Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Sep 28;111(8):1012-26.
doi: 10.1161/CIRCRESAHA.112.274142. Epub 2012 Aug 17.

Mitofusins 1 and 2 are essential for postnatal metabolic remodeling in heart

Kyriakos N Papanicolaou  1 , Ryosuke Kikuchi, Gladys A Ngoh, Kimberly A Coughlan, Isabel Dominguez, William C Stanley, Kenneth Walsh
Affiliations

Mitofusins 1 and 2 are essential for postnatal metabolic remodeling in heart

Kyriakos N Papanicolaou et al. Circ Res. .

Abstract

Rationale: At birth, there is a switch from placental to pulmonary circulation and the heart commences its aerobic metabolism. In cardiac myocytes, this transition is marked by increased mitochondrial biogenesis and remodeling of the intracellular architecture. The mechanisms governing the formation of new mitochondria and their expansion within myocytes remain largely unknown. Mitofusins (Mfn-1 and Mfn-2) are known regulators of mitochondrial networks, but their role during perinatal maturation of the heart has yet to be examined.

Objective: The objective of this study was to determine the significance of mitofusins during early postnatal cardiac development.

Methods and results: We genetically inactivated Mfn-1 and Mfn-2 in midgestational and postnatal cardiac myocytes using a loxP/Myh6-cre approach. At birth, cardiac morphology and function of double-knockout (DKO) mice are normal. At that time, DKO mitochondria increase in numbers, appear to be spherical and heterogeneous in size, but exhibit normal electron density. By postnatal day 7, the mitochondrial numbers in DKO myocytes remain abnormally expanded and many lose matrix components and membrane organization. At this time point, DKO mice have developed cardiomyopathy. This leads to a rapid decline in survival and all DKO mice die before 16 days of age. Gene expression analysis of DKO hearts shows that mitochondria biogenesis genes are downregulated, the mitochondrial DNA is reduced, and mitochondrially encoded transcripts and proteins are also reduced. Furthermore, mitochondrial turnover pathways are dysregulated.

