Sang-Ging Ong

Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorff-perfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 μM). Leptin reduced infarct size significantly (control, 60.05 ± 7.41% vs. leptin treated, 29.9 ± 3.24%, P < 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% (P < 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P < 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening (P < 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required.

Effect of human donor cell source on differentiation and function of cardiac induced pluripotent stem cells.

BACKGROUND Human-induced pluripotent stem cells (iPSCs) are a potentially unlimited source for generation of cardiomyocytes (iPSC-CMs). However, current protocols for iPSC-CM derivation face several challenges, including variability in somatic cell sources and inconsistencies in cardiac differentiation efficiency. OBJECTIVES This study aimed to assess the effect of epigenetic memory on differentiation and function of iPSC-CMs generated from somatic cell sources of cardiac versus noncardiac origins. METHODS Cardiac progenitor cells (CPCs) and skin fibroblasts from the same donors were reprogrammed into iPSCs and differentiated into iPSC-CMs via embryoid body and monolayer-based differentiation protocols. RESULTS Differentiation efficiency was found to be higher in CPC-derived iPSC-CMs (CPC-iPSC-CMs) than in fibroblast-derived iPSC-CMs (Fib-iPSC-CMs). Gene expression analysis during cardiac differentiation demonstrated up-regulation of cardiac transcription factors in CPC-iPSC-CMs, including NKX2-5, MESP1, ISL1, HAND2, MYOCD, MEF2C, and GATA4. Epigenetic assessment revealed higher methylation in the promoter region of NKX2-5 in Fib-iPSC-CMs compared with CPC-iPSC-CMs. Epigenetic differences were found to dissipate with increased cell passaging, and a battery of in vitro assays revealed no significant differences in their morphological and electrophysiological properties at early passage. Finally, cell delivery into a small animal myocardial infarction model indicated that CPC-iPSC-CMs and Fib-iPSC-CMs possess comparable therapeutic capabilities in improving functional recovery in vivo. CONCLUSIONS This is the first study to compare differentiation of iPSC-CMs from human CPCs versus human fibroblasts from the same donors. The authors demonstrate that although epigenetic memory improves differentiation efficiency of cardiac versus noncardiac somatic cell sources in vitro, it does not contribute to improved functional outcome in vivo.

Development of poly(β-amino ester)-based biodegradable nanoparticles for nonviral delivery of minicircle DNA.

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

Mitochondrial-Shaping Proteins in Cardiac Health and Disease - the Long and the Short of It!

Mitochondrial health is critically dependent on the ability of mitochondria to undergo changes in mitochondrial morphology, a process which is regulated by mitochondrial shaping proteins. Mitochondria undergo fission to generate fragmented discrete organelles, a process which is mediated by the mitochondrial fission proteins (Drp1, hFIS1, Mff and MiD49/51), and is required for cell division, and to remove damaged mitochondria by mitophagy. Mitochondria undergo fusion to form elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins (Mfn1, Mfn2 and OPA1), and which enables the replenishment of damaged mitochondrial DNA. In the adult heart, mitochondria are relatively static, are constrained in their movement, and are characteristically arranged into 3 distinct subpopulations based on their locality and function (subsarcolemmal, myofibrillar, and perinuclear). Although the mitochondria are arranged differently, emerging data supports a role for the mitochondrial shaping proteins in cardiac health and disease. Interestingly, in the adult heart, it appears that the pleiotropic effects of the mitochondrial fusion proteins, Mfn2 (endoplasmic reticulum-tethering, mitophagy) and OPA1 (cristae remodeling, regulation of apoptosis, and energy production) may play more important roles than their pro-fusion effects. In this review article, we provide an overview of the mitochondrial fusion and fission proteins in the adult heart, and highlight their roles as novel therapeutic targets for treating cardiac disease.

Parkinson's disease proteins: Novel mitochondrial targets for cardioprotection.

