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Comment
. 2011 Sep 13;21(3):469-78.
doi: 10.1016/j.devcel.2011.08.008.

The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation

Jennifer R Hom  1 , Rodrigo A Quintanilla, David L Hoffman, Karen L de Mesy Bentley, Jeffery D Molkentin, Shey-Shing Sheu, George A Porter Jr
Affiliations
Comment

The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation

Jennifer R Hom et al. Dev Cell. .

Erratum in

  • Dev Cell. 2011 Nov 15;21(5):975

Abstract

Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.

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Figures

Figure 1
Figure 1. Mitochondrial morphology changes during cardiac development
(A) Epifluorescence microscopy of MTG in live cultured ventricular myocytes at ages E9.5 and 13.5. (B) Fixed myocytes stained for α-actinin in the contractile apparatus and AIF in mitochondria. (C) Fixed myocytes stained for cyt c in mitochondria. (D) Measurements of mitochondrial length in cultured WT E9.5, 11.5, and 13.5 myocytes (*P<0.05 compared to E9.5). (E) Transmission electron micrographs from WT E9.5 and 13.5 hearts. (F) A histogram of mitochondrial ultrastructure classes from electron micrographs from ventricular myocytes of WT E9.5, 11.5, 13.5 hearts. The weighted classifications were significantly different among the three ages (P<0.05).
Figure 2
Figure 2. Mitochondrial function changes as the embryonic heart ages
(A and B) Epifluorescence microscopy of TMRE with MTG (A) and DCF with MTR (B) in cultured myocytes at E9.5 and 13.5. (C and D) Measurements of Δψm (C, TMRE normalized to MTG) and ROS (D, DCF fluorescence) in cultured E9.5, 11.5, and 13.5 myocytes (*P<0.05 compared to E9.5). (E and F) Representative traces of Δψm in myocytes treated with 1 µg/mL oligomycin (oligo), then either 1 µM rotenone (rot, E) or 2 mM malonate (mal, F) followed by 1 µM FCCP. Data is shown as ratio of fluorescence at any time point to first time point. Bar graphs represent the % change in TMRE after addition of rotenone or malonate compared to the total change after addition of the protonophore, FCCP (*P<0.05 compared to E9.5).
Figure 3
Figure 3. Mitochondrial structure and function changes after closure of the mPTP
(A) Epifluorescence microscopy of MTG in live E9.5 cultured ventricular myocytes: WT, WT with 500 nM Cyclosporin A (CsA) for 2 hours, and CyP-D null (CyP-D KO) myocytes. (B) Mitochondrial length in cultured WT E9.5 myocytes with and without treatment with 500 nM CsA or 500 nM FK-506 for 2 hours, CyP-D null E9.5 myocytes, and WT E11.5 and 13.5 myocytes (*P<0.05 compared to WT E9.5). (C) Electron micrographs of WT E9.5 and CyP-D null E9.5 hearts. (D) A histogram of mitochondrial ultrastructure classes in electron micrographs from ventricular myocytes of WT E9.5, 11.5, 13.5, or CyP-D null E9.5 hearts. (E) Images of E9.5 cultured ventricular myocytes stained with calcein-AM in the presence of CoCl2. Quantification showed increased calcein fluorescence after treatment with 500 nM CsA (*P<0.05). (F and G) Measurements of Δψm (F) and ROS (G) in cultured WT E9.5 myocytes with and without treatment with 500 nM CsA or 500 nM FK-506 for 2 hours, CyP-D null E9.5 myocytes, and WT E11.5 and 13.5 myocytes (*P<0.05 compared to WT E9.5). Expanded graphical data is provided in Figure S2C–E.
Figure 4
Figure 4. Closure of the embryonic mPTP enhances myocyte differentiation
(A) Cultured WT E9.5 myocytes with and without treatment with 500 nM CsA or 500 nM FK-506 for 2 hours, CyP-D null (KO) E9.5 myocytes, and WT E13.5 myocytes were stained with antibodies to α-actinin and AIF to label Z-bands and mitochondria, respectively. (B) Quantification of Z-band number in WT E9.5 myocytes with and without treatment with 500 nM CsA or 500 nM FK-506 for 2 hours, CyP-D null E9.5 myocytes, and WT E11.5 and 13.5 myocytes (P<0.05 compared to WT E9.5). Expanded graphical data and details of quantification are provided in Figures S2F and S3A.
Figure 5
Figure 5. CyP-D deletion accelerates myocyte differentiation of E9.5, but not E11.5, myocytes in vivo
Images (3,500 × magnification) taken of the left ventricular wall of E9.5 (25–26 somites) and E11.5 hearts and trabeculae of E11.5 hearts from WT and CyP-D null embryos. Arrows: examples of Z-bands of the myofibrils; En: endothelial cell; L: lumenal side of the wall; Ep: epicardial surface.
Figure 6
Figure 6. Myocyte differentiation is regulated by changes in oxidative stress downstream of mPTP activity
(A) Cultured WT E9.5 and 13.5 myocytes were treated with 100 µM bongkrekic acid (BKA), 100 µM carboxyatractyloside (CAT), 100 µM 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), or 10 µM tert-butyl hydroperoxide (tBHP) from 24–48 hours in culture and stained with antibodies to α-actinin to label Z-bands. (B) Quantification of Z-band number in WT E9.5 and 13.5 myocytes treated with combinations of BKA, CAT, Trolox, or tBHP from 24–48 hours in culture (P<0.05 compared to WT *E9.5 or **E13.5).
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Comment in

  • Mitochondria in control of cell fate.
    Folmes CD, Dzeja PP, Nelson TJ, Terzic A. Folmes CD, et al. Circ Res. 2012 Feb 17;110(4):526-9. doi: 10.1161/RES.0b013e31824ae5c1. Circ Res. 2012. PMID: 22343555 Free PMC article.

Comment on

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