Abstract
Every heartbeat originates from a tiny tissue in the heart called the sinoatrial node (SAN). The SAN harbors only ≈10 000 cardiac pacemaker cells, initiating an electrical impulse that captures the entire heart, consisting of billions of cardiomyocytes for each cardiac contraction. How these rare cardiac pacemaker cells (the electrical source) can overcome the electrically hyperpolarizing and quiescent myocardium (the electrical sink) is incompletely understood. Due to the scarcity of native pacemaker cells, this concept of source–sink mismatch cannot be tested directly with live cardiac tissue constructs. By exploiting TBX18 induced pacemaker cells by somatic gene transfer, 3D cardiac pacemaker spheroids can be tissue‐engineered. The TBX18 induced pacemakers (sphTBX18) pace autonomously and drive the contraction of neighboring myocardium in vitro. TBX18 spheroids demonstrate the need for reduced electrical coupling and physical separation from the neighboring ventricular myocytes, successfully recapitulating a key design principle of the native SAN. β‐Adrenergic stimulation as well as electrical uncoupling significantly increase sphTBX18s' ability to pace‐and‐drive the neighboring myocardium. This model represents the first platform to test design principles of the SAN for mechanistic understanding and to better engineer biological pacemakers for therapeutic translation.
Highlights
Every heartbeat originates from a tiny tissue in the heart called the initiating each and every heartbeat.[2]
We have previously demonstrated reprogramming of chamber cardiac myocytes to induced pacemaker cells by a transient expression of a single transcription factor, TBX18.[30,31,32,33,34] The de novo TBX18-induced pacemaker cells recapitulate hallmarks of native sinoatrial node (SAN) pacemaker cells in their electrophysiology with their automaticity driven by the ion channels on the sarcolemma and the rhythmic releases of intracellular Ca2+.[31]
The Neonatal rat ventricular myocytes (NRVMs) were freshly isolated from 1–3 d old pups and transduced with adenoviral vectors expressing either GFP or TBX18 to enable somatic gene transfer
Summary
Neonatal rat ventricular myocytes (NRVMs) represent the best characterized and validated platform to study cardiac physiology in vitro in a longitudinal manner.[36] The NRVMs were freshly isolated from 1–3 d old pups and transduced with adenoviral vectors expressing either GFP (control) or TBX18 (experimental) to enable somatic gene transfer. The transduced NRVMs were seeded as either 2D monolayers or 3D cell aggregates by seeding them into an array of microwells (1000 cells per well) shaped in the form of inverted pyramids (Figure 1a). Expression of the fluorescent reporter proteins, GFP in control NRVMs and ZsGreen in TBX18 induced pacemaker cells was robust in most myocytes, suggesting high efficiency of viral gene transfer (Figure 1b).
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