Frontiers in CardioVascular Biology

Transcription

1 Frontiers in CardioVascular Biology Young Investigator Award Mitofusin 2 controls calcium transmission between the SR and mitochondria and regulates the bioenergetic feedback response in cardiac myocytes Maxie Meiser Klinik für Innere Medizin III Universitätsklinikum des Saarlandes Homburg, Germany Conflict of Interest: none

2 Physiology: Excitation-contraction coupling and mitochondrial energetics Na + I Na Na + Mito Oxidative Phosphorylation NCX Sarcoplasmic Reticulum (SR) SERCA SERCA T-tubulus I Ca ATP ADP RyR2 Cytosol Myofilaments

3 ph Intermembrane Space Inner Mitochondrial Membrane Mitochondrial Matrix I H + Q III c IV V II H + H + H + Ψ m -180 mv e - NADH NAD + ATP ADP Krebs Cycle + FADH 2 FAD + MCU Cytosol

4 Privileged communication between SR and mitochondria K d ( ) µm Kohlhaas & Maack, Circulation 2010;122:

5 Physical tethers between SR and mitochondria 3D-electron tomography in mouse hearts jsr Hayashi et al., J Cell Sci 2009;122: Mito removed Tether jsr

6 Mitofusins 1 and 2 Redundant roles in mitochondrial fusion Outer mitochondrial membrane Mfn2 Mfn1 Mfn2 Mfn2 Mfn1 Mfn1

7 Mitofusins 1 and 2 Redundant roles in mitochondrial fusion Mfn2 Mfn1 Mfn2 Mfn2 Mfn1 Mfn1

8 Mitofusins 1 and 2 Redundant roles in mitochondrial fusion Mfn2 Mfn1 Mfn2 Mfn2 Mfn1 Mfn1

9 Mitofusin 2: Tethers ER to mitochondria in fibroblasts Mitochondria Endoplasmic reticulum De Brito & Scorrano; Nature 2008;456:

10 Mitofusin 2 Possible linker protein between SR and mitochondria in cardiac myocytes? Mfn2 MCU Mfn1 MCU Mfn2 RyR RyR RyR Mfn2 Mfn2 Mfn2 Sarcoplasmic reticulum

11 Mitofusin 2 Possible linker protein between SR and mitochondria in cardiac myocytes? Mfn2 MCU Mfn1 MCU RyR RyR RyR Mfn2 Mfn2 Mfn2 Mfn2 Mfn1-KO

12 Mitofusin 2 Possible linker protein between SR and mitochondria in cardiac myocytes? Mfn2 MCU Mfn1 RyR RyR RyR MCU Mfn2 Mfn2 Mfn2 Mfn2 Mfn1-KO

13 Mfn2 deficiency impairs the spatial association of mitochondria to the SR WT

14 Experimental Protocol Wild Type (WT) Mfn1-KO Isolated cardiac myocytes Electrical Field Stimulation Isoproterenol (30 nm) 0.5 Hz 5 Hz 0.5 Hz Sarcomere Shortening

15 ph H + H + H + H + I Q III c IV V II NADH/NAD + FADH 2 /FAD Autofluorescence e - NADH Krebs Cycle NAD + + FADH 2 FAD + Rhod-2 ATP ADP MCU Indo-1

16 Unchanged cytosolic and mitochondrial signaling in Mfn1-KO myocytes [ ] c (F 405/485 ) [ ] c Mfn1-KO WT Time (s) [ ] m (F/F 0 ) [ ] m Mfn1-KO WT Time (s) Mfn1-KO mitoch. matrix (Rhod-2) [ ] m (F/F 0 ) 3.0 Mfn1-KO WT [ ] c (F 405/485 ) Diastolic [ ] m (F/F 0 ) Hz 0.5 Hz 0.5 Hz 0 Isoproterenol 30 nm WT Mfn1-KO Time (s) cytosol MCU IMM* (Indo-1)

