Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death.

Increased inducible nitric oxide synthase (iNOS) expression is a component of the immune response and has been demonstrated in cardiomyocytes in septic shock, myocarditis, transplant rejection, ischemia, and dilated cardiomyopathy. To explore whether the consequences of such expression are adaptive or pathogenic, we have generated a transgenic mouse model conditionally targeting the expression of a human iNOS cDNA to myocardium. Chronic cardiac-specific upregulation of iNOS in transgenic mice led to increased production of peroxynitrite. This was associated with a mild inflammatory cell infiltrate, cardiac fibrosis, hypertrophy, and dilatation. While iNOS-overexpressing mice infrequently developed overt heart failure, they displayed a high incidence of sudden cardiac death due to bradyarrhythmia. This dramatic cardiac phenotype was rescued by specific attenuation of transgene activity. These data implicate cardiomyocyte iNOS overexpression as sufficient to cause cardiomyopathy, bradyarrhythmia, and sudden cardiac death.

Hypoxemic reperfusion exacerbates the neurological injury sustained during neonatal deep hypothermic circulatory arrest: a model of cyanotic surgical repair.

OBJECTIVE: Deep hypothermic circulatory arrest (DHCA) is frequently used in infants undergoing the Norwood procedure. These infants are necessarily hypoxemic after separation from CPB. Considerable energy has been spent characterizing the physiological and histological consequences of DHCA, but these have largely focused on a normoxemic period of reperfusion. Furthermore, evidence has accumulated to suggest that the cerebral vascular autoregulatory mechanisms are dysfunctional following DHCA. In particular, the vasodilatation that elevates cerebral blood flow (CBF) in response to hypoxemia is absent. This study therefore aimed to investigate whether post-CPB hypoxemia exacerbates brain injury resulting from DHCA. METHODS: Twelve neonatal piglets were subjected to 2h DHCA and then separated from CPB. They were then randomized to either: Group 1, normoxic ventilation (n=5); or Group 2, hypoxemia (n=7), in which the arterial PaO(2) was reduced to 40-50 mmHg for the duration of reperfusion. Following a 20 h period of warm reperfusion, the animals were perfusion fixed and the brain analyzed for histological evidence of injury. Nine additional animals were studied in one of three control groups. RESULTS: All animals survived the protocol. Post-operative parameters - including mean arterial pressure, acid-base status, inotrope requirements and arterial PaCO(2) - were similar. None of the control animals had any evidence of ischemia. Group 1 animals had moderate injury (total score 7.4+/-1.6). In Group 2, three animals sustained irretrievable brain injury evidenced by gross edema and early liquefactive necrosis. The remaining four had severe ischemic histological changes (score 14.5+/-1.6, p<0.03). CONCLUSIONS: Hypoxemic reperfusion after prolonged DHCA results in increased neuronal loss. The use of DHCA for staged palliation may confer disproportionately greater cerebral risk than other patient groups. Alternatively, methods to augment oxygen delivery - such as by ECMO - may be of particular benefit in the early re-perfusion window.

Retrospective analysis comparing hollow fiber and silicone membrane oxygenators for neonates on ECMO.

There is little information showing the use of microporous polypropylene hollow fiber oxygenators during extra-corporeal life support (ECLS). Recent surveys have shown increasing use of these hollow fibers amongst ECLS centers in the United States. We performed a retrospective analysis comparing the Terumo BabyRx hollow fiber oxygenator to the Medtronic 800 silicone membrane oxygenator on 14 neonatal patients on extracorporeal membrane oxygenation (ECMO). The aim of this study was to investigate the similarities and differences when comparing pressure drops, prime volumes, oxygenator endurance, and gas transfer capabilities between the two groups.

Conditional expression of a dominant-negative c-Myb in vascular smooth muscle cells inhibits arterial remodeling after injury.

