Mild hypothermia during cardiopulmonary bypass assisted CABG is associated with improved short- and long-term survival, a 18-year cohort study.

Data substantiating the optimal patient body temperature during cooling procedures in cardiac operations are currently unavailable. To explore the optimal temperature strategy, we examined the association between temperature management and survival among patients during cardiopulmonary bypass assisted coronary artery bypass grafting (CABG) procedures on 30-days and 5-year postoperative survival. Adult patients (n = 5,672, 23.6% female and mean (SD) age of 66 (10) years) operated between 1997 and 2015 were included, with continuous measured intraoperative nasopharyngeal temperatures. The association between mortality and patient characteristics, laboratory parameters, the lowest intraoperative plateau temperature and intraoperative cooling/rewarming rates were examined by multivariate Cox regression analysis. Machine learning-based cluster analysis was used to identify patient subgroups based on pre-cooling parameters and explore whether specific subgroups benefitted from a particular temperature management. Mild hypothermia (32-35°C) was independently associated with improved 30-days and 5-year survival compared to patients in other temperature categories regardless of operation year. 30 days and 5-year survival were 98% and 88% in the mild hypothermia group, whereas it amounted 93% and 80% in the severe hypothermia (<30°C). Normothermia (35-37°C) showed the lowest survival after 30 days and 5 years amounting 93% and 72%, respectively. Cluster analysis identified 8 distinct patient subgroups principally defined by gender, age, kidney function and weight. The full cohort and all patient subgroups displayed the highest survival at a temperature of 32°C. Given these associations, further prospective randomized controlled trials are needed to ascertain optimal patient temperatures during CPB.

Homozygous whole body Cbs knockout in adult mice features minimal pathology during ageing despite severe homocysteinemia.

Deficiencies in Cystathionine-ß-synthase (CBS) lead to hyperhomocysteinemia (HHCy), which is considered a risk factor for cardiovascular, bone and neurological disease. Moreover, CBS is important for the production of cysteine, hydrogen sulfide (H2 S) and glutathione. Studying the biological role of CBS in adult mice has been severely hampered by embryological disturbances and perinatal mortality. To overcome these issues and assess the effects of whole-body CBS deficiency in adult mice, we engineered and characterized a Cre-inducible Cbs knockout model during ageing. No perinatal mortality occurred before Cbs-/- induction at 10 weeks of age. Mice were followed until 90 weeks of age and ablation of Cbs was confirmed in liver and kidney but not in brain. Severe HHCy was observed in Cbs-/- (289 ± 58 µM) but not in Cbs+/- or control mice (<10 µM). Cbs-/- showed impaired growth, facial alopecia, endothelial dysfunction in absence of increased mortality, and signs of liver or kidney damage. CBS expression in skin localized to sebaceous glands and epidermis, suggesting local effects of Cbs-/- on alopecia. Cbs-/- showed increased markers of oxidative stress and senescence but expression of other H2 S producing enzymes (CSE and 3-MST) was not affected. CBS deficiency severely impaired H2 S production capacity in liver, but not in brain or kidney. In summary, Cbs-/- mice presented a mild phenotype without mortality despite severe HHCy. The findings demonstrate that HHCy is not directly linked to development of end organ damage.

Brain inflammatory cytokines and microglia morphology changes throughout hibernation phases in Syrian hamster.

Hibernators tolerate low metabolism, reduced cerebral blood flow and hypothermia during torpor without noticeable neuronal or synaptic dysfunction upon arousal. Previous studies found extensive changes in brain during torpor, including synaptic rearrangements, documented both morphologically and molecularly. As such adaptations may represent organ damage, we anticipated an inflammatory response in brain during specific hibernation phases. In this study, signs of inflammation in the brain were investigated in the Syrian hamster hippocampus (Mesocricetus Auratus) both during hibernation (torpor and arousal phases) and in summer and winter euthermic animals. mRNA expression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß was quantified by RT-qPCR. Morphological changes of microglia were studied by immunohistochemistry staining for IBA-1. Activation of microglia based on retraction and thickening of the dendritic branches and an increase in cell body size was quantified by calculation of cell body size to total cell size ratio. Expression of pro-inflammatory cytokines was upregulated early in arousal (90 min), and normalized after 8 h of arousal. Substantial loss of microglia ramification was found throughout torpor and early arousal together with a 2-fold increase in the cell body size to total cell size ratio. Notably, microglia changes were fully reversed in late arousal (8 h) to euthermic levels. These results demonstrate an upregulation of inflammatory cytokines and signs of microglia activation during hibernation, which completely resolves by late arousal. Activation of this response may serve to prevent or offset brain damage resulting from the substantial physiological changes accompanying torpor and their rapid change during early arousal.

