Exercise training promotes cardioprotection through oxygen-sparing action in high fat-fed mice.

Although exercise training has been demonstrated to have beneficial cardiovascular effects in diabetes, the effect of exercise training on hearts from obese/diabetic models is unclear. In the present study, mice were fed a high-fat diet, which led to obesity, reduced aerobic capacity, development of mild diastolic dysfunction, and impaired glucose tolerance. Following 8 wk on high-fat diet, mice were assigned to 5 weekly high-intensity interval training (HIT) sessions (10 × 4 min at 85-90% of maximum oxygen uptake) or remained sedentary for the next 10 constitutive weeks. HIT increased maximum oxygen uptake by 13%, reduced body weight by 16%, and improved systemic glucose homeostasis. Exercise training was found to normalize diastolic function, attenuate diet-induced changes in myocardial substrate utilization, and dampen cardiac reactive oxygen species content and fibrosis. These changes were accompanied by normalization of obesity-related impairment of mechanical efficiency due to a decrease in work-independent myocardial oxygen consumption. Finally, we found HIT to reduce infarct size by 47% in ex vivo hearts subjected to ischemia-reperfusion. This study therefore demonstrated for the first time that exercise training mediates cardioprotection following ischemia in diet-induced obese mice and that this was associated with oxygen-sparing effects. These findings highlight the importance of optimal myocardial energetics during ischemic stress.

The cardiokine secreted Frizzled-related protein 3, a modulator of Wnt signalling, in clinical and experimental heart failure.

OBJECTIVES: Experimental studies have shown involvement of Wnt signalling in heart failure (HF). We hypothesized that secreted frizzled-related protein 3 (sFRP3), a modulator of Wnt signalling, is related to the progression of HF. DESIGN: Circulating sFRP3 was measured in 153 HF patients and compared with 25 healthy controls. The association of sFRP3 with mortality was evaluated in 1202 patients (GISSI-HF trial). sFRP3 mRNA expression was assessed in failing human and murine left ventricles (LV), and cellular localization was determined after fractioning of myocardial tissue. In vitro studies were carried out in cardiac fibroblasts subjected to cyclic mechanical stretch. RESULTS: (i) Heart failure patients had significantly raised serum sFRP3 levels compared with controls, (ii) during a median follow-up of 47 months, 315 patients died in the GISSI-HF substudy. In univariable Cox regression, tertiles of baseline sFRP3 concentration were significantly associated with all-cause and cardiovascular mortality. After adjustment for demographic and clinical variables, but not for CRP and NT-proBNP, the associations with mortality remained significant for the third tertile (all-cause, HR 1.45, P = 0.011; cardiovascular, HR 1.66, P = 0.003), (iii) sFRP3 mRNA expression was increased in failing human LV, with a decline following LV assist device therapy. LV from post-MI mice showed an increased sFRP3 mRNA level, particularly in cardiac fibroblasts, and (iv) mechanical stretch enhanced sFRP3 expression and release in myocardial fibroblasts. CONCLUSION: There is an association between increased sFRP3 expression and adverse outcome in HF, suggesting that the failing myocardium itself contributes to an increase in circulating sFRP3.

Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization.

beta-Adrenergic receptor kinase (beta ARK) and beta-arrestin function in the homologous or agonist-activated desensitization of G protein-coupled receptors. The isoforms beta ARK-2 and beta-arrestin-2 are highly enriched in and localized to the dendritic knobs and cilia of the olfactory receptor neurons where the initial events of olfactory signal transduction occur. Odorants induce a rapid and transient elevation of adenosine 3',5'-monophosphate (cAMP), which activates a nonspecific cation channel and produces membrane depolarization. Preincubation of rat olfactory cilia with antibodies raised against beta ARK-2 and beta-arrestin-2 increased the odorant-induced elevation of cAMP and attenuated desensitization. These results suggest that beta ARK-2 and beta-arrestin-2 mediate agonist-dependent desensitization in olfaction.

Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats.