Inhibiting activity of the c-Myb transcription factor attenuates G1 to S phase cell cycle transitions in vascular smooth muscle cells (SMCs) in vitro. To determine the effects of arterial SMC-specific expression of a dominant-negative c-Myb molecule (Myb-Engrailed) on vascular remodeling in vivo, we performed carotid artery wire-denudation in 2 independent lines of binary transgenic mice with SM22alpha promoter-defined Doxycycline-suppressible expression of Myb-Engrailed. Adult mice with arterial SMC-specific expression of Myb-Engrailed were overtly normal in appearance and did not display any changes in cardiovascular structure or physiology. However, bromodeoxyuridine-defined arterial SMC proliferation, neointima formation, medial hyperplasia, and arterial remodeling were markedly decreased in mice expressing arterial SMC-restricted Myb-Engrailed after arterial injury. These data suggest that c-Myb activity in arterial SMCs is not essential for arterial structure or function during development, but is involved in the proliferation of arterial SMCs as occurs in vascular pathology, and that the expression of a dominant-negative c-Myb can dramatically reduce adverse arterial remodeling in an in vivo model of restenosis. As such, this model represents a novel tissue-specific strategy for the potential gene therapy of diseases characterized by arterial SMC proliferation.

Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure.

In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.

Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension.

BACKGROUND: Increased serine elastase activity has been implicated in the vascular remodeling associated with chronic hypoxia-related pulmonary hypertension in rats. METHODS AND RESULTS: In this study we determined the time course of hypoxia-induced serine elastase activity in the murine lung and related this to initiation of a proteolytic cascade characterized by an increase in matrix metalloproteinases (MMPs). We then used transgenic mice in which overexpression of the selective serine elastase inhibitor elafin was targeted to the cardiovascular system to determine whether upregulation of a naturally occurring serine elastase inhibitor suppresses MMPs and the hemodynamic and structural response to chronic hypoxia (air at 380 mm Hg). In nontransgenic but not in elafin-transgenic mice, we documented a transient increase in serine elastase activity after 12 hours of hypoxic exposure attributed to a 30-kDa protein as determined by elastin zymography and fluorophosphonate/fluorophosphate-biotin labeling. Two days after hypoxia, the pro-forms of MMP-2 and MMP-9 were induced in the nontransgenic mice, but MMP-9 was suppressed in elafin-transgenic mice. Acute hypoxic vasoconstriction was similar in nontransgenic and elafin-transgenic littermates. Chronic hypoxia for 26 days resulted in >1-fold increase in right ventricular pressure (P<0.004) in nontransgenic compared with control or elafin-transgenic littermates. In the latter mice, normalization of the right ventricular pressure was associated with reduced muscularization and preservation of the number of distal vessels (P<0.04 for both comparisons). CONCLUSIONS: Modulation of the severity of chronic hypoxia-induced pulmonary vascular disease could be a function of endogenously expressed serine elastase inhibitors.

Rat cardiopulmonary bypass model: application of a miniature extracorporeal circuit composed of asanguinous prime.

A clinically relevant rat cardiopulmonary bypass (CPB) model would be a valuable tool for investigating pathophysiological and therapeutic strategies on bypass. Previous rat CPB models have been described in the literature; however, they have many limitations, including large circuit surface area, the inability to achieve full bypass, and donor blood requirements for prime. Therefore, we have established a rat CPB model designed to overcome these limitations. The miniature circuit consisted of a filtered reservoir, heat exchanger, membrane oxygenator (surface area = 0.02 m2) with a static priming volume of 2.8 mL, and an inline blood gas monitor. The circuit was primed with 9.5+/-0.5 mL of crystalloid solution and CPB was established on male Sprague-Dawley rats (430-475 g, n = 5) by cannulating the left common carotid artery and the right external jugular vein. The animals were placed on CPB at full flow (111+/-13 mL/kg/ min) for 1 hour and were monitored for and additional 2 hours after the CPB procedure. Hemodynamics, hemoglobin concentration (Hb), and blood gases were analyzed at three time intervals: before, during, and after CPB. The circuit performance was evaluated according to prime volume, compliance, hemodynamic parameters, and gas and heat exchange as described by modified AMMI standards. Data are expressed as mean+/-SD and a repeated-measures analysis of variance with post-Hoc test was used for data comparison between the three time intervals. The ratio of oxygenator surface area to subject body weight for this model is comparable with that of current human adult CPB practice (0.05 m2/kg vs 0.057 m2/kg) Full CPB was achieved and we observed clinically acceptable PaO2, PaCO2, and SvO2 values (209+/-86 mmHg, 25+/-2 mmHg, 78+/-8%, respectively) while on CPB. The use of asanguinous prime did produce statistically significant Hg reduction (15.7+/-0.76 vs. 9.2+/-0.59 g/dL) comparable with clinical practice. No statistically significant differences between pre- and post-CPB hemodynamics and blood gases were found in our study. We have established a miniature circuit consisting of asanquineous prime for a rat CPB model that maintains clinically acceptable results regarding hemodynamic parameters, blood gases, and hemodilution. This model would be valuable for further use in clinically relevant research studies.

