Cardiovascular disease remains the leading cause of mortality worldwide, and at its molecular core lies a silent disruptor: oxidative stress. This imbalance between reactive oxygen species (ROS) and antioxidant defenses not only damages cellular components but also orchestrates a cascade of pathological events across diverse cardiac cell types. In cardiomyocytes, ROS overload impairs contractility and survival, contributing to heart failure and infarction. Cardiac fibroblasts respond by promoting fibrosis through excessive collagen deposition. Macrophages intensify inflammatory responses, such as atherosclerosis, via ROS-mediated lipid oxidation—acting both as mediators of damage and targets for antioxidant intervention. This review examines how oxidative stress affects cardiac cell types and evaluates antioxidant-based therapeutic strategies. Therapeutic approaches include natural antioxidants (e.g., polyphenols and vitamins) and synthetic agents (e.g., enzyme modulators), which show promise in experimental models by improving myocardial remodeling. However, clinical trials reveal inconsistent outcomes, underscoring translational challenges (e.g., clinical biomarkers). Emerging strategies—such as targeted antioxidant delivery, activation of endogenous pathways, and disease modeling using 3D organoids—aim to enhance efficacy. In conclusion, we spotlight innovative technologies—like lab-grown heart tissue models—that help scientists better understand how oxidative stress affects heart health. These tools are bridging the gap between early-stage research and personalized medicine, opening new possibilities for diagnosing and treating heart disease more effectively.

Metabolic syndrome (MetS) represents a constellation of interrelated metabolic disturbances, including insulin resistance, central obesity, dyslipidemia, and hypertension, which collectively increase the risk of cardiovascular disease and type 2 diabetes mellitus ([Alberti et al., 2009][1]). With

Fibrosis, characterised by excessive extracellular matrix deposition, contributes to both organ failure and significant mortality worldwide. Whereas fibroblasts are activated into myofibroblasts, marked by phenotypic factors such as α-smooth muscle actin (α-SMA), periostin, fibroblast activation protein (FAP) and heat shock protein 47 (HSP47), the cellular processes of trans-differentiation for fibrosis development remain poorly understood. Herein, we hypothesised that the molecular signalling of geranylgeranyl pyrophosphate (GGPP), a crucial biochemical molecule for protein prenylation, is essential in the regulation of profibrotic mechanisms for fibroblast-to-myofibroblast activation. To test this hypothesis, we demonstrated pharmacological inhibition of geranylgeranyl pyrophosphate synthase (GGPS1) significantly decreased TGF-β1-dependent myofibroblast differentiation assessed by reduced α-SMA, periostin, FAP and HSP47 expression. Exogenous GGPP in the presence of GGPS1 inhibition restored TGF-β1-induced differentiation, supporting posttranslational requirements of GGPP modification during myofibroblast differentiation. Selective inhibition of either geranylgeranyl transferase or farnesyl transferase significantly impacted TGF-β1-induced myofibroblast α-SMA and HSP47 expression. The importance of protein prenylation as a key regulator of myofibroblast differentiation was remarkably revealed by an unexpected decrease in HSP47 expression. In contrast, direct HSP47 inhibition not only suppressed TGF-β1-induced α-SMA expression but surprisingly could not be rescued using exogenous GGPP. A selective role for the ER-resident chaperone HSP47 expression downstream of GGPP was suggested when the effects of GGPS1 inhibition on periostin expression were counteracted by GGPP and geranylgeranyl transferase inhibition. Taken together, our findings underscore for the first time the functional role of cholesterol synthesis-independent GGPP-dependent pathway in fibroblast-to-myofibroblast transition and open new potential therapeutic targets for antifibrosis therapies.

