Stem cells from human cardiac adipose tissue depots show different gene expression and functional capacities

Background The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity. Methods EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis. Results ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration. Conclusion Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep ( n = 8) contained 24 ± 2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430 ± 20 μmol/kg (with SR Ca2+ likely <250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.

Sodium-independent inward chloride pumping in rat cardiac ventricular cells

The intracellular Cl concentration ([Cl]i) in rat cardiac ventricular muscle, measured with double-barreled microelectrodes in vitro, was 21.3 +/- 1.5 (SD) mM [number of observations (n) = 46]. With the Na-K-Cl cotransport inhibitor bumetanide (10 microM), it fell to 13.4 +/- 1.4 mM (n = 27), and with 1 mM acetazolamide, it fell further, to 7.2 +/- 1.5 mM (n = 5), close to equilibrium with the membrane potential. In the absence of Na, [Cl]i was 15.9 +/- 1.4 mM (n = 8), and with 1 mM acetazolamide, it fell to 6.5 +/- 0.6 mM (n = 4), again close to equilibrium. The bumetanide- and Na-insensitive components of inward Cl pumping were inhibited by chlorothiazide and ethacrynic acid but were unaffected by the Na-Cl cotransport inhibitor metolazone. There was inhibition of Na-K-Cl cotransport by chlorothiazide = acetazolamide > metolazone. The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and HCO3 had no effect on [Cl]i in any condition. Thus Cl accumulation in the rat ventricle is fully accounted for by two systems, namely, Na-K-Cl cotransport and an Na-independent, possibly primary active, process.