BACKGROUND: Increased plasma adrenomedullin (ADM) levels have been reported in congestive heart failure (HF). The present study was designed to investigate myocardial regulation of the different components of the ADM signaling system (ADM, ADM receptor, and receptor-activity-modifying protein-2, RAMP-2) during ischemic HF in rats and to identify the cells in the myocardium displaying ADM-like immunoreactivity (ADM-ir). Furthermore, the effects of endothelin (ET) receptor antagonism on expression of the myocardial ADM system during HF were investigated. METHODS AND RESULTS: Northern blot analysis revealed increased ADM mRNA expression in the nonischemic left ventricle, with maximal levels 28 days after induction of myocardial infarction (1.5-fold, P<0.05) compared with the sham group. Parallel elevations of myocardial ADM receptor and RAMP-2 mRNA levels were also observed (2.3- and 1.5-fold increase, respectively; P<0.05). In addition, high levels of ADM mRNA were seen in the ischemic region. Immunohistochemical analysis revealed a substantial increase of ADM-ir in microvascular endothelium and perivascular interstitial cells of myocardial tissue contiguous to the ischemic region. In addition, radioligand binding studies demonstrated a 1.6-fold increase of specific ADM binding sites in the failing left ventricle (P<0.05). Intervention with the mixed ET(A)/ET(B) receptor antagonist bosentan (100 mg. kg(-1). day(-1) PO) for 15 days prevented the increase of RAMP-2 mRNA. CONCLUSIONS: The study demonstrates a concerted induction of several components of the myocardial ADM signaling system during postinfarction failure and that the vessels are the main source of myocardial ADM. Our observations indicate a role for ADM as an autocrine/paracrine factor during ventricular remodeling after myocardial infarction.

Different mechanisms are involved in cAMP-mediated induction of mRNAs for subunits of cAMP-dependent protein kinases.

The present study addresses possible mechanisms through which cAMP mediates its effects on mRNA levels for the subunits of protein kinase A (PKA) and the cellular protooncogene, c-fos. Messenger RNAs for the PKA subunits (RI alpha, RII alpha, RII beta, and C alpha) were regulated by cAMP with similar kinetics in Sertoli cells. However, effects of cAMP on the PKA mRNAs were slow compared to a well characterized cAMP responsive gene, c-fos. The magnitude of stimulation was dramatically different between the various PKA subunits, in that RII beta mRNA increased more than 50-fold while the mRNAs for the other subunits were induced only two to four times. Separation of nuclear and cytoplasmic RNA demonstrated that mRNAs for PKA subunits were stimulated to the same extent in these two cellular compartments. The more rapid induction of c-fos mRNA by cAMP, compared to the mRNA for RII beta, was also seen at the level of transcription. Maximal transcription rate for c-fos, RI alpha, and C alpha were observed after 30 min, whereas that for RII beta was increasing during the 2-h period examined. Transcriptional activation of the RI alpha gene also appeared faster than that for RII beta. When Sertoli cells were incubated with 8-(4-chlorophenylthio) cAMP and cycloheximide, a potent inhibitor of protein synthesis, we observed a super-induction of the mRNAs for c-fos (10-fold) and RI alpha (2-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased cardiac expression of endothelin-1 mRNA in ischemic heart failure in rats.

