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Background: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetrical growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Identification of the mechanisms governing asymmetrical myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure. Methods: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)–mediated gene delivery in mice, and human tissue samples were used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin immunoprecipitation assays with sequencing and precision nuclear run-on sequencing were used to define a transcriptional mechanism. Results: We report that asymmetrical cardiac myocyte hypertrophy is modulated by SRF (serum response factor) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser 103 phosphorylation is bidirectionally regulated by RSK3 (p90 ribosomal S6 kinase type 3) and PP2A (protein phosphatase 2A) at signalosomes organized by the scaffold protein mAKAPβ (muscle A-kinase anchoring protein β), such that increased SRF phosphorylation activates AP-1 (activator protein-1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPβ-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPβ scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling induced by pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser 103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPβ-SRF signalosome could be a new therapeutic approach for human heart failure. Conclusions: We have identified a new molecular switch, namely mAKAPβ signalosome–regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.

Ischemic cardiomyopathy is a leading cause of death and an unmet clinical need. Adeno-associated virus (AAV) gene-based therapies hold great promise for treating and preventing heart failure. Previously we showed that muscle A-kinase Anchoring Protein β (mAKAPβ, AKAP6β), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy. Here, we show that inhibition of mAKAPβ expression in stressed adult cardiomyocytes in vitro was cardioprotective, while conditional cardiomyocyte-specific mAKAP gene deletion in mice prevented pathological cardiac remodeling due to myocardial infarction. We developed a new self-complementary serotype 9 AAV gene therapy vector expressing a short hairpin RNA for mAKAPβ under the control of a cardiomyocyte-specific promoter (AAV9sc.shmAKAP). This vector efficiently downregulated mAKAPβ expression in the mouse heart in vivo. Expression of the shRNA also inhibited mAKAPβ expression in human induced cardiomyocytes in vitro. Following myocardial infarction, systemic administration of AAV9sc.shmAKAP prevented the development of pathological cardiac remodeling and heart failure, providing long-term restoration of left ventricular ejection fraction. Our findings provide proof-of-concept for mAKAPβ as a therapeutic target for ischemic cardiomyopathy and support the development of a translational pipeline for AAV9sc.shmAKAP for the treatment of heart failure.

Fibroblast growth factor (FGF) 21, a hormone that increases insulin sensitivity, has shown promise as a therapeutic agent to improve metabolic dysregulation. Here we report that FGF21 directly targets cardiac myocytes by binding β-klotho and FGF receptor (FGFR) 4. In combination with high glucose, FGF21 induces cardiac myocyte growth in width mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. While short-term FGF21 elevation can be cardio-protective, we find that in type 2 diabetes (T2D) in mice, where serum FGF21 levels are elevated, FGFR4 activation induces concentric cardiac hypertrophy. As T2D patients are at risk for heart failure with preserved ejection fraction (HFpEF), we propose that induction of concentric hypertrophy by elevated FGF21-FGFR4 signaling constitutes a novel mechanism promoting T2D-associated HFpEF and that FGFR4 blockade might serve as a cardio-protective therapy in T2D. In addition, potential adverse cardiac effects of FGF21 mimetics currently in clinical trials should be investigated.

The impairment of neuronal nitric oxide synthase (nNOS) signaling contributes to disease pathology in the muscle wasting disorder Duchenne muscular dystrophy (DMD). nNOS signal propagation occurs through nitric oxide sensitive soluble guanylate cyclase (sGC), a critical source of cyclic guanosine monophosphate (cGMP) in muscle. Although both nNOS and sGC activity are impaired in DMD patients, little is known about sGC as a therapeutic target. In this study, we tested the hypothesis that stimulating sGC activity with the allosteric agonist BAY41-8543 mitigates striated muscle pathology in the mdx4cv mouse model of DMD. In contrast to DMD patients, mdx mice exhibited greater basal sGC activity than wild type controls with preservation of cGMP levels due partly to upregulation of sGC in some muscles. Stimulating sGC activity in mdx mice with BAY41-8543 substantially reduced skeletal muscle damage, macrophage densities and inflammation and significantly increased resistance to contraction-induced fatigue. BAY41-8543 also enhanced in vivo diaphragm function while reducing breathing irregularities suggesting improved respiratory function. BAY41-8543 attenuated cardiac hypertrophic remodeling, fibrosis and diastolic dysfunction including left atrium enlargement in aged mdx mice. Overall, sGC stimulation significantly mitigated skeletal and cardio-respiratory dysfunction in mdx4cv mice. Importantly, this study provides compelling pre-clinical evidence supporting sGC as a novel target in DMD and the repurposing of FDA-approved sGC stimulators, such as riociguat and veraciguat, as a novel therapeutic approach for DMD.

Preterm infants with bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) have accelerated lung aging and poor long-term outcomes. Klotho is an antiaging protein that modulates oxidative stress, angiogenesis and fibrosis. Here we test the hypothesis that decreased cord Klotho levels in preterm infants predict increased BPD–PH risk and early Klotho supplementation prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. In experiment 1, Klotho levels were measured in cord blood of preterm infants who were enrolled in a longitudinal cohort study. In experiment 2, using an experimental BPD–PH model, rat pups exposed to room air or hyperoxia (85% O2) were randomly assigned to receive every other day injections of recombinant Klotho or placebo. The effect of Klotho on lung structure, PH and cardiac function was assessed. As compared to controls, preterm infants with BPD or BPD–PH had decreased cord Klotho levels. Early Klotho supplementation in neonatal hyperoxia-exposed rodents preserved lung alveolar and vascular structure, attenuated PH, reduced pulmonary vascular remodeling and improved cardiac function. Together, these findings have important implications as they suggest that perinatal Klotho deficiency contributes to BPD–PH risk and strategies that preserve Klotho levels, may improve long-term cardiopulmonary outcomes in preterm infants.