Ischemic heart disease (IHD) is the leading cause of death and disability worldwide. Therefore, novel therapeutic targets for protecting the heart against acute ischemia/reperfusion injury (IRI) are required to attenuate cardiomyocyte death, preserve myocardial function, and prevent the onset of heart failure. In this regard, a specific group of mitochondrial proteins, which have been linked to familial forms of Parkinsons disease (PD), may provide novel therapeutic targets for cardioprotection. In dopaminergic neurons of the substantia nigra, these PD proteins, which include Parkin, PINK1, DJ-1, LRRK2, and α-synuclein, play essential roles in preventing cell death—through maintaining normal mitochondrial function, protecting against oxidative stress, mediating mitophagy, and preventing apoptosis. These rare familial forms of PD may therefore provide important insights into the pathophysiology underlying mitochondrial dysfunction and the development of PD. Interestingly, these PD proteins are also present in the heart, but their role in myocardial health and disease is not clear. In this article, we review the role of these PD proteins in the heart and explore their potential as novel mitochondrial targets for cardioprotection.

Exosomes as Potential Alternatives to Stem Cell Therapy in Mediating Cardiac Regeneration

Cardiovascular diseases represent a significant cause of death and disability worldwide.1 The decline of cardiovascular mortality as a result of modern medicine and surgery has in turn led to a rapid increase of patients having heart failure, with the only definite cure being heart transplantation. However, many patients are unable to undergo transplantation surgery because of complications from existing comorbidities, and among suitable patients, the procedure is plagued by limited donor supply, high costs, and the need for chronic immunosuppressant therapy. Hence, recent advances in cardiac regenerative therapy have emerged as an attractive alternative. Article, see p 52 Currently, there are several methods to achieve cardiac regeneration. Endogenous cardiac repair that involves generation of new cardiomyocytes from differentiation of cardiac progenitor cells (CPCs) or renewal of pre-existing adult cardiomyocytes is one such approach, albeit a rare and inefficient process to cope with the loss of cardiomyocytes after myocardial infarction or other cardiac diseases.2 Alternatively, functional myocardium may be salvaged or replenished through transplantation of exogenous stem cells.3,4 However, the poor long-term engraftment and survival in current transplantation have largely precluded substantial cell replacement, and instead supports the paracrine hypothesis that the release of external factors contributes to myocardial salvage or repair. Historically, the paracrine hypothesis is thought to be mediated primarily by chemical and physical signals, such as soluble proteins, gene products, lipids, and gases. Indeed, various studies have demonstrated that stem cells produce and secrete a broad range of cytokines, chemokines, and growth factors that are involved in cardiac repair.5 Strong support of a paracrine hypothesis came from experimental studies in which the administration of conditioned medium from stem cells was able to confer beneficial effects without the physical presence of stem cells within the infarcted heart.5,6 There is a …

Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy

Significance We have found that long telomeres protect mice from genetic cardiac diseases analogous to those found in humans, such as Duchenne muscular dystrophy (DMD). Mice lacking dystrophin, similar to patients with DMD, exhibit only mild disease. In contrast, mice that lack dystrophin and have “humanized” telomere lengths (mdx4cv/mTRG2) fully manifest both the severe human skeletal muscle wasting and cardiac failure typical of DMD. Remarkably, telomere shortening accompanies cardiac development even after cardiomyocyte division has ceased. This chronic proliferation-independent shortening in dystrophin-deficient cardiomyocytes is associated with induction of a DNA damage response, mitochondrial dysfunction, increased oxidative stress, and metabolic failure. Our findings highlight an interplay between telomere length and mitochondrial homeostasis in the etiology of dystrophic heart failure. Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx4cv/mTRG2 mouse model with “humanized” telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.