OBJECTIVES: Plasma endothelin (ET) concentrations are increased in heart failure. The aims of the present study were to investigate to what extent cardiac ET mRNA expression is induced in ischemic heart failure and whether there may be compensatory downregulation of myocardial mRNA levels for the ETA and ETB receptor subtypes. METHODS: In rats with ischemic heart failure (left ventricular end-diastolic pressure > 15 mmHg) due to left coronary artery ligation. Northern blot analyses were performed on mRNA isolated from cardiac tissues. RESULTS: A substantial upregulation was revealed in all chambers of the failing hearts. Up to 27-fold upregulation (mean 10.6 +/- 4.0, P = 0.002) of left ventricular ET-1 mRNA levels was measured 1 week after myocardial infarction, whereas only a modest upregulation was detected after 6 weeks (mean 2.7 +/- 0.5, P < 0.05). Ribonuclease protection assay revealed 2.8 +/- 0.4-fold higher levels of ET-1 mRNA in the left ventricular area subjected to myocardial infarction compared to the non-infarcted tissue after 1 week. Left ventricular ET-1 mRNA correlated significantly with left ventricular end-diastolic pressure after 1 week (r2 = 0.86, P = 0.007). The ETA and ETB receptor mRNA levels tended to increase 1 week after myocardial infarction although these changes were not statistically significant. CONCLUSIONS: Cardiac ET-1 mRNA levels are increased in ischemic heart failure and correlate significantly with left ventricular end-diastolic pressure 1 week after myocardial infarction. The increase in cardiac ET-1 mRNA is not accompanied by a decrease in ET receptor mRNA.

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure.

OBJECTIVES: Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS: We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS: Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION: The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.

Mechanisms of glucagon-induced homologous and heterologous desensitization of adenylate cyclase in membranes and whole Sertoli cells of the rat.

In the present work the molecular mechanisms of glucagon-induced desensitization of adenylate cyclase in cultured Sertoli cells have been studied in both whole cells and in a cell-free system. 1) Pretreatment of both whole Sertoli cells and membranes with glucagon induces a time- and dose-dependent desensitization of adenylate cyclase response that is primarily homologous and similar in the two systems. The component of heterologous desensitization, estimated by the reduced responsiveness to other hormones and NaF, accounted for only 12-20% loss of glucagon-responsive adenylate cyclase activity (P less than 0.01). 2) Glucagon-induced desensitization is ATP-dependent. Half maximal desensitization was achieved between 0.1 and 0.2 mM of ATP. 3) The typical time lag in the maximal activation of adenylate cyclase by GMPP(NH)P in the absence of hormone reappeared upon desensitization in spite of the presence of glucagon. The lag, however, was eliminated by FSH, showing that the homologous desensitization is due to a receptor-specific alteration. 4) The heterologous component of glucagon-induced desensitization is largely cAMP/protein kinase dependent. cAMP/protein kinase-induced desensitization was heterologous and caused approximately 30% loss of both hormonal and fluoride-stimulated enzyme activity. 5) Glucagon-induced desensitization is not due to altered activity of Ni since it proceeded equally well in membranes of cells pretreated with pertussis toxin (100 ng/ml) which eliminates Ni-mediated effects. It is concluded that glucagon induces both homologous and heterologous desensitization of the Sertoli cell adenylate cyclase. The locus of homologous desensitization appears to be at the level of the receptor and most probably involves a cAMP-independent phosphorylation reaction, whereas the heterologous desensitization appears to be cAMP-mediated and at least involves impaired functional activity of the Ns component.

Gene expression of the renal endothelin system in renal transplant recipients on cyclosporine A based immunosuppression.

BACKGROUND: Immunosuppressive therapy based on cyclosporine A (CsA) is potentially nephrotoxic, and each dose of CsA is followed by a transient increase in plasma endothelin (ET)-1. The aim of this study was to investigate the effect of CsA based immunosuppressive therapy on renal gene expression of the ET(A) and ET(B) receptor subtypes and preproET-1 in human transplant needle biopsies. METHODS: Twelve living donor renal transplant recipients, median age 51.5 years (range 24-63 years) were included in the study. Immunosuppressive therapy consisted of CsA, azathioprine, and prednisolone. Baseline renal cortical needle biopsies from the living donor kidneys were obtained just before nephrectomy. Follow-up biopsies were obtained from the same transplanted kidneys after 2-6 weeks of immunosuppressive therapy. We used a quantitative, competitive reverse transcriptase polymerase chain reaction assay to measure renal ET(A) and ET(B) receptor subtype mRNAs as well as preproET-1 mRNA levels in each of the biopsies. RESULTS: The renal ET system was not significantly altered by CsA-based immunosuppressive therapy. Median ET(A) mRNA level was 185 (range 35-244) at baseline, and 120 (11-189) amol/microg total RNA after CsA based immunosuppressive therapy (P=0.11). ET(B) mRNA level was 506 (209-1411) at baseline, and 463 (267-1609) amol/microg total RNA at follow-up (P=0.44) and preproET-1 mRNA level was 160 (112-392) before and 221 (187-361) amol/microg total RNA after immunosuppressive therapy based on CsA (P=0.58). CONCLUSION: This study indicates that 2-6 weeks of CsA-based immunosuppression neither significantly influences renal gene expression of the ET(A) or ET(B) receptor subtypes nor preproET-1 in living donor renal transplant kidneys.

