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... Show moreElectronic 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. Show less
Schwach, V.; Verkerk, A.O.; Mol, M.; Monshouwer-Kloots, J.J.; Devalla, H.D.; Orlova, V.V.; ... ; Passier, R. 2017
Cardiovascular disease is responsible for 17 million deaths globally each year. Research aimed at understanding the form and function of this vital organ will be key to improving patient care.... Show moreCardiovascular disease is responsible for 17 million deaths globally each year. Research aimed at understanding the form and function of this vital organ will be key to improving patient care. Although animal models such as rat, rabbit and dog have proven to be valuable to study heart physiology, there are a number of important species-specific differences, for e.g. heart rate, when compared with the human heart. In the recent years, research on cardiomyocytes(CMs) derived from human pluripotent stem cells has demonstrated that they resemble native human CMs and hence, make excellent models to study heart development and disease in vitro.In this dissertation,we present studies that describe the use of hPSC-CMs for pharamacological testing and for modeling inherited arrhythmogenic disorders.Combined with other novel technologies in the fields of genetic medicine, tissue engineering,and genome editing, hPSC-CM models will be valuable for 1) understanding lineage decisions determining CM specification, 2) unraveling molecular basis of disease, 3) translational applications such as target/drug discovery, diagnostic medicine and developing effective treatment strategies. Show less
Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed... Show moreMaximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to iden- tify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyop- athy (HCM). A simple screen revealed the collabora- tive effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the elec- trophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condi- tion, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of hap- loinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM Show less
Background-Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene... Show moreBackground-Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene expression data for the developing human conduction system has precluded valid extrapolations from experimental studies to the human situation. Methods and Results-We performed immunohistochemical analyses of the expression of key transcription factors, such as ISL1, TBX3, TBX18, and NKX2-5, ion channel HCN4, and connexins in the human embryonic heart. We supplemented our molecular analyses with 3-dimensional reconstructions of myocardial TBX3 expression. TBX3 is expressed in the developing conduction system and in the right venous valve, atrioventricular ring bundles, and retro-aortic nodal region. TBX3-positive myocardium, with exception of the top of the ventricular septum, is devoid of fast-conducting connexin40 and connexin43 and hence identifies slowly conducting pathways. In the early embryonic heart, we found wide expression of the pacemaker channel HCN4 at the venous pole, including the atrial chambers. HCN4 expression becomes confined during later developmental stages to the components of the conduction system. Patterns of expression of transcription factors, known from experimental studies to regulate the development of the sinus node and atrioventricular conduction system, are similar in the human and mouse developing hearts. Conclusions-Our findings point to the comparability of mechanisms governing the development of the cardiac conduction patterning in human and mouse, which provide a molecular basis for understanding the functioning of the human developing heart before formation of a discrete conduction system. (Circ Arrhythm Electrophysiol. 2011;4:532-542.) Show less