17 Unchanged bioenergetic feedback response in Mfn1-KO myocytes Isoproterenol 5 Hz 0.5 Hz 0.5 Hz Mfn1-KO NADH (%) WT Mfn1-/- I Q II III 60 FAD + (%) e - NADH NAD + NADH / FAD Krebs Cycle + FADH 2 FAD Time (s)

18 Unchanged sarcomere shortening in Mfn2 KO myocytes Intact cardiac myocytes 5 Hz 0.5 Hz 0.5 Hz Isoproterenol 30 nm Sarcomere shortening Cytosolic [ ] FS (%) Hz 0.5 Hz 0.5 Hz Isoproterenol 30 nm WT Time (s) [ ] c (F 405/485 ) [ ] c (F 405/485 ) * sys. dias Time (s) *p<0.05 vs. WT

19 Decreased mitochondrial accumulation in myocytes [ ] c (F 405/485 ) [ ] c WT Time (s) [ ] m (F/F 0 ) [ ] m WT Time (s) mitoch. matrix (Rhod-2) MCU IMM* [ ] m (F/F 0 ) 2.0 WT [ ] c (F 405/485 ) cytosol (Indo-1)

20 Decreased mitochondrial accumulation in myocytes 5 Hz 0.5 Hz 0.5 Hz Isoproterenol 30 nm Diastolic [ ] m (F/F 0 ) inset WT mitoch. matrix (Rhod-2) Time (s) MCU IMM* Min 0-3 F/F Hz Iso * WT cytosol (Indo-1) sec *p<0.05

21 Impaired bioenergetic feedback response in myocytes NADH / FAD + FAD + (%) NADH (%) Hz 0.5 Hz 0.5 Hz ** ** ** Isoproterenol WT Mfn2-/ Time (s) e - NADH I Krebs Cycle Q II NAD + + III FADH 2 FAD +

22 Impaired bioenergetic feedback response in myocytes NADH / FAD + FAD + (%) NADH (%) Hz 0.5 Hz 0.5 Hz ** ** ** Isoproterenol WT Mfn2-/ Time (s) [ ] m (F/F 0 ) e - NADH I 0.5 Hz Iso Krebs Cycle Q II NAD + + # III WT sec Mfn2-/- FADH 2 FAD +

23 ph Intermembrane Space FCCP H + I H + H + H + Q III c IV V H + Inner Mitochondrial Membrane II Oligomycin Mitochondrial Matrix H + e - NADH NAD + ATP ADP Krebs Cycle + λ exc =540 nm λ em =605 nm TMRM MCU Ψ m -180 mv Cytosol

24 Unchanged mitochondrial membrane potential ( Ψ m ) in myocytes Ψ m (F/F 0 ) FCCP & Oligomycin WT Ψ m (A.U.) 1.8 WT Time (s) 0.0 Basal FCCP +Oligom.

25 Deletion of Mfn2, but not Mfn1, induces dilated cardiomyopathy Mfn1-KO

26 Conclusions Mfn2, but not Mfn1 tethers mitochondria to the SR in cardiac myocytes is a molecular determinant of a mitochondrial microdomain in cardiac myocytes important for energy supply and demand matching Defects in SR-mitochondria transmission may play a causal role for the development of heart failure.

27 Acknowledgments Maack lab Financial Support DFG - KFO 196 DFG - SFB 894 HOMFOR Washington University, St. Louis, USA Gerald Dorn Yun Chen Thomas Jefferson University, Philadelphia, USA Gyorgy Csordas

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29 Specific localization of rhod-2 and indo-1 in mitochondria and cytosol, respectively [ ] c (F 405/485 ) [ ] m (F/F 0 ) Isoproterenol systolic 5 Hz systolic diastolic diastolic Con Ru360 Ru360 Con * * [ ] c (F 405/485 ) [ ] m (F/F 0 ) Patch-clamped cardiac myocytes Ru360 Con Con Ru360 Ru360 mitoch. matrix cytosol MCU Rhod-2 IMM* Indo Time (s) Time (s) *IMM= inner mitochondrial membrane

30 Postnatal deletion of Mfn1 or Mfn2 Mfn1-KO