Gabriel Peinkofer

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.

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 ReninAngiotensin-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.

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.

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.

Cell persistence and electrical integration of transplanted fetal cardiomyocytes from different developmental stages

Cell replacement therapy is a promising approach to overcome thelimited regenerative potential of damaged myocardium. Persistenceand functional integration of transplanted cardiomyocytes are crucialfor a safe and efficient cell replacement therapy. The mechanismspromoting cell persistence and integration are poorly understood. Thedevelopmental stage of transplanted cells appears to be an importantfactor, since adult cardiomyocytes do not survive transplantation [1],while immature cardiomyocytes do survive and integrate functionally[1,2]. During fetal development, cardiomyocytes undergo substantialstructural and electrophysiological alterations [3–5], but it has notbeen studied yet, whether different stages of fetal developmentinfluence persistence and functional integration of transplantedcardiomyocytes, and if there is a distinct immature developmentalstagethatpromotesanoptimalpersistenceandfunctionoftransplantedcardiomyocytes.Therefore,weinvestigatedcellpersistenceandelectro-physiological integration of transplanted murine fetal cardiomyocytes(FCM) from different developmental stages.FetalventriclesfromtransgenicCrl:OF1miceexpressingeGFPundercontrol of the alpha-actin promoter were harvested at days 9.5(earlyFCM),14.5(interFCM)and18.5(lateFCM)p.c.Injectionsofdisso-ciated FCM were performed at two different sites of the left ventricularwall (2 × 450.000 cells) in adult Crl:OF1 mice. Six days after surgery,viable slices of recipient hearts (150 μm thickness) were producedwith a microtome (Leica, Germany). Intracellular action potentialswererecordedwithglassmicroelectrodes(WPI,USA).Sliceswerestim-ulated focally in host tissue. Cell preparation, surgery and microelec-trode recordings were performed as described before [2].Cellpersistence was quantified using a score ranging from “0” to “3”(0 = no graft cells visible; 1 = few and small clusters of transplantedcells (Fig. 1A + C); 2 = larger clusters of transplanted cells; 3 = largeareas of transplanted cells (Fig. 1B)). Quantitative analysis of cell persis-tence by quantitative TaqMan real-time PCR (qPCR) was performed sim-ilarlyasdescribedbefore [6,7].Sliceswerelysated,andgenomicDNAwasprepared, which was then used for qP CR with primers against the genesfor eGFP (grafted cells) and for beta-actin (total cell count). Data weretested for statistical significance by one-way ANOVA with post test or, ifnormality test failed, with Kruskal–Wallis test. Data are presentedas mean ± SEM. All experiments were approved by the local animalwelfare committee, and all animals received humane care.Persistence score was 2.91 ± 0.09 (n = 11) in interFCM, whichwas significantly higher compared to lateFCM (1.04 ± 0.12, n = 23,p b 0.001)and earlyFCM(0.40 ± 0.11,n = 20, p b 0.001vs.interFCM,p b 0.05 vs. lateFCM, Fig. 1D). The qPCR analysis confirmed thesefindings with 15,189 ± 4791 transgenic cells per mg detected inrecipient heart slices after injection of interFCM (n = 11), 3972 ±1730 cells/mg for lateFCM (n = 20, p b 0.05 vs. interFCM) and2290 ± 1851 cells/mg for earlyFCM (n = 17, p b 0.01 vs. interFCM,p = n.s. vs. lateFCM; Fig. 1E). Persistence of earlyFCM was insufficientfor electrophysiological analyses.The maximal stimulation frequency without conduction blocks,which was used to indicate the quality of integration, was8.62 ± 0.42 Hz in interFCM (n = 13) and 4.60 ± 0.67 Hz in lateFCM(n = 10, p b 0.001, Fig. 2A).Host cellsfollowed higherstimulation fre-quencies(11.00 ± 0.30 Hz;n = 21).Toquantifythevelocityofexcita-tion spread, we analyzed the delay between stimulus and actionpotential upstroke in host and graft cardiomyocytes. This delay waslower in interFCM (15.14 ± 1.03 ms, n = 18) than in lateFCM(28.49 ± 1.98 ms,n = 16,p b 0.001, Fig.2B + D),butwassignificant-ly higher in all transplanted FCM than in host cardiomyocytes(7.71 ± 0.21 ms, n = 28, pb 0.001 vs. interFCM and lateFCM). Thedistance between stimulation and recording electrode was equal inthese measurements (stimulation electrode to host cardiomyocytes1.51 ± 0.09 mm, to transplanted interFCM 1.55 ± 0.02 mm, totransplanted lateFCM 1.29 ± 0.16 mm; p = n.s.). Action potential

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.

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 Tyrodes 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.