Effects of time-restricted exercise on activity rhythms and exercise-induced adaptations in the heart.

Circadian rhythms play a crucial role in the regulation of various physiological processes, including cardiovascular function and metabolism. Exercise provokes numerous beneficial adaptations in heart, including physiological hypertrophy, and serves to shift circadian rhythms. This study investigated the impact of time-restricted exercise training on exercise-induced adaptations in the heart and locomotor activity rhythms. Male mice (n = 45) were allocated to perform voluntary, time-restricted exercise in the early active phase (EAP), late active phase (LAP), or remain sedentary (SED) for 6 weeks. Subsequently, mice were allowed 24-h ad libitum access to the running wheel to assess diurnal rhythms in locomotor activity. Heart weight and cross-sectional area were measured at sacrifice, and cardiac protein and gene expression levels were assessed for markers of mitochondrial abundance and circadian clock gene expression. Mice rapidly adapted to wheel running, with EAP mice exhibiting a significantly greater running distance compared to LAP mice. Time-restricted exercise induced a shift in voluntary wheel activity during the 24-h free access period, with the acrophase in activity being significantly earlier in EAP mice compared to LAP mice. Gene expression analysis revealed a higher expression of Per1 in LAP mice. EAP exercise elicited greater cardiac hypertrophy compared to LAP exercise. These findings suggest that the timing of exercise affects myocardial adaptations, with exercise in the early active phase inducing hypertrophy in the heart. Understanding the time-of-day dependent response to exercise in the heart may have implications for optimizing exercise interventions for cardiovascular health.

Correction: STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

[This corrects the article DOI: 10.1371/journal.pone.0179835.].

Smad3 promotes adverse cardiovascular remodeling and dysfunction in doxorubicin-treated hearts.

Many anticancer therapies cause serious cardiovascular complications that degrade quality of life and cause early mortality in treated patients. Specifically, doxorubicin is known as an effective anticancer agent that causes cardiomyopathy in treated patients. There has been growing interest in defining the role of endothelial cells in cardiac damage by doxorubicin. We have shown in the present study that endothelial nuclei accumulate more intravenously administered doxorubicin than other cardiac cell types. Doxorubicin enhanced cardiac production of the transforming growth factor-ß (TGF-ß) ligands and nuclear translocation of phospho-Smad3 in both cultured and in vivo cardiac endothelial cells. To examine the role of the TGF-ß/mothers against decapentaplegic homolog 3 (Smad3) pathway in cardiac damage by doxorubicin, we used both Smad3 shRNA stable endothelial cell lines and Smad3-knockout mice. We demonstrated using endothelial transcriptome analysis that upregulation of the TGF-ß and inflammatory cytokine/cytokine receptor pathways, as well as suppression of cell cycle and angiogenesis by doxorubicin, were alleviated in Smad3-deficient endothelial cells. The results of transcriptomic analysis were validated using qPCR, immunoblotting, and ex vivo aortic ring sprouting assays. Similarly, increased cardiac expression of cytokines and chemokines observed in treated wild-type mice was diminished in treated Smad3-knockout animals. We also detected increased end-diastolic diameter and depressed systolic function in doxorubicin-treated wild-type but not Smad3-knockout mice. This work provides evidence for the critical role of the canonical TGF-ß/Smad3 pathway in cardiac damage by doxorubicin.NEW & NOTEWORTHY Microvascular endothelial cells in the heart accumulate more intravenously administered doxorubicin than nonendothelial cardiac cell types. The treatment enhanced the TGF-ß/Smad3 pathway and elicited endothelial cell senescence and inflammatory responses followed by adverse cardiac remodeling and dysfunction in wild-type but not Smad3-deficient animals. Our study suggests that the TGF-ß/Smad3 pathway contributes to the development of doxorubicin cardiomyopathy and the potential value of novel approaches to ameliorate cardiotoxicity by targeting the Smad3 transcription factor.

Vitamin D3 induces mesenchymal-to-endothelial transition and promotes a proangiogenic niche through IGF-1 signaling.

Although vitamin D3 (VitD3) prevents angiogenesis in cancer, VitD3 deficiency is associated with greater incidence of cardiovascular events in patients. We examined the influence of VitD3 on the angiogenic potential of mesenchymal stem cells (MSCs). VitD3 treatment increased the expression of proangiogenic molecules in MSCs, which exhibited an endothelial cell-like phenotype and promoted vascularization in vitro and in vivo. VitD3 activated the IGF-1 promoter and boosted IGF-1 receptor (IGF-1R) signaling, which was essential for the mesenchymal-to-endothelial transition (MEndoT) of MSCs. VitD3-treated MSCs created a proangiogenic microenvironment for co-cultured arterial endothelial cells, as well as aortic rings. The induction of MEndoT and angiogenesis promotion by VitD3-stimulated MSCs was attenuated by IGF-1R inhibitor picropodophyllin. We conclude that VitD3 promotes MEndoT in MSCs, and VitD3-treated MSCs augment vascularization by producing a proangiogenic niche through continued IGF-1 secretion. These results suggest a potential therapeutic role of VitD3 toward enhancing MSC-induced angiogenesis.