Non-coding RNAs as therapeutic targets for preventing myocardial ischemia-reperfusion injury

ABSTRACT Introduction: New treatments are required to improve clinical outcomes in patients with acute myocardial infarction (AMI), for reduction of myocardial infarct (MI) size and preventing heart failure. Following AMI, acute ischemia/reperfusion injury (IRI) ensues, resulting in cardiomyocyte death and impaired cardiac function. Emerging studies have implicated a fundamental role for non-coding RNAs (microRNAs [miRNA], and more recently long non-coding RNAs [lncRNA]) in the setting of acute myocardial IRI. Areas covered: In this article, we discuss the roles of miRNAs and lncRNAs as potential biomarkers and therapeutic targets for the detection and treatment of AMI, review their roles as mediators and effectors of cardioprotection, particularly in the settings of interventions such as ischemic pre- and post-conditioning (IPC & IPost) as well as remote ischemic conditioning (RIC), and highlight future strategies for targeting ncRNAs to reduce MI size and prevent heart failure following AMI. Expert opinion: Investigating the roles of miRNAs and lncRNAs in the setting of AMI has provided new insights into the pathophysiology underlying acute myocardial IRI, and has identified novel biomarkers and therapeutic targets for detecting and treating AMI. Pharmacological and genetic manipulation of these ncRNAs has the therapeutic potential to improve clinical outcomes in AMI patients.

Cortical Bone–Derived Stem Cells: A Novel Class of Cells for Myocardial Protection

Acute myocardial infarction (MI) remains a significant cause of mortality and morbidity worldwide, despite steady advances in our understanding and treatment of coronary heart disease.1 After MI, lost myocardium is replaced with scar tissue, leading to left ventricular (LV) remodeling and ultimately culminating with ischemic cardiomyopathy and congestive heart failure. We now know that the adult heart is not a postmitotic organ lacking capacity for self-renewal after injury. This paradigm shift occurred after identification of stem cell niches in the adult heart and isolation of cardiac progenitor cells, including c-kit+ cardiac stem cells (CSCs), side population cells, and cardiospheres.2–7 However, the endogenous proliferation of cardiac progenitor cells after MI is inadequate for full replacement of the large number of depleted cells.8 To overcome this problem, extensive efforts have been made in developing cell-based therapies to promote tissue repair by introduction of exogenous cells, such as bone marrow–derived mononuclear cells (BM-MNCs), bone marrow–derived mesenchymal stem cells (BM-MSCs), adipose tissue–derived mesenchymal stem cells, CD34+ stem cells, c-kit+ CSCs, and cardiosphere-derived cells, as evidenced by recent clinical trials.9–16 Article, see p 539 For now, the BM-MNCs are the most widely used source of cells in cardiovascular regenerative therapy trials. However, despite some successful early-stage clinical trials, several recent studies involving the use of BM-MNCs have not been overwhelmingly positive. For example, in the placebo-controlled Transplantation in Myocardial Infarction Evaluation (TIME) trial in which investigators assessed whether differential timing of BM-MNCs delivery affected LV ejection fraction (LVEF) after acute MI (3–7 days), no significant differences between the BM-MNCs and placebo groups were observed for either primary or secondary end points.17 In fact, the TIME trial was the third study in a series of studies (Late-TIME,18 Effectiveness of Stem Cell Treatment for …

Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia‐Inducible Factor 1‐α (HIF1‐α) in an Ovine Model of Acute Myocardial Infarction

Background Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia‐inducible factor 1‐α (HIF1‐α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen‐resistant HIF1‐α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. Methods and Results Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1‐α (BMMSC‐HIF) were injected in the peri‐infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2‐fold (P<0.001) in the presence of markedly decreased end‐systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo‐treated animals (n=6), neither parameters changed over time. HIF1‐α‐transfected BMMSCs (BMMSC‐HIF) induced angio‐/arteriogenesis and decreased apoptosis by HIF1‐mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin‐1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long‐term retention of BMMSC‐HIF. Conclusions Intramyocardial delivery of BMMSC‐HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1‐mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.