Neuroprotective hypothermia - Why keep your head cool during ischemia and reperfusion.

BACKGROUND: Targeted temperature management (TTM) is the induced cooling of the entire body or specific organs to help prevent ischemia and reperfusion (I/R) injury, as may occur during major surgery, cardiac resuscitation, traumatic brain injury and stroke. Ischemia and reperfusion induce neuronal damage by mitochondrial dysfunction and oxidative injury, ER stress, neuronal excitotoxicity, and a neuroinflammatory response, which may lead to activation of apoptosis pathways. SCOPE OF REVIEW: The aim of the current review is to discuss TTM targets that convey neuroprotection and to identify potential novel pharmacological intervention strategies for the prevention of cerebral ischemia and reperfusion injury. MAJOR CONCLUSIONS: TTM precludes I/R injury by reducing glutamate release and oxidative stress and inhibiting release of pro-inflammatory factors and thereby counteracts mitochondrial induced apoptosis, neuronal excitotoxicity, and neuroinflammation. Moreover, TTM promotes regulation of the unfolded protein response and induces SUMOylation and the production of cold shock proteins. These advantageous effects of TTM seem to depend on the clinical setting, as well as type and extent of the injury. Therefore, future aims should be to refine hypothermia management in order to optimize TTM utilization and to search for pharmacological agents mimicking the cellular effects of TTM. GENERAL SIGNIFICANCE: Bundling knowledge about TTM in the experimental, translational and clinical setting may result in better approaches for diminishing I/R damage. While application of TTM in the clinical setting has some disadvantages, targeting its putative protective pathways may be useful to prevent I/R injury and reduce neurological complications.

Local gene therapy with indoleamine 2,3-dioxygenase protects against development of transplant vasculopathy in chronic kidney transplant dysfunction.

Chronic transplant dysfunction (CTD) is the primary cause of late allograft loss in kidney transplantation. Indoleamine 2,3-dioxygenase (IDO) is involved in fetomaternal tolerance and IDO gene therapy inhibits acute rejection following kidney transplantation. The aim of this study is to investigate whether gene therapy with IDO is able to attenuate CTD. Transplantation was performed in a rat Dark-Agouti to Wistar-Furth CTD model. Donor kidneys were incubated either with an adenovirus carrying IDO gene, a control adenovirus or saline. During the first 10 days recipients received low-dose cyclosporine. Body weight, blood pressure, serum creatinine and proteinuria were measured every 2 weeks. Rats were killed after 12 weeks. IDO had a striking beneficial effect on transplant vasculopathy at week 12. It also significantly improved body weight gain; it reduced blood pressure and decreased proteinuria during the follow-up. However, it did not affect the kidney function. In addition, IDO therapy significantly decreased the number of graft-infiltrating macrophages at week 12. The messenger RNA levels of forkhead box p3 and transforming grow factor-ß were elevated in the IDO treated group at week 12. Here we show for first time a clear beneficial effect of local IDO gene therapy especially on transplant vasculopathy in a rat model of renal CTD.

Hippocampal cystathionine beta synthase in young and aged mice.