Fibrosis, characterized by excessive extracellular matrix (ECM) components deposition, is a common pathological process underlying numerous chronic diseases. Differentiating fibroblasts into myofibroblasts leads to the deposition of fibrous proteins like collagen in the ECM. Heat shock protein 47 (HSP47) is a molecular chaperone protein that assists in the folding of collagen. Recent studies show that inhibition of cholesterol synthesis-independent HMGCoA-reductase pathway enzyme geranylgeranyl pyrophosphate synthase 1 (GGPS1) decreases fibroblast differentiation and its bioenergetics. However, the role of HSP47 in this pathway is unknown, although recent observations show that HSP47 is decreased when GGPS1 is inhibited. Hence, we tested the hypothesis that HSP47 promotes cellular respiration in myofibroblasts. Seahorse assays were performed on different fibroblasts/myofibroblasts repeatedly and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in the presence and absence of HSP47 inhibitor (HY124817). Complex V of the electron transport chain was measured by immunoblotting ATP-5A. HY124817 tends to decrease the OCR, basal respiration rate, ATP-linked, and spare respiratory capacity. Co-incubation with HY124817 and digeranyl bisphosphonate (DGBP), an inhibitor of GGPS1, significantly (P = 0.043) inhibited further the ATP-linked OCR compared to the TGF group. Immunoblotting showed that Complex V did not change significantly across treatments. These results suggest that HSP47 is involved in fibroblast bioenergetics related to ATP-linked OCR in both differentiated myofibroblasts and normal fibroblasts. Inhibition of HSP47 complements the decrease in myofibroblast cellular respiration caused by the GGPS1 inhibition. This finding is novel in that HSP47 plays a role in fibroblast bioenergetics regulated by the cholesterol synthesis-independent HMGCoA-reductase pathway. Further research is necessary to identify what HSP47 targets and regulatory elements involved in this process. This would provide more insight into the mechanisms that underlie critical fibrosis conditions like myocardial fibrosis and potential therapeutics. This experience was supported by the Medical College of Wisconsin's Advancing Student Potential for Inclusion with Research Experiences (ASPIRE) program. The program is funded by a federal grant from the National Heart, Lung, and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

We recently described a subgroup of autopsied COVID-19 subjects (~40%), termed ‘profibrotic phenotype,’ who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. Recruitment of CD163+ macrophages (Mφs) and collagen α1(l)(COLα1) were also identified within HSP47+ “hot spots” relative to controls and other COVID subjects. We now characterize profibrotic extracellular matrix (ECM) phenotypes, identifying increases in and regional distribution of periostin (POSTN) and fibroblast activating proteins (FAP) as well as COLα1. These data support induction of the endoplasmic reticulum (ER) stress response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction. ECM shifts occur without significant increases in either trichrome positive staining or myocardial injury based quantitively on standard H&E scoring among controls, non-profibrotic or profibrotic COVID-19 subjects. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside hot spots in COVID-19 subjects. Taken together, the matrix remodeling of human COVID-19 hearts in situ is characterized by pro-fibrotic/-inflammatory mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., Colα1(I), POSTN, FAP). Given the established associations of viral infection (e.g., HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Background Cardiac fibrosis complicates SARS‐CoV‐2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress‐inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection. Methods and Results Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47 + cells and collagen α 1(l) in hearts from people with SARS‐CoV‐2 infections. Because macrophages are also linked to inflammation, we measured CD163 + cells in the same tissues. We observed irregular groups of spindle‐shaped HSP47 + and CD163 + cells as well as increased collagen α 1(I) deposition, each proximate to one another in “hot spots” of ≈40% of hearts after SARS‐CoV‐2 infection (HSP47 + P <0.05 versus nonfibrotics and P <0.001 versus controls). Because HSP47 + cells are consistent with myofibroblasts, subjects with hot spots are termed “profibrotic.” The remaining 60% of subjects dying with COVID‐19 without hot spots are referred to as “nonfibrotic.” No control subject exhibited hot spots. Conclusions Colocalization of myofibroblasts, M2(CD163 + ) macrophages, and collagen α 1(l) may be the first evidence of a COVID‐19–related “profibrotic phenotype” in human hearts in situ. The potential public health and diagnostic implications of these observations require follow‐up to further define mechanisms of viral‐mediated cardiac fibrosis.

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed 'profibrotic phenotype,' who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Background Age‐related heart diseases are significant contributors to increased morbidity and mortality. Emerging evidence indicates that mitochondria within cardiomyocytes contribute to age‐related increased reactive oxygen species (ROS) generation that plays an essential role in aging‐associated cardiac diseases. Methods and Results The present study investigated differences between ROS production in cardiomyocytes isolated from adult (6 months) and aged (24 months) Fischer 344 rats, and in cardiac tissue of adult (18–65 years) and elderly (>65 years) patients with preserved cardiac function. Superoxide dismutase inhibitable ferricytochrome c reduction assay (1.32±0.63 versus 0.76±0.31 nMol/mg per minute; P=0.001) superoxide and H2O2 production, measured as dichlorofluorescein diacetate fluorescence (1646±428 versus 699±329, P=0.04), were significantly higher in the aged versus adult cardiomyocytes. Similarity in age‐related alteration between rats and humans was identified in mitochondrial‐electron transport chain‐complex‐I‐associated increased oxidative‐stress by MitoSOX fluorescence (53.66±18.58 versus 22.81±12.60; P=0.03) and in 4‐HNE adduct levels (187.54±54.8 versus 47.83±16.7 ng/mg protein, P=0.0063), indicative of increased peroxidation in the elderly. These differences correlated with changes in functional enrichment of genes regulating ROS homeostasis pathways in aged human and rat hearts. Functional merged collective network and pathway enrichment analysis revealed common genes prioritized in human and rat aging‐associated networks that underlay enriched functional terms of mitochondrial complex I and common pathways in the aging human and rat heart. Conclusions Aging sensitizes mitochondrial and extramitochondrial mechanisms of ROS buildup within the heart. Network analysis of the transcriptome highlights the critical elements involved with aging‐related ROS homeostasis pathways common in rat and human hearts as targets.

