The adipocyte-derived adipokine leptin exerts pleiotropic effects, which are essential for the regulation of energy balance and cell metabolism, for controlling inflammatory and immune responses, and ...for the maintenance of homeostasis of the cardiovascular system. Leptin resistance in obese or type 2 diabetes mellitus (T2DM) patients is defined as a decrease in tissue response to leptin. In the cardiovascular system, leptin resistance exhibits the adverse effect on the heart's response to stress conditions and promoting cardiac remodeling due to impaired cardiac metabolism, increased fibrosis, vascular dysfunction, and enhanced inflammation. Leptin resistance or leptin signaling deficiency results in the risk increase of cardiac dysfunction and heart failure, which is a leading cause of obesity- and T2DM-related morbidity and mortality. Animal studies using leptin- and leptin receptor- (Lepr) deficient rodents have provided many useful insights into the underlying molecular and pathophysiological mechanisms of obese- and T2DM-associated metabolic and cardiovascular diseases. However, none of the animal models used so far can fully recapitulate the phenotypes of patients with obese or T2DM. Therefore, the role of leptin in the human cardiovascular system, and whether leptin affects cardiac function directly or acts through a leptin-regulated neurohumoral pathway, remain elusive. As the prevalence of obesity and diabetes is continuously increasing, strategies are needed to develop and apply human cell-based models to better understand the precise role of leptin directly in different cardiac cell types and to overcome the existing translational barriers. The purpose of this review is to discuss the mechanisms associated with leptin signaling deficiency or leptin resistance in the development of metabolic and cardiovascular diseases. We analyzed and comprehensively addressed substantial findings in pathophysiological mechanisms in commonly used leptin- or Lepr-deficient rodent models and highlighted the differences between rodents and humans. This may open up new strategies to develop directly and reliably applicable models, which resemble the human pathophysiology in order to advance health care management of obesity- and T2DM-related cardiovascular complications.
Metabolic diseases and diabetes represent an increasing global challenge for human health care. As associated with a strongly elevated risk of developing atherosclerosis, kidney failure and death ...from myocardial infarction or stroke, the treatment of diabetes requires a more effective approach than lowering blood glucose levels. This review summarizes the evidence for the cardioprotective benefits induced by antidiabetic agents, including sodium-glucose cotransporter 2 inhibitor (SGLT2i) and glucagon-like peptide-1 receptor agonist (GLP1-RA), along with sometimes conversely discussed effects of dipeptidyl peptidase-4 inhibitor (DPP4i) and metformin in patients with high cardiovascular risk with or without type 2 diabetes. Moreover, the proposed mechanisms of the different drugs are described based on the results of preclinical studies. Recent cardiovascular outcome trials unexpectedly confirmed a beneficial effect of GLP-1RA and SGLT2i in type 2 diabetes patients with high cardiovascular risk and with standard care, which was independent of glycaemic control. These results triggered a plethora of studies to clarify the underlying mechanisms and the relevance of these effects. Taken together, the available data strongly highlight the potential of repurposing the original antidiabetics GLP1-RA and SGLT2i to improve cardiovascular outcome even in non-diabetic patients with cardiovascular diseases.
Abstract In vitro generation of cardiomyocytes (CMs) from human cells opens the possibility to develop patient-specific therapies to various cardiomyopathies. By establishing the in vitro ...reprograming methods that produce human CMs, we learn about what is involved in the development of specific CM subtypes. In this review, we summarize the latest achievements in CM generation technologies, emphasizing the differentiation methods of specific CM subtypes. We also relate the biological properties and functions of the in vitro -generated CMs to those of their in vivo counterparts. Furthermore, we describe the main problem of current CM derivation methods – maturation of CMs. We subsequently discuss biochemical and physical stimuli that are used to overcome the maturation problems of in vitro -derived CMs. As a result, a more holistic approach with controllable environment and timing of specific stimuli for creation of more mature engineered heart tissues is described as well. Finally, we propose a novel approach in which enhancing energy transfer mechanisms in the immature CMs might help to overcome the current hurdle of incomplete in vitro differentiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
•Blebbistatin improves single cardiomyocyte preparation for electrophysiological studies.•Blebbistatin terminates cardiomyocyte contraction to prevent calcium-paradox damage.•Blebbistatin shows no ...side-effects on action potentials and field potentials.•Blebbistatin improves INa and action potential recording using automated patch-clamp.•Phenotypes of Brugada syndrome could be recapitulated using automated patch-clamp.
Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation–contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage ...may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The increase in activity of the two-pore potassium-leak channel Kcnk5b maintains allometric juvenile growth of adult zebrafish appendages. However, it remains unknown how this channel maintains ...allometric growth and how its bioelectric activity is regulated to scale these anatomical structures. We show the activation of Kcnk5b is sufficient to activate several genes that are part of important development programs. We provide in vivo transplantation evidence that the activation of gene transcription is cell autonomous. We also show that Kcnk5b will induce the expression of different subsets of the tested developmental genes in different cultured mammalian cell lines, which may explain how one electrophysiological stimulus can coordinately regulate the allometric growth of diverse populations of cells in the fin that use different developmental signals. We also provide evidence that the post-translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity to scale the fin. Thus, we show how an endogenous bioelectric mechanism can be regulated to promote coordinated developmental signaling to generate and scale a vertebrate appendage.
Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of ...this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes.
We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated.
Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.
Patient-specific induced pluripotent stem cells (ps-iPSCs) and their differentiated cell types are a powerful model system to gain insight into mechanisms driving early developmental and ...disease-associated regulatory networks. In this study, we use ps-iPSCs to gain insights into Tetralogy of Fallot (TOF), which represents the most common cyanotic heart defect in humans. iPSCs were generated and further differentiated to cardiomyocytes (CMs) using standard methods from two well-characterized TOF patients and their healthy relatives serving as controls. Patient-specific expression patterns and genetic variability were investigated using whole genome and transcriptome sequencing data. We first studied the clonal mutational burden of the derived iPSCs. In two out of three iPSC lines of patient TOF-01, we found a somatic mutation in the DNA-binding domain of tumor suppressor P53, which was not observed in the genomic DNA from blood. Further characterization of this mutation showed its functional impact. For patient TOF-02, potential disease-relevant differential gene expression between and across cardiac differentiation was shown. Here, clear differences at the later stages of differentiation could be observed between CMs of the patient and its controls. Overall, this study provides first insights into the complex molecular mechanisms underlying iPSC-derived cardiomyocyte differentiation and its transcriptional alterations in TOF.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Calpains are calcium-activated neutral proteases involved in the regulation of key signaling pathways. Junctophilin-2 (JP2) is a Calpain-specific proteolytic target and essential structural ...protein inside Ca
2+
release units required for excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in heart failure has been reported, the precise molecular identity of the Calpain cleavage sites and the (patho-)physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently at three additional secondary Calpain cleavage sites. Moreover, we identified the Calpain-specific primary cleavage products for the first time in human iPSC-derived cardiomyocytes. Knockout of RyR2 in hiPSC-cardiomyocytes destabilized JP2 resulting in an increase of the Calpain-specific cleavage fragments. The primary N-terminal cleavage product NT
1
accumulated in the nucleus of mouse and human cardiomyocytes in a Ca
2+
-dependent manner, closely associated with euchromatic chromosomal regions, where NT
1
is proposed to function as a cardio-protective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT
1
by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Myocardial insulin resistance contributes to heart failure in response to pathological stresses, therefore, a therapeutic strategy to maintain cardiac insulin pathways requires further investigation. ...We demonstrated that insulin receptor substrate 1 (IRS1) was reduced in failing mouse hearts post-myocardial infarction (MI) and failing human hearts. The mice manifesting severe cardiac dysfunction post-MI displayed elevated
in the myocardium. Ischemia-upregulated
promoted
degradation. Using rat cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes, we elucidated that mitogen-activated protein kinase 7 (MAPK7, also known as ERK5)-mediated CCAAT/enhancer-binding protein beta (CEBPβ) transcriptionally represses
under hypoxia. Therapeutically, functional studies demonstrated gene therapy-delivered cardiac-specific MAPK7 restoration or overexpression of CEBPβ impeded cardiac injury after MI, at least partly due to normalization of
. Furthermore, inhibition of
preserved
and ameliorated cardiac dysfunction post-MI. In conclusion, we reveal that targeting
mitigates myocardial insulin resistance, thereafter slowing down the progression of heart failure post-ischemia.
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