Assessment of mitophagy in human iPSC-derived cardiomyocytes.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.Abbreviations: AAVS1: adeno-associated virus integration site 1; AKT/protein kinase B: AKT serine/threonine kinase; CAG promoter: cytomegalovirus early enhancer, chicken ACTB/ß-actin promoter; CIS: cisplatin; CRISPR: clustered regularly interspaced short palindromic repeats; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; hiPSC: human induced pluripotent stem cell; hiPSC-CMs: human induced pluripotent stem cell-derived cardiomyocytes; ISO: isoproterenol; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RT: room temperature; SB: SBI-0206965; ULK1: unc-51 like autophagy activating kinase 1.

Effect of neutrophil-to-lymphocyte ratio on short-term prognosis of elderly patients with hip fracture.

OBJECTIVE: To investigate the effect of neutrophil-to-lymphocyte ratio (NLR) on short-term prognosis in elderly patients with hip fracture. METHODS: Altogether, 124 elderly patients with hip fractures who underwent surgery in our hospital were retrospectively studied, and they were divided into survival group (n=98) and death group (n=26) according to their 1-year survival. General data of both groups were collected and compared, and indicators with statistical differences in univariate analysis were further examined by logistic regression analysis. Venous blood samples were drawn from all patients 1 day after the surgery to detect and compare NLR, serum procalcitonin (PCT) and C-reactive protein (CRP) levels between both groups. ROC curve was used to analyze the clinical value of NLR in predicting the prognosis of patients. NLR cutoff value obtained by the ROC curve analysis was adopted to divide the patients into high and low ratio groups, and Kaplan-Meier (K-M) curves were used to assess the survival rate of patients in both groups. RESULTS: There were significant differences in age, gender, marital status, medical history and American Society of Anesthesiologists (ASA) grades between both groups. Logistic regression analysis showed that advanced age (≥85 years), male gender, and higher ASA grades (III-IV) were risk factors for short-term poor prognosis in elderly patients with hip fracture. Compared with survival group, NLR, PCT and CRP levels were higher in death group. ROC curve showed that the AUC of NLR predicting patients' prognosis was 0.804 at a cutoff value of 6.939%. K-M curves showed that the overall survival was lower in high-ratio group than in low-ratio group. CONCLUSION: Advanced age (overall survival was lower in high-ratio group than in low-ratio group), male gender, and higher ASA grades (III-IV) were risk factors for short-term poor prognosis in elderly patients with hip rifracture. NLR has some clinical value in predicting and evaluating the prognosis of patients.

Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenge in heart regeneration. Efforts have focused on improving the efficiency by understanding fundamental mechanisms. One major challenge is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in primary cell cultures. Both forskolin, which increases cAMP levels, and TGFß inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c, and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblasts could improve their plasticity for reprogramming and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold more iCMs than untreated control cells. In conclusion, suppression of CREB-CBP signaling improves fibroblast plasticity for direct cardiac reprogramming.

Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.

Over the last decade, great achievements have been made in the field of direct epigenetic reprogramming, which converts one type of adult somatic cells into another type of differentiated cells, such as direct reprogramming of fibroblasts into cardiomyocytes, without passage through an undifferentiated pluripotent stage. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate cardiac fibrotic remodeling in a diseased heart. Furthermore, in vitro reprogramming of fibroblasts into cardiomyocyte-like cells provides new avenues to conduct basic mechanistic studies, to test drugs, and to model cardiac diseases in a dish. Here, we describe a detailed step-by-step protocol for in vitro production of induced cardiomyocyte-like cells (iCMs) from fibroblasts. The related procedures include high-quality fibroblast isolation of different origins (neonatal cardiac, tail-tip, and adult cardiac fibroblasts), retroviral preparation of reprogramming factors, and iCM generation by fibroblast reprogramming via retroviral transduction of Gata4, Mef2c, and Tbx5. A detailed written protocol will help many other laboratories, inexperienced in this area, to use and further improve this technology in their studies of cardiac regenerative medicine.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

BACKGROUND: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. METHODS: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. RESULTS: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1ß (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1ß or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. CONCLUSIONS: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

A Singular Role of IK1 Promoting the Development of Cardiac Automaticity during Cardiomyocyte Differentiation by IK1 -Induced Activation of Pacemaker Current.

The inward rectifier potassium current (IK1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that IK1 could promote the spontaneously-firing automaticity induced by upregulation of pacemaker funny current (If) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of IK1 to activate If and thereby promote automaticity has not been explored. In this study, we combined mathematical and experimental assays and found that only IK1 and If, at a proper-ratio of densities, were sufficient to generate rhythmic MP-oscillations even in unexcitable cells (i.e. HEK293T cells and undifferentiated mouse embryonic stem cells [ESCs]). We termed this effect IK1-induced If activation. Consistent with previous findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESC-differentiated CMs could spontaneously fire action potentials (APs). We found that spontaneously-firing ESC-CMs displayed more hyperpolarized maximum diastolic potential and more outward IK1 current than quiescent-yet-excitable m/hESC-CMs. Rather than classical depolarization pacing, quiescent mESC-CMs were able to fire APs spontaneously with an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was also promoted in quiescent hESC-CMs by an IK1-specific agonist zacopride. In addition, we found that the number of spontaneously-firing m/hESC-CMs was significantly decreased when If was acutely upregulated by Ad-CGI-HCN infection. Our study reveals a novel role of IK1 promoting the development of cardiac automaticity in m/hESC-CMs through a mechanism of IK1-induced If activation and demonstrates a synergistic interaction between IK1 and If that regulates cardiac automaticity.