Background: The Ca 2+ /calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the β isoform of the calcineurin catalytic A-subunit (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca 2+ drives excitation–contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Methods: Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca 2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus–mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo. Results: CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast 2-hybrid screen. Cardiac myocyte–specific CIP4 gene deletion in mice attenuated pressure overload–induced pathological cardiac remodeling and heart failure. Blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca 2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca 2+ levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca 2+ and calcineurin signal transduction. Conclusions: These results support a structural model for Ca 2+ and CaNAβ compartmentation in cells based on an isoform-specific mechanism for calcineurin protein–protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation.

Rationale Myocardial infarction (MI) triggers a dynamic microRNA response with the potential of yielding therapeutic targets. Objective We aimed to identify novel aberrantly expressed cardiac microRNAs post-MI with potential roles in adverse remodeling in a rat model, and to provide post-ischemic therapeutic inhibition of a candidate pathological microRNA in vivo. Methods and results Following microRNA array profiling in rat hearts 2 and 14 days post-MI, we identified a time-dependent up-regulation of miR-31 compared to sham-operated rats. A progressive increase of miR-31 (up to 91.4 ± 11.3 fold) was detected in the infarcted myocardium by quantitative real-time PCR. Following target prediction analysis, reporter gene assays confirmed that miR-31 targets the 3´UTR of cardiac troponin-T (Tnnt2), E2F transcription factor 6 (E2f6), mineralocorticoid receptor (Nr3c2) and metalloproteinase inhibitor 4 (Timp4) mRNAs. In vitro, hypoxia and oxidative stress up-regulated miR-31 and suppressed target genes in cardiac cell cultures, whereas LNA-based oligonucleotide inhibition of miR-31 (miR-31i) reversed its repressive effect on target mRNAs. Therapeutic post-ischemic administration of miR-31i in rats silenced cardiac miR-31 and enhanced expression of target genes, while preserving cardiac structure and function at 2 and 4 weeks post-MI. Left ventricular ejection fraction (EF) improved by 10% (from day 2 to 30 post-MI) in miR-31i-treated rats, whereas controls receiving scrambled LNA inhibitor or placebo incurred a 17% deterioration in EF. miR-31i decreased end-diastolic pressure and infarct size; attenuated interstitial fibrosis in the remote myocardium and enhanced cardiac output. Conclusion miR-31 induction after MI is deleterious to cardiac function while its therapeutic inhibition in vivo ameliorates cardiac dysfunction and prevents the development of post-ischemic adverse remodeling.

Supplemental oxygen (O2) therapy in preterm infants impairs lung development, but the impact of O2 on long‐term systemic vascular structure and function has not been well‐explored. The present study tested the hypothesis that neonatal O2 therapy induces long‐term structural and functional alterations in the systemic vasculature, resulting in vascular stiffness observed in children and young adults born preterm. Newborn Sprague‐Dawley rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) for 1 and 3 weeks. A subgroup exposed to 3 weeks hyperoxia was recovered in normoxia for an additional 3 weeks. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound and pressure myography. Aorta remodeling was assessed by collagen deposition and expression. Left ventricular (LV) function was assessed by echocardiography. We found that neonatal hyperoxia exposure increased vascular stiffness at 3 weeks, which persisted after normoxic recovery at 6 weeks of age. These findings were accompanied by increased PWV, aortic remodeling, and altered LV function as evidenced by decreased ejection fraction, cardiac output, and stroke volume. Importantly, these functional changes were associated with increased collagen deposition in the aorta. Together, these findings demonstrate that neonatal hyperoxia induces early and sustained biomechanical alterations in the systemic vasculature and impairs LV function. Early identification of preterm infants who are at risk of developing systemic vascular dysfunction will be crucial in developing targeted prevention strategies that may improve the long‐term cardiovascular outcomes in this vulnerable population., Prolonged neonatal oxygen exposure causes persistent aortic structural and biomechanical alterations, which was even more pronounced after recovery in normoxia. This was accompanied by a marked impairment in left ventricular function. These findings have important implications as it suggests that premature babies exposed to oxygen may be predisposed to vascular dysfunction, leading to increased risk of cardiovascular disease in later life. As this affected population begins to age, there will be a critical need to identify the underlying pathophysiological mechanisms for these vascular morbidities in preterm survivors.

The family of p90 ribosomal S6 kinases (RSKs) are pleiotropic effectors for extracellular signal-regulated kinase signaling pathways. Recently, RSK3 was shown to be important for pathological remodeling of the heart. Although cardiac myocyte hypertrophy can be compensatory for increased wall stress, in chronic heart diseases, this nonmitotic cell growth is usually associated with interstitial fibrosis, increased cell death, and decreased cardiac function. Although RSK3 is less abundant in the cardiac myocyte than other RSK family members, RSK3 appears to serve a unique role in cardiac myocyte stress responses. A potential mechanism conferring the unique function of RSK3 in the heart is anchoring by the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ). Recent findings suggest that RSK3 should be considered as a therapeutic target for the prevention of heart failure, a clinical syndrome of major public health significance.