Regulation of hormone-responsive Sertoli cell adenylyl cyclase in a cell-free system.

Homologous desensitization of either FSH- or isoproterenol-responsive adenylyl cyclases in Sertoli cell membranes can be achieved in a cell-free system. Incubation of membrane particles from cultured immature Sertoli cells with either FSH or isoproterenol resulted in a time- and concentration-dependent loss of subsequent adenylyl cyclase response to the homologous hormone. Half-maximal refractoriness was achieved within 20-30 min of incubation. The concentration of FSH required to obtain half-maximal loss of adenylyl cyclase response (400 ng/ml) was in the same order of magnitude as the apparent Km for FSH-stimulated adenylyl cyclase activity (300 ng/ml). Hormone-induced homologous desensitization was dependent on the presence of both ATP and Mg2+. Increasing concentrations of ATP in the presence of FSH caused a concentration-dependent loss of subsequent response to the homologous hormone. Half-maximal desensitization was achieved at an ATP concentration of 0.2 mM. Increasing concentrations of Mg2+ in the presence of either FSH or isoproterenol caused desensitization for the homologous hormone. The concentration of Mg2+ giving half-maximal effect was approximately 5 mM in excess of ATP and EDTA. However, higher concentrations of free Mg2+, in the absence of hormone, caused desensitization of both FSH- and isoproterenol-sensitive adenylyl cyclase with half-maximal effect at approximately 30 mM. Hormone-specific desensitization was obtained in the presence of GTP. However, when GTP was substituted with the non-hydrolysable analogue GMPP(NH)P the hormonal activation remained constant throughout 90 min of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic regulation of Sertoli cell adenylyl cyclase: desensitization by homologous hormone.

Incubation of Sertoli cell-enriched cultures with D,L-isoproterenol caused a time- and concentration-dependent, homologous desensitization of isoproterenol-responsive adenyl cyclase, whereas the response to FSH was unaffected. Half-maximal desensitization was achieved within 1 h of preincubation, after which a more gradual loss of response was observed. Preincubation of Sertoli cells for 24 h with increasing concentrations of D,L-isoproterenol demonstrated that the concentration required to obtain half-maximal densensitization was approximately 10-fold lower than the Km for activation of adenylyl cyclase. The function of the guanine nucleotide regulatory component (N-component) of the adenylyl cyclase complex in hormonally desensitized Sertoli cells, as evaluated by activation of adenylyl cyclase by GTP, GMPP(NH)P, fluoride and Mg2+, was not affected by the hormone pretreatment. Preincubation of Sertoli cells with a high concentration of dbcAMP (10(-3) M) for 24 h was associated with a 45% reduction in adenylyl cyclase activation by both FSH and isoproterenol. Also in this case fluoride- and GTP-stimulated adenylyl cyclase activities were normal. However, the effects of dibutyryl cyclic AMP occurred much more slowly than agonist-induced desensitization, indicating that cAMP may not be the primary mediator of homologous desensitization of Sertoli cell adenylyl cyclase by isoproterenol.

Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats.