Expandable Cardiovascular Progenitor Cells Reprogrammed from Fibroblasts.

Stem cell-based approaches to cardiac regeneration are increasingly viable strategies for treating heart failure. Generating abundant and functional autologous cells for transplantation in such a setting, however, remains a significant challenge. Here, we isolated a cell population with extensive proliferation capacity and restricted cardiovascular differentiation potentials during cardiac transdifferentiation of mouse fibroblasts. These induced expandable cardiovascular progenitor cells (ieCPCs) proliferated extensively for more than 18 passages in chemically defined conditions, with 10(5) starting fibroblasts robustly producing 10(16) ieCPCs. ieCPCs expressed cardiac signature genes and readily differentiated into functional cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) in vitro, even after long-term expansion. When transplanted into mouse hearts following myocardial infarction, ieCPCs spontaneously differentiated into CMs, ECs, and SMCs and improved cardiac function for up to 12 weeks after transplantation. Thus, ieCPCs are a powerful system to study cardiovascular specification and provide strategies for regenerative medicine in the heart.

Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.

Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK119ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Electrophysiological properties of human induced pluripotent stem cells.

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC(50) = 3.3 +/- 2.7 mM) delayed rectifier K(+) currents (I(KDR)) in 105 of 110 single iPSCs (15.4 +/- 0.9 pF). I(KDR) in iPSCs displayed a current density of 7.6 +/- 3.8 pA/pF at +40 mV. The voltage for 50% activation (V(1/2)) was -7.9 +/- 2.0 mV, slope factor k = 9.1 +/- 1.5. However, Ca(2+)-activated K(+) current (I(KCa)), hyperpolarization-activated pacemaker current (I(f)), and voltage-gated sodium channel (Na(V)) and voltage-gated calcium channel (Ca(V)) currents could not be measured. TEA inhibited iPSC proliferation (EC(50) = 7.8 +/- 1.2 mM) and viability (EC(50) = 5.5 +/- 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC(50) = 4.5 +/- 0.5 mM) but had less effect on proliferation (EC(50) = 0.9 +/- 0.5 mM). Cell cycle analysis further revealed that K(+) channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of I(KCa) in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.

Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression.

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca(2+) handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca(2+) transient amplitudes, slower kinetics, and reduced Ca(2+) content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca(2+) handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca(2+)-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQDelta-IRES-GFP (Ad-CSQDelta, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca(2+) content but did not alter L-type Ca(2+) current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQDelta-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca(2+) transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca(2+) handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes.

Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms.

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca(2+) concentrations ([Ca(2+)](i)). Because the sarcoplasmic reticulum (SR) and Na(+)/Ca(2+) exchanger (NCX) play crucial roles in regulating [Ca(2+)](i) and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca(2+) overload by maintaining Ca(2+) homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca(2+) transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca(2+) transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca(2+)](i) levels and attenuated the depression of the Ca(2+) transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca(2+) release channels and/or ryanodine receptors (RyRs) and SR Ca(2+) pump ATPase (SERCA2) and SR Ca(2+) release and uptake. In addition, a delayed decay rate time constant of Ca(2+) transients and cell shortening of Ca(2+) transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca(2+) homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R.

Protective effect of estrogen on intestinal ischemia-reperfusion injury in pubertal rats.

PURPOSE: This study investigated the role of 17beta-estradiol (E(2)) in intestinal ischemia-reperfusion (I/R) injury and its possible mechanism. METHODS: Rats of pubertal age were ovariectomized and injected subcutaneously with vehicle (vehicle group) or E(2) (100 or 500 mug/kg/every other day, E(2) or 5E(2) group) for 4 weeks. Other rats of the same age underwent sham ovariectomy as a control group. The rats in each group (n = 15) were subjected to superior mesenteric artery occlusion followed by 1 hour (n = 5), 6 hours (n = 5), or 24 hours (n = 5) reperfusion. Intestine specimens then were obtained for the determination of histopathologic score, inducible nitric oxide synthase (iNOS) mRNA expression, and iNOS activity. RESULTS: In vehicle, control, E(2), and 5E(2) groups, the histopathologic scores were 3.31 +/- 0.12, 3.00 +/- 0.09, 2.57 +/- 0.12, and 2.98 +/- 0.08, respectively. The expression levels of iNOS mRNA were 3.85 +/- 0.42, 4.86 +/- 0.76, 5.17 +/- 0.34, and 4.25 +/- 0.41 log copies, respectively. Lower histopathologic score but higher iNOS mRNA expression were found in E(2) group than in the other groups (P < .01). The level of iNOS activity paralleled the expression of iNOS mRNA. CONCLUSIONS: Estrogen may exert a protective effect on intestinal I/R injury in pubertal rats, probably by enhancing iNOS expression.