Noninvasive biomarker-based risk stratification for development of new onset atrial fibrillation after coronary artery bypass surgery.

BACKGROUND: Postoperative atrial fibrillation (PoAF) is a common complication after cardiac surgery. A pre-existing atrial substrate appears to be important in postoperative development of dysrhythmia, but its preoperative estimation is challenging. We tested the hypothesis that a combination of clinical predictors, noninvasive surrogate markers for atrial fibrosis defining abnormal left atrial (LA) mechanics, and biomarkers of collagen turnover is superior to clinical predictors alone in identifying patients at-risk for PoAF. METHODS: In patients without prior AF undergoing coronary artery bypass grafting, concentrations of biomarkers reflecting collagen synthesis and degradation, extracellular matrix, and regulatory microRNA-29s were determined in serum from preoperative blood samples and correlated to atrial fibrosis extent, alteration in atrial deformation properties determined by 3D speckle-tracking echocardiography, and AF development. RESULTS: Of 90 patients without prior AF, 34 who developed PoAF were older than non-PoAF patients (72.04 ± 10.7 y; P = 0.043) with no significant difference in baseline comorbidities, LA size, or ventricular function. Global (P = 0.007) and regional longitudinal LA strain and ejection fraction (P = 0.01) were reduced in PoAF vs. non-PoAF patients. Preoperative amino-terminal-procollagen-III-peptide (PIIINP) (103.1 ± 39.7 vs. 35.1 ± 19.3; P = 0.041) and carboxy-terminal-procollagen-I-peptide levels were elevated in PoAF vs. non-PoAF patients with a reduction in miR-29 levels and correlated with atrial fibrosis extent. Combining age as the only significant clinical predictor with PIIINP and miR-29a provided a model that identified PoAF patients with higher predictive accuracy. CONCLUSIONS: In patients without a previous history of AF, using age and biomarkers of collagen synthesis and regulation, a noninvasive tool was developed to identify those at risk for new-onset PoAF.

A Multiwell Cardiac µGMEA Platform for Action Potential Recordings from Human iPSC-Derived Cardiomyocyte Constructs.

Multielectrode array (MEA) technology has been extensively used for field potential recordings from excitable cells. However, its application for action potential (AP) measurements has not been harnessed. Here, we report a novel platform for high-resolution intracellular AP recordings from induced pluripotent stem cell-cardiomyocyte constructs derived from human cardiac fibroblasts. To gain intracellular access, micro-gold MEAs were used to electroporate multiple constructs simultaneously. High-throughput AP measurements were obtained from 41 multicellular constructs. Repeated electroporations of the same cells did not affect the signal stability. Our model has the capability to distinguish subtle differences in AP morphology to characterize the network profile. Furthermore, we confirm the reliability of the system by recapitulating known drug-induced physiological and arrhythmogenic responses. Overall, the model provides a unique cardio-electronic interface for non-invasive measurements of AP dynamics for drug screening and disease modeling. This technology opens the door for identifying novel cardio-factors to enhance electrophysiological maturation.

Aging-related increase in store-operated Ca2+ influx in human ventricular fibroblasts.

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 SOCE-related ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥50 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 SOCE-related 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. NEW & NOTEWORTHY Human ventricular fibroblasts exhibit an age-related increase in store-operated Ca2+ influx induced by angiotensin II, an endogenous vasoactive hormone, or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, independent of receptor mechanisms or genes encoding store-operated Ca2+ entry-related ion channel subunits. Selective inhibition of this augmented store-operated Ca2+ entry could therapeutically limit aging-related cardiac fibrosis.

Effect of Aging on Mitochondrial Energetics in the Human Atria.

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.

Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart.

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 Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ 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/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC 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 ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 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 Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.