Addition of glucagon to the incubation medium of cultured Sertoli cells isolated from immature (19-day-old) rats resulted in a time- and concentration-dependent stimulation of cAMP accumulation measured both in the cells and in the medium. Maximal intracellular levels of cAMP were reached after 30 min, after which the levels decreased. In the medium cAMP levels reached a plateau after 6 h. The magnitude and kinetics of the responses were comparable to those observed with FSH in the same culture preparations. 1-Methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, greatly potentiated the magnitude of the effects of glucagon and FSH. Glucagon stimulated adenylate cyclase activity in isolated membrane preparations from similar cultures, and the concentration causing half-maximal stimulation (EC50) was approximately 300 ng/ml. Glucagon also stimulated aromatization in cultured Sertoli cells to the same extent as FSH. It is concluded that cultured Sertoli cells isolated from immature rats contain receptors for glucagon, coupled to adenylate cyclase, and that glucagon also stimulates aromatization of testosterone to estradiol.

Beta-adrenoceptor mediated responses and subtypes of beta-adrenoceptors in cultured rat Sertoli cells.

Membrane particles from Sertoli cell cultures were examined for subtypes of beta-adrenoceptors with a radioligand binding technique using [125I]iodocyanopindolol and a beta 1-selective antagonist (Sandoz 204 545) or a beta 2-selective antagonist (ICI 118 551). Biphasic competition curves and modified Eddie-Hofstee plots revealed a relative distribution of approx. 80% beta 1-adrenoceptors and 20% beta 2-adrenoceptors. Only 45% of the adrenoceptor mediated stimulation of adenylyl cyclase activity was associated with beta 1-adrenoceptors, whereas the remaining 55% was mediated via beta 2-adrenoceptors. The subtype selective antagonists inhibited isoproterenol stimulated aromatization of testosterone to estradiol-17 beta in a concentration-dependent manner. Complete inhibition of beta 1-adrenoceptors resulted in a 45% reduction of estradiol-17 beta formation, whereas similar inhibition of beta 2-adrenoceptors resulted in only a 35% reduction. It is concluded that cAMP-dependent effects of beta-adrenergic agonists in Sertoli cells are mediated by activation of both beta 1- and beta 2-adrenoceptors. The discrepancy between the relative number of beta 1- and beta 2-adrenoceptors and their relative contribution to cAMP production and aromatization indicates that beta 2-adrenoceptors in Sertoli cells are more tightly coupled to the adenylyl cyclase system than beta 1-adrenoceptors. Furthermore, complete inhibition of either beta 1- or beta 2-adrenoceptors by subtype selective antagonists, demonstrates a substantial fraction of spareness between agonist activation of the adenylyl cyclase complex and aromatization.

Modulation of gonadotropic and purinergic responsiveness by islet activating protein in sertoli cells from immature rats.

In the present study we have examined the effects of islet activating protein (IAP) on the regulatory effects of FSH, glucagon and (-)N6-(R)-phenyl-isopropyladenosine (PIA), an adenosine A1 receptor agonist, on the formation of cAMP and estradiol-17 beta (E2) in Sertoli cell cultures isolated from immature (19-day-old) rats. FSH (NIH-FSH-S-15) (1.25 micrograms/ml) caused a more than 10-fold stimulation of the level of both cAMP and E2 in the spent media from Sertoli cell cultures during an 18 h incubation. Both responses were reduced by 80% in the presence of PIA (10(-6) M). When the cultures were preincubated for 24 h with increasing concentration of IAP, the inhibitory effects of PIA were counteracted in a concentration-dependent manner. Moreover, preincubation with IAP (> 20 ng/ml) caused a significant stimulation of FSH-stimulated cAMP production even in the absence of PIA. PIA inhibited FSH-stimulated cAMP production in a concentration dependent manner. However, when the cells were preincubated with IAP (100 ng/ml) for 24 h, the inhibitory effects of PIA were completely abolished, and PIA now actually caused a slight stimulation of cAMP production. Both FSH and glucagon stimulated cAMP production in a concentration-dependent manner. Preincubation with IAP (100 ng/ml for 24 h) resulted in an increase in maximal stimulation of cAMP production for both FSH and glucagon. When adenylyl cyclase (AC) activity was measured directly in isolated membrane particles from Sertoli cells cultured in the presence of IAP (100 ng/ml) for 24 h, both basal and FSH-stimulated AC activity were significantly higher than in membrane particles from control cells. These results provide a further characterization of the functional Gi component coupled to the AC complex in cultured rat Sertoli cells, mediating the inhibitory effects of adenosine and possibly other endogenous substances on cAMP production.