Metformin is the first-line medication for treatment of type 2 diabetes and has been shown to reduce heart damage and death. However, mechanisms by which metformin protects human heart remain debated. The aim of the study was to evaluate the cardioprotective effect of metformin on cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) and mitochondria isolated from human cardiac tissue. At concentrations ≤2.5 mM, metformin significantly increased oxygen consumption rate (OCR) in the hiPSC-CMs by activating adenosine monophosphate activated protein kinase (AMPK)-dependent signaling and enhancing mitochondrial biogenesis. This effect was abrogated by compound C, an inhibitor of AMPK. At concentrations >5 mM, metformin inhibited the cellular OCR and triggered metabolic reprogramming by enhancing glycolysis and glutaminolysis in the cardiomyocytes. In isolated cardiac mitochondria, metformin did not increase the OCR at any concentrations but inhibited the OCR starting at 1 mM through direct inhibition of electron-transport chain complex I. This was associated with reduction of superoxide production and attenuation of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in the mitochondria. Thus, in human heart, metformin might improve cardioprotection due to its biphasic effect on mitochondria: at low concentrations, it activates mitochondrial biogenesis via AMPK signaling and increases the OCR; at high concentrations, it inhibits the respiration by directly affecting the activity of complex I, reduces oxidative stress and delays mPTP formation. Moreover, metformin at high concentrations causes metabolic reprogramming by enhancing glycolysis and glutaminolysis. These effects can be a beneficial adjunct to patients with impaired endogenous cardioprotective responses.

Introduction: Acute postoperative heart failure (PoHF) is encountered in 20-35% of elderly patients after cardiac surgery but predictors are not well defined. Circulating microRNAs (miRNAs) predict HF or postoperative atrial fibrillation, yet, their role in identifying those at risk for PoHF with preserved LV function and gender-related differences is not known. Hypothesis: miRNAs involved in CVD can identify patients who develop PoHF in gender-specific manner. Methods: Preoperative blood samples from patients with preserved LV function undergoing cardiac surgery with no prior history of HF, supraventricular, or ventricular tachycardia was used for RNA isolation. Differences in relative plasma levels of miRNA between PoHF vs. No-PoHF were assessed using qPCR. Univariate and multiple logistic regressions were performed to assess risk factors for PoHF. Results: Out of 68 patients (mean age 68.3±12.4), 13 (19%) developed PoHF (54% males; mean age 64.1±15.1; p=0.23), while 55 (51% males; mean age 69.3y ± 11.6) remained free of PoHF. Although all patients had preserved LV function, those who developed PoHF had lower LVEF, (52 vs 58; P<0.05). No significant differences were observed in the prevalence of hypertension, diabetes, myocardial infarction, heart failure, stroke, COPD, or the use of cardiac medications. Out of 13 miRNAs analyzed, stepwise discriminant analysis showed miR-423 (11-fold), -187 (6-fold), -26a2 (4-fold), and -15b (-1.9-fold) were significantly (p<0.05) altered in patients who developed PoHF. When miRNAs were assessed for gender differences, levels of only four miRNAs [miR-423(P<0.01), -23b2(p<0.05), -15b1(p<0.01), and -29b (p<0.05)] principally involved in TGFβ1-SMAD 2/3 regulated extracellular matrix (ECM) turnover pathways were significantly altered in male but not in female patients who developed PoHF compared to No-PoHF group. Conclusions: The differences in preoperative miRNA expression in pathways involved in ECM turnover in male patients who develop PoHF is suggestive of pathophysiological substrate differences between males and females and identifies a group at risk of PoHF with preserved LV function.

Background: Advancements in cardiac surgical techniques have led to decreasing operative risk. However, postoperative heart failure (PoHF) continues to be a major risk factor for adverse cardiac events in 20-35% of patients after cardiac surgery, with a 10-fold increase in 30-day mortality. Prediction of PoHF is challenging, particularly in patients with preserved ventricular function. Circulating microRNAs (miRNAs) recently were identified to predict HF or AF after surgery, but their role in predicting PoHF is not known. This study aimed to find novel noninvasive circulating biomarkers along with clinical factors that can identify patients at risk of developing PoHF immediately after surgery. Methods: Patients undergoing CABG surgery with no previous history of HF, ventricular or supraventricular tachycardia were recruited, and preoperative blood assessed for circulating levels of protein biomarkers using ELISA. Differences in relative plasma levels of 13 miRNAs between the PoHF and no-PoHF groups were assessed by qPCR. Preoperative echocardiography was obtained. SAS was used for statistical analysis and ROC curve. Results: Out of 68 patients, 13 developed PoHF (19.1%, mean age 64.1±11.6y, 53.8% males), whereas 55 (mean age 68.3±12.4y) remained free of HF. Patients who developed PoHF had lower LVEF (51.4±13.7 vs 58.2±9.9, P<0.05) with no differences in prevalence of hypertension, diabetes, hyperlipidemia, obesity, previous myocardial infarction, stroke, COPD, sleep apnea, or use of cardiac medications. The correlation matrix of all 13 miRNAs was transformed in a principal component (PC), resulting in 3 main clusters with eigenvalue >1. PC cluster2 consisted of miR-23a, -23b, -25 and -26a2, principally involved in oxidatives stress, fibrosis and contractility, and had the strongest association (AUC=0.797; P<0.01) with PoHF. A model combining PC cluster2 with age and LVEF improved sensitivity and specificity of the model to identify patients at risk of PoHF (AUC=0.880; 95% CL, 0.761-0.991; P<0.001) Conclusion: Our study demonstrates that miR-23a, -23b, -25 and -26a2 may be useful predictors of PoHF. Circulating miRNA as biomarkers may have diagnostic potential to preoperatively, noninvasively identify patients at risk of developing PoHF.