Cystathionine beta synthase (CBS) is the main contributor to the production of hydrogen sulfide (H2S) in the brain. Exogenously administered H2S has been reported to protect neurons against hypoxic injury, ischemia and LPS-induced neuro-inflammation and in the facilitating of long term potentiation (LTP). Dysregulation of CBS leads to different diseases, which all have mental retardation in common. Although multiple studies have implicated a link between the CBS/H2S pathway and neurodegeneration, no studies have been performed examining the pathway in healthy aging animals. We hypothesize that CBS/H2S pathway plays an important role in the protection of learning and memory functions in the brain at the level of the hippocampus. Thus, we studied a set of 8 young (4 months) and 14 aged (24 months (n=6) and 28 months (n=8)) C57Bl6 mice. The 24-month-old mice displayed a significant decrease of CBS immunoreactivity in the MoDG only, compared to 4-month-old mice. In 28-month-old mice, we observed a significant increase of CBS immunoreactivity in the MoDG, compared to 4-month-old mice. When comparing 28-month-old mice to 24-month-old mice, all areas showed a significant increase of CBS immunoreactivity. Thus, throughout aging, CBS expression is maintained in the hippocampus, and many other forebrain regions as well. Mice at the unusual age of 28 months even have a higher hippocampal CBS expression than young mice. Maintenance (and increase) of CBS levels may sustain memory and learning by precluding neuronal loss in areas of the hippocampus.

Hydrogen sulfide restores a normal morphological phenotype in Werner syndrome fibroblasts, attenuates oxidative damage and modulates mTOR pathway.

Werner syndrome (WS) protein is involved in DNA repair and its truncation causes Werner syndrome, an autosomal recessive genetic disorder with a premature aging phenotype. WRN protein mutation is currently known as the primary cause of WS. In cultured WS fibroblasts, we found an increase in cytosolic aggregates and hypothesized that the phenotype is indirectly related to an excess activation of the mTOR (mammalian target of rapamycin) pathway, leading to the formation of protein aggregates in the cytosol with increasing levels of oxidative stress. As we found that the expression levels of the two main H2S producing enzymes, cystathionine ß synthase and cystathionine γ lyase, were lower in WS cells compared to normal, we investigated the effect of administration of H2S as NaHS (50µM). NaHS treatment blocked mTOR activity, abrogated protein aggregation and normalized the phenotype of WS cells. Similar results were obtained by treatment with the mTOR inhibitor rapamycin. This is the first report suggesting that hydrogen sulfide administered as NaHS restores proteostasis and cellular morphological phenotype of WS cells and hints to the importance of transsulfuration pathway in WS.

α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor.

BACKGROUND AND PURPOSE: High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α(1) -adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α(1) -adrenoceptors. EXPERIMENTAL APPROACH: Thoracic aorta was isolated from 12-14 week old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine were characterized in the absence and presence of the EGFR kinase inhibitors, AG1478 and DAPH, in intact and endothelium-denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, matrix metalloproteinase (MMP), extracellular signal-regulated kinase (ERK)1/2 and phosphatidyl inositol 3-kinase (PI3K). Phosphorylation of EGFR and ERK1/2 was measured after short-term phenylephrine or EGF stimulation in aorta segments in the presence of AG1478 and the PI3K inhibitor, wortmannin. KEY RESULTS: AG1478 and DAPH concentration dependently attenuated phenylephrine-induced contractile responses in intact or endothelium-denuded aortic rings. Inhibition of PI3K (wortmannin and LY294002) but not heparin or inhibitors of Src or MMP, prevented the effect of AG1478 on the responses to phenylephrine. Phenylephrine induced phosphorylation of EGFR, which was partially blocked by AG1478. Phenylephrine also increased phosphorylation of ERK1/2, time-dependently and was blocked by AG1478 and wortmannin. CONCLUSIONS AND IMPLICATIONS: Contractions of rat thoracic aorta mediated by α(1) -adrenoceptors involved transactivation of EGFR, mediated via a PI3K and ERK1/2 dependent pathway.