Endothelin-1 is upregulated during skeletal muscle ischemia and reperfusion.

The aim of the present study was to test the hypothesis that the vasoconstrictive peptide endothelin-1 is upregulated in ischemia and reperfusion in skeletal muscle. Sixty-eight Wistar rats were included in the series: 12 served as controls that did not undergo the procedure, 16 underwent sham operations, and 40 were subjected to a modified tourniquet ischemia for 3 hours and 20 minutes. Of the 40 rats, 16 were killed at the end of the ischemic period, 16 underwent reperfusion for 2 hours, and eight underwent reperfusion for 72 hours. Areas of necrosis were measured by morphometry in hematoxylin and eosin-stained cross sections of the anterior tibial muscles that had been reperfused for 72 hours. Sections from the controls, the muscles that had not been reperfused, and the reperfused muscles were immunostained for endothelin-1. Serum endothelin-1 levels in blood samples from the aorta were determined with a commercial enzyme immunoassay kit. The anterior tibial muscle was harvested for preproendothelin-1 mRNA analysis with RNase protection assay. The hematoxylin and eosin-stained sections showed extensive necrosis with an acellular core of no reperfusion. The muscular core demonstrated weak immunostaining for endothelin-1 in all sections, a subfascial narrow brim of fibers showed enhanced immunoreactivity at the end of ischemia, and all fibers outside the core stained by 2 hours after the start of reperfusion. After 72 hours of reperfusion, the fibers outside the core stained positive in a checkerboard-like pattern. There were no differences in serum endothelin-1 levels between the groups. Preproendothelin-1 mRNA analysis with RNase protection assay showed 2-fold upregulation at the end of ischemia and 4-fold upregulation after 2 hours of reperfusion (p = 0.001). This study supports the hypothesis that both ischemia and reperfusion upregulate endothelin-1 in skeletal muscle.

Protecting the heart through delivering DNA encoding for heme oxygenase-1 into skeletal muscle.

AIM: To evaluate if remote gene delivery of HMOX-1 prior to myocardial infarction can prevent cardiac remodeling and preserve function, without causing general angiogenesis. MAIN METHODS: Right quadriceps muscles of mice were treated with DNA encoding for HMOX-1 or empty vector (pcDNA) and electroporated to enhance nuclear uptake, while a third group received saline. Transfection efficacy was evaluated by real time PCR and situ hybridization in transfected muscle, contralateral muscle, and heart. Seven days after transfection baseline echocardiography was performed. Myocardial infarction was induced by ligation of the left coronary artery. Six weeks later heart function was reassessed by echocardiography. Hearts were extracted for evaluation of infarct size. Immunoflorescent staining was used to evaluate angiogenesis using the endothelial marker CD31 in cross-sections of the transfected quadriceps muscle, the untreated muscle, and hearts. KEY FINDINGS: Gene delivery of HMOX-1 leads to a local expression of HMOX-1 in the treated muscle, but not in any other organ. HMOX-1 treated mice had reduced infarct size (p=0.03) and improved function evident as higher ejection fraction (p=0.001), improved fractional shortening (p<0.0001) and higher stroke volume (p=0.002). HMOX-1 did not cause angiogenesis in the heart or skeletal muscle. SIGNIFICANCE: Remote delivery of DNA encoding for HMOX-1 was cardioprotective, as evidenced by preserved cardiac structure and function. Angiogenesis was not induced by HMOX-1 treatment.

Induction of myocardial connective tissue growth factor in pacing-induced heart failure in pigs.