Introduction: Heart failure (HF) morbidity and mortality remain significant, mandating the continued development of new therapeutics. The scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) is a regulator of pathological remodeling. Our unpublished data show that induction before myocardial infarction (MI) of cardiac myocyte-specific mAKAPβ gene deletion decreases infarct size and preserves cardiac function long-term in mice. To develop a therapeutic providing the benefit of mAKAPβ targeting, we designed a new gene therapy vector, a self-complementary, serotype 9 adeno-associated virus (scAAV) that expresses a mAKAP-specific small hairpin RNA (shRNA) under the control of the cardiac troponin T promoter. We have tested whether post-MI suppression of mAKAPβ expression in the cardiomyocyte as provided by this new biologic prevents HF. Methods: MI was induced by permanent ligation of the left coronary artery in adult C57BL/6 mice. Mice with ejection fraction (EF) ≤40% 2 days post-MI and sham-operated controls were randomized and injected the following day with 5x10 11 vg I.V. of either mAKAP- or control-shRNA scAAV. Results: EF pre-treatment was 31.7% and 32.8% for mAKAP-MI (n=10) and control-MI (n=6) cohorts, respectively. mAKAP-shRNA treatment resulted in amelioration of systolic dysfunction as evidenced by normalization of EF from day 2 to weeks 2, 4 and 8 post-MI, while control-MI mice displayed progressive EF deterioration. At 8 weeks post MI, EF for the mAKAP-MI cohort was comparable to mAKAP- and control-Sham animals (44.3±3.3% vs. 54.6±1.7% & 54.5±2.0%), whereas it was markedly higher than for control-MI animals (18.0±4.3%, P Conclusion: Inhibition of mAKAPβ expression using a novel gene therapy vector ameliorates post-MI cardiac dysfunction and prevents the development of HF.

Visual Abstract, Highlights • Targeted profiling of ceramides identified a 12-ceramide plasma signature that predicted 12-month cardiovascular death, MI, and stroke in 2 prospective cohorts of AMI patients. • Among coronary artery bypass grafting patients, plasma ceramides were higher in those with recent AMI compared with those without recent acute MI. • Analysis of rat ischemic myocardium revealed a consistent increase in ceramide levels and overexpression of 3 enzymes in ceramide biosynthesis., Summary We identified a plasma signature of 11 C14 to C26 ceramides and 1 C16 dihydroceramide predictive of major adverse cardiovascular events in patients with acute myocardial infarction (AMI). Among patients undergoing coronary artery bypass surgery, those with recent AMI, compared with those without recent AMI, showed a significant increase in 5 of the signature’s 12 ceramides in plasma but not simultaneously-biopsied aortic tissue. In contrast, a rat AMI model, compared with sham control, showed a significant increase in myocardial concentrations of all 12 ceramides and up-regulation of 3 ceramide-producing enzymes, suggesting ischemic myocardium as a possible source of this ceramide signature.

Background: Summary Left main disease (LMD) and associated cardiac risk factors are often perceived as a limiting factor for the outcome of off-pump coronary artery bypass (OPCAB) grafting. In this study, we assess whether the outcome of OPCAB surgery is affected in such patients. Methods: We retrospectively compared perioperative parameters in 66 OPCAB patients (group A) with LMD and 216 OPCAB patients without (group B) LMD. The patients were operated in the time frame between 2002 and 2007. LMD was defined as a stenosis >50%. Results: Patients in group A had a higher EuroSCORE (logistic: 3.7 ± 0.1 vs 6.3 ± 0.3, p = 0.027), increased coronary artery disease (CAD) family history (p = 0.015) and cerebrovascular accidents (p = 0.027), increased history of congestive heart failure (p = 0.013), more urgent surgery (p = 0.008), previous percutaneous transluminal coronary angioplasties (PTCAs) (p = 0.05) and previous stent implantation (p = 0.023). An intra-aortic balloon pump (IABP) was inserted more frequently in the LMD group preoperatively (p = 0.004). There were two conversions to on-pump during OPCAB surgery. There were no differences in the postoperative outcomes in the LMD group A versus group B, such as cardiac-related events, neurological deficits, cardiac enzyme course, arrhythmias, blood loss, infections and renal failure. Conclusions: The presence of LMD and higher EuroSCORE does not yield adverse outcomes in OPCAB patients

p90 Ribosomal S6 Kinase 3 (RSK3) is required for the induction of concentric hypertrophy in hearts subjected to pressure overload and catecholamine infusion, as well as in a model for Noonan Syndrome-associated hypertrophic cardiomyopathy. Serving important roles in both cardiac development and adult function, the transcription factor Serum Responsive Factor (SRF) regulates genes expression involved in myocyte growth and sarcomeric assembly. It has been reported that SRF can be phosphorylated by RSK protein kinases on residue Ser-103, but the function of that post-translational modification has remained uncertain. We now show that SRF is a substrate for RSK3 associated with muscle A-Kinase-Anchoring-Protein (mAKAP) scaffold in cardiac myocytes, contributing to the regulation of concentric myocyte growth. By co-immunoprecipitation assay, SRF and RSK3 were both associated with the mAKAP scaffold in heart extracts, such that a ternary complex could be detected when recombinant proteins were expressed in cells. Silencing RSK3 or mAKAP expression by siRNA transfection reduced phenylephrine-induced SRF(S103) phosphorylation in neonatal cardiac myocytes. Similar results were acquired following expression of a peptide comprising the mAKAP RSK3-binding domain (RBD) that competed the binding of the kinase to the scaffold. Overexpression of either a SRF S103A phosphoablative mutant or the RBD peptide diminished the increase in width of adult cardiac myocytes stimulated by phenylephrine. In contrast, overexpression of RSK3 or a SRF S103D phosphomimetic mutant promoted adult cardiac myocyte concentric hypertorphy in vitro. While baseline SRF S103 phosphorylation in the heart was inhibited by mAKAP or RSK3 gene knock-out in mice, acute transverse aortic constriction significantly increased SRF S103 phosphorylation in the heart. Based upon these results, we porpose that RSK3 phosphorylation of SRF at mAKAP signalosomes is an important regulator of concentric cardiac myocyte growth. These results are consistent with additional findings that an adeno-associated virus gene therapy vector that expresses RBD in the cardiac mycoyte attenuates pathological hypertrophy and prevents heart failure in response to pressure overload.

