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Switching off to switch pacemaker cells on 

What if we could stop implanting pacemakers and create biological pacemakers by turning existing cardiomyocytes into specialized pacemaker cells? And how could suppressing rebellious microRNAs help?
By Alomone Team • June 2023

Your heart relies on its pacemaker cells to get it through the three billion beats it’ll make over the course of your life. But sometimes things go wrong, resulting in the need for an electrical pacemaker. And while we’ve been improving pacemaker implants for around 60 years, they’re still not ideal. New work could be changing that. A US team of scientists have been refining biological pacemakers, turning cardiomyocytes in the heart into pacemaker cells. But they also discovered a somewhat rebellious pair of microRNAs (miRs) resistant to such a change that must first be silenced.  

Building a biological pacemaker 

Specialized pacemaker cells are found in the sinoatrial node (SAN) and these set your heartbeat frequency. These pacemaker cells express HCN4, which enables the pacemaker current, known as the funny current (If). Dysfunction in these cells can cause cardiac arrhythmias, specifically bradyarrhythmia. An electrical pacemaker implant essentially fills the role these cells would normally play.  

The researchers wanted to expand on the idea of a biological pacemaker, whereby an ectopic area of cells is reprogrammed to work as a replacement for the dysfunctional SAN. They have previously demonstrated successful conversion of cardiomyocytes to pacemaker cells using the human embryonic T-box transcription factor 18 (TBX18) gene. In this new study, the researchers built on their previous work by using a chemically modified mRNA (CMmRNA) to deliver the TBX18 gene and sustain protein expression. The benefit of delivering a transgene mRNA directly rather than with a viral vector is that it bypasses any possible host immune response to viral proteins as well as potential insertional mutagenesis. 

Pacemaker cells switch on if microRNAs are switched off 

Initial transfection attempts in rat ventricular myocytes (NRVMs) were unsuccessful – they couldn’t maintain TBX18 expression beyond 24 hours. Testing ruled out mRNA decay as the culprit, so the researchers modified the uridine residues in human TBX18 mRNA (CMmTBX18). They also pretreated cells with acriflavine (ACF), an inhibitor of argonaute 2 (AGO2), which binds small non-coding RNAs (including miRs).  

After the adjustments, TBX18 expression was steady for at least 72 hours. This suggested that miRs were in some way suppressing TBX18 protein expression in the cells. Additional work identified miR-1-3p and miR-1b as the responsible miRs, since they were binding the TBX18 3’ UTR to suppress translation. This was confirmed by co-transfecting NRVMs with CMmTBX18 and an antagonist of these two miRs (antagomiRs), at which point the researchers observed sustained TBX19 expression for at least 42 hours. HCN4 was also expressed for 72 hours, visualized via Western blot with Alomone’s anti-HCN4 antibody, a marker of pacemaker cells (Figure 1). 

Suppression of TBX18 Protein Expression 

Figure 1. Argonaute 2-dependent suppression of TBX18 protein expression: miR-1-3p and miR-1b arrest TBX18 mRNA translation. Representative western blot demonstrating HCN4 protein expression by co-transfecting NRVMS with TBX18 chemically modified RNA and antagomiRs to miR-1-3p and miR-1b. Image from Sanchez L, et al. Cell Rep Med. 2022;3(12):100871.  

The team wanted to be sure that the cells they were creating were indeed functional induced SANs (iSANs). Patch clamp recordings of CMmTBX18-transfected NRVMs demonstrated action potentials typical of pacemaker cells. Moreover, NRVMs co-transfected with CMmTBX18 plus both two antagomiRs had iSAN morphology 72 h post-transfection, along with (HCN2, HCN4, SCN5a, and Nkx2.5) expression as determined by immunocytochemistry, consistent with successful somatic reprogramming (Figure 2). These changes weren’t seen without the antagomiRs. 

TBX18, HCN4, and HCN2 staining of NRVMs 

Figure 2. In vitro immunocytochemical micrographs demonstrate TBX18, HCN4, and HCN2 staining of NRVMs post-TBX18 chemically-modified mRNA and AntagomiRs against miR-1-3p and miR-1b. (A) Representative immunofluorescence images of TBX18 chemically-modified mRNA and AntagomiRs, eGFP chemically-modified mRNA AntagomiRs, TBX18 chemically-modified mRNA only and TBX18 adenovirus of NRVMS 72 hrs post-transfection. Images our Anti-HCN2 Antibody (#APC-030)  and Anti-HCN4 Antibody (#APC-052). (B) Quantification of iSAN-like morphology 72hrs post-transfection with TBX18 chemically modified mRNA and antagomiRs to miR-1-3p and miR-1b in TBX18 staining (n=15 in quantification of percent SAN-like cells for CMmTBX18 + AntagomiRs and CMmeGFP + AntagomiRs [biological replicates]) Scale bar: 20μm. (C) Quantification of HCN4 positive cells 72 hrs post-transfection with TBX18 chemically modified mRNA and antagomiRs to miR-1-3p and miR-1b (n=15 in quantification of percent HCN4 positive cells for CMmTBX18+AntagomiRs and CMmeGFP+ AntagomiRs [biological replicates]) Scale bar: 20μm (D) RT-qPCR demonstrates 72 hrs post-transfection (n=6 in CMmTBX18+AntagomiRs, n=6 CMmeGFP+AntagomiRs and TBX18 adenovirus [biological replicates]) Data are represented as mean ± SEM. Statistical significance was determined using an independent t-test.  Image from Sanchez L, et al. Cell Rep Med. 2022;3(12):100871.  

Perfecting the cardiac cocktail 

The initial plan was to develop a biological pacemaker without the need for a viral vector. To do this, the researchers injected CMmTBX18 into rat hearts but found it didn’t induce pacemaker activity. However, creating a cocktail of CMmTBX18 plus miR-1-3p and miR-1b antagomiRs was successful in transforming cells, something they determined by simultaneous field electrocardiography (ECG) and optical mapping of hearts 72 hours post-injection. 

At the site of he CMmTBX18 and dual antagomiR cocktail injection, the team observed increased TBX18 mRNA along with appropriate changes in the expression of genes known to be influenced by TBX18 (Cx43, SCN5a, and Nkx2.5). Immunohistochemistry confirmed iSAN morphology of cells at the injection site, something that was absent in control hearts. 

To put their iSAN cocktail to the final test, the researchers transfected rat hearts with CMmTBX18 and the two antagomiRs. After two weeks, they observed the expected changes in electrical activity. However, while TBX18 mRNA expression and protein expression were decreased, they did observe sustained expression of reprogramming markers such as HCN4, HCN2, and Cx45.  

Suppressing miRs to induce pacemaker activity 

This fascinating research shows how CMmTBX18 is effective at creating iSANs in vivo and in vitro, but only when both miR-1-3p and miR-1b – miRs that would otherwise fight against the transgene being inserted – are suppressed at the same time. In addition to highlighting why Alomone’s HCN antibodies are valuable and reliable tools for cardiac research, this research has driven an intriguing new insight into how miRs can fight to resist change, and how suppressing them may improve CMmRNA therapies. 

We also recently discussed how researchers are using a new model to investigate pacemaker cell dysfunction and lend insights into primate longevity. 

Supporting your cardiac research 

If you’re interested in pacemaker cell research, we have several reagents you might like: 

Antibodies 

Explorer kits