Cardiac function in mice lacking the glucagon-like peptide-1 receptor.

Glucagon-like peptide-1 (GLP-1) acts via its G protein-coupled receptor (GLP-1R) to regulate blood glucose. Although the GLP-1R is widely expressed in peripheral tissues, including the heart, and exogenous GLP-1 administration increases heart rate and blood pressure in rodents, the physiological importance of GLP-1R action in the cardiovascular system remains unclear. We now show that 2-month-old mice with genetic deletion of the GLP-1R (GLP-1R(-/-)) exhibit reduced resting heart rate and elevated left ventricular (LV) end diastolic pressure compared with CD-1 wild-type controls. At the age of 5 months, echocardiography and histology demonstrate increased LV thickness in GLP-1R(-/-) mice. Although baseline hemodynamic parameters of GLP-1R(-/-) did not differ significantly from those of wild type, GLP-1R(-/-) mice displayed impaired LV contractility and diastolic function after insulin administration. The defective cardiovascular response to insulin was not attributable to a generalized defect in the stress response, because GLP-1R(-/-) mice responded appropriately to insulin with increased c-fos expression in the hypothalamus and increased circulating levels of glucagon and epinephrine. Furthermore, LV contractility after exogenous epinephrine infusion was also reduced in GLP-1R(-/-) mice. These findings provide new evidence implicating an essential role for GLP-1R in the control of murine cardiac structure and function in vivo.

A role for the aryl hydrocarbon receptor in cardiac physiology and function as demonstrated by AhR knockout mice.

The aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, is the receptor for the polycyclic aromatic hydrocarbons found in tobacco smoke, polychlorinated biphenyls, and the environmental pollutant, dioxin. To better understand the role of the AhR in the heart, echocardiography, invasive measurements of aortic and left ventricular pressures, isolated working heart preparations, as well as morphological and molecular analysis were used to investigate the impact of AhR inactivation on the mouse heart using the AhR knockout as a model. Cardiac hypertrophy is an early phenotypic manifestation of the AhR knockout. Although the knockout animals were not hypertensive at the ages examined, cardiomyopathy accompanied by diminished cardiac output developed. Despite the structural left ventricular remodeling, the hearts of these animals exhibit minimal fibrosis and do not have the expected increases in surrogate molecular markers of cardiac hypertrophy. The anatomic remodeling without typical features of molecular remodeling is not consistent with hypertrophic growth secondary to pressure or volume overload, suggesting that increased cardiomyocyte size may be a direct consequence of the absence of the AhR in this cell type.

Argatroban usage for anticoagulation for ECMO on a post-cardiac patient with heparin-induced thrombocytopenia.

We report a post-Norwood Stage I patient requiring ECMO support using Argatroban as an anticoagulant following diagnosis of heparin-induced thrombocytopenia (HIT). A 2.6 kg female was born with hypoplastic left heart syndrome and underwent a Norwood Stage I operation on day 4 of life. The patient weaned off cardiopulmonary bypass with no complications and was routinely placed on a ventricular assist device (VAD) for 3 days. Heparin was infused at a rate of 16-32 IU/ kg/h to maintain an ACT of 160-180 seconds. Two days after VAD termination, the patient was placed on continuous veno-veno hemofiltration (CVVH). Shortly after CVVH, the patient was diagnosed with HIT and placed on an Argatroban infusion. Five days later, a VAD and subsequent ECMO was used because of decreasing left ventricular function, gross body edema, and poor renal function. This case report summarizes the use of Argatroban during VAD and ECMO support for a patient diagnosed with HIT.

High-volume, zero balanced ultrafiltration improves pulmonary function in a model of post-pump syndrome.