Changes in glomerular filtration rate after cardiac surgery with cardiopulmonary bypass in patients with mild preoperative renal dysfunction.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CPB) is commonly perceived as a risk factor for decline in renal function, especially in patients with preoperative renal dysfunction. There are few data on the effects of CPB on renal function in patients with mild preoperative renal dysfunction. The purpose of this study was to evaluate renal function in patients with pre-existing mild renal dysfunction undergoing cardiac surgery with CPB. METHODS: In a multicentre study cohort we measured prospectively the glomerular filtration rate (GFR) by radioactive markers both before operation and on the 7th postoperative day in cardiac surgical patients with preoperative serum creatinine >120 micromol l(-1) (n=56). In a subgroup of patients (n=14) in addition to the GFR, the effective renal plasma flow (ERPF) and the filtration fraction (FF) were measured. RESULTS: While preoperative GFR [77.9 (25.5) ml min(-1)] increased to 84.4 (23.7) ml min(-1) (P=0.005) 1 week after surgery, ERPF did not change [295.8 (75.2) ml min(-1) and 295.9 (75.9) ml min(-1), respectively; P=0.8]. In accordance, the FF increased from 0.27 (0.05) (before operation) to 0.30 (0.04) (Day 7, P=0.01). CONCLUSION: Our results oppose the view that cardiac surgery with CPB adversely affects renal function in patients with preoperative mild renal dysfunction and an uncomplicated clinical course.

Systemic gene therapy with interleukin-13 attenuates renal ischemia-reperfusion injury.

Ischemia-reperfusion injury is a leading cause of acute renal failure and a major determinant in the outcome of kidney transplantation. Here we explored systemic gene therapy with a modified adenovirus expressing Interleukin (IL)-13, a cytokine with strong anti-inflammatory and cytoprotective properties. When ischemia was induced we found that the IL-13 receptor is expressed in both the normal and experimental kidneys. Prior to the induction of ischemia, rats received adenovirus-IL-13, control adenovirus or saline. IL-13 plasma levels increased more than 50-fold in adenovirus-IL-13 treated animals, confirming successful IL-13 gene delivery. Histological analysis showed decreased tubular epithelial cell damage with adenovirus-IL-13 therapy, accompanied by reduced kidney injury molecule-1 expression. Interstitial infiltration by neutrophils and macrophages was reduced by half as was interstitial fibrosis and expression of alpha-smooth muscle actin. IL-13 treatment significantly diminished the expression of E-selectin, IL-8, MIP-2, TNF-alpha and MCP-1 mRNA. These results suggest that the use of systemic IL-13 gene therapy may be useful in reducing renal tubulointerstitial damage and inflammation caused by ischemia-reperfusion.

Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure.

Rats with chronic heart failure (CHF) develop increased myogenic constriction in mesenteric resistance arteries. Here we investigated increased myogenic constriction in relation to alterations in EDHF- and NO-mediated dilatation in CHF-rats. Male Spraque-Dawley rats were subjected to myocardial-infarction or sham-surgery. At 9-10 weeks after surgery, isolated mesenteric artery ring preparations were studied in a wire-myograph. Stretch-induced myogenic constriction was obtained by stepwise increase of the internal circumference diameter (0.5-1.2 L100). Cyclooxygenase- and eNOS-inhibitors were employed to study NO- and EDHF-mediated dilatation in response to acetylcholine. Rats with CHF (n=8), but not sham-rats (n=6), developed significant myogenic constriction. In addition, the contribution of endothelial dilator mediators was significantly altered in CHF-rats, with increased dependency on NO and decreased EDHF-mediated dilatation. Moreover, EDHF-mediated dilatation was inversely correlated with myogenic constriction in individual CHF-rats (r=-0.74, p=0.04). These data demonstrate increased myogenic constriction in mesenteric arteries of rats with CHF post-MI to be correlated to decreased EDHF-mediated dilatation. These findings extend the previous observation that myogenic constriction antagonizes EDHF-mediated dilatation in rat coronary artery under normal conditions, and suggests this relationship also to become functional in mesenteric arteries under pathophysiological conditions of CHF.

Enhanced transduction of fibroblasts in transplanted kidney with an adenovirus having an RGD motif in the HI loop.

Application of gene therapy to the renal graft has a powerful potential to improve the outcome of kidney transplantation and eliminate detrimental side effects associated with systemic therapy, through local expression of immunoregulatory or other protective molecules. However, the search for the optimal vector is still ongoing. In this study, we used a modified adenovirus that has an Arg-Gly-Asp (RGD) motif inserted in the HI loop of the fiber knob, as a successful strategy to transduce the renal graft. Donor Lewis rat kidneys were infused via the renal artery with a solution containing either a fiber-modified adenovirus (AdTL-RGD) or an unmodified adenovirus (AdTL), or with saline. Syngeneic recipients were killed after 3, 7 or 14 days. Efficiency, selectivity, localization, time course of gene expression and side effects were studied using biochemical and immunohistological techniques. Enhanced gene expression was achieved selectively in the transplanted kidney by AdTL-RGD, when compared to AdTL. Transgene expression lasted for at least 2 weeks. With the AdTL-RGD vector, the transgene was abundantly expressed in the renal interstitial fibroblasts. An increase in the number of cytotoxic T lymphocytes accompanied the use of either vector, when compared to saline. These data convincingly show enhanced and selective gene transfer to the fibroblasts of transplanted kidneys using an RGD-modified adenovirus, providing a highly efficient vector system for future therapeutic interventions.

