Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing.

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (-20 pA/pF at -140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function.

The epicardium as modulator of the cardiac autonomic response during early development.

The cardiac autonomic nervous system (cANS) modulates heart rate, contraction force and conduction velocity. The embryonic chicken heart already responds to epinephrine prior to establishment of the cANS. The aim of this study was to define the regions of the heart that might participate in modulating the early autonomic response to epinephrine. Immunofluorescence analysis reveals expression of neural markers tubulin beta-3 chain and neural cell adhesion molecule in the epicardium during early development. In addition, expression of the ß2 adrenergic receptor, the receptor for epinephrine, was found in the epicardium. Ex-ovo micro-electrode recordings in hearts with inhibition of epicardial outgrowth showed a significantly reduced response of the heart rate to epinephrine compared to control hearts. This study suggests a role for the epicardium as autonomic modulator during early cardiac development.

Declaration of common standards for the preregistration of animal research-speeding up the scientific progress.

Preregistration of studies is a recognized tool in clinical research to improve the quality and reporting of all gained results. In preclinical research, preregistration could boost the translation of published results into clinical breakthroughs. When studies rely on animal testing or form the basis of clinical trials, maximizing the validity and reliability of research outcomes becomes in addition an ethical obligation. Nevertheless, the implementation of preregistration in animal research is still slow. However, research institutions, funders, and publishers start valuing preregistration, and thereby level the way for its broader acceptance in the future. A total of 3 public registries, the OSF registry, preclinicaltrials.eu, and animalstudyregistry.org already encourage the preregistration of research involving animals. Here, they jointly declare common standards to make preregistration a valuable tool for better science. Registries should meet the following criteria: public accessibility, transparency in their financial sources, tracking of changes, and warranty and sustainability of data. Furthermore, registration templates should cover a minimum set of mandatory information and studies have to be uniquely identifiable. Finally, preregistered studies should be linked to any published outcome. To ensure that preregistration becomes a powerful instrument, publishers, funders, and institutions should refer to registries that fulfill these minimum standards.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a "funny current" and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Human Cardiomyocyte Progenitor Cells in Co-culture with Rat Cardiomyocytes Form a Pro-arrhythmic Substrate: Evidence for Two Different Arrhythmogenic Mechanisms.

Background: Cardiomyocyte progenitor cells (CMPCs) are a promising cell source for regenerative cell therapy to improve cardiac function after myocardial infarction. However, it is unknown whether undifferentiated CMPCs have arrhythmogenic risks. We investigate whether undifferentiated, regionally applied, human fetal CMPCs form a pro-arrhythmic substrate in co-culture with neonatal rat ventricular myocytes (NRVMs). Method: Unipolar extracellular electrograms, derived from micro-electrode arrays (8 × 8 electrodes) containing monolayers of NRVMs (control), or co-cultures of NRVMs and locally seeded CMPCs were used to determine conduction velocity and the incidence of tachy-arrhythmias. Micro-electrodes were used to record action potentials. Conditioned medium (Cme) of CMPCs was used to distinguish between coupling or paracrine effects. Results: Co-cultures demonstrated conduction slowing (5.6 ± 0.3 cm/s, n = 50) compared to control monolayers (13.4 ± 0.4 cm/s, n = 26) and monolayers subjected to Cme (13.7 ± 0.6 cm/s, n = 11, all p < 0.001). Furthermore, co-cultures had a more depolarized resting membrane than control monolayers (-47.3 ± 17.4 vs. -64.8 ± 7.7 mV, p < 0.001) and monolayers subjected to Cme (-64.4 ± 8.1 mV, p < 0.001). Upstroke velocity was significantly decreased in co-cultures and action potential duration was prolonged. The CMPC region was characterized by local ST-elevation in the recorded electrograms. The spontaneous rhythm was faster and tachy-arrhythmias occurred more often in co-cultured monolayers than in control monolayers (42.0 vs. 5.4%, p < 0.001). Conclusion: CMPCs form a pro-arrhythmic substrate when co-cultured with neonatal cardiomyocytes. Electrical coupling between both cell types leads to current flow between a, slowly conducting, depolarized and the normal region leading to local ST-elevations and the occurrence of tachy-arrhythmias originating from the non-depolarized zone.

Part and Parcel of the Cardiac Autonomic Nerve System: Unravelling Its Cellular Building Blocks during Development.

The autonomic nervous system (cANS) is essential for proper heart function, and complications such as heart failure, arrhythmias and even sudden cardiac death are associated with an altered cANS function. A changed innervation state may underlie (part of) the atrial and ventricular arrhythmias observed after myocardial infarction. In other cardiac diseases, such as congenital heart disease, autonomic dysfunction may be related to disease outcome. This is also the case after heart transplantation, when the heart is denervated. Interest in the origin of the autonomic nerve system has renewed since the role of autonomic function in disease progression was recognized, and some plasticity in autonomic regeneration is evident. As with many pathological processes, autonomic dysfunction based on pathological innervation may be a partial recapitulation of the early development of innervation. As such, insight into the development of cardiac innervation and an understanding of the cellular background contributing to cardiac innervation during different phases of development is required. This review describes the development of the cANS and focuses on the cellular contributions, either directly by delivering cells or indirectly by secretion of necessary factors or cell-derivatives.