The systemic inflammatory response syndrome (SIRS), which may develop following cardiopulmonary bypass (CPB), can cause postoperative complications that contribute to the morbidity and mortality associated with open-heart surgery. Inflammatory mediators such as cytokines, are thought to play an important role in SIRS. Zero Balance Ultrafiltration (Z-BUF) is thought to reduce the quantity of inflammatory mediators associated with CPB and may attenuate the adverse effects of bypass. Following ethics committee approval, both an unfiltered experimental group and Z-BUF treatment group consisting of Yorkshire pigs (41 +/- 19 kg) were anesthetized, ventilated, instrumented, cannulated and placed on CPB for 60 minutes. Following CPB, an infusion of low-dose endotoxin (1 microg/kg) was administered I.V. and the animals were monitored for 3.5 hours. The Z-BUF treatment group (n = 5) received high-volume Z-BUF (122 ml/kg +/- 41) and the unfiltered experimental group (n = 5) did not. Hemodynamics, blood gases, and pulmonary functions were measured before, during, and after CPB. Following euthanasia, the middle lobe of the lung was prepared for histological analysis. Necropsy of the lung sample was weighed before and after dehydration to evaluate lung water content. During the experimental time course, plasma samples were evaluated for Interleukin-8 (IL-8) concentrations. Arterial PO2's (mmHg) in the unfiltered experimental group showed a significant reduction at 3.5 hours post CPB when compared to baseline while the Z-BUF treatment group PaO2 did not significantly change. There was a significant difference in the PaO2 between the unfiltered experimental and Z-BUF group at the final 3.5 hour time point (78 +/- 32 vs. 188 +/- 92 mmHg respectively). Pulmonary compliance (ml/cmH2O) was significantly reduced in both the unfiltered experimental and Z-BUF treatment groups with the unfiltered experimental group being the most significant. Lung wet/dry ratios were established and results found the unfiltered experimental group ratio significantly greater than that of the Z-BUF treatment group. Morphometric analysis of histologic lung sections confirmed pulmonary injury in the unfiltered experimental group and protection in the Z-BUF treatment group. This study suggests that Z-BUF provides higher arterial PO2's and lung compliances while reducing pulmonary edema and lung injury in a porcine model of PPS.

Lipopolysaccharide preconditioning induces robust protection against brain injury resulting from deep hypothermic circulatory arrest.

OBJECTIVE: Delayed preconditioning genetically reprograms the response to ischemic injury. Subclinical bacterial lipopolysaccharide acts through preconditioning, powerfully protecting against experimental stroke. We investigated the potential for lipopolysaccharide to protect against brain injury related to cardiopulmonary bypass. METHODS: Neonatal piglets were blindly and randomly preconditioned with lipopolysaccharide (n = 6) or saline (n = 6). Three days later, they experienced 2 hours of deep hypothermic circulatory arrest before being weaned and supported anesthetized for 20 hours in an intensive care setting. Controls included cardiopulmonary bypass without deep hypothermic circulatory arrest (n = 3) and no cardiopulmonary bypass (n = 3). Brain injury was quantified by light and fluorescent microscopy (Fluoro-Jade; Histo-Chem, Inc, Jefferson, Ark). RESULTS: All animals were clinically indistinguishable before surgery. Perioperative and postoperative parameters between experimental groups were similar. No control animal scored falsely positive. Histologic scores were 0.33 +/- 0.21, 0.66 +/- 0.42, and 0.5 +/- 0.24 in the cortex, basal ganglia, and hippocampus, respectively, in the lipopolysaccharide-treated animals but significantly worse in all saline control animals (1.33 +/- 0.21, P < .01; 1.66 +/- 0.33, P = .09; and 6.0 +/- 1.5, P < .01). One lipopolysaccharide-treated brain was histologically indistinguishable from controls. CONCLUSION: This is the first evidence that lipopolysaccharide can precondition against cardiopulmonary bypass-related injury. Because lipopolysaccharide preconditioning is a systemic phenomenon offering proven protection against myocardial, hepatic, and pulmonary injury, this technique offers enormous potential for protecting against systemic neonatal injury related to cardiopulmonary bypass.

Hawley H. Seiler Resident Award paper. The use of a miniaturized circuit and bloodless prime to avoid cerebral no-reflow after neonatal cardiopulmonary bypass.