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.

ObjectiveMetformin (Metf), an oral antihyperglycemic agent, is widely used to treat type 2 diabetes mellitus. The primary action of the drug is to lower hepatic glucose synthesis and enhance peripheral glucose uptake via activation of the insulin receptor. It also exerts a cardioprotective effect independent of its glucose lowering action; however, mechanisms underlying cardioprotection in the human heart remain poorly defined. We hypothesize that the cardioprotective effect of Metf is associated with mild inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and reduction of oxidative stress that protect mitochondria against permeability transition pore (PTP) opening.MethodsLeft atrial appendage tissue was collected from middle‐aged and elderly patients (n=21) undergoing open‐heart surgery. Mitochondria were isolated from fresh tissue using differential centrifugation and exposed to different concentrations of Metf (0–10 mM). The functional activity of mitochondrial OXPHOS complexes (CI‐V) was measured spectrophotometrically using specific OXPHOS inhibitors, electron donors, and acceptors. Superoxide production was measured by monitoring changes in fluorescence intensity of dihydroethidium (DHE) in the absence and presence of antimycin A (AA, 5 μM). The sensitivity of mitochondria toward PTP opening was assessed by exposing isolated mitochondria to 10 μM of Ca2+ pulses and monitoring abrupt mitochondrial Ca2+ release (Fluo‐5N fluorescence) and swelling of the matrix (decrease in absorbance). The study was approved by the Aurora Institutional Review Board and adhered to the Health Insurance Portability and Accountability Act and Aurora Health Care patient privacy and security guidelines. The study followed the principles of the Declaration of Helsinki.ResultsMetf had a dose‐dependent mild inhibitory effect on the activity of CI, CIV, and CV without affecting CII and CIII (Figure A). This was associated with reduction of superoxide production before and after exposure of the mitochondria to AA (Figure B). Metf in a dose‐dependent manner also attenuated Ca2+‐induced mPTP opening (Figure C) with delayed swelling of mitochondrial matrix (Figure D).ConclusionMetf improves cardioprotection by increasing tolerance to stress‐induced PTP opening due to its mild inhibitory effect on the OXPHOS and reduction of superoxide production. This can be a beneficial alternative to patients with impaired endogenous cardioprotective responses.Support or Funding InformationN 570‐3657, Cardiac Research Award, Aurora Health CareThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract Aims Fibroblast to myofibroblast trans‐differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de‐differentiation could mitigate CF‐related pathologies. We determined whether 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase inhibitors—statins, commonly prescribed to patients at risk of heart failure (HF)—can de‐differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. Methods and results Either in vitro statin treatment of differentiated myofibroblasts (n = 3–6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4–12). In vitro, hVFs were differentiated by transforming growth factor‐β1 (TGF‐β1) for 72 h (TGF‐72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α‐smooth muscle actin (α‐SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration‐dependently de‐differentiated the myofibroblasts. The respective half‐maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 μmol/L), the reduced product of HMG‐CoA, prevented the statin‐induced de‐differentiation (α‐SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 μmol/L), a cholesterol synthesis‐independent HMG‐CoA reductase pathway intermediate, completely prevented the statin‐induced de‐differentiation (α‐SMA/GAPDH ratios: 0.89 ± 0.05 [TGF‐72 h + 72 h], 0.63 ± 0.02 [TGF‐72 h + simvastatin], and 1.2 ± 0.08 [TGF‐72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co‐incubation of simvastatin (200 nmol/L) with glibenclamide (10 μmol/L), a KATP channel inhibitor, attenuated the simvastatin‐induced de‐differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de‐differentiation effect (0.33 ± 0.02). OCR (pmol O2/min/μg protein) was significantly decreased in the simvastatin‐treated hVFs, including basal (P = 0.002), ATP‐linked (P = 0.01), proton leak‐linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP‐linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (α‐SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01). Conclusions This study demonstrates the de‐differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of KATP channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.