Effect of dexamethasone on perioperative renal function impairment during cardiac surgery with cardiopulmonary bypass.

BACKGROUND: In cardiac surgery with cardiopulmonary bypass (CPB), corticosteroids are administered to attenuate the physiological changes caused by the systemic inflammatory response. The effects of corticosteroids on CPB-associated renal damage have not been documented. The purpose of this study was to evaluate the effects of dexamethasone on perioperative renal dysfunction in patients undergoing cardiac surgery with CPB. METHODS: Renal damage was prospectively studied in 20 patients without concomitant morbidity undergoing coronary artery surgery with CPB. Patients were randomized in a double-blind fashion to receive dexamethasone or placebo. Markers of glomerular function (creatinine clearance) and damage (microalbuminuria), and markers of tubular function (fractional excretion of sodium and free water clearance) and damage (N-acetyl-beta-D glucosaminidase (NAG)) were evaluated in addition to plasma and urinary glucose levels. Plasma and urinary specimens were obtained at the following time periods: (1) baseline, during the 12 h before surgery; (2) skin incision before heparinization; (3) from heparinization until the end of CPB; (4) during the 2 h following weaning from CPB; (5) in the intensive care unit from 2 to 6 h after weaning of CBP; (6) and from 36 to 60 h after weaning of CPB. RESULTS: CPB was associated with an increase in markers in the placebo group, which returned to baseline during the second postoperative day, demonstrating a transient impairment of glomerular and tubular renal function. Similar patterns were observed in patients treated with dexamethasone. While postoperative glycosuria was significantly higher in the dexamethasone-treated group, no other differences between groups were observed. CONCLUSION: Dexamethasone administration before CPB has no protective effect on perioperative renal dysfunction in low-risk cardiac surgical patients.

Recombinant Semliki Forest virus as a vector system for fast and selective in vivo gene delivery into balloon-injured rat aorta.

Previously, we demonstrated that recombinant Semliki Forest virus (SFV) vector rapidly and selectively transfers genes into cultured vascular smooth muscle cells (VSMC), leaving endothelial cells (EC) unaffected. From this, we hypothesized that recombinant SFV in vivo only transfers genes into the media of balloon-injured but not intact vessel, that gene expression in VSMC is fast, and that the specificity of SFV for VSMC is caused by specific binding sites. To address these hypotheses, we studied the time course of in vivo SFV-LacZ and Ad-LacZ expression in balloon-injured rat aorta. In addition, the fusion characteristics of fluorescent pyrene-labeled SFV were explored in cultured VSMC and EC. In intact aorta, no LacZ expression was found in the intima or media at 24 h. In contrast, in denuded aorta, LacZ expression was detected in as early as 12 h after incubation. LacZ expression was predominantly present in the media. Ad-LacZ expression started after 12 h, but was predominantly present in the adventitia. Ad-LacZ expression in the media started after 72 h. In vitro transfection with SFV showed that fusion was higher and, moreover, saturable in VSMC as compared with EC, indicating the presence of specific SFV binding sites on VSMC, but not EC. From this we conclude that in vivo selectivity of SFV in balloon-injured vessels is based on the removal of the endothelium, which results in accessibility of VSMC in the media that carry specific binding sites for the SFV vector.

Effects of preoperative treatment with diltiazem on diastolic ventricular function after coronary artery bypass graft surgery.

