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. 2012 Sep 14;111(7):863-75.
doi: 10.1161/CIRCRESAHA.112.266585. Epub 2012 Jul 9.

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk

Yun Chen  1 , György Csordás, Casey Jowdy, Timothy G Schneider, Norbert Csordás, Wei Wang, Yingqiu Liu, Michael Kohlhaas, Maxie Meiser, Stefanie Bergem, Jeanne M Nerbonne, Gerald W Dorn 2nd, Christoph Maack
Affiliations

Mitofusin 2-containing mitochondrial-reticular microdomains direct rapid cardiomyocyte bioenergetic responses via interorganelle Ca(2+) crosstalk

Yun Chen et al. Circ Res. .

Abstract

Rationale: Mitochondrial Ca(2+) uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca(2+) uptake despite low Ca(2+) affinity of the mitochondrial Ca(2+) uniporter. However, the existence of such mitochondrial Ca(2+) microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, whereas Mfn2 but not Mfn1 tethers endoplasmic reticulum to mitochondria in noncardiac cells.

Objective: To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca(2+) signaling, and bioenergetic regulation in cardiac myocytes.

Methods and results: Fruit fly heart tubes deficient of the Drosophila Mfn ortholog MARF had increased contraction-associated and caffeine-sensitive Ca(2+) release, suggesting a role for Mfn in SR Ca(2+) handling. Whereas cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca(2+) cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca(2+) uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca(2+) release. Accordingly, Ca(2+)-induced stimulation of Krebs cycle dehydrogenases during β-adrenergic stimulation was hampered in Mfn2-KO but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)(+) and FADH(2)/FAD.

Conclusions: Physical tethering of SR and mitochondria via Mfn2 is essential for normal interorganelle Ca(2+) signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca(2+) signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