Background: Cardiomyocytes (CM), derived from human induced-pluripotent stem cells (iPSC-CM), are promising tools for in vitro proarrhythmia screening for drug safety. However, among various challenges with interpretation of iPSC-CM experimental results, the influence of varied culture periods post-differentiation on the electrophysiological properties of iPSC-CMs is unclear. Objective: To test the hypothesis that prolonged post-differentiation culture of iPSC-CMs will influence the biophysical characteristics of Na + - and Ca 2+ -channels. Methods: Using whole-cell patch clamp, we compared the electrophysiological properties of differentiated ventricular-like iPSC-CMs (APD 30-40 /APD 70-80 >1.5) cultured for 10 to 15 days (D10-15) to those cultured for more than 30 days (≥ D30) both under current- and voltage- clamp configurations. Quantitative real-time PCR was used to quantify the mRNA expression of voltage-gated Na channel subunits SCN1A, SCN5A and Ca channel subunits CACNA1C, CACNA1G and CACNA1I. Fold change was calculated by 2 -ΔΔCt method. Data were analyzed by Student’s t test, and P value <0.05 was considered significant. Results: Post-differentiation prolonged culture imparts increased cellular excitability with high frequency spontaneous action potentials, robust increase in the magnitude of peak Na + -current level, relatively shallow inactivation kinetics of Na + channels, faster recovery from inactivation, and augmented Ca 2+ current density compared to early cultured iPSC-CM. This was associated with a greatly enhanced mRNA expression of α-subunit transcripts of SCN1A (7.5 fold) and SCN5A (38 fold) Na channel subtypes while less robust elevation in the mRNA expression of CACNA1C (0.5 fold), CACNA1G (2 fold) and CACNA1I (2 fold) subunits in prolonged culture compared to early cultured iPSC-CM. Conclusion: Prolonged culture of ventricular-like iPSC-CMs affects the excitability, single-cell electrophysiological properties and differential ion channels subunit expression. Standardization of post-differentiation period for ventricular-like iPSC-CMs culture is crucial for in vitro proarrhythmia screening, health/disease modelling to study cellular mechanisms or test high-throughput drugs’ efficacy and toxicity.

Excessive fibrosis underlies many critical organ dysfunctions.1, 2 Fibrosis emanates from fibroblast trans-differentiation into myofibroblasts,3 marked by increased α-smooth muscle actin (α-SMA) expression and excessive collagen secretion, initiated as a reparative process of normal wound healing and tissue repair in response to injury.4 However, activated myofibroblasts accumulate within pathological lesions of various fibrotic disorders,5 including patchy and interstitial fibrosis in progressive heart failure and cardiac hypertrophy.6 Therefore, attenuation of differentiation to myofibroblasts is expected to mitigate fibrosis. We attempted to find a potential target to extenuate the fibroblast differentiation by analysing the transcription factors in human fibroblasts/myofibroblasts, as transcriptome changes occur in fibroblasts during differentiation.7 Here, we report a novel molecular target, transcription factor AP-2α (TFAP2A), to reduce fibroblasts trans-differentiation. Informed consents were obtained from all participants, and the study was carried out according to the World Medical Association Declaration of Helsinki. Human ventricular fibroblasts (hVFs)-Control hVFs from disease-free trauma victims (Lonza Inc, Allendale, NJ; ScienCell, Carlsbad, CA); hVFs were isolated from Heart Patients (Aurora Health Care, Milwaukee, WI), (HF) as reported earlier.8 NIH/3T3 fibroblasts (ATCC, Manassas, VA), Transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ), angiotensin II (Abcam, Cambridge, MA), miRNeasy Mini Kit, RT2 Profiler PCR Array, RT2 First Strand Kit, RT2 SYBR Green PCR master mix, miScript II RT kit (QIAGEN, Venlo, the Netherlands); Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA), Antibodies: Anti-α-SMA, Anti-TFAP2A (Abcam, Cambridge, MA), Anti-α/β-tubulin and Anti-GAPDH (Cell Signaling, Danvers, MA) were purchased. The isolated hVFs were grouped into fibroblasts-less differentiated (HF-LD) and fibroblasts-highly differentiated (HF-HD) based on their α-SMA expression (immunoblot), compared to the control hVFs (Figure 1A,B). Polymerase chain reaction (PCR) array was performed with RT2 Profiler™ PCR Array-Human Transcription Factors and compared between HF-LD (n = 3) and HF-HD (n = 3). Mature RNA (miRNeasy Mini kit) was reverse transcribed using RT2 First strand cDNA synthesis kit. The cDNA was used on the real-time RT2 Profiler PCR Array (QIAGEN, Cat# PAHS-075Z) in combination with RT2 SYBR® Green qPCR Mastermix (Roche LightCycler® 480 Instrument). Threshold cycle (CT) values (excel file) were uploaded onto the data analysis centre web portal (http://www.qiagen.com/geneglobe). CT