Pathological cardiac remodeling is the primary mechanism underlying the development of heart failure. The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin is a key regulator of pathological myocyte hypertrophy. Previously, it was shown that the β-isoform of the calcineurin catalytic A-subunit (CaNAβ) was uniquely required for hypertrophy. In order to determine the mechanism of CaN A-subunit specificity, we performed a yeast two-hybrid screen using the CaNAβ-specific N-terminal polyproline domain. We now show that a new binding partner for CaNAβ is the F-Bar (Fes-CIP4 homology [ F CH] - B in/ A mphyphysin/ R vs) scaffold protein Cdc42-interacting protein 4 (CIP4/TRIP10). The CaNAβ polyproline domains binds the SH3 domain of CIP4. Using a new CIP4 conditional knock-out mouse, we show that CIP4 contributes to the hypertrophy induced in vivo by pressure overload. After induction of conditional gene deletion using tamoxifen, CIP4 fl/fl ;Tg(Myh6-cre/Esr1*) mice exhibited decreased hypertrophy and improved cardiac function over a period of 16 weeks following transverse aortic constriction survival surgery when compared to control tamoxifen-treated Tg(Myh6-cre/Esr1*) and CIP4 fl/fl mice. In order to show the relevance of CaNAβ-CIP4 binding, we also developed an adeno-associated virus (AAV) gene therapy vector that expresses a CaNAβ polyproline domain - GFP fusion protein under the control of the cardiac myocyte-specific troponin T promoter. Disruption of CIP4-CaNAβ binding in vivo by the AAV vector attenuates cardiac hypertrophy following two weeks of pressure overload and improved systolic function. Together these results both demonstrate the role of a F-Bar scaffold protein in cardiac remodeling and reveal a novel mechanism for specificity in calcineurin signaling. Identification of this new signaling pathway also suggests a new therapeutic approach to heart failure with reduced ejection fraction.

Aim: To determine the therapeutic effect of thy- mosin β4 (Tβ4) for treatment of ischemic limb disease in a mouse model. Methods: A mouse model of hindlimb ischemia was created by permanent ligation of femoral arteries and internal iliac artery. Tβ4 was dissolved in sterile saline and intramuscularly injected into the centre and periphery of ligation area in the treatment group (n = 10) starting from the surgery day until 4 weeks after surgery, while control animals received saline injection only (n = 9). All animals were sacrificed at 6 weeks after surgery and used for immunohistochemistry studies. Results: Tβ4 stimulated angiogenesis was evidenced by increased vascular density based on CD31 immunostaining, which was sig- nifycantly increased in Tβ4 group (562.5 ± 78.4/mm2) as compared with control group (371.1 ± 125.7/mm2; p 0.05) groups. Tβ4 increased Pax3/7+ skeletal muscle progenitor cell density. Pax3/7+ cell density of Tβ4 group (13.7% ± 2%) was significantly higher than that of the control group (4.3% ± 1.6%, p < 0.05). However, the numbers of central nuclei fiber and central nuclei per fiber were insignificantly increased in Tβ4 group as compared to control group. The numbers of central nuclei fiber were 8.9 ± 2.1 and 9.5 ± 1.6, and the central nuclei per fiber were 0.25 ± 0.07 and 0.48 ± 0.09 for control and Tβ4 groups, respectively. Conclusions: This preliminary study suggests that localized delivery of Tβ4 increased angiogenesis and skeletal muscle progenitor cell density in ischemic skeletal muscle, but failed to promote skeletal muscle regeneration.

Angiogenesis plays a key role in post-ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)-enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP-luciferase-expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG-treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham-operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post-implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end-systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p < 0.05; LV end-diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p < 0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p < 0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p < 0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven-fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p < 0.0001). Implantation of AA-enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function.

Cell therapy and tissue engineering attract increasing attention as a potential approach for cardiac repair. Adult stem cells from autologous origin are a practically safe and appealing source for cell-based regenerative therapies that may hold realistic clinical potential. A plethora of interesting concepts have been introduced aiming at regenerating ischemic myocardium through adult stem cell-based bioartificial cardiac tissue supplements. Yet, current pre-clinical concepts have not reached translational applicability, and successes are only episodic. This review will provide a brief overview of the latest concepts and breakthroughs in the emerging field of cardiac adult stem cell-based tissue engineering, and discuses the challenges that this field needs to overcome to achieve realistic therapeutic translation into the clinical arena. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Introduction: The applicability of off-pump coronary-artery bypass (OPCAB) in patients who present as emergency remains controversial. Herein, we explore the efficacy and safety of OPCAB in patients who were indicated for emergency surgery. Materials and Methods: Between 2002 and 2007, a total of 282 patients underwent OPCAB, of which 68 were presented as emergency. This cohort (group A) was compared to 68 patients who had traditional on-pump coronary artery bypass grafting (CABG, group B) under emergency indications during the same period of time. Baseline demographics, intraoperative data and postoperative outcomes were analysed. Results: Preoperative demographics were comparable in both groups. Mortality during the first 30 days was comparable in both groups and no stroke occurred in the whole series. Patients in group A had significantly less pulmonary complications (4.4% vs 14.7%, P= 0.04), less ventilation time (30.3 ± 33.6 hours vs 41.5 ± 55.4 hours, P = 0.18) and were less likely to have prolonged ventilation, (19.1% vs 35.3%, P = 0.03). Similarly, OPCAB patients had less postoperative renal-failure/dysfunction (5.9% vs 8.8%, P = 0.51) and required less inotropic support (66.2% vs 88.2%, P = 0.002), bloodtransfusions (23% vs 86.8%, P