Conclusions: Our findings establish that Mfn-1 and Mfn-2 are essential in mediating mitochondrial remodeling during postnatal cardiac development, a time of dramatic transitions in the bioenergetics and growth of the heart.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Mitofusins are transcriptionally activated in postnatal WT but not DKO hearts
A) Quantitative assessment of cardiac Mfn-1 mRNA in different developmental stages. The quantitation was performed in real time according to the ΔΔCt method where the levels of Mfn-1 in WT E15.5 hearts were used as a reference (value=1). B) Quantitative assessment of cardiac Mfn-2 mRNA in the indicated developmental stages. In both A and B, Rpl30 was used as the housekeeping gene. Two or three hearts per group per developmental stage were used and error bars represent standard error. C) The growth of DKO hearts during the developmental window E15.5-P03 is not hampered. Overall morphology in freshly-dissected WT and DKO hearts at the indicated developmental stages. Hearts were photographed in ice cold PBS.
Figure 2
Figure 2. The heart function at birth (P0) is similar in DKO compared to WT mice
A) Representative M-mode echocardiograms taken from parasternal short axis views of the left ventricle of WT and DKO pups. B) Representative flow patterns at the mitral valve of WT and DKO mice. The bright arcs appearing in pairs above the baseline are the E and A waves. Note that at this age the A wave peaks higher than the E wave. C��J) Values of different echocardiographic parameters in conscious WT and DKO mice at P0 (n=10 per group). FS; fractional shortening, LVIDd; Left ventricle internal diameter in diastole, LVPWs; LV ventricle posterior wall thickness in systole, LV Vol.d; Left ventricle volume in diastole, E/A ratio between the peak velocity of the early (A) and late (D) mitral inflow.
Figure 3
Figure 3. The heart function at P07 is impaired in DKO mice
A) Motion of the ventricular walls is monitored by echocardiography in WT and DKO mice. Note the poor movement of the walls in the DKO. B) Pattern of the inward (above baseline) and outward (below baseline) flow of blood through the mitral valve in WT and DKO hearts. C–G) Morphometric analysis of WT and DKO hearts at P07. For definitions of acronyms see legend in Fig. 2. The thresholds of significance are indicated for each pairwise comparison. H) Representative ECG patterns (lead II configuration) of WT and DKO mice at P07. A single arrow indicates the QRS complex in WT and a double arrow the QRS complex in DKO mice. †indicates the P waves. mV; millivolts, s; seconds
Figure 4
Figure 4. Diminished lifespan and features of cardiac pathology in DKO mice
A) Survival curves of WT, monoallelic and DKO mice. Pairwise comparisons (Log Rank) reveal P=0.024 in the difference in survival between WT and monoallelic mice. Difference in survival between DKO and WT or DKO and monoallelic is significant for P<0.001. The median survival time of DKO mice is 9.83 days. B) Representative picture of WT and DKO littermates at P06. C) Gross morphology of freshly-dissected WT and DKO hearts at P04. The arrowhead shows thinning of the right ventricular (RV) wall and the arrow thrombus formation in the left atrium. Scale bar is 500 μm. D–E) Metrics of WT and DKO mice at age P06–P07. F) Sagittal sections of WT and DKO hearts at P04 stained with Masson’s trichrome. White arrow indicates focal fibrosis in the DKO heart. Black arrow and arrowhead indicate atrial congestion and interventricular septum thinning respectively.
Figure 5
Figure 5. Aberrant accumulation of spherical mitochondria in DKO hearts at P07
A) Low power image showing the typical intracellular structure of WT cardiac myocytes. B) Low power image of DKO cardiac myocytes containing spherical mitochondria of variable size that occupy a large portion of the cell. Z; sarcomeric banding, M; mitochondria, L; lipid droplet. C) Structural arrangement of mitochondria and myofibrils in WT cardiac myocyte. D) Altered proportions between mitochondria and myofibrils in DKO cardiac myocytes. Arrowheads in C and D indicate the distance between myofibrils. E) Morphologies of intermyofibrillar/perinuclear mitochondria in WT cardiac myocytes. F) Abnormally patterned mitochondria in DKO cardiac myocytes. Mitochondria with unusual sizes (small and large) are indicated by arrows. These representative images were taken from two animals per genotype. G–H) Quantitation of volume density of mitochondria and myofibrils. Each quantified field (×6300 magnification) had dimensions 12.7×13.9 μm.
Figure 6
Figure 6. Structural abnormalities of P07 DKO mitochondria
A and A′) Sample image of WT intermyofibrillar mitochondria. The boxed region in A is shown in greater detail in A′. B) Image of DKO intermyofibrillar mitochondria and select region (white box, B′) presenting mitochondria undergoing unpacking of cristae and loss of matrix density. C) Image of DKO mitochondria and select region (white box, C′) presenting extreme degeneration of cristae, mitochondrial swelling and loss of matrix density. D) Sample region of WT mitochondria and select region (white box, D′) presenting the boundary relationship between WT mitochondria. E) Image of DKO mitochondria forming atypical protrusions and select region (white box, E′) showing in detail the local curvature of the mitochondrial membrane (*). F) Sample image from DKO cells. ‡indicates mitochondria with poor internal organization, arrows indicate mitochondrial voids. G–I) Sample DKO regions containing chains of mitochondria with atypical membrane protrusions (*) and omega-like membrane structures (#).
Figure 7
Figure 7. Repression of transcription factors/coactivators of mitochondrial biogenesis and accumulation of early mediators of autophagy in DKO hearts at P07
A) Relative abundance of target mRNAs at P07. Abbreviations: Nrf-1;Nuclear respirator factor-1, Err-α; Estrogen receptor related-α, Pgc-1α;PPARγ coactivator-1α, Tfam; Transcription factor α mitochondrial B) Western blot analysis of the early autophagic mediator p62. C) Protein levels of p62 in WT and DKO hearts at P07. The arrow on A indicates that only the upper band was quantified in this analysis. D) Protein levels of LC3 (early autophagy marker) and cathepsin-D (late autophagy/lysosomal marker). E) Densitometry-based quantification of LC3-II and cathepsin-D.
Figure 8
Figure 8. Defects in the maintenance and expression of mtDNA in DKO hearts
A–B) Transcript levels of genes located in the mtDNA. Nd-5; NADH dehydrogenase subunit- 5, Cyt-b; cytochrome-b, subunit of the bc1 complex. C) Temporal pattern of mtDNA expansion in WT and DKO hearts. The quantitation was performed in real time according to the ΔΔCt method where the levels of COX-I in WT P03 hearts were used as a reference (value=1). D) Western blot analysis assessing the abundance of COX-I and F1, subunit-α. E) Dual COX/SDH staining in situ in freshly-cut heart sections. Arrows indicate cardiac myocytes where SDH- specific staining predominates. These fields are magnified × 40 and the hearts were from P09 mice. Arrows indicate myocytes with low COX activity and more prevalent SDH activity.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lopaschuk GD, Collins-Nakai RL, Itoi T. Developmental changes in energy substrate use by the heart. Cardiovasc Res. 1992;26:1172–1180. - PubMed
    1. Girard J, Ferre P, Pegorier JP, Duee PH. Adaptations of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition. Physiol Rev. 1992;72:507–562. - PubMed
    1. Alaynick WA, Kondo RP, Xie W, He W, Dufour CR, Downes M, Jonker JW, Giles W, Naviaux RK, Giguere V, Evans RM. Errgamma directs and maintains the transition to oxidative metabolism in the postnatal heart. Cell Metab. 2007;6:13–24. - PubMed
    1. Lai L, Leone TC, Zechner C, Schaeffer PJ, Kelly SM, Flanagan DP, Medeiros DM, Kovacs A, Kelly DP. Transcriptional coactivators pgc-1alpha and pgc-lbeta control overlapping programs required for perinatal maturation of the heart. Genes Dev. 2008;22:1948–1961. - PMC - PubMed
    1. Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol. 1996;28:1737–1746. - PubMed

Publication types

MeSH terms