values were normalized based on a Manual Selection of reference genes. The fold change/regulation (2^(-ΔΔCT)) was calculated using ΔΔCT method [ΔCT was calculated between target gene and an average of reference genes (HKG), followed by ΔΔCT calculations (ΔCT (Test Group)-ΔCT (Control Group))]. Total RNA was isolated from hVFs (miRNeasy Mini kit) and reverse transcribed (miScript RT II kit) with the supplied HiFlex buffer. qPCR was performed on the LightCycler 480 Instrument II, using the Power SYBR Green PCR Master Mix and 10 ng diluted cDNA per well. The following human primers were used: TFAP2A—F:5′-GACCTCTCGATCCACTCCTTAC-3′ R: 5′-GAGACGGCATTGCTGTTGGACT-3′; β-2-microglobulin (B2M)- F: 5′-CCACTGAAAAAGATGAGTATGCCT-3′ and R: 5′-CCAATCCAAATGCGGCATCTTCA-3′. The following PrimeTime qPCR mouse primer assays were used: α-SMA (Mm.PT.58.16320644); COL1A1 (Mm.PT.58.7562513); COL2A1 (Mm.PT.58.5206680); COL3A1 (Mm.PT.58.13848686), TGFBR1 (Mm.PT.58.28402453), TGFBR2 (Mm.PT.58.6358355) and B2M (Mm.PT.39a.22214835). The cycling conditions were 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 s, 1 minute at 60°C, and 72°C for 40 s. Melt curve analysis was performed by an additional dissociation step of 1 cycle at 95°C for 5 s followed by 65°C for 1 min and ramping data collection to 97°C. Relative expression values (ΔCt) were obtained by normalizing Ct values (Roche Lightcycler 480 Software v1.5.1.62) of the tested genes with that of B2M. The TFAP2A knockout cell line with NIH/3T3 fibroblasts (TFAP2A-KO) was established using CRISPR/CAS9 technology through Creative Biogene, Shirley, NY. Fibroblasts from wild-type or TFAP2A-KO groups were plated at 4000 cells/cm2 with DMEM media (10% BCS) and incubated at 37°C under 5%CO2. Following 24 hours, hVFs were either treated with TGF-β1 (5 ng/mL), angiotensin II (100 nM) or kept as control in DMEM media (2.5% BCS). After 48-72 hours, the fibroblasts/myofibroblasts were rinsed with Dulbecco's PBS and assayed. Standard western protocols were followed8 with respective primary (dilutions: α-SMA, 1:500, TFAP2A, 1:100) and secondary antibodies (1:2000). All samples were immunoblotted simultaneously and repeated at least twice. Both WT and KO fibroblasts were plated as stated before in triplicate (per time-point) in 6-well plates and counted by Cellometer Auto 2000 (Nexcelom Bioscience, Lawrence, MA) at 24, 48, and 72 hours post-plating. Doubling time was calculated by [t − t0]/{[log(Nt)-log(N0)]/log(2)}, where t0 refers time (initial count), t represents time (second count), N0 refers count at time t0, and Nt represents count at time t. From left ventricle of human heart, fibroblasts were isolated and grouped into less differentiated (HF-LD) and highly differentiated (HF-HD) based on their α-SMA expression, compared to control hVFs as shown in Figure 1A,B. PCR array of human transcription factors uncovered that the TFAP2A expression, along with ELK1, was decreased with decrease in differentiation as visualized in the heat map (Figure 1C) and fold regulation data (Figure 1D) (n = 3). This decreased expression of TFAP2A in HF-LD fibroblasts compared to HF-HD myofibroblasts noticed in PCR array was validated by quantitative reverse transcriptase-PCR (n = 5) (Figure 1E). Based on these data, we have suggested that TFAP2A is crucial for the trans-differentiation of fibroblasts into myofibroblasts. We applied CRISPR/Cas9-based gene editing to knockout TFAP2A from NIH/3T3 fibroblasts (Figure 2A) and analysed the differentiation- and pro-fibrotic parameters at both basal level and following TGF-β1 treatment. TGF-β1 significantly increased the mRNA expression of α-SMA (Figure 2B), collagen (COL) 1A1 (Figure 2C), COL2A1 (Figure 2D) in the wild-type while the TGF-β1 effect was significantly low in the TFAP2A-KO fibroblasts. Even at basal level, the expressions of α-SMA (Figure 2B) and COL3A1 (Figure 2E) were significantly decreased in the TFAP2A-KO fibroblasts compared to the wild-type. This suggests that TFAP2A is important for the trans-differentiation of fibroblasts to myofibroblasts. This reduced differentiation of TFAP2A-KO fibroblasts observed in qPCR was further confirmed at protein level by immunoblotting where α-SMA expression was significantly low both at basal level and after TGF-β1 administration (Figure 2F,G). The blunted effect of TGF-β1 in the TFAP2A-KO fibroblasts does not appear to be due to changes in the upstream TGF-β1 receptor levels, as the mRNA levels of TGF-β1 receptor type1 (TGFBR1) is increased in TFAP2A-KO fibroblasts (Figure 2H) without any significant difference in the type2 receptors (TBFBR2) compared to the wild-type (Figure 2I). Interestingly, deletion of TFAP2A gene attenuates not only TGF-β1-induced fibroblast differentiation, but also angiotensin II (Ang II)-induced differentiation as well, as evident from lack of increase in α-SMA expression in the