Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand

Abstract

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

Fig. 1. Higher resistance of TBX18-iPMs to metabolic stresses.

a TBX18- and GFP-NRVMs were incubated in normoxia or 1% oxygen (Hypoxia) with or without 2-DG treatment for 3 days from D3 to D6, followed by ethidium homodimer-1 (EthD-1) staining on D6. The % cell death was calculated based on the number of EthD-1⁺ cells per total cells. Representative pictures of EthD-1 and Hoechst 33342 staining of D6 GFP- and TBX18-NRVMs. b Quantitative graphs of % cell death (n ≥ 8 for each condition). Scale bar: 10 µm *p < 0.05

Fig. 2. Smaller mitochondria in TBX18-iPMs and native pacemaker cells.

a Representative EM images of mitochondria with two different magnifications from GFP-NRVMs and TBX18-iPMs. b Mitochondria size distribution of GFP-NRVMs and TBX18-iPMs. Representative images of MitoTracker staining (left) and percentage of mitochondria according to their size distribution in three groups: >10 .m², 1–10 µm², and 0.1–1 µm² (right) (n = 10 for GFP-NRVMs and n = 14 for TBX18-iPMs). c Representative superresolution images of mitochondria stained with MitoTracker from GFP-NRVMs and TBX18-iPMs. d Representative superresolution images of mitochondria stained with MitoTracker from freshly isolated mouse ventricular myocytes and pacemaker cells. Scale bar: 2 µm (a: right panels of each group), 5 μm (a: left panels of each group and b), and 10 μm (c and d) *p < 0.05

Fig. 3. TBX18-iPMs show a slower oxygen consumption rate and a lower glycolytic capacity.

a Oxygen consumption rate (OCR) of TBX18-iPMs and GFP-NRVMs was measured with a SeaHorse XFp analyzer at baseline and after sequential treatments with oligomycin (an ATP-synthase inhibitor), FCCP (an uncoupler of mitochondrial oxidative phosphorylation), and rotenone/AA (an inhibitor of mitochondrial electron transport chain) on D3, D5, and D7. Basal and maximal OCR are plotted for each time point (n = 6). b Glycogen contents were determined in the cell lysates of D6 TBX18-iPMs and GFP-NRVMs under normoxia, after 1 hour of incubation in 1% O₂ or upon treatment with 20 mm 2-deoxy-d-glucose (2-DG) (n = 8). The lactate content in the media from D6 TBX18-iPMs and GFP-NRVMs was quantitated under normoxia or after incubating the myocytes in 1% O₂ for 1 hour (n = 8). *p < 0.05

Fig. 4. The reduced level of Opa1 in TBX18-iPMs correlates with smaller mitochondria.

a Proteomic quantification of mitochondrial fusion factors, Mfn1, Mfn2, Opa1, Oma1, and Yme1l1 in the TBX18-iPM proteome (n = 3). b Validation of Opa1 protein expression levels in D3 GFP-NRVMs and TBX18-iPMs (n = 6). c Relative mRNA expression levels of fusion-related factors, including Mfn1, Mfn2, Opa1, Oma1, and Yme1l, normalized to Gapdh in GFP-NRVMs and TBX18-iPMs (n = 6). d mRNA level of Opa1 after siOpa1 transfection in TBX18-iPMs (n = 4). e Reduced protein level of Opa1 with siOpa1 transfection in TBX18-iPMs. Representative immunoblot (left) and quantitative graph (right) (n = 4). f Mitochondrial size distribution of GFP-NRVMs and TBX18-iPMs with siScramble or siOpa1 transfection. Representative pictures of Mitotracker staining (left) and % mitochondrial size distribution divided as >10 μm², 1–10 μm², and 0.1–1 μm² (right) (n = 4 for GFP-NRVMs transfected with siScramble or siOpa1 and n = 6 for TBX18-iPMs transfected with siScramble or siOpa1). g Mitochondrial density in given cytoplasmic area of GFP-NRVMs and TBX18-iPMs with siScramble or siOpa1 transfection (n = 4 for GFP-NRVMs transfected with siScramble or siOpa1 and n = 6 for TBX18-iPMs transfected with siScramble or siOpa1). *p < 0.05

Fig. 5. Mitochondrial dynamics regulate the synchronous automaticity of TBX18-iPMs.

a BPMs of synchronous beats from siScramble- or siOpa1-transfected TBX18-iPMs on D2, D4, and D6 after transfection (n = 5). Representative distribution of synchronous beats from siScramble- or siOpa1-transfected TBX18-iPMs on D2, D4, and D6 after transfection (right three panels). b Conduction velocity of spontaneous synchronous beatings from GFP- and TBX18-NRVMs with or without Opa1 knockdown on D2, D4, and D6 after transfection (n = 5). c Relative protein levels of selected gap junctions, including phosphorylated Cx43 (p-Cx43), Cx43, and Cx45, were analyzed in D4 GFP-NRVMs and TBX18-iPMs (n = 4). Representative immunoblots are shown on the left and a quantitative graph is presented on the right. BPM: Beats per minute. Scale bar: 5 μm *p < 0.05