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Senescence-related fibrosis contributes to cardiac dysfunction. Profibrotic processes are Ca2+ dependent. The effect of aging on the Ca2+ mobilization processes of human ventricular fibroblasts (hVFs) is unclear. Therefore, we tested whether aging altered intracellular Ca2+ release and store-operated Ca2+ entry (SOCE). Disease-free hVFs from 2- to 63-yr-old trauma victims were assessed for cytosolic Ca2+ dynamics with fluo 3/confocal imaging. Angiotensin II or thapsigargin was used to release endoplasmic reticulum Ca2+ in Ca2+-free solution; CaCl2 (2 mM) was then added to assess SOCE, which was normalized to ionomycin-induced maximal Ca2+. The angiotensin II experiments were repeated after phosphoenolpyruvate pretreatment to determine the role of energy status. The expression of genes encoding SOCErelated ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥h50 yr were compared using unpaired t-test or regression analysis. Ca2+ release by angiotensin II or thapsigargin was not different between the groups, but SOCE was significantly elevated in the ≥50-yr group. Regression analysis showed an age-dependent phosphoenolpyruvate-sensitive increase in SOCE of hVFs. Aging did not alter the mRNA expression of SOCE-related genes. The profibrotic phenotype of hVFs was evident by sprouty1 downregulation with age. Thus, an age-associated increase in angiotensin II- and thapsigargin-induced SOCE occurs in hVFs, independent of receptor mechanisms or alterations of mRNA expression level of SOCErelated ion channel subunits but related to the cellular bioenergetics status. Elucidation of mechanisms underlying enhanced hVF SOCE with aging may refine SOCE targets to limit aging-related progression of Ca2+-dependent cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Energy production in myocardial cells occurs mainly in the mitochondrion. Although alterations in mitochondrial functions in the senescent heart have been documented, the molecular bases for the aging-associated decline in energy metabolism in the human heart are not fully understood. In this study, we examined transcription profiles of genes coding for mitochondrial proteins in atrial tissue from aged (≥65 years old) and comorbidities-matched adult (<65 years old) patients with preserved left ventricular function. We also correlated changes in functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes with gene expression changes. There was significant alteration in the expression of 10% (101/1,008) of genes coding for mitochondrial proteins, with 86% downregulated (87/101). Forty-nine percent of the altered genes were confined to mitochondrial energetic pathways. These changes were associated with a significant decrease in respiratory capacity of mitochondria oxidizing glutamate and malate and functional activity of complex I activity that correlated with the downregulation of NDUFA6, NDUFA9, NDUFB5, NDUFB8, and NDUFS2 genes coding for NADH dehydrogenase subunits. Thus, aging is associated with a decline in activity of OXPHOS within the broader transcriptional downregulation of genes regulating mitochondrial energetics, providing a substrate for reduced energetic efficiency in the senescent human atria. [ABSTRACT FROM AUTHOR]

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca 2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca 2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro , hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/10 6 cells vs 58.5±8.8 µg/10 6 cells, P <0.05; (Sircol™ assay)]. However, collagen gene expressions ( COL1A1 and COL1A2 ; RT-PCR) were not different. Ca 2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca 2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca 2+ influx via store-operated Ca 2+ entry/Ca 2+ release-activated channels (SOCE/CRAC) was significantly ( P ≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for I CRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly ( P <0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective I CRAC inhibitor, for 48 h (66.78±5.87 µg/10 6 cells vs 55.81±7.09 µg/10 6 cells, P =0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca 2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF., (© 2017. Published by The Company of Biologists Ltd.)