TFAP2A-KO fibroblasts (Supplemental Figure). This suggests that TFAP2A could serve as a common downstream regulator of genes associated with fibroblast differentiation. Importantly, the knockdown of TFAP2A did not adversely affect the basal proliferation capacity (Figure 2J). The TFAP2A-KO fibroblasts proliferated like that of the wild-type with a doubling time of 21 ± 6 hr (TFAP2A-KO) vs 25 ± 7 hr (wild-type) (n = 3). TFAP2A is a known DNA-binding transcription factor to have both repressive and facilitating effects9 on various genes and complete knockout of which is embryonically lethal.10 The exact mechanism for the reduced trans-differentiation of TFAP2A-KO fibroblasts in response to TGF-β1 is unclear. Chromatin immunoprecipitation studies of Smad2/3, important factors in TGF-β1 signalling, revealed abundant TFAP2A binding elements in Smad2/3 binding sites of the promoter regions of various genes in keratinocytes and knockdown of TFAP2A changed the TGF-β1- induced transcriptions.11 Whether similar mechanisms underlie in fibroblasts is not known. In human Sertoli cells, Bone Morphogenetic Protein (BMP) 6, a member of TGF-β superfamily, targets TFAP2A to positively regulate their growth.12 In contrast, the basal proliferation of fibroblasts did not reduce following TFAP2A knockdown in our study. This is in accordance with the observation in another study where TFAP2A can induce cell cycle arrest13 while reduced TFAP2A expression was suggested to impair p21cip-mediated growth arrest resulting in increased proliferation.14 These properties found in the TFAP2A-KO fibroblasts suggest that TFAP2A could emerge as a useful molecular target to mitigate excessive fibrosis by inhibiting fibroblast differentiation. As evident from the isolated human cardiac fibroblasts from left ventricles, the decrease in TFAP2A expression when cardiac fibroblast differentiation is decreased, suggest that TFAP2A is crucial for the trans-differentiation of cardiac fibroblasts into myofibroblasts which can lead to excessive cardiac fibrosis underlying many cardiac dysfunctions. Therefore, selective inhibition of TFAP2A could develop as a novel therapeutic strategy to reduce cardiac fibroblast differentiation into myofibroblast, mitigate cardiac fibrosis and preserve cardiac function. Aurora Health Care Cardiovascular Surgery Research Award (#570-5028) to GRR. There is no conflict of interest. GRR initiated, designed, executed, analysed the study and wrote the manuscript; SE executed the real-time PCR and PCR array; CW and PH implemented the cell culture, immunoblotting and proliferation assays; FXD, LE, FR and AJ interpreted data and proof-read the manuscript. All data sets are publicly available from the Dryad Digital Repository at https://doi.org/10.6084/m9.figshare.7898168. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: Postoperative atrial fibrillation (PoAF) is a common complication occurring in 35-50% patients within 2-3 days after cardiac surgery. Identification of patients, especially those with no prior history of atrial fibrillation before surgery, is a challenge. A pre-existing atrial substrate appears to be an important factor in the development of PoAF. The aim of this study was to assess the role of biomarkers in identifying patients at risk of PoAF in a pathophysiology-based risk predictive model by combining clinical and biochemical risk factors. Methods: Preoperative blood from patients undergoing cardiac surgery with no previous history of AF was assessed for circulating levels of biomarkers reflecting collagen synthesis/degradation and extracellular matrix remodeling using ELISA. Reverse transcriptase polymerase chain reaction assessed microRNA29 in the serum and correlated to extent of atrial fibrosis. Echocardiographic evaluation of LA mechanics was performed preoperatively using M-mode, 2D Doppler, and 3D Speckle tracking. Results: Out of 55 patients, 31 patients (56.4%) who developed PoAF after surgery during their hospital stay were older in age (70.0 ± 4.0 years vs.63.4±9.9; p<0.01) with abnormal global longitudinal stain (6.9±0.69 vs.10.9±0.93, p=0.007), higher amino-terminal peptide procollagen III (PIIINP) levels (101.1±42.7 vs.36.6±20.0; p=0.043) with increased collagen to myocardial ratio (0.20±0.09 vs. 0.09±0.01, p= 0.026), and reduced preoperative circulating microRNA-29a, -29b and -29c levels. By combining the clinical risk factors, circulating biochemical and molecular biomarkers, we developed a model that identified PoAF patients (AUC=0.7987; 95% CI, 0.6174—0.98) with reduced preoperative atrial ejection fraction (32±2% vs. 42±2 %; p=0.01) as an independent risk factor for PoAF. Conclusion: Our study developed a noninvasive tool to identify those who are at risk for new-onset PoAF in patients with no previous history of AF when combining age, biomarkers of collagen synthesis and microRNA-29a.