Background: A severely decreased ejection fraction (EF) of ?25% is an established risk factor for a worse outcome after heart surgery and therefore has been incorporated into the EuroSCORE risk-stratification model. We compare clinical outcomes after off-pump coronary artery bypass grafting (OPCAB) and on-pump coronary artery bypass grafting in patients with a severely compromised EF.Methods: We compared 112 patients with a low EF (?25%) who underwent myocardial revascularization between 2003 and 2008. Forty-four patients underwent OPCAB (group A), and 68 patients underwent on-pump surgery (group B). We compared demographics, intraoperative parameters, intraoperative outcomes, and the completeness of revascularization for the 2 groups.Results: Demographic and EuroSCORE data were comparable for groups A and B. The 2 groups appeared to be similar with respect to mortality rate during the first 30 days (2.2% and 8.8%, respectively; P = .11) and the rate of major complications such as stroke (2.2% and 2.9%, respectively; P = 0.83). The patients in group A had fewer pulmonary complications (7% versus 25%, P < .01), received fewer blood transfusions (15.9% versus 47.0%, P < .01), required fewer postoperative pacing procedures (atrial, 11.4% versus 39.7%; ventricular, 13.6% versus 47.1%; P < .01), and had fewer wound infections (2.2% versus 16.1%, P = .02). The numbers of diseased vessels were comparable, and although the OPCAB patients received more arterial grafts (1.05 ± 0.43 versus 0.84 ± 0.37, P < .01), the total number of grafts per patient was lower among these patients (2.50 ± 0.88 versus 3.53 ± 0.92, P = .03). Similarly, complete revascularization was achieved less frequently within this group (80% versus 94%, P = .02).Conclusions: A standardized OPCAB approach in patients with a severely decreased EF is safe and may benefit this subset of patients with respect to fewer postoperative complications. Although complete revascularization is the optimal approach for these patients, they benefit from avoiding cardiopulmonary bypass.

Noonan syndrome (NS) is a congenital disorder resulting from mutations of the Ras-Raf signaling pathway. Hypertrophic cardiomyopathy associated with RAF1 "RASopathy" mutations is a major risk factor for heart failure and death in NS and has been attributed to activation of MEK1/2-ERK1/2 mitogen-activated protein kinases. We recently discovered that type 3 p90 ribosomal S6 kinase (RSK3) is an ERK effector that is required, like ERK1/2, for concentric myocyte hypertrophy in response to pathological stress such as pressure overload. In order to test whether RSK3 also contributes to NS-associated hypertrophic cardiomyopathy, RSK3 knock-out mice were crossed with mice bearing the Raf1(L613V) human NS mutation. We confirmed that Raf1(L613V) knock-in confers a NS-like phenotype, including cardiac hypertrophy. Active RSK3 was increased in Raf1(L613V) mice. Constitutive RSK3 gene deletion prevented the Raf1(L613V)-dependent concentric growth in width of the cardiac myocyte and attenuated cardiac hypertrophy in female mice. These results are consistent with RSK3 being an important mediator of ERK1/2-dependent growth in RASopathy. In conjunction with previously published data showing that RSK3 is important for pathological remodeling of the heart, these data suggest that targeting of this downstream MAP-kinase pathway effector should be considered in the treatment of RASopathy-associated hypertrophic cardiomyopathy.

Introduction: Circulating microRNA (miRNAs) profiles are dysregulated in heart failure (HF) suggesting possible diagnostic and prognostic applications. In addition, miRNAs may yield insights into the pathophysiology of HF and provide targets for therapeutic intervention. We sought to identify miRNAs diagnostic utility in HF and to further investigate selected miRNAs as potential targets for therapeutic intervention. Hypothesis: We hypothesized that distinct circulating miRNAs are dysregulated in HF and may be potentially important targets of therapy to improve outcomes for HF. Methods: Plasma miRNAs levels were profiled from 338 well-characterized HF patients and 208 age-matched controls without HF. We also examined selected miRNAs expression in a rat post-myocardial infarction (MI) model. Target predictive databases were used to identify potential miRNA targets. Results: Comparison between HF and non-HF controls was carried out by multivariate analysis. Seventy five miRNAs differed significantly between HF and non-HF controls after adjustment for age, hypertension, diabetes and atrial fibrillation (p-value after FDR0.65 , pNPPB . The roles of miR-503 and miR-374b-5p in HF warrant further investigation using both in-vitro and in-vivo preclinical HF models. Conclusion: We provide a detailed study of circulating miRNA profiling in both clinical HF and experimental model. We demonstrated that circulating miR-503 and miR-374b-5p are associated with HF.