Transplantation of Human Umbilical Cord Blood-Derived Cellular Fraction Improves Left Ventricular Function and Remodeling After Myocardial Ischemia/Reperfusion.

Rationale: Human umbilical cord blood (hUCB) contains diverse populations of stem/progenitor cells. Whether hUCB-derived nonhematopoietic cells would induce cardiac repair remains unknown. Objective: To examine whether intramyocardial transplantation of hUCB-derived CD45-Lin- nonhematopoietic cellular fraction after a reperfused myocardial infarction in nonimmunosuppressed rats would improve cardiac function and ameliorate ventricular remodeling. Methods and Results: Nonhematopoietic CD45-Lin- cells were isolated from hUCB. Flow cytometry and quantitative polymerase chain reaction were used to characterize this subpopulation. Age-matched male Fischer 344 rats underwent a 30-minute coronary occlusion followed by reperfusion and 48 hours later received intramyocardial injection of vehicle or hUCB CD45-Lin- cells. After 35 days, compared with vehicle-treated rats, CD45-Lin- cell-treated rats exhibited improved left ventricular function, blunted left ventricular hypertrophy, greater preservation of viable myocardium in the infarct zone, and superior left ventricular remodeling. Mechanistically, hUCB CD45-Lin- cell injection favorably modulated molecular pathways regulating myocardial fibrosis, cardiomyocyte apoptosis, angiogenesis, and inflammation in postinfarct ventricular myocardium. Rare persistent transplanted human cells could be detected at both 4 and 35 days after myocardial infarction. Conclusions: Transplantation of hUCB-derived CD45-Lin- nonhematopoietic cellular subfraction after a reperfused myocardial infarction in nonimmunosuppressed rats ameliorates left ventricular dysfunction and improves remodeling via favorable paracrine modulation of molecular pathways. These findings with human cells in a clinically relevant model of myocardial ischemia/reperfusion in immunocompetent animals may have significant translational implications.Visual Overview: An online visual overview is available for this article.

Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis.

Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and ß-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and ß-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.

Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction.

RATIONALE: The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. OBJECTIVE: To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy and to elucidate the underlying molecular pathways. METHODS AND RESULTS: Wild-type and IL-6 knockout (IL-6(-/-)) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6(-/-) mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6(-/-) hearts after TAC. Transcriptional and protein assays of myocardial tissue identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 3 (STAT3) activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from wild-type and IL-6(-/-) mice exposed to prohypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together, these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. CONCLUSIONS: Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension.

FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertrophy.

Fibroblast growth factor 23 (FGF23) is a hormone released primarily by osteocytes that regulates phosphate and vitamin D metabolism. Recent observational studies in humans suggest that circulating FGF23 is independently associated with cardiac hypertrophy and increased mortality, but it is unknown whether FGF23 can directly alter cardiac function. We found that FGF23 significantly increased cardiomyocyte cell size in vitro, the expression of gene markers of cardiac hypertrophy, and total protein content of cardiac muscle. In addition, FGFR1 and FGFR3 mRNA were the most abundantly expressed FGF receptors in cardiomyocytes, and the coreceptor α-klotho was expressed at very low levels. We tested an animal model of chronic kidney disease (Col4a3(-/-) mice) that has elevated serum FGF23. We found elevations in common hypertrophy gene markers in Col4a3(-/-) hearts compared with wild type but did not observe changes in wall thickness or cell size by week 10. However, the Col4a3(-/-) hearts did show reduced fractional shortening (-17%) and ejection fraction (-11%). Acute exposure of primary cardiomyocytes to FGF23 resulted in elevated intracellular Ca(2+) ([Ca(2+)](i); F/F(o) + 86%) which was blocked by verapamil pretreatment. FGF23 also increased ventricular muscle strip contractility (67%), which was inhibited by FGF receptor antagonism. We hypothesize that although FGF23 can acutely increase [Ca(2+)](i), chronically this may lead to decreases in contractile function or stimulate cardiac hypertrophy, as observed with other stress hormones. In conclusion, FGF23 is a novel bone/heart endocrine factor and may be an important mediator of cardiac Ca(2+) regulation and contractile function during chronic kidney disease.