Secretogranin II; a Protein Increased in the Myocardium and Circulation in Heart Failure with Cardioprotective Properties

Background: Several beneficial effects have been demonstrated for secretogranin II (SgII) in non-cardiac tissue. As cardiac production of chromogranin A and B, two related proteins, is increased in heart failure (HF), we hypothesized that SgII could play a role in cardiovascular pathophysiology. Methodology/Principal Findings: SgII production was characterized in a post-myocardial infarction heart failure (HF) mouse model, functional properties explored in experimental models, and circulating levels measured in mice and patients with stable HF of moderate severity. SgII mRNA levels were 10.5 fold upregulated in the left ventricle (LV) of animals with myocardial infarction and HF (p<0.001 vs. sham-operated animals). SgII protein levels were also increased in the LV, but not in other organs investigated. SgII was produced in several cell types in the myocardium and cardiomyocyte synthesis of SgII was potently induced by transforming growth factor-beta and norepinephrine stimulation in vitro. Processing of SgII to shorter peptides was enhanced in the failing myocardium due to increased levels of the proteases PC1/3 and PC2 and circulating SgII levels were increased in mice with HF. Examining a pathophysiological role of SgII in the initial phase of post-infarction HF, the SgII fragment secretoneurin reduced myocardial ischemia-reperfusion injury and cardiomyocyte apoptosis by 30% and rapidly increased cardiomyocyte Erk1/2 and Stat3 phosphorylation. SgII levels were also higher in patients with stable, chronic HF compared to age-and gender-matched control subjects: median 0.16 (Q1-3 0.14-0.18) vs. 0.12 (0.10-0.14) nmol/L, p<0.001. Conclusions: We demonstrate increased myocardial SgII production and processing in the LV in animals with myocardial infarction and HF, which could be beneficial as the SgII fragment secretoneurin protects from ischemia-reperfusion injury and cardiomyocyte apoptosis. Circulating SgII levels are also increased in patients with chronic, stable HF and may represent a new cardiac biomarker

Glycosyalted chromogranin A in heart faiure. Implications for processing and cardiomyocyte calcium homeostasis

Background—Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. Methods and Results—CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca2+ spark and wave frequency, reduced Ca2+ spark dimensions, increased sarcoplasmic reticulum Ca2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca2+ transients and contractions. Conclusions—CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca2+ handling because of reduced CaMKIIδ inhibition

SgII production outside of the left ventricle in heart failure.

<p>SgII levels were decreased in pulmonary tissue during HF development, while levels were unchanged in the other tissues examined. SgII levels in the (A) right ventricle, (B) pulmonary tissue, (C) liver, (D) spleen, (E) kidney, (F) stomach, (G) colon, and (H) skeletal muscle were measured by RIA and are presented as fold change ± SEM (n = 6 for both groups, except pulmonary tissue: HF: n = 14, sham: n = 13). # p<0.05.</p

Descriptive statistics of heart failure patients and control subjects.

<p>NYHA class indicates New York Heart Association functional class; Q1–3, quartile 1–3; LVEF, left ventricular ejection fraction; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blocker; ASA, acetyl salicylic acid; PPI, proton pump inhibitor; CRT, cardiac resynchronization therapy; ICD, implantable cardioverter-defibrillator; CgA, chromogranin A; CgB, chromogranin B; and BNP, B-type natriuretic peptide. Biomarker levels are presented as median (quartile 1–3).</p

Descriptive statistics of animals.

<p>LV indicates left ventricle; RV, right ventricle; CgA, chromogranin A; CgB, chromogranin B; BNP, B-type natriuretic peptide; IVSd, intraventricular septum thickness in diastole; IVSs, intraventricular septum thickness in systole; LVDd, LV diameter in diastole; LVDs, LV diameter in systole; LVFS, LV fractional shortening; PWd, posterior wall thickness in diastole; PWs, posterior wall thickness in systole; and LAD, left atrial diameter.</p><p>mRNA levels were investigated in a subset of animals (n = 9 HF, n = 8 sham) and are presented as fold change±SEM. Echocardiographic data are reported as mean±SEM and are obtained from a representative subset of animals (13 HF animals, 6 sham animals).</p

The secretogranin II fragment secretoneurin has protective effects during myocardial ischemia and cardiomyocyte stress.