Aims Myocardial infarction followed by adverse left ventricular (LV) remodeling is the most frequent proximate cause of heart failure. Hydrogen sulfide (H 2 S) is an important endogenous modulator of diverse physiological and pathophysiological processes. Its role in post-ischemic ventricular remodeling and the associated neurohormonal responses has not been defined. Here, we aimed at evaluating whether the slow-releasing water-soluble H 2 S donor GYY4137 (GYY) exerts cardioprotective effects and modulates the neurohormonal response to cardiac ischemic injury. Methods and results Treatment for 2 or 7 days with GYY (100 mg/Kg/48 h, IP) after acute myocardial infarction (MI) in rats preserved LV dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and dl -propargylglycine- (PAG, an inhibitor of endogenous H 2 S synthesis) treated animals (n = 9/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts had significantly less LV fibrosis than MI, placebo and PAG hearts. A higher density of blood vessels was found in the LV scar area of GYY-treated animals compared to all other infarcted groups. Despite preserved LV structure and function, treatment with GYY increased the levels of the natriuretic peptides ANP and BNP in association with enhanced cyclic GMP levels, paralleled by higher cGMP-dependent protein kinase type I (cGKI) protein levels. Conclusions Our data suggest that the slow-releasing H 2 S donor, GYY4137, preserves cardiac function, attenuates adverse remodeling and may exert post-ischemic cardioprotective (pro-angiogenic, anti-apoptotic, anti-hypertrophic and anti-fibrotic) effects in part through enhanced early post-ischemic endogenous natriuretic peptide activation.

Heightened expression of the S100 calcium–binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100β, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT 1B ), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT–induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal–regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4–mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT 1B receptor and SERT are codependent in regulating S100A4/Mts1.

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.

Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.

The limited efficacy of cardiac cell-based therapy is thought to be due to poor cell retention within the myocardium. Hence, there is an urgent need for biomaterials that aid in long-term cell retention. This study describes the development of injectable microcapsules for the delivery of mesenchymal stem cells (MSCs) into the infarcted cardiac wall. These microcapsules comprise of low concentrations of agarose supplemented with extracellular matrix (ECM) proteins collagen and fibrin. Dextran sulfate, a negatively charged polycarbohydrate, was added to mimic glycosaminoglycans in the ECM. Cell viability assays showed that a combination of all components is necessary to support long-term survival and proliferation of MSCs within microcapsules. Following intramyocardial transplantation, microcapsules degraded slowly in vivo and did not induce a fibrotic foreign body response. Pre-labeling of encapsulated MSCs with iron oxide nanoparticles allowed continued cell-tracking by MRI over several weeks following transplantation into infarcted myocardium. In contrast, MSCs injected as cell suspension were only detectable for two days post transplantation by MRI. Histological analysis confirmed integration of transplanted cells at the infarct site. Therefore, microcapsules proved to be suitable for stem cell delivery into the infarcted myocardium and can overcome current limitations of poor cell retention in cardiac cell-based therapy.

Aims Cell-based myocardial restoration has not penetrated broad clinical practice yet due to poor cell retention and survival rates. In this study, we attempt a translational, large-scale restorative but minimally invasive approach in the pig, aiming at both structurally stabilizing the left ventricular (LV) wall and enhancing function following ischemic injury. Methods and results A myocardial infarction (MI) was created by permanent ligation of left circumflex coronary artery through a small lateral thoracotomy. Thirty-six Yorkshire pigs were randomized to receive transthoracic intramyocardial injection into both infarct and border zone areas with different compounds: 1) Hyaluronic acid-based hydrogel; 2) autologous platelet-rich plasma (PRP); 3) ascorbic acid-enriched hydrogel (50 mg/L), combined with IV ibuprofen (25 mg/kg) and allopurinol (25 mg/kg) (cocktail group); 4) PRP and cocktail (full-compound); or 5) saline (control). The latter two groups received daily oral ibuprofen (25 mg/kg) for 7 days and allopurinol (25 mg/kg) for 30 days, postoperatively. Hemodynamic and echocardiographic studies were carried out at baseline, immediately after infarction and at end-point. Eight weeks after MI, the full-compound group had better LV fractional area change, ejection fraction and smaller LV dimensions than the control group. Also, dp/dt max was significantly higher in the full-compound group when the heart rate increased from 100 bpm to 160bpm in stress tests. Blood vessel density was higher in the full-compound group, compared to the other treatment groups. Conclusions A combination of PRP, anti-oxidant and anti-inflammatory factors with intramyocardial injection of hydrogel has the potential to structurally and functionally improve the injured heart muscle while attenuating adverse cardiac remodeling after acute myocardial infarction.

Myocardial infarction is the most frequent proximate cause of heart failure, which remains a leading cause of death in the developed world. Hydrogen sulfide (H 2 S) is emerging as an important endogenous modulator in diverse physiological and pathophysiological events. We hypothesized that the slow-releasing water-soluble H 2 S donor GYY4137 (GYY) may exert cardioprotective effects through modulation of neurohormonal response to cardiac injury. We have found that treatment for 1 week with GYY (100mg/Kg/48hr, IP) after acute myocardial infarction in rats, provides powerful sustained preservation of left ventricular (LV) dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and D-propargylglycine- (PAG, an inhibitor of endogenous H 2 S synthesis) treated animals 2 and 7 days after infarct (n=6/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts displayed a significantly lower percentage of LV fibrosis than MI, placebo and PAG hearts, whereas PAG treated animals had significantly bigger scar size relative to GYY, at days 2 and 7 after MI. A higher density of blood vessels was found in the scar area of GYY-treated animals compared to all other infarcted groups at days 2 (P