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Figures

Figure 1
Figure 1. SR Ca2+ handling is altered by dMfn/MARF suppression in Drosophila heart tubes
A. (left) Representative Ca2+ transients monitored by fluorescence of GCaMP3.0 expressed specifically in cardiac myocytes of spontaneously contracting Drosophila heart tubes in situ. Black tracings are tincΔ-Gal4-driven GCaMP3 (controls); green tracings are tincΔ-Gal4 driven mitoGFP (which is Ca2+-insensitive), demonstrating minimal effects of heart tube contraction on fluorescence signals. (right) Representative tracing of RyR-deficient Drosophila heart tube, showing decreased amplitude and delayed normalization of Ca2+ transient, which is typical of heart failure. B. Ca2+ transients of control (ctrl; tincΔ-Gal4) and mitofusin/MARF deficient (dMfn RNAi) expressing RNAi for dMfn. Representative tracings are shown to the left and group data from n=11 individual flies are to the right. C. SR Ca2+ content measured as caffeine-stimulated cardiomyocyte Ca2+ release in heart tubes of dMfn deficient (RNAi) and ctrl flies. Representative tracings are shown to the left with arrows marking the time of caffeine (10 mM) addition; group data from n=5 or 6 individual flies are to the right. *p<0.05 vs ctrl. D. Heart tube dimensions and contractile function in control and dMfn RNAi Drosophila, assessed by optical coherence tomography (OCT). Representative b-mode OCT scans are shown on the left, with group data from the same set of flies that underwent Ca2+ measurements shown to the right. *p<0.05 vs ctrl.
Figure 2
Figure 2. Postnatal heart-specific ablation of Mfn1 and Mfn2 using Myh6-directed nuclear-localized Cre
A. and B, Schematic representations of Cre-Lox strategy for cardiomyocyte-specific deletion of mfn1 exon 4 and mfn2 exon 6. Representative immunoblot analyses of cardiac Mfn1 and Mfn2 expression for each knockout (KO) is shown below; each column is a separate mouse heart. α-tub, α-tubulin loading control. C. Time of cardiomyocyte gene recombination by Myh6-nuclear-directed “turbo” Cre assessed by ROSA-26 LacZ reporter line. Recombination (blue staining) at 13.5 days p.c. (E13.5), the second day post birth (P2) and three weeks of age (P21). Top, whole embryos; middle and bottom, isolated hearts. Representative of at least four specimens per group. D. and E., Representative hearts and M-mode echocardiograms of mouse left ventricles. Group echocardiographic data are shown below (ctrl, white bars; Mfn KO, black bars; n=15–25 per group). LV %FS=left ventricular % fractional shortening; LVEDD=left ventricular end diastolic dimension. F. and G., Invasive hemodynamic studies of cardiac contraction (peak rate of increase in LV pressure; +dP/dt max) and inotropic response to dobutamine-induced β1-adrenergic receptor stimulation. Dobutamine was infused intravenously at increasing doses from 4 to 256 ng/g/min. ctrl, white circles; Mfn KO, black squares. Data are of 3–5 per group.
Figure 3
Figure 3. Ca2+ cycling and SR Ca2+ release in Mfn1 and Mfn1-deficient murine cardiac myocytes
A. and B., Phasic Ca2+ transients in Fura-2 loaded field-stimulated isolated Mfn1- (A) and Mfn2 (B) null ventricular cardiac myocytes (−/−) and respective floxed allele controls (ctrl). Group quantitative data for transient amplitude and time constant for normalization (tau) are shown to the right. Data are averaged from n=5 paired hearts, averaging 8–12 cardiomyocytes per heart. C. Representative immunoblot analysis of SR calcium handling protein expression in Mfn1−/− and Mfn2−/− cardiac homogenates and their respective controls. SERCA=SR Ca2+-ATPase; RyR=ryanodine receptor 2; NCX1=Na+/Ca2+ exchanger; PLN=phospholamban. D. Representative whole-cell L-type Ca2+ currents (normalized to cell capacitance), evoked in response to 400 ms depolarizing voltage steps to test potentials between −30 to +50 mV from a holding potential of −40 mV, recorded from control (CTL; top) and Mfn2−/− (bottom) ventricular myocytes. Peak ICa,L densities in CTL (n=11) and KO (n=16) LV myocytes are plotted as a function of the test potential on the right. E. and F. SR Ca2+ release by caffeine (10 mM) in isolated Mfn1 (E) and Mfn2 (F) null and control cardiac myocytes. Group data for Ca2+ signal amplitude are shown to the right. G. RyR content in mitochondrial-associated membranes from Mfn1 and Mfn2 KO mouse hearts. Cytochrome oxidase (Cox) is a mitochondrial protein loading control. *p<0.05 vs ctrl.
Figure 4
Figure 4. Cardiomyocyte mitochondrial-SR architecture is altered by Mfn2 ablation
A. Transmission electron micrographs of longitudinal sections of myocardium derived from control (top) and cardiac Mfn2-null (Mfn2-KO) mice (bottom). Lower magnification overview images on the left show the overall mitochondrial distribution and morphology. 5-fold higher magnification of the framed areas are shown on the right with arrows pointing to SR-mitochondrial associations. B and C, Cumulative analysis of mean perimeter (top, left) and area of mitochondria (top, right), and of the transverse side length (bottom, left) and contact length with jSR (bottom, right, respectively) in Mfn1- (B) and Mfn2-KO mice (C) compared to their respective controls. Mfn1-KO (B): n=8 and 6 cellular areas analyzed from 2 different hearts each for Mfn1-KO and control, respectively; each cellular area represents the means/sum of 117–377 and 144–299 individual mitochondria. Mfn2-KO (C): n=8 and 7 cellular areas from 4 different hearts each for Mfn2-KO and control, respectively; each cellular area represents the means/sum of 44–173 and 65–304 individual mitochondria. *p<0.05 (Mann-Whitney rank sum test).
Figure 5
Figure 5. Impaired mitochondrial Ca2+ accumulation and bioenergetic feedback response in Mfn2-deficient myocytes
Experiments were performed on intact cardiac myocytes with acute isoproterenol and pacing stress (see Supplemental Figure 4); Mfn1-KO on left; Mfn2-KO on right. A. and C. Averaged original traces of [Ca2+]c (top) and [Ca2+]m transients (middle) in WT and Mfn-KO myocytes after isoproterenol (30 nM) for 1 min at 0.5 Hz. Bottom panels show dynamic changes of [Ca2+]m plotted against [Ca2+]c in the same cell in the presence of isoproterenol for 1 min at 0.5 Hz (Mfn1: n= 4 control, n=7 KO; Mfn2: n=14 control, n=12 KO). B. and D. Time-dependent changes in diastolic [Ca2+]m with pacing and isoproterenol stress. Inset in D shows change of diastolic [Ca2+]m in the first 2 minutes after application of isoproterenol. E. and F. Autofluorescence of NAD(P)H (top), FAD (middle) and the ratio of NAD(P)H/FAD (bottom; Mfn1-KO, n=17; control, n=7; Mfn2-KO, n=24; control, n=15). *p<0.05 and **p<0.01 WT vs. KO, respectively (ANOVA for repeated measures).
Figure 6
Figure 6. Effects of Mfn2 deletion on cardiomyocyte mitochondrial superoxide formation, ΔΨm integrity and Ca2+ permeability
A. Superoxide (·O2−) was measured using MitoSOX during studies performed as in Figure 5. Left, time-dependence of ·O2− production; right, rate of mitochondrial ·O2− production one minute after isoproterenol (Minute 1) and after antimycin A (Anti-A; n=16 ctrl, n=9 KO). B. Absolute fluorescence of TMRM indicating ΔΨm at baseline and after mitochondrial uncoupling with FCCP (5 µmol/L) and oligomycin (1.26 µmol/L) in control and Mfn2-KO myocytes (n=10 WT, 4 KO). C. Ca2+-induced swelling of isolated heart mitochondria was induced by addition of 250 µM CaCl2 and quantified as the time-dependent decrease in absorbance at 540 nm. Results from ctrl are on the left, Mfn2 null on the right.
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