAT MYOCARDIUM: A MECHANISM FOR PROTECTION FROM ISCHEMIA AND REPERFUSION INJURY
(2014-03) Downey, H. Fred; Manukhina, Eugenia B.; Goryacheva, Anna V.; Belkina, Ludmila M.; Terekhina, Olga L.; Mallet, Robert T.
During a heart attack, the heart produces excessive amounts of harmful chemicals. This study showed that prior exposure of rats to short cycles of a low oxygen atmosphere reduced production of these chemicals and lessened the damage to muscle and blood vessels of the heart. This procedure may benefit humans at risk of heart attack. Purpose (a): Recently we demonstrated that intermittent, normobaric hypoxia conditioning (IHC) prevented injuries of myocardium and coronary blood vessels induced by myocardial ischemia and reperfusion (IR). This cardio- and vasoprotection of was associated with alleviation of nitric oxide overproduction. The aim of this study was to identify specific NO synthases responsible for the IR-induced NO overproduction and to determine the effects of IHC on these NO synthases. Methods (b): This research was approved by the Animal Care and Use Committee of the Institute of General Pathology and Pathology. IHC of rats was performed in a normobaric chamber (5-8 cycles/d for 20 d, FIO2 9.5 - 10% for 5 - 10 min/cycle, with intervening 4-min normoxia). IR was produced by ligation of the left anterior descending coronary artery for 30 min followed by 60-min reperfusion. The protein nitration marker, nitrotyrosine (3-NT) and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthases were measured by immunoblot. Results (c): IR induced appreciable 3-NT accumulation in the left ventricular free wall, increasing the 3-NT content by 42% (p<0.01), but not in septum. In IHC rats, 3-NT after IR was similar to that of control rats without IR. IHC decreased iNOS by 71% (p<0.05) and eNOS by 41% (p<0.05) in the left ventricular myocardium; the myocardial content of nNOS remained unchanged. Conclusions (d): IHC prevents IR-induced NO overproduction in myocardium by restricting myocardial expression of iNOS and eNOS.
PEDIATRIC CARDIOVASCULAR SCREENING RESEARCH
(2014-03) Mou, Margaret; Wilson, Dr. Don
According to current NIH guidelines, there should be universal screening of children ages 9-11 for cholesterol. By doing so, pediatricians can often detect familial hypercholesterolemia or high cholesterol that leads to future heart disease. However, many pediatricians currently are not following these guidelines. This project is to determine why that is the case and what changes can be implemented to help universal screening. Purpose (a): The objective of this project is to understand general pediatricians’ screening and treatment processes in children with a variety of cardiovascular disease risk factors, including what steps could be taken to ensure universal cholesterol screening of children ages 9-11. Methods (b): 1. Recruitment: Participants will be recruited via NLA pediatricians, contacted via email directly from the directors of these networks in order to maintain anonymity. 2. Data and Storage: Only the primary investigator will have access to the participating pediatrician’s responses to the questionnaire. The information will be imported directly from the submitted questionnaires, collated and stored on a secure online excel spreadsheet. 3. Procedures to Maintain Confidentiality: Because the directors of the participating networks are sending out the questionnaire on behalf of the study investigators, there is no direct trail from the primary investigator to the respondents. Thus, anonymity is maintained, and all the submitted information will remain confidential to the individual. 4. Data Security: Access to the excel spreadsheet will be password restricted to only the study’s primary investigator(s). Results (c): Briefly speaking, the majority of respondents do not currently order cholesterol screenings for their patients. However, 83% of the respondents do think that universal screening of cholesterol of children ages 9-11 years old is important. The major barriers seen by providers included family hesitancy and financial restrictions. Conclusions (d): There are opportunities to capitalize on the barriers for universal cholesterol screening in children ages 9-11, including provider education on current NIH guidelines, insurance policy, and family education.
BASELINE SYMPATHETIC ACTIVITY IS ELEVATED IN PATIENTS WITH ATRIAL FIBRILLATION
(2014-03) Hanson, Gabriel S.; Cielonko, Luke A.; Smith, Michael L.
Atrial fibrillation, or AF, is an abnormal rhythm of the heart that is common in many people, particularly in individuals over 60 years of age. The association of AF with other cardiovascular diseases and the potential relation to sudden cardiac death (or cardiac arrest) is not known. This study investigated the baseline activity of the sympathetic nervous system. The sympathetic nervous system is the primary branch of the nervous system that mediates the response to a stress. The findings of this study strongly suggest that sympathetic nervous system activity is increased in AF which may also increase incidence of progression of other cardiovascular disease states including potentially fatal ventricular dysrhythmias. Purpose (a): Most cardiovascular diseases are associated with high sympathetic nerve activity (SNA), and elevations in SNA are known to increase the progression of many forms of cardiovascular disease. Atrial fibrillation (AF) is a serious cardiac dysrhythmia that afflicts a substantial percentage of the population and is known to result in increased risk of blood clotting. This results in a higher risk for pulmonary emboli and stroke. These complications tend to be associated with a generalized fatigue and reduced exercise capacity due to decreased cardiac output. However, the effect of atrial fibrillation on SNA is unknown, thus the purpose of this study was to determine the effect of AF on baseline SNA independent of other factors. Methods (b): Two studies were performed in patients with AF. First, eight patients with drug-refractory AF were studied before and after completion of an AV nodal ablation to normalize the ventricular rate. Microneurographic recordings of SNA were obtained continuously during and after completion of the procedure. Upon effective AV nodal ablation, the patient was immediately paced with a temporary pacemaker inserted in the ventricular apex via vascular access. Ventricular pacing was conducted at the same rate as the patients ventricular rate during their episodes of AF. Baseline SNA was determined prior to ablation and during ventricular pacing. A comparison of SNA was made between the AF state and during the artificially paced ventricular rate, which as stated above was conducted at the mean of their ventricular rate during AF. Second, nine patients with paroxysmal AF were studied during AF and after cardioversion of AF during sinus rhythm. Microneurographic recordings of SNA were obtained continuously during AF and during sinus rhythm. Statistical comparisons were performed for each study with a paired Student's T test to determine the difference in SNA between conditions with AF and without AF. Results (c): For study 1, mean ventricular rates during AF were 93 + 4 bpm (range= 82-128 bpm) and the post-ablation pacing rates were the same and were maintained at a constant regularity with no inter-beat difference. Baseline SNA decreased significantly from 2836 + 332 units to 2291 + 298 units (p < 0.03). For study 2, mean ventricular rates during AF were 98 + 3 bpm (range= 78-140 bpm) and the post-cardioversion sinus rhythm rates were 77 + 2 bpm (range= 62-91 bpm). The baseline SNA decreased significantly from 3755 + 401 units to 2329 + 339 units (p < 0.01). In each case, the measurement of SNA was based on 100 heartbeats, and thus was independent of the rate. Conclusions (d): These data support the hypothesis that baseline SNA is elevated in atrial fibrillation and appears to be independent of rate. These findings support the hypothesis that AF may also increase the risk of progression of other cardiovascular disease states including potentially fatal ventricular dysrhythmias.

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