MicroRNAs (miRNA), small sequences of non-coding RNA which interact with complementary sequences on the 3’untranslated region of target messenger RNAs to modulate translation, have a pivotal role in the development of the heart and its response to injury. Myocardial infarction (MI) triggers a dynamic miRNA response with the potential of yielding therapeutic targets. Following miRNA array profiling in rat hearts 2, 7 and 14 days after MI induced by coronary ligation, we identified a progressive time-dependent up-regulation of miR-31 compared to sham rats. Increase of miR-31 in heart tissue in the acute and subacute phases after MI (up to 90-fold) was also detected by Real-Time PCR (P=0.02 at day 2; P

Natriuretic peptide receptor 3 (NPR3) is the clearance receptor for the cardiac natriuretic peptides (NPs). By modulating the level of NPs, NPR3 plays an important role in cardiovascular homeostasis. Although the physiological functions of NPR3 have been explored, little is known about its regulation in health or disease. MicroRNAs play an essential role in the post-transcriptional expression of many genes. Our aim was to investigate potential microRNA-based regulation of NPR3 in multiple models. Hypoxic challenge elevated levels of NPPB and ADM mRNA, as well as NT-proBNP and MR-proADM in human left ventricle derived cardiac cells (HCMa), and in the corresponding conditioned medium, as revealed by qRT-PCR and ELISA. NPR3 was decreased while NPR1 was increased by hypoxia at mRNA and protein levels in HCMa. Down-regulation of NPR3 mRNA was also observed in infarct and peri-infarct cardiac tissue from rats undergoing myocardial infarction. From microRNA microarray analyses and microRNA target predictive databases, miR-100 was selected as a candidate regulator of NPR3 expression. Further analyses confirmed up-regulation of miR-100 in hypoxic cells and associated conditioned media. Antagomir-based silencing of miR-100 enhanced NPR3 expression in HCMa. Furthermore, miR-100 levels were markedly up-regulated in rat hearts and in peripheral blood after myocardial infarction and in the blood from heart failure patients. Results from this study point to a role for miR-100 in the regulation of NPR3 expression, and suggest a possible therapeutic target for modulation of NP bioactivity in heart disease.

A crucial question in post-ischemic cell therapy refers to the ideal method of cell delivery to the heart. We hypothesized that epicardial implantation of subamnion-cord-lining mesenchymal stem cells (CL-MSC) angiogenic spheroids embedded within fibrin grafts (SASG) facilitates donor cell survival and enhances cardiac function in failing rat hearts. Furthermore, we compared the efficacy of this approach applied through two delivery methods. Spheroids made of 1.5×10(4) human CL-MSC coated with 2×10(3) human umbilical vein endothelial cells were self-assembled in hanging drops. SASG were constructed by embedding 150 spheroids in fibrin matrix. Except for untreated rats (MI, n=8), grafts were implanted 2 weeks after myocardial infarction upon confirmation of ensued heart failure through thoracotomy: SASG (n=8) and fibrin graft (FG, n=8); or video-assisted thoracoscopic surgery (VATS): SASG-VATS (n=8) and FG-VATS (n=7). In vivo CL-MSC survival was comparable between both SASG-treated groups throughout the study. SASG and SASG-VATS animals had decreased left ventricular end-diastolic pressure relative to untreated animals, and increased fractional shortening compared to MI and FG controls, 4 weeks after treatment. A 14.1% and 6.2% enhancement in ejection fraction from week 2 to 6 after injury was observed in SASG/SASG-VATS, paralleled by improvement in cardiac output. Treated hearts had smaller scar size, and more blood vessels than MI, while donor CL-MSC contributed to arteriogenesis within the graft and infarct areas. Taken together, our data suggest that SASG treatment has the potential to restore failing hearts by preserving cardiac function and inducing myocardial revascularization, while attenuating cardiac fibrosis. Furthermore, we introduce a method for minimally invasive in situ graft assembly.

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×106 CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p

Angiogenesis plays a key role in post-ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)-enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP-luciferase-expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG-treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham-operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post-implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end-systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p0.05; LV end-diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven-fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p0.0001). Implantation of AA-enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function.

Diabetes and habitual smoking cause advanced coronary artery disease (CAD) in Asian patients at a younger age. No definite data exist as to whether off-pump (OPCAB) is better than conventional on-pump coronary artery bypass grafting (CCAB) in terms of postoperative renal complications. Thus, we aimed to compare the renal outcomes of on-pump and off-pump coronary artery bypass grafting (CABG) on our patients, which constituted a predominantly Asian population.A cohort of 395 patients following CCAB were compared with 293 patients who underwent OPCAB. Baseline demographics, comorbidities, intraoperative data, intensive care unit stay, number of grafts, New York Heart Association (NYHA) score, American Society of Anesthesiologists (ASA) score, EuroSCORE risk assessment model, and postoperative complications particularly renal, were collected and analysed.The off-pump group consisted of significantly older patients with higher Canadian Cardiovascular Society (CCS) and ASA scores. Additionally, the off-pump group involved a significantly greater number of smokers and chronic obstructive pulmonary disease (COPD) patients. Other demographic parameters were not different between the groups. Postoperative investigations showed a significantly elevated serum creatinine (100.3 +/- 42.5 vs 127.6 +/- 114.2 micromol/L; off-pump vs on-pump; P = 0.039) and urea levels (5.9 +/- 3.1 vs 10.6 +/- 15.6 mg/dL; off-pump vs on-pump; P = 0.006) in the on-pump group. Moreover, there was a high tendency towards a higher rate of renal dysfunction associated death in this group.OPCAB is a safe and equally efficient operative method compared to CCAB, and has a significant lower risk for postoperative renal complications as a treatment modality for surgical coronary revascularisation.