BACKGROUND: Our miniaturized bloodless prime circuit for neonatal cardiopulmonary bypass (CPB) has previously been shown to elicit significantly reduced systemic inflammation. We studied the effects of this circuit on cerebral reperfusion because the pathophysiology of "no-reflow" is believed to have an inflammatory component. METHODS: Twenty neonatal piglets were randomized to CPB with miniaturized circuitry using either blood (group 1) or bloodless (group 2) prime. At 18 degrees C, piglets underwent 60 minutes of either (A) deep hypothermic circulatory arrest (DHCA) or (B) continuous low-flow bypass (DHCLF). Analysis of cerebral blood flow (CBF) was undertaken before and after CPB in addition to quantification of circulating tumor necrosis factor-alpha (TNFalpha) and intracerebral TNFalpha messenger RNA (mRNA). RESULTS: The final hematocrit in group 2 was 22% versus 28% (p < 0.05). The CBF fell in every animal in group 1A, but increased in every animal in group 2A (p < 0.001), despite no overall change in total cardiac output. The use of DHCLF was not associated with pronounced trends in either prime group. Final serum TNFalpha concentrations were significantly higher in group 1B (3166 +/- 843 pg/mL) than group 2B (439 +/- 192 pg/mL; p < 0.05). Irrespective of the CPB strategy used, the use of a blood prime generated significantly higher levels of intracerebral TNFalpha mRNA. CONCLUSIONS: We attribute the hyperemic cerebrovascular response to reduced inflammation through avoiding allogeneic whole blood. The analysis of circulating and intracerebral TNFalpha in this study suggests that DHCLF in conjunction with a bloodless prime might offer advantages through avoiding ischemia, no-reflow, and in addition, resulting in a significantly reduced cerebral inflammatory response.

Effects of age, gender, and blood pressure on myogenic responses of mesenteric arteries from C57BL/6 mice.

The myogenic response (MR) may represent an important physiological parameter underlying arterial blood pressure (BP). We studied the effects of age, gender, and BP on the MR of mesenteric arteries from 8- to 52-wk-old mice. Increasing age and BP are associated with an increase in the perfusion pressure at which tone develops (myogenic set point). An inverse correlation exists between age and extent (magnitude) of the MR in male (r(2) = 0.93, P = 0.0087) and female mice (r(2) = 0.90, P = 0.013) as well as between BP and extent of the MR in male (r(2) = 0.96, P = 0.0036) and female (r(2) = 0.90, P = 0.014) mice. In contrast, the strength of the MR (slope of active diameter-pressure relationship) and phenylephrine-mediated constriction did not differ among these groups. Although gender had no effect on MR at any perfusion pressure or age, only male mice showed significant salt-induced hypertension and an associated increase in the set point and reduction in the extent of the MR. The set point and extent of the MR is linked to the in vivo pressure during development and experimental hypertension.

Elafin-overexpressing mice have improved cardiac function after myocardial infarction.

Elevated serine elastase activity after myocardial infarction can contribute to remodeling associated with left ventricular dilatation and dysfunction. We therefore assessed the effects of overexpressing the selective serine elastase inhibitor elafin in transgenic mice in which a myocardial infarction was caused by ligation of the left anterior descending coronary artery (LAD). Elevated serine elastase activity was observed in nontransgenic littermates as early as 6 h after LAD ligation and persisted at 4 and 7 days but not in sham-operated or elafin-overexpressing transgenic mice. Myeloperoxidase activity (index of inflammatory cells) and matrix metalloproteinase 2 were also increased but only at 4 and 7 days and only in nontransgenic mice (P < 0.05 for both comparisons), and this increase correlated with inflammatory cell infiltration. Echocardiographic study at 4 days revealed indexes of diastolic dysfunction in nontransgenic versus elafin-overexpressing mice (P < 0.05). Morphometric and biochemical analyses at 28 days indicated impairment in cardiac performance, with greater scar thinning and infarct expansion in nontransgenic versus elafin transgenic littermates (P < 0.05 for all comparisons). Thus serine elastase inhibition appears to suppress inflammation, cardiac dilatation, and dysfunction after myocardial infarct.