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. 2015 Jul 21;112(29):9129-34.
doi: 10.1073/pnas.1504705112. Epub 2015 Jul 7.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

Tyler P Rasmussen  1 , Yuejin Wu  2 , Mei-ling A Joiner  3 , Olha M Koval  3 , Nicholas R Wilson  2 , Elizabeth D Luczak  2 , Qinchuan Wang  2 , Biyi Chen  3 , Zhan Gao  3 , Zhiyong Zhu  3 , Brett A Wagner  4 , Jamie Soto  3 , Michael L McCormick  4 , William Kutschke  3 , Robert M Weiss  3 , Liping Yu  5 , Ryan L Boudreau  3 , E Dale Abel  6 , Fenghuang Zhan  3 , Douglas R Spitz  4 , Garry R Buettner  4 , Long-Sheng Song  3 , Leonid V Zingman  3 , Mark E Anderson  7
Affiliations

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart

Tyler P Rasmussen et al. Proc Natl Acad Sci U S A. .

Abstract

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Keywords: ischemia-reperfusion injury; mitochondrial calcium uniporter; myocardium.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Increased myocardial oxygen consumption in DN-MCU hearts. (A) Schematic of the DN-MCU construct and expressed mutant channel in the transgenic mice. (B) Quantitative PCR measurement for WT and DN Mcu transcript expression. (C, Top) Western blot detection of MYC-tagged protein in heart (H), liver (L), and skeletal muscle (S) tissues from DN-MCU and WT mice. GAPDH was used as a loading control. (Bottom) Western blot detection of MYC-tagged protein in mitochondria (M) or cytosolic (C) isolates from DN-MCU and WT hearts. GAPDH confirmed purity of cytosolic isolates, and COXIV confirmed purity of mitochondrial isolates. (D) Oxygen consumption rates in Langendorff-perfused and paced hearts (beats/min). (E) Representative isolated hearts. (Scale bar, 200 μm.) (F) Summary of heart weight (HW) to body weight (BW) ratio measurements. (G) Left ventricular ejection fraction measured by echocardiography in unanesthetized mice. (H) Representative transmission electron microscopy images (5,000× and 10,000×). (I) Summary data of mitochondrial injury scores. (J) Mitochondrial protein (mg) measurements normalized to heart weight (g). (K) Mitochondrial:nuclear DNA. All error bars represent SEM. *P < 0.05, **P < 0.01, Student’s t test. Sample size (n) indicated for each group in parentheses.
Fig. S1.
Fig. S1.
MCU protein is increased in DN-MCU hearts. Western blot detecting MCU in mitochondrial isolates from DN-MCU and WT hearts. Coomassie stain was used as a loading control.
Fig. S2.
Fig. S2.
Echocardiographic analysis. Heart rate (HR), left ventricular (LV) mass, stroke volume, end systolic volume (ESV), and end diastolic volume (EDV), as measured by echocardiography. All error bars represent SEM. Sample size (n) is indicated for each group in parentheses.
Fig. S3.
Fig. S3.
COXIV protein expression is similar in WT and DN-MCU hearts. (Top) DN-MCU and WT whole-heart lysates were blotted for cyclooxygenase IV (COXIV) and Actin. (Bottom) The ratio of COXIV:Actin was used to compare mitochondrial protein content to cytosolic protein. Error bars represent SEM. Sample size (n) is indicated for each group in parentheses. No significant differences were detected.
Fig. 2.
Fig. 2.
DN-MCU expression reduced left ventricular pressure responses to pacing. (A and B) Representative waveform of left ventricular pressure (LVP) (mmHg) in WT and DN-MCU at 400 bpm, respectively. LVDP indicated by arrows. (C and D) Representative LVP changes in WT and DN-MCU hearts when increasing pacing rate from 400 to 600 bpm. (E and F) Representative LVP changes in WT and DN-MCU hearts when increasing pacing rate from 600 to 750 bpm. (G) LVDP (mmHg) at 400, 600, and 750 bpm. (H) +dP/dtMAX (mmHg/s) at 400, 600, and 700 bpm. (I) −dP/dtMAX (mmHg/s) at 400, 600, and 700 bpm. (J) OCR [μmol/min/g(Wet)] in WT and DN-MCU hearts. All error bars represent SEM. Sample size (n) indicated for each group in parentheses. *P < 0.05, **P < 0.01, Student’s t test. #P < 0.05, ##P < 0.01, and ###P < 0.001 comparing 750 to 400 bpm; $P < 0.05 comparing 750 to 600 bpm, Tukey’s post hoc multiple comparison test.
Fig. S4.
Fig. S4.
In vivo left ventricular pressure recordings. (A) Baseline +dP/dtMAX (mmHg/s). (B) +dP/dtMAX (mmHg/s) 2 min after isoproterenol administration (10 μg/kg, i.p.). (C) Percentage increase in +dP/dtMAX from baseline to isoproterenol condition. (D) Baseline −dP/dtMAX (mmHg/s). (E) −dP/dtMAX (mmHg/s) 2 min after isoproterenol administration (10 μg/kg, i.p.). (F) Percentage decrease in −dP/dtMAX from baseline to isoproterenol condition. (G) Baseline heart rate (HR) (bpm) measured by pressure waveform. (H) HR 2 min after isoproterenol administration (10 μg/kg, i.p.). (I) Percentage increase in HR from baseline to isoproterenol condition. All error bars represent SEM. Sample size (n) is indicated for each group in parentheses. *P < 0.05, ***P < 0.001, Student’s t-test.
Fig. 3.
Fig. 3.
DN-MCU expression alters cytoplasmic Ca2+ dynamics. (A) Normalized kinetic tracings for CaGr5N-loaded, cell membrane-permeabilized ventricular myocytes. Arrows represent addition of 100 μM Ca2+. (B) Western blot detection of phosphorylated (pPDH) and total pyruvate dehydrogenase (PDH). (C) Summary data for the pPDH to total PDH ratio. (D) Summary data for PDH activity normalized to mitochondrial protein. (E) Representative Ca2+ transient traces from WT (black) and DN-MCU (red) ventricular myocytes stimulated by field stimulation. Summary data for (F) diastolic and (G) systolic [Ca2+] measurements made with Fura-2–loaded cells stimulated by field stimulation. All error bars represent SEM. *P < 0.05, ***P < 0.001, Student’s t test. Sample size (n) indicated for each group in parentheses.
Fig. S5.
Fig. S5.
Mitochondrial Ca2+ uptake measurement. (A) Assessment of Ca2+ uptake by isolated mitochondria using mitochondrial impermeant dye Calcium Green-5N. (B) Assessment of Ca2+ uptake by isolated mitochondria preloaded with intramitochondrial calcium indicator Fura-FF-AM.
Fig. S6.
Fig. S6.
DN-MCU expression does not alter glucose metabolism in the heart. (A) Total peak intensity (in arbitrary units), a measure of total 13C incorporation obtained by summation of the peak intensities present in the heteronuclear multiple quantum coherence (HMQC) spectra. (B) Measured NMR peak intensity (in arbitrary units) of glucose metabolites in the perfused heart extracts. The peak intensities were measured from the assigned HMQC spectra (**P < 0.01 compared with WT, Student’s t test). (C) Overlay of the HMQC spectra of glucose metabolites of WT (black) and DN-MCU (red) perfused heart extracts. Select cross-peaks are labeled: lactate (Lac), alanine (Ala), glutamate (Glu), aspartate (Asp), and phosphocreatine (PCr). The