OBJECTIVE: To examine whether preoperative treatment with diltiazem could ameliorate left ventricular (LV) diastolic dysfunction in patients after coronary artery bypass graft (CABG) surgery. DESIGN: Prospective, nonrandomized clinical study. SETTING: University hospital. PARTICIPANTS: Thirty-four patients with preserved LV function undergoing elective CABG surgery. INTERVENTIONS: According to medical history, patients were divided into 2 groups: patients not receiving diltiazem (n = 17) and patients treated with once-daily oral diltiazem for at least 2 weeks (n = 17). All patients received preoperative beta-blockers. MEASUREMENTS AND MAIN RESULTS: After induction of anesthesia, after sternal closure, and 4 hours after cardiopulmonary bypass (CPB), mitral and pulmonary venous flow velocities were measured with pulsed Doppler. LV short-axis end-diastolic area by Doppler transesophageal echocardiography (TEE) and hemodynamic variables were obtained simultaneously at comparable pulmonary capillary wedge pressures. Postoperatively, increased peak E and A velocities were observed in patients with diltiazem and controls and returned to baseline 4 hours post-CPB in controls. Changes in these velocities did not result in a decreased E/A ratio. Peak A velocity, E/A ratio, and E wave deceleration time were significantly dependent on heart rate, not peak E velocity. End-diastolic area at comparable pulmonary capillary wedge pressure remained unchanged. In relation to diltiazem, only peak A velocity and time velocity integral of the A wave (TVI-A) at 4 hours post-CPB differed from controls. CONCLUSION: Diastolic function is preserved after CABG surgery and is not altered by diltiazem in patients with preserved LV systolic function. The persistence of increased peak A velocity and TVI-A into the postoperative period suggests improved atrial systolic function with diltiazem.

Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels.

OBJECTIVES: Our purpose was to determine whether patients with persistent atrial fibrillation (AF) and patients with paroxysmal AF show alterations in potassium channel expression. BACKGROUND: Persistent AF is associated with a sustained shortening of the atrial action potential duration and atrial refractory period. Underlying molecular changes have not been studied in humans. We investigated whether a changed gene expression of specific potassium channels is associated with these changes in patients with persistent AF and in patients with paroxysmal AF. METHODS: Right atrial appendages were obtained from 8 patients with paroxysmal AF, 10 with persistent AF and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass surgery, whereas most AF patients underwent Cox's MAZE surgery (atrial arrhythmia surgery to cure AF) (n = 12). All patients had normal left ventricular function. mRNA (ribonucleic acid) levels were measured by semiquantitative polymerase chain reaction and protein content by Western blotting. RESULTS: mRNA levels of transient outward channel (Kv4.3), acetylcholine-dependent potassium channel (Kir3.4) and ATP-dependent potassium channel (Kir6.2) were reduced in patients with persistent AF (-35%, -47% and -36%, respectively, p < 0.05), whereas only Kv4.3 mRNA level was reduced in patients with paroxysmal AF (-29%, p = 0.03). No changes were found for Kv1.5 and HERG mRNA levels in either group. Protein levels of Kv4.3, Kv1.5 and Kir3.1 were reduced both in patients with persistent AF (-39%, -84% and -47%, respectively, p < 0.05) and in those with paroxysmal AF (-57%, -64%, and -40%, respectively, p < 0.05). CONCLUSIONS: Persistent AF is accompanied by reductions in mRNA and protein levels of several potassium channels. In patients with paroxysmal AF these reductions were observed predominantly at the protein level and not at the mRNA level, suggesting a post-transcriptional regulation.

Early expression of natriuretic peptides and SERCA in mild heart failure: association with severity of the disease.

