Ellen O. Weinberg

Identification of Serum Soluble ST2 Receptor as a Novel Heart Failure Biomarker

Background—Using genomic technology, we previously identified an interleukin-1 receptor family member, ST2, as a gene markedly induced by mechanical strain in cardiac myocytes. The soluble receptor form of ST2 is secreted and detectable in human serum. This study tested the hypothesis that soluble ST2 levels in the serum of patients with severe chronic heart failure are increased in patients with neurohormonal activation. Methods and Results—Serum samples, clinical variables, and neurohormone levels from the PRAISE-2 heart failure trial (NYHA functional class III-IV; end point, mortality or transplantation) were analyzed. ST2 serum measurements were performed with ELISA on samples from 161 patients obtained at trial enrollment and from 139 of the same patients obtained 2 weeks after trial enrollment. Baseline ST2 levels were correlated with baseline B-type natriuretic peptide (BNP) levels (r =0.36, P <0.0001), baseline proatrial natriuretic peptide (ProANP) levels (r =0.36, P <0.0001), and baseline norepinephrine levels (r =0.39, P <0.0001). The change in ST2 was significant as a univariate predictor of subsequent mortality or transplantation (P =0.048), as was baseline BNP (P <0.0001) and baseline ProANP (P <0.0001). In multivariate models including BNP and ProANP, the change in ST2 remained significant as a predictor of mortality or transplantation independent of BNP and ProANP. Conclusions—Serum soluble ST2 is a novel biomarker for neurohormonal activation in patients with heart failure. In patients with severe chronic NYHA class III to IV heart failure, the change in ST2 levels is an independent predictor of subsequent mortality or transplantation.

Expression and Regulation of ST2, an Interleukin-1 Receptor Family Member, in Cardiomyocytes and Myocardial Infarction

Background—We identified an interleukin-1 receptor family member, ST2, as a gene markedly induced by mechanical strain in cardiac myocytes and hypothesized that ST2 participates in the acute myocardial response to stress and injury. Methods and Results—ST2 mRNA was induced in cardiac myocytes by mechanical strain (4.7±0.9-fold) and interleukin-1&bgr; (2.0±0.2-fold). Promoter analysis revealed that the proximal and not the distal promoter of ST2 is responsible for transcriptional activation in cardiac myocytes by strain and interleukin-1&bgr;. In mice subjected to coronary artery ligation, serum ST2 was transiently increased compared with unoperated controls (20.8±4.4 versus 0.8±0.8 ng/mL, P <0.05). Soluble ST2 levels were increased in the serum of human patients (N=69) 1 day after myocardial infarction and correlated positively with creatine kinase (r =0.41, P <0.001) and negatively with ejection fraction (P =0.02). Conclusions—These data identify ST2 release in response to myocardial infarction and suggest a role for this innate immune receptor in myocardial injury.

Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding.

Left ventricular hypertrophy (LVH) is associated with reinduction of the fetal program of gene expression. It is unclear whether this pattern of cardiac gene expression changes with the development of left ventricular decompensation and failure. To answer these questions, we quantified steady-state levels of mRNA by the polymerase chain reaction in the left ventricular myocardium of rats 8 and 20 weeks after ascending aortic banding. Clinical and hemodynamic assessment identified two distinct groups of animals 20 weeks after aortic banding. The first group (20-week nonfailed LVH) demonstrated substantial LVH but no depression in systolic developed pressure per gram left ventricular weight compared with the age-matched control group. In contrast, a second group of rats exhibited clinical signs of congestive failure as well as a marked diminution in left ventricular developed pressure per gram. Assessment of the levels of mRNA encoding a panel of cardiac proteins demonstrated a greater than twofold increase in beta-myosin heavy chain mRNA and an approximately sixfold increase in atrial natriuretic factor mRNA in left ventricular myocardium of all three groups (8-week LVH, 20-week nonfailed LVH, 20-week failed LVH) when compared with appropriate age-matched control groups. In contrast, Ca(2+)-ATPase mRNA levels were decreased by 50% only in the left ventricular myocardium of animals with both clinical signs and hemodynamic indexes consistent with cardiac decompensation (20-week failed LVH). These results suggest that in rats with ascending aortic banding the hypertrophic phenotype is associated with a selective reinduction of the fetal gene program, which persists even after the development of left ventricular failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum Levels of the Interleukin-1 Receptor Family Member ST2 Predict Mortality and Clinical Outcome in Acute Myocardial Infarction

