Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling.

RATIONALE: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. OBJECTIVE: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. METHODS AND RESULTS: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. CONCLUSIONS: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes.

Electrophysiological maturation and integration of transplanted cardiomyocytes are essential to enhance safety and efficiency of cell replacement therapy. Yet, little is known about these important processes. The aim of our study was to perform a detailed analysis of electrophysiological maturation and integration of transplanted cardiomyocytes. Fetal cardiomyocytes expressing enhanced green fluorescent protein were transplanted into cryoinjured mouse hearts. At 6, 9 and 12 days after transplantation, viable slices of recipient hearts were prepared and action potentials of transplanted and host cardiomyocytes within the slices were recorded by microelectrodes. In transplanted cells embedded in healthy host myocardium, action potential duration at 50% repolarization (APD50) decreased from 32.2 ± 3.3 ms at day 6 to 27.9 ± 2.6 ms at day 9 and 19.6 ± 1.6 ms at day 12. The latter value matched the APD50 of host cells (20.5 ± 3.2 ms, P=0.78). Integration improved in the course of time: 26% of cells at day 6 and 53% at day 12 revealed no conduction blocks up to a stimulation frequency of 10 Hz. APD50 was inversely correlated to the quality of electrical integration. In transplanted cells embedded into the cryoinjury, which showed no electrical integration, APD50 was 49.2 ± 4.3 ms at day 12. Fetal cardiomyocytes transplanted into healthy myocardium integrate electrically and mature after transplantation, their action potential properties after 12 days are comparable to those of host cardiomyocytes. Quality of electrical integration improves over time, but conduction blocks still occur at day 12 after transplantation. The pace of maturation correlates with the quality of electrical integration. Transplanted cells embedded in cryoinjured tissue still possess immature electrophysiological properties after 12 days.

Spironolactone prevents aldosterone induced increased duration of atrial fibrillation in rat.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice. The Renin-Angiotensin-Aldosterone-System plays a major role for the atrial structural and electrical remodelling. Recently elevated aldosterone levels have been suggested to increase the risk for the development of AF. METHODS: Rats were treated with aldosterone by means of an osmotic minipump (0.5µg/h) over a period of 4 weeks. AF was induced by transesophageal burst pacing. Action potentials (AP) were recorded from left atrial preparations with microelectrodes. Atrial collagen was quantified by histological studies. RESULTS: Aldosterone treatment resulted in hypertrophy as indicated by an increased ratio of heart weight/tibia length and doubled the time until the AF converted spontaneously into sinus rhythm (85.8±13.4 s vs. 38.3±6.9 s, p<0.01). This was associated with a significant shortening of the AP (APD90 26.2±1.1 vs. 31.2±1.9, p<0.05) and an increased protein expression of Kir2.1 and Kv1.5. Atrial collagen deposition was significantly greater in aldosterone-treated rats. The alterations could be prevented by additional application spironolactone. CONCLUSIONS: The results of the present study suggest that in addition to the structural remodelling aldosterone also promotes AF by altering repolarising potassium currents leading to action potential shortening.

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels.

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell-cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testingin vitro(organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapiesin vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells inpoly-N-isopropylacrylamid(PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages.

BACKGROUND: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. METHODS: To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6-7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. RESULTS: The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. CONCLUSION: The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations.

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.

Nitro-fatty acids suppress ischemic ventricular arrhythmias by preserving calcium homeostasis.

Nitro-fatty acids are electrophilic anti-inflammatory mediators which are generated during myocardial ischemic injury. Whether these species exert anti-arrhythmic effects in the acute phase of myocardial ischemia has not been investigated so far. Herein, we demonstrate that pretreatment of mice with 9- and 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA) significantly reduced the susceptibility to develop acute ventricular tachycardia (VT). Accordingly, epicardial mapping revealed a markedly enhanced homogeneity in ventricular conduction. NO2-OA treatment of isolated cardiomyocytes lowered the number of spontaneous contractions upon adrenergic isoproterenol stimulation and nearly abolished ryanodine receptor type 2 (RyR2)-dependent sarcoplasmic Ca2+ leak. NO2-OA also significantly reduced RyR2-phosphorylation by inhibition of increased CaMKII activity. Thus, NO2-OA might be a novel pharmacological option for the prevention of VT development.