<p>A, Secretoneurin (SN) perfusion reduces infarct size by 30% (upper left) as demonstrated in representative TTC stained images (upper right) after global ischemia in the isolated perfused rat heart. B, Secretoneurin perfusion also improves myocardial function as assessed by LV end-diastolic pressure (LVEDP) after I/R injury. C, Cardiomyocyte apoptosis <i>in vitro</i> after H₂O₂ exposure was attenuated by secretoneurin stimulation. Cells were extracted from 5 different cell isolations (n = 5 for all groups). D, Short-term stimulation of cardiomyocytes with 10 µg/mL secretoneurin activated protective intracellular pathways as reflected by increased Stat3 and Erk1/2 phosphorylation (n = 5 for all groups). **p<0.001, *p<0.01, # p<0.05.</p

Circulating SgII levels are elevated in patients with chronic, stable HF.

<p>SgII levels were significantly increased in HF patients (n = 58) compared to healthy age- and gender-matched control subjects (n = 20): Median 0.16 (Q1–3 0.14–0.18) vs. 0.12 (0.10–0.14) nmol/L, p<0.001. HF patients are presented according to NYHA functional class. The horizontal line within the box represents the median level, the boundaries of the box the 25^th and 75^th percentile levels, and the whiskers the 10^th–90^th percentile. **p<0.001.</p

Regulation of cardiomyocyte SgII expression by important hormonal and paracrine factors in HF.

<p>SgII mRNA levels were measured by RT-qPCR after stimulating neonatal rat cardiomyocytes for 24 h with either PBS (Ctr, n = 9), forskolin (FSK n = 5), norepinephrine (NE, n = 5), angiotensin II (AngII, n = 4), endothelin-1 (ET-1, n = 5), transforming growth factor-β (TGF-β, n = 6), or tumor necrosis factor-α (TNF-α, n = 6). SgII mRNA levels are presented as fold change ± SEM vs. PBS-stimulated cells. **p<0.001, *p<0.01, # p<0.05.</p

Left ventricular SgII gene expression in heart failure.

<p>A, SgII mRNA levels in non-infarcted left ventricular tissue during HF development. SgII mRNA levels were 10.5 fold increased (p<0.001) in non-infarcted LV tissue in HF animals (n = 9) compared to sham-operated animals (n = 8). Gene expression was measured by RT-qPCR and is presented as fold change ± SEM. B, LV SgII mRNA levels were closely correlated with CgA mRNA levels in both HF (r = 0.68, p = 0.04) and sham animals (r = 0.81, p = 0.02). **p<0.001.</p

SgII is produced by cardiomyocytes and increased in the left ventricle during HF development.

<p>A, SgII protein levels as measured by RIA were increased in both the non-infarcted and infarcted region of the LV in HF animals compared to levels in the myocardium of sham-operated animals (n = 9 for both groups). B, Representative photomicrographs of myocardial tissue sections of a HF mouse demonstrating SgII immunoreactivity (brown staining) in cardiomyocytes of the non-infarcted LV (upper left image). Images of the area bordering the infarcted zone (border zone) are presented in the upper right image (magnification: ×100) and the lower left image (magnification: ×400) and demonstrate SgII immunoreactivity also in non-cardiomyocyte cardiac cells, including fibroblasts. In the upper right image, the infarct area is seen on the left side with granulation tissue in between non-infarcted tissue in the center. Bottom right picture demonstrates very weak staining after use of non-immune rabbit serum as control (ctr). Magnification: ×100 except lower left image (×400). C, SgII mRNA levels were measured in fractions of cardiomyocytes (n = 5), endothelial cells (n = 2), and non-cardiomyocytes, non-endothelial cells (n = 5) extracted from LV tissue. Gene expression was measured by RT-qPCR and is presented as fold change ± SEM vs. levels in the cardiomyocyte fraction. **p<0.001, *p<0.01.</p