Background—Mechanically overloaded cardiomyocytes secrete a soluble interleukin-1 receptor family member called ST2. Serum levels of ST2 are associated with prognosis in nonischemic heart failure, but the predictive value of ST2 in patients with acute myocardial infarction is unknown. Methods and Results—ST2 levels were measured in serum from 810 patients with acute myocardial infarction in the Thrombolysis In Myocardial Infarction (TIMI) 14 (362 patients) and Enoxaparin and TNK-tPA With or Without GPIIb/IIIa Inhibitor as Reperfusion Strategy in STEMI (ENTIRE)-TIMI 23 (448 patients) clinical trials. Baseline levels of ST2 were significantly higher in those patients who died (0.379 versus 0.233 ng/mL, P = 0.0001) or developed new congestive heart failure (0.287 versus 0.233 ng/mL, P = 0.009) by 30 days. In an analysis of outcomes at 30 days by ST2 quartiles, both death (P = 0.001) and the combined death/heart failure end point (P = 0.001) showed a significant graded association with levels of ST2; furthermore, in-hospital death (P = 0.003) and death/heart failure (P = 0.004) were also significantly associated with higher ST2 levels. In a logistic regression analysis that controlled for important clinical factors, increasing levels of ST2 remained associated with death at 30 days (P = 0.047). ST2 levels rose during the first day after infarction and were maximal at 12 hours; ST2 levels at 12 hours were also independently associated with death at 30 days (P < 0.001). Conclusions—Serum levels of the interleukin-1 receptor family member ST2 predict mortality and heart failure in patients with acute myocardial infarction. These data suggest that ST2 may be a useful biomarker and that this novel inflammatory receptor may play a role in cardiac pathophysiology.

Serial Echocardiographic-Doppler Assessment of Left Ventricular Geometry and Function in Rats With Pressure-Overload Hypertrophy Chronic Angiotensin-Converting Enzyme Inhibition Attenuates the Transition to Heart Failure

BACKGROUND Although chronic pressure overload may progress to left ventricular (LV) failure, the pathophysiology of this transition is not well understood. In addition, the effects of chronic angiotensin-converting enzyme (ACE) inhibition on this transition are largely undefined. METHODS AND RESULTS To examine changes in LV structure and function during the transition to heart failure, rats with LV hypertrophy due to banding of the ascending aorta (LVH, n = 22) and age-matched sham-operated rats (n = 6) were studied 6, 12, and 18 weeks after aortic banding. Two-dimensionally guided transthoracic M-mode echocardiograms and transmitral Doppler spectra were recorded for assessment of LV geometry and systolic and diastolic functions. LVH rats were randomized to no treatment (n = 10) or treatment with the ACE inhibitor fosinopril (50 mg/kg per day, n = 12) after the baseline echocardiogram. Six weeks after banding, LVH rats had increased LV wall thickness with normal cavity dimensions and supranormal endocardial systolic shortening. However, midwall shortening was mildly depressed, and a restrictive diastolic filling pattern was present. After 18 weeks of untreated pressure overload, LV wall thickness was unchanged, but cavity dilation, a fall in endocardial shortening, and further deterioration of diastolic filling were evident. In contrast to untreated LVH rats, the fosinopril-treated rats showed no change in LV diastolic cavity dimension, and systolic and diastolic functions did not deteriorate or improved. Closed chest LV systolic pressures at 18 weeks were not different in LVH or LVH-fosinopril rats (197 versus 198 mm Hg), although end-diastolic pressure was higher in the untreated rats (18 versus 11 mm Hg). Calculated LV systolic wall stress was lower in fosinopril-treated than untreated LVH rats. The severity of LV diastolic filling abnormalities correlated strongly with operating LV chamber stiffness (r = .88, P < .0001). CONCLUSIONS This model of pressure overload is characterized initially by concentric LV hypertrophy with compensated LV chamber performance; however, markedly abnormal diastolic filling is present. The transition from compensated hypertrophy to early failure is heralded by LV dilation, impairment of systolic function, and progression of the abnormalities in LV filling. Chronic ACE inhibition in rats with supravalvular aortic banding (1) does not change in vivo LV systolic pressure but prevents increased LV cavity size and increased LV wall stress and (2) attenuates impairment of (or improves) both systolic and diastolic functions. The effects of fosinopril could be explained in part by inhibition of an intracardiac renin-angiotensin system.