Organ restoration via cell therapy and tissue transplantation is limited by impaired graft survival. We tested the hypothesis that ascorbic acid (AA) reduces cell death in myocardial grafts both in vitro and in vivo and introduced a new model of autologous graft vascularization for later transplantation. Luciferase (Fluc)- and green fluorescent protein (GFP)-expressing H9C2 cardiomyoblasts were seeded in gelatin scaffolds to form myocardial artificial grafts (MAGs). MAGs were supplemented with AA (5 or 50 mumol/L) or plain growth medium. Bioluminescence imaging showed increased cell photon emission from day 1 to 5 in grafts supplemented with 5 mumol/L (p < 0.001) and 50 mumol/L (p < 0.01) AA. The amount of apoptotic cells in plain MAGs was significantly higher than in AA-enriched grafts. In our in vitro model, AA also enhanced H9C2 cell myogenic differentiation. For in vivo studies, MAGs containing H9C2-GFP-Fluc cells and enriched with AA (n = 10) or phosphate-buffered saline (n = 10) were implanted in the renal pouch of Wistar rats. At day 6, postimplantation bioluminescence signals decreased by 74% of baseline in plain MAGs versus 36% in AA-enriched MAGs (p < 0.0001). AA grafts contained significantly higher amounts of blood vessels, GFP(+) donor cells, and endothelial cells. In this study, we identified AA as a potent supplement that improves cardiomyoblast survival and promotes neovascularization in bioartificial grafts.

There has been an intense and competitive quest to manufacture bioartificial heart muscle in the last decade. Numerous biocompatible scaffolds and scaffold-free systems, enriched with various cell types, have been used to fabricate 3D grafts for myocardial repair. In spite of the impressive achievements in the myocardial tissue-engineering field, many issues remain to be addressed before clinical application of this strategy becomes feasible. This is largely due to the uniqueness of the heart's structure and function. This review provides a survey upon the reported strategies, and indicates caveats and perspectives in the field of myocardial tissue engineering.

During emergency repair of acute Stanford type A aortic dissections, surgical compromises in the form of incomplete arch replacement are made due to the unstable condition of the patient and safety issues of the performing team. We report a case of delayed reoperation after previous incomplete surgery for acute type A aortic dissection in a young patient with Marfan's syndrome. He presented again with repetitive chest pain five years after initial surgical treatment. Extensive aneurysmal dilatation of the aorta and remaining dissection led to the decision to replace the ascending aorta and the aortic arch. After a good progress during the first days after surgery, the patient died due to a ruptured thoraco-abdominal aneurysm on the fifth postoperative day. Extensive surgical reconstruction including aortic arch replacement should be considered in patients with Marfan's syndrome who present with aortic dissections type A to avoid unnecessary reoperations and their complications.

Background— Implantation of bioartificial patches distorts myocardial geometry, and functional improvement of the recipient heart is usually attributed to reactive angiogenesis around the graft. With the liquid bioartificial tissue compound used in this study, we achieved targeted large-scale support of the infarcted left ventricular wall and improvement of heart function. Methods and Results— A liquid compound consisting of growth factor-free Matrigel and 10 6 green fluorescent protein (GFP)-positive mouse (129sv) embryonic stem cells (ESCs) was generated and injected into the area of ischemia after ligation of the left anterior descending artery in BALB/c mice (group I). Left anterior descending artery-ligated mice (group II) and mice with Matrigel (group III) or ESC treatment alone (group IV) were used as the control groups (n=5 in all groups). The hearts were harvested for histology 2 weeks later after echocardiographic assessment with a 15-MHz probe. The liquid injectable tissue solidified at body temperature and retained the geometry of the infarcted lateral wall. Immunofluorescence stains revealed voluminous GFP grafts. The quality of restoration (graft/infarct area ratio) was 45.5±10.8% in group I and 29.1±6.7% in group IV ( P =0.034). ESCs expressed connexin 43 at intercellular contact sites. The mice treated with the compound had a superior heart function compared with the controls ( P Conclusions— Injectable bioartificial tissue restores the heart’s geometry and function in a targeted and nondistorting fashion. This new method paves the way for novel interventional approaches to myocardial repair, using both stem cells and matrices.

Background— We previously reported that administration of elastase inhibitors reverses fatal pulmonary arterial hypertension (PAH) in rats by inducing smooth muscle cell (SMC) apoptosis. We showed in pulmonary artery (PA) organ culture that the mechanism by which elastase inhibitors induce SMC apoptosis involves repression of matrix metalloproteinase (MMP) activity and subsequent signaling through α v β 3 -integrins and epidermal growth factor receptors (EGFRs). This suggests that blockade of these downstream effectors may also induce regression of PAH. Methods and Results— In this study, we first showed in PA organ culture that MMP inhibition or α v β 3 -integrin blockade with agents in clinical and preclinical use (SC-080 and cilengitide, respectively) mediates SMC apoptosis and regression of medial hypertrophy. We also documented similar results with an EGFR tyrosine kinase inhibitor. We then induced PAH in rats by injection of monocrotaline and, at day 21, began a 2-week treatment with SC-080, cilengitide, or the EGFR inhibitor PKI166. No vehicle- or cilengitide-treated animal survived beyond 2 weeks. Administration of SC-080 resulted in 44% survival at 2 weeks, and PKI166 therapy resulted in 78% and 54% survival in daily or 3-times-weekly treated animals, respectively. Four weeks after cessation of PKI166, we documented survivals of 50% and 23% in the 2 treatment groups, associated with reductions in pulmonary pressure, right ventricular hypertrophy, and abnormally muscularized distal arteries. Conclusion— We propose that selective blockade of EGFR signaling may be a novel strategy to reverse progressive, fatal PAH.