Robert J. Vincent

Transplantation of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells Attenuates Left Ventricular Dysfunction and Remodeling After Myocardial Infarction

Adult bone marrow (BM) contains Sca‐1+/Lin−/CD45− very small embryonic‐like stem cells (VSELs) that express markers of several lineages, including cardiac markers, and differentiate into cardiomyocytes in vitro. We examined whether BM‐derived VSELs promote myocardial repair after a reperfused myocardial infarction (MI). Mice underwent a 30‐minute coronary occlusion followed by reperfusion and received intramyocardial injection of vehicle (n= 11), 1 × 105 Sca‐1+/Lin−/CD45+ enhanced green fluorescent protein (EGFP)‐labeled hematopoietic stem cells (n= 13 [cell control group]), or 1 × 104 Sca‐1+/Lin−/CD45− EGFP‐labeled cells (n= 14 [VSEL‐treated group]) at 48 hours after MI. At 35 days after MI, VSEL‐treated mice exhibited improved global and regional left ventricular (LV) systolic function (echocardiography) and attenuated myocyte hypertrophy in surviving tissue (histology and echocardiography) compared with vehicle‐treated controls. In contrast, transplantation of Sca‐1+/Lin−/CD45+ cells failed to confer any functional or structural benefits. Scattered EGFP+ myocytes and capillaries were present in the infarct region in VSEL‐treated mice, but their numbers were very small. These results indicate that transplantation of a relatively small number of CD45− VSELs is sufficient to improve LV function and alleviate myocyte hypertrophy after MI, supporting the potential therapeutic utility of these cells for cardiac repair.

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.

Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction

Adult bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) exhibit a Sca‐1+/Lin–/CD45– phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 106) of freshly isolated, non‐expanded VSEL‐SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL‐SCs in the marrow is very low, we examined whether VSEL‐SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n= 11), 1 × 105 enhanced green fluorescent protein (EGFP)‐labelled expanded untreated VSEL‐SCs (group II, n= 7), or 1 × 105 EGFP‐labelled expanded VSEL‐SCs pre‐incubated in a cardiogenic medium (group III, n= 8). At 35 days after myocardial infarction (MI), mice treated with pre‐incubated VSEL‐SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle‐treated controls. In contrast, transplantation of expanded but untreated VSEL‐SCs did not produce appreciable reparative benefits. Scattered EGFP+ cells expressing α‐sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)‐1, or von Willebrand factor were present in VSEL‐SC‐treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL‐SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre‐incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL‐SCs for cardiac repair.

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.

Atorvastatin Therapy during the Peri-Infarct Period Attenuates Left Ventricular Dysfunction and Remodeling after Myocardial Infarction

Although statins impart a number of cardiovascular benefits, whether statin therapy during the peri-infarct period improves subsequent myocardial structure and function remains unclear. Thus, we evaluated the effects of atorvastatin on cardiac function, remodeling, fibrosis, and apoptosis after myocardial infarction (MI). Two groups of rats were subjected to permanent coronary occlusion. Group II (n = 14) received oral atorvastatin (10 mg/kg/d) daily for 3 wk before and 4 wk after MI, while group I (n = 12) received equivalent doses of vehicle. Infarct size (Massons trichrome-stained sections) was similar in both groups. Compared with group I, echocardiographic left ventricular ejection fraction (LVEF) and fractional area change (FAC) were higher while LV end-diastolic volume (LVEDV) and LV end-systolic and end-diastolic diameters (LVESD and LVEDD) were lower in treated rats. Hemodynamically, atorvastatin-treated rats exhibited significantly higher dP/dtmax, end-systolic elastance (Ees), and preload recruitable stroke work (PRSW) and lower LV end-diastolic pressure (LVEDP). Morphometrically, infarct wall thickness was greater in treated rats. The improvement of LV function by atorvastatin was associated with a decrease in hydroxyproline content and in the number of apoptotic cardiomyocyte nuclei. We conclude that atorvastatin therapy during the peri-infarct period significantly improves LV function and limits adverse LV remodeling following MI independent of a reduction in infarct size. These salubrious effects may be due in part to a decrease in myocardial fibrosis and apoptosis.

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.

The beneficial effects of postinfarct cytokine combination therapy are sustained during long-term follow-up

We have previously reported that administration of granulocyte colony-stimulating factor (G-CSF)+Flt-3 ligand (FL) or G-CSF+stem cell factor (SCF) improves left ventricular (LV) function and halts LV remodeling at 35 d after myocardial infarction (MI). In the current study, we investigated whether these beneficial effects are sustained in the long term - an issue of fundamental importance for clinical translation. Mice undergoing a 30-min coronary occlusion followed by reperfusion received vehicle (group I), G-CSF+FL (group II), G-CSF+SCF (group III), or G-CSF alone (group IV) starting 4 h after reperfusion and were euthanized 48 wk later. LV structure and function were assessed by serial echocardiography before and at 48 h and 4, 8, 16, 32, and 48 wk after MI. During follow-up, mice in group I exhibited worsening of LV function and progressive LV remodeling. Compared with group I, both groups II and III exhibited improved LV EF at 4 wk after MI; however, only in group II was this improvement sustained at 48 wk. Group II was also the only group in which the decrease in infarct wall thickening fraction, the LV dilatation, and the increase in LV mass were attenuated vs. group I. We conclude that the beneficial effect of G-CSF+FL on postinfarction LV dysfunction and remodeling is sustained for at least 11 months, and thus is likely to be permanent. In contrast, the effect of G-CSF+SCF was not sustained beyond the first few weeks, and G-CSF alone is ineffective. To our knowledge, this is the first long-term study of cytokines in postinfarction LV remodeling. The results reveal heretofore unknown differential actions of cytokines and have important translational implications.

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 β-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 β-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.

BORON BASED HGF MEMETIC MOLECULE HAS CARDIOPROTECTIVE ROLE AGAINST ISCHEMIA-REPERFUSION INJURY

Background: Synthetic boron based hepatocyte growth factor memetic (HGF-m) may have a ischemic preconditioning effect on cardiomyocytes. Methods: Freshly isolated adult cardiomyocytes (ACM) were incubated overnight with/or without different concentrations of HGF-m. The cells were then subjected to

PRESSURE-OVERLOAD INDUCED LV HYPERTROPHY AND DYSFUNCTION: CRITICAL ROLES OF CAMKII AND P38 MAP KINASE IN ER STRESS SIGNALING PATHWAY

The role of endoplasmic reticulum (ER) stress in pressure overload-induced LV hypertrophy and dysfunction is poorly understood. The specific roles of CaMKII and p38 signaling remain unclear. Transverse aortic constriction (TAC) was performed in age-matched CHOP knockout (KO) mice and controls.