Hypertrophic remodeling: gender differences in the early response to left ventricular pressure overload

OBJECTIVES To identify gender differences in left ventricular remodeling, hypertrophy, and function in response to pressure overload due to ascending aortic banding in rats. BACKGROUND Gender may influence the adaptation to pressure overload, as women with aortic stenosis have greater degrees of left ventricular hypertrophy and better left ventricular function than men. METHODS Fifty-two weanling rats underwent ascending aortic banding (16 males, 18 females), or sham surgery (9 males, 9 females). At 6 and 20 weeks, rats underwent transthoracic echo Doppler studies, and closed-chest left ventricular pressures with direct left ventricular puncture. Perfusion-fixed tissues from eight rats were examined morphometrically for myocyte cross-sectional area and percent collagen volume. RESULTS At 6 weeks after aortic banding, left ventricular remodeling, extent of hypertrophy, and function appeared similar in male and female rats. At 20 weeks, male but not female rats showed an early transition to heart failure, with onset of cavity dilatation (left ventricular diameter=155% vs. 121% of same-sex sham), loss of concentric remodeling (relative wall thickness=102% vs. 139% of sham), elevated wall stress (systolic stress=266% vs. 154% of sham), and diastolic dysfunction (deceleration of rapid filling=251% vs. 190% of sham). Left ventricular systolic pressures were higher in female compared with male rats (186+/-20 vs. 139+/-13 mm Hg), while diastolic pressures tended to be lower (14+/-4 vs. 17+/-4 mm Hg). CONCLUSIONS Gender significantly influences the evolution of the early response to pressure overload, including the transition to heart failure in rats with aortic stenosis.

Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis.

BackgroundWe tested the hypotheses that long-term administration of the angiotensin-converting enzyme (ACE) inhibitor fosinopril will regress hypertrophy, modify the transition to heart failure, and prolong survival in rats with chronic left ventricular (LV) pressure overload due to ascending aortic stenosis. Methods and ResultsAortic stenosis was created in weanling male Wistar rats by a stainless steel clip placed on the ascending aorta. Age-matched control animals underwent a sham operation (Sham group, n=57). Six weeks after surgery, rats with aortic stenosis were randomized to receive either oral fosinopril 50 mg · kg−1 · d−1 (Fos/LVH group, n=38) or no drug (LVH group, n=36) for 15 weeks. Pilot studies confirmed that this dosage produced significant inhibition of LV tissue ACE in vivo. Animals were monitored daily, and survival during the 15-week treatment period was assessed by actuarial analysis. At 15 weeks, in vivo LV systolic and diastolic pressures and heart rate were measured. To assess contractile function, the force-calcium relation was evaluated by use of the isovolumic buffer-perfused, balloon-in-LV heart preparation at comparable coronary flow rates per gram LV weight. Quantitative morphometry was performed. Mortality during the 15-week trial was significantly less in the Fos/LVH group than in the LVH group (3% versus 31%, P<.005). No deaths occurred in the Sham group. In vivo LV systolic pressure was similar between Fos/LVH and LVH hearts (223±10 versus 232±9 mm Hg) and significantly higher than the Sham group (99±3 mm Hg, P<.05). In vivo LV diastolic pressure was significantly lower in Fos/LVH hearts than in LVH hearts (10±2 versus 15±2 mm Hg), and both were significantly higher than in the Sham group (5±1 mm Hg, P<.05). Heart rate was similar among all groups. Despite equivalent elevation of LV systolic pressure, fosinopril resulted in regression of myocyte hypertrophy in Fos/LVH versus LVH (myocyte cell width, 14.8±0.5 versus 20.8±2.2 μm, P<.05) to normal levels (Sham, 16.3±0.9 μm). Quantitative morphometry demonstrated that the regression of LV myocyte hypertrophy in the Fos/LVH group was associated with a relative increase in the fractional volume of fibrillar collagen and noncollagen interstitium. In the isolated heart experiments, LV systolic developed pressure relative to perfusate [Ca2+] was significantly higher in Fos/LVH hearts than in LVH hearts. The improvement in systolic function was not related to any difference in myocardial high-energy phosphate levels, since LV ATP and creatine phosphate levels were similar in Fos/LVH and LVH hearts. ConclusionsIn rats with ascending aortic stenosis, chronic ACE inhibition with fosinopril improved survival, decreased the extent of LV hypertrophy, and improved cardiac function despite persistent elevation of LV systolic pressure. The favorable effects of fosinopril may be related in part to inhibition of the effects of cardiac ACE on myocyte hypertrophy rather than to systemic hemodynamic mechanisms.