AIMS: Connective tissue growth factor (CTGF) is a secreted, heparin-binding, and extracellular matrix associated protein shown to stimulate many of the cellular events underlying fibrosis. Previous investigations have revealed that myocardial CTGF is substantially induced in ischaemic heart failure, particularly in the ischaemic and peri-ischaemic region. The purpose of the present study was to investigate to what extent myocardial induction of CTGF is a general response to congestive heart failure (CHF) and to what extent CTGF is a decisive effector of fibrosis. METHODS: Experimental heart failure in pigs was induced by rapid pacing at 220-240 beats min(-1) for 3 weeks (CHF pigs; n = 12). RESULTS: The CHF pigs exhibited significant left ventricular (LV) dilatation, reduced contractility, and increased cardiac filling pressures. Northern blot analysis demonstrated that myocardial CTGF mRNA levels in CHF pigs were fivefold higher (P < 0.05) than those in control pigs (n = 10). Similar elevations of immunoreactive CTGF (sixfold; P < 0.05) were observed in myocardial tissue samples prepared for Western blot analysis. Immunohistochemical analysis of myocardial tissue sections revealed predominant expression in interstitial and perivascular fibroblasts and endothelial cells. Myocardial procollagen alpha1(I) mRNA levels were also significantly elevated (sixfold; P < 0.05) in CHF pigs compared with controls, whereas myocardial tissue contents of collagen were not statistically different between the groups. CONCLUSION: Induction of myocardial CTGF in heart failure is not just a response to ischaemia, but rather a general response to evolving heart failure. Yet, induction of myocardial CTGF was clearly not a sufficient effector of fibrosis.

[Gene therapy in cardiovascular diseases]. / Genterapi ved sykdommer i det kardiovaskulaere system.

Cardiovascular disease is the most frequent cause of death in the western hemisphere. Although significant advances in pharmacotherapy have been achieved, the morbidity and mortality of cardiovascular disease will probably remain a scourge of affluent societies for many years to come. This situation gives the scientific community and the pharmaceutical industry an impetus to develop novel therapeutical strategies. Gene therapy is a principle which may provide new means of targeting the pathophysiological mechanisms of cardiovascular disease and has recently drawn a lot of scientific attention, as the endothelial cells of the vasculature are in direct contact with the blood. Thus, many of the obstacles which are thought to hamper gene delivery may not apply in this system. Although there are many ongoing gene therapy trials in this patient group, the results that have so far been reported are from early phase studies (phase I and II studies) only. The published reports have investigated to what extent the growth factors vascular endothelial growth factor and fibroblast growth factor may improve revascularization in ischaemic myocardial tissue. Although the preliminary results are promising, it should be pointed out that phase III trials have not yet been started in this disease group.

Quantification of mRNA levels of endothelin receptor subtypes and preproEndothelin-1 in renal needle biopsies by competitive reverse transcriptase polymerase chain reaction.

The vasoconstrictive peptide endothelin-1 (ET-1) is an autocrine/paracrine peptide of putative pathophysiological importance in renal transplant medicine. The aim of the present study was to develop a method for analysis of gene expression of the renal endothelin system in humans. Only small amounts of tissue are available from renal cortical needle biopsies. Thus, in the present study we developed a quantitative assay based on the competitive reverse transcriptase polymerase chain reaction (RT-PCR) technology. We quantified endothelin A (ET(A)) and B (ET(B)) receptor subtype mRNAs and preproET-1 mRNA levels in renal cortex biopsies obtained before nephrectomy of healthy kidney donors. Mean (+/- SEM) mRNA levels of the ET(A) and ET(B) receptor subtypes in 26 living donors were 212 +/- 23 and 368 +/- 56 amol/microg total RNA, respectively. The preproET-1 mRNA level in 19 living donors was 213 +/- 28 amol/microg total RNA. The inter-assay coefficient of variation (CV) for the assay was 10%; the intra-assay CV was 6-13%. The competitive RT-PCR assay described provides an accurate tool for gene expression investigation of the human endothelin system in renal cortical needle biopsies.