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 µm are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode's solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 °C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Intramyocardially Transplanted Neonatal Cardiomyocytes (NCMs) Show Structural and Electrophysiological Maturation and Integration and Dose-Dependently Stabilize Function of Infarcted Rat Hearts.

Cardiac cell replacement therapy is a promising therapy to improve cardiac function in heart failure. Persistence, structural and functional maturation, and integration of transplanted cardiomyocytes into recipients' hearts are crucial for a safe and efficient replacement of lost cells. We studied histology, electrophysiology, and quantity of intramyocardially transplanted rat neonatal cardiomyocytes (NCMs) and performed a detailed functional study with repeated invasive (pressure-volume catheter) and noninvasive (echocardiography) analyses of infarcted female rat hearts including pharmacological stress before and 3 weeks after intramyocardial injection of 5 × 106 (low NCM) or 25 × 106 (high NCM) syngeneic male NCMs or medium as placebo (Ctrl). Quantitative real-time polymerase chain reaction (PCR) for Y-chromosome confirmed a fivefold higher persisting male cell number in high NCM versus low NCM after 3 weeks. Sharp electrode measurements within viable slices of recipient hearts demonstrated that transplanted NCMs integrate into host myocardium and mature to an almost adult phenotype, which might be facilitated through gap junctions between host myocardium and transplanted NCMs as indicated by connexin43 in histology. Ejection fraction of recipient hearts was severely impaired after ligation of left anterior descending (LAD; pressure-volume catheter: 39.2 ± 3.6%, echocardiography: 39.9 ± 1.4%). Repeated analyses revealed a significant further decline within 3 weeks in Ctrl and a dose-dependent stabilization in cell-treated groups. Consistently, stabilized cardiac function/morphology in cell-treated groups was seen in stroke volume, cardiac output, ventricle length, and wall thickness. Our findings confirm that cardiac cell replacement is a promising therapy for ischemic heart disease since immature cardiomyocytes persist, integrate, and mature after intramyocardial transplantation, and they dose-dependently stabilize cardiac function after myocardial infarction.

From Early Embryonic to Adult Stage: Comparative Study of Action Potentials of Native and Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPS-CMs) are promising candidates for cell therapy, drug screening, and developmental studies. It is known that iPS-CMs possess immature electrophysiological properties, but an exact characterization of their developmental stage and subtype differentiation is hampered by a lack of knowledge of electrophysiological properties of native CMs from different developmental stages and origins within the heart. Thus, we sought to systematically investigate action potential (AP) properties of native murine CMs and to establish a database that allows classification of stem cell-derived CMs. Hearts from 129S2PasCrl mice were harvested at days 9-10, 12-14, and 16-18 postcoitum, as well as 1 day, 3-4 days, 1-2 weeks, 3-4 weeks, and 6 weeks postpartum. AP recordings in left and right atria and at apical, medial, and basal left and right ventricles were performed with sharp glass microelectrodes. Measurements revealed significant changes in AP morphology during pre- and postnatal murine development and significant differences between atria and ventricles, enabling a classification of developmental stage and subtype differentiation of stem cell-derived CMs based on their AP properties. For iPS-CMs derived from cell line TiB7.4, a typical ventricular phenotype was demonstrated at later developmental stages, while there were electrophysiological differences from atrial as well as ventricular native CMs at earlier stages. This finding supports that iPS-CMs can develop AP properties similar to native CMs, but points to differences in the maturation process between iPS-CMs and native CMs, which may be explained by dissimilar conditions during in vitro differentiation and in vivo development.

Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro.

The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.

The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.

Expansion and differentiation of germline-derived pluripotent stem cells on biomaterials.

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells.

AIMS: Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS: Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION: Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.