Gender differences in molecular remodeling in pressure overload hypertrophy

OBJECTIVES The objective of this study was to examine gender differences in left ventricular (LV) function and expression of cardiac genes in response to LV pressure overload due to ascending aortic stenosis in rats. BACKGROUND Clinical studies have documented gender differences in the pattern of adaptive LV hypertrophy. Whether these differences result from intrinsic differences in molecular adaptation to pressure overload between men and women, or are related to other factors is not known. METHODS Male (n = 8) and female (n = 8) Wistar rats underwent ascending aortic stenosis and were studied 6 weeks after banding with gender-matched control rats (male n = 7; female n = 7). The LV contractile reserve was examined in isolated hearts from each group. We compared LV messenger ribonucleic acid (mRNA) levels of atrial natriuretic factor (ANF), beta-myosin heavy chain, sarcoplasmic reticulum Ca2+-adenosine triphosphatase (ATPase) and Na+-Ca2+ exchanger. Reverse transcriptase polymerase chain reaction was used to identify estrogen receptor transcript in cardiac myocytes and LV tissue. RESULTS The magnitude of LV hypertrophy (LVH) and systolic wall stress were similar in male and female animals with LVH. Male LVH hearts demonstrated a depressed contractile reserve; in contrast, contractile reserve was preserved in female LVH hearts. The expression of beta-myosin heavy chain and ANF mRNA was greater in male versus female LVH hearts. Sarcoplasmic reticulum Ca2+-ATPase mRNA levels were depressed in male LVH but not in female LVH compared with control rats, and Na+-Ca2+ exchanger mRNA levels were increased similarly in both male and female LVH hearts. Estrogen receptor transcript was detected in both adult male and female cardiac myocytes and LV tissue. CONCLUSIONS There are significant gender differences in the LV adaptation to pressure overload despite a similar degree of LVH and systolic wall stress in male and female rats. There is the potential for estrogen signaling through the adult myocyte estrogen receptor in both male and female rats to contribute to gender differences in gene expression in pathologic hypertrophy.

Angiotensin II–Induced Growth Responses in Isolated Adult Rat Hearts Evidence for Load-Independent Induction of Cardiac Protein Synthesis by Angiotensin II

Cardiac myocyte hypertrophy often occurs in response to both hemodynamic and neurohumoral factors. To study whether activation of the renin-angiotensin system by itself may induce a cardiac growth response, the acute effects of angiotensin II on cardiac protein synthesis were studied in isolated rat hearts. New protein synthesis in isolated buffer-perfused adult rat hearts was measured by incorporation of [3H]phenylalanine into cardiac proteins during a 3-hour perfusion protocol. Angiotensin II (1 x 10(-8) mol/L), administered alone or in combination with the alpha 1-blocker prazosin (1 x 10(-7) mol/L), stimulated protein synthesis in both ventricles. The rate of [3H]phenylalanine incorporation into cardiac proteins was 3.9-fold (P < .005) and 2.6-fold (P < .01) higher in angiotensin II-perfused (n = 6) than in vehicle-perfused (n = 6) left and right ventricles, respectively. The induction of new protein synthesis by angiotensin II was blocked by the angiotensin II type 1 (AT1) receptor antagonist losartan (1 x 10(-7) mol/L, n = 5). To study the pathways of angiotensin signal transduction, protein kinase C (PKC)-epsilon as well as cardiac c-fos and c-jun mRNA levels were analyzed. Angiotensin II (1 x 10(-8) mol/L, n = 20) resulted in a transient translocation of PKC-epsilon from the cytosol to the cellular membrane. However, compared with phorbol ester stimulation (phorbol 12-myristate 13-acetate [PMA], 1 x 10(-7) mol/L; n = 20), angiotensin II effects on PKC translocation were significantly less pronounced and required a more prolonged stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressure Overload Induces Severe Hypertrophy in Mice Treated With Cyclosporine, an Inhibitor of Calcineurin

Cardiac hypertrophy is the fundamental adaptation of the adult heart to mechanical load. Recent work has shown that inhibition of calcineurin activity with cyclosporine suppresses the development of hypertrophy in calcineurin transgenic mice and in in vitro systems of neonatal rat cardiocytes stimulated with peptide growth factors. To test the hypothesis that the calcineurin signaling pathway is critical for load-induced hypertrophy in vivo, we examined the effects of cyclosporine treatment on left ventricular hypertrophy induced by experimental ascending aortic stenosis for 4 weeks in mice. Left ventricular systolic pressure was elevated to a similar level in aortic stenosis mice that were treated with cyclosporine versus no drug. Left ventricular mass and myocyte size were similar in treated and untreated aortic stenosis animals and significantly greater than control animals, showing that cyclosporine treatment does not suppress hypertrophic growth. Both treated and untreated animals showed increased left ventricular expression of the load-sensitive gene atrial natriuretic factor. Calcineurin activity was measured in the left ventricle and the spleen from control mice and aortic stenosis mice treated with cyclosporine versus no drug. Levels of calcineurin activity were similar in the spleens of control and untreated aortic stenosis mice. However, calcineurin activity was severely depressed in left ventricular tissue of untreated aortic stenosis mice compared with control mice and was further reduced by cyclosporine treatment. Thus, pathological hypertrophy and cardiac-restricted gene expression induced by pressure overload in vivo are not suppressed by treatment with cyclosporine and do not appear to depend on the elevation of left ventricular calcineurin activity.