Mechanisms by which metformin (Mtf) protects human heart in type 2 diabetes remain debated. We hypothesize that Mtf has a biphasic effect on mitochondria: it causes activation of respiration at low doses via AMPK signaling and inhibition at high doses by directly affecting the activity of complex I and reducing oxidative stress. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and mitochondria isolated from human cardiac tissue were exposed to different doses of Mtf (0-20 mM) for 24h and acutely. Oxygen consumption rate (OCR) was assessed in the cells and mitochondria energized by glutamate and malate (complex I substrates) using XF96 Extracellular Flux Analyzer. Superoxide production was measured by monitoring changes in fluorescence intensity of dihydroethidium (DHE). At low doses (0.1 - 1 mM), Mtf significantly increased OCR in hiPSC-CMs (Fig. 1A). The highest OCR was observed at 0.5 mM Mtf. This effect was abrogated by compound C, an inhibitor of AMP-kinase (Fig. 1A). Mtf did not increase OCR in isolated mitochondria (Fig. 1B). At higher doses (1-20 mM), it inhibited OCR both in the cells and isolated mitochondria. This was associated with reduction of superoxide production in the mitochondria (Fig. 1C). In the human heart, Mtf improves cardioprotection due to its biphasic effect on mitochondria. This can be a beneficial alternative to patients with impaired endogenous cardioprotective responses. Disclosure L. Emelyanova: None. X. Bai: None. T. Rudic: None. C. Warner: None. F. Rizvi: None. G. Ross: None. D. Kress: None. A. Jahangir: None. Funding Aurora Health Care (N-570-3657)

BackgroundFibroblast to myofibroblast trans‐differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Promotion of de‐differentiation could mitigate CF‐related pathologies; however, no specific remedial therapeutics for CF is clearly defined. Therefore, we determined whether statins, a hypolipidemic class of drugs commonly prescribed in patients at risk of heart failure (HF), can alter cellular bioenergetics and de‐differentiate myofibroblasts.MethodsPrimary cultures of differentiated human ventricular fibroblasts (hVFs) were randomly subjected to in vitro statin treatment (n = 3–6). Differentiation status was determined by α‐smooth muscle actin (α‐SMA) expression while cellular respiration was measured by Seahorse Extracellular Flux Analyzer XF‐96, as oxygen consumption rate (OCR) at baseline and following application of each well with mitochondrial modulators: Oligomycin (1 μg/ml), FCCP (0.1 μM) and antimycin A (1 μg/ml), normalized to total protein. Data were analyzed by either unpaired t test or one‐Way Analysis of Variance.ResultsIn vitro treatment of already differentiated myofibroblasts for 72 hours with both lipophilic (atorvastatin, simvastatin) and hydrophilic (rosuvastatin) statins concentration – dependently reduced α‐SMA/α‐β‐tubulin expression, normalized to % maximal differentiation. The respective IC50 values were 729 ± 13 nM (atorvastatin), 3.6 ± 1 μM (rosuvastatin) and 185 ± 13 nM (simvastatin); n = 3 per concentration of each statin. Basal OCR was significantly decreased in the simvastatin‐treated hVFs vs the differentiated control hVFs (TGF) (0.144 ± 0.026 vs. 0.278 ± 0.104, p = 0.002). Simvastatin reduced the ATP‐linked (0.083 ± 0.018 vs. 0.160 ± 0.059, p = 0.01), the proton leak‐linked (0.017 ± 0.008 vs. 0.074 ± 0.041, p = 0.01) and the maximal (0.250 ± 0.031 vs. 0.501 ± 0.142, p<0.001) OCR, but did not significantly affect spare capacity or non‐mitochondrial OCR. The inhibitory effect of simvastatin on the mitochondrial OCR was reversed by geranylgeranyl pyrophosphate (GGPP, 20 μM ) which significantly increased the basal (0.193 ± 0.053, p = 0.020), the spare‐capacity (0.233 ± 0.091, p = 0.008), and the maximal (0.377 ± 0.131, p = 0.003) OCR, without any change in the non‐mitochondrial OCR. The simvastatin treatment of the differentiated hVFs in vitro reduced ECAR after addition of FCCP (0.066 ± 0.011 vs. 0.093 ± 0.010, p<0.001) and AA (0.056 ± 0.014 vs. 0.097 ± 0.008, p<0.001). GGPP reversed ECAR to a similar level as control differentiated hVFs (FCCP: 0.081 ± 0.015, p = 0.005; AA: 0.080 ± 0.014, p = 0.018). Both atorvastatin (100 nM, 300 nM) and rosuvastatin (300 nM, 1 μM) also increased the ADP/ATP ratio in these cells, confirming the pan‐statin effect of decreased bioenergetics during de‐differentiation. Therefore, statins altered hVF bioenergetics and de‐differentiated the myofibroblasts which involved GGPP‐sensitive mechanisms, reduced cellular respiration with potential activation of KATP channels, as GGPP supplementation or KATP‐channel inhibition by glibenclamide countered the de‐differentiation.ConclusionStatin induced de‐differentiation of myofibroblasts via GGPP‐sensitive signaling, lowered bioenergetics, and KATP channels.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.