BACKGROUND: We investigated changes in genetic expression of atrial and brain natriuretic peptides (ANP and BNP) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in patients with stable mild to moderate chronic heart failure (CHF), since data on this topic were primarily obtained in end-stage CHF. METHODS: We studied tissue from 25 patients with idiopathic dilated cardiomyopathy (IDC) in New York Heart Association (NYHA) class II (n=12) and III-IV (n=13). Myocardial tissue from normal hearts (n=10) served as controls. Messenger RNA (mRNA) expression of ANP, BNP, and SERCA was isolated, and correlated with severity of CHF, left ventricular function (LVEF), peak oxygen uptake (peak VO(2)), and wedge pressure. RESULTS: A significant trend for gradual changes in mRNA expression according to increasing NYHA class was found for ANP, BNP (P<0.0001) and SERCA (P=0.04), with a marked increase in patients with more advanced CHF (ANP and BNP: P<0.01 vs. controls; SERCA: NS) and less pronounced changes in patients with mild CHF. mRNA of ANP and BNP correlated strongly with LVEF (-0.621 and -0.621, respectively, both P<0.01) and peak VO(2) (-0.625 and -0.555, respectively, both P<0.01) and, to a lesser extent, with wedge pressure (0.440 and 0.488, respectively, both P<0.05). SERCA correlated most strongly with wedge pressure (-0.623, P<0.01), and weak, non-significant correlations with LVEF and peak VO(2) were found. CONCLUSIONS: Genetic expression of ANP, BNP, and SERCA is progressively altered in proportion to the severity of CHF, although this is more marked for ANP and to a lesser extent BNP, than for SERCA. These changes support the concept that already early in CHF, genetic expression is affected, which has implications for the understanding of the pathophysiology of CHF.

Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation.

OBJECTIVE: Persistent atrial fibrillation (AF) results in an impairment of atrial function. In order to elucidate the mechanism behind this phenomenon, we investigated the gene expression of proteins influencing calcium handling. METHODS: Right atrial appendages were obtained from eight patients with paroxysmal AF, ten with persistent AF (> 8 months) and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass grafting, whereas most AF patients underwent Cox's MAZE surgery (n = 12). All patients had a normal left ventricular function. Total RNA was isolated and reversely transcribed into cDNA. In a semi-quantitative polymerase chain reaction the cDNA of interest and of glyceraldehyde-3-phosphate dehydrogenase were coamplified and separated by ethidium bromide-stained gel electrophoresis. Slot blot analysis was performed to study protein expression. RESULTS: L-type calcium channel alpha 1 and sarcoplasmic reticulum Ca(2+)-ATPase mRNA (-57%, p = 0.01 and -28%, p = 0.04, respectively) and protein contents (-43%, p = 0.02 and -28%, p = 0.04, respectively) were reduced in patients with persistent AF compared to the controls. mRNA contents of phospholamban, ryanodine receptor type 2 and sodium/calcium exchanger were comparable. No changes were observed in patients with paroxysmal AF. CONCLUSIONS: Alterations in gene expression of proteins involved in the calcium homeostasis occur only in patients with long-term persistent AF. In the absence of underlying heart disease, the changes are rather secondary than primary to AF.

Alterations in gene expression of proteins involved in the calcium handling in patients with atrial fibrillation.

INTRODUCTION: Atrial fibrillation (AF) leads to a loss of atrial contraction within hours to days. During persistence of AF, cellular dedifferentiation and hypertrophy occur, eventually resulting in degenerative changes and cell death. Abnormalities in the calcium handling in response to tachycardia-induced intracellular calcium overload play a pivotal role in these processes. METHODS AND RESULTS: The purpose was to investigate the mRNA expression of proteins and ion channels influencing the calcium handling in patients with persistent AF. Right atrial appendages were obtained from 18 matched controls in sinus rhythm (group 1) and 18 patients with persistent AF undergoing elective cardiac surgery. Previous duration of AF was < or = 6 months in 9 (group 2) and > 6 months in 9 patients (group 3). In a single semiquantitative polymerase chain reaction, the mRNA of interest and of glyceraldehyde-3-phosphate dehydrogenase, were coamplified and separated by gel electrophoresis. L-type calcium channel alpha1 subunit mRNA content was inversely related to the duration of AF: -26% in group 2 compared to group 1 (P = 0.2), and -49% in group 3 compared to group 1 (P = 0.01). Inhibitory guanine nucleotide binding protein ialpha2 mRNA content was reduced in group 3 compared to group 1 (-30%, P = 0.01). Sarcoplasmic reticulum calcium ATPase, phospholamban and sodium-calcium exchanger mRNA contents were not affected by AF. CONCLUSIONS: AF-induced alterations in mRNA contents of proteins and ion channels involved in the calcium handling seem to occur in relation to the previous duration of AF. In the present patient population, these changes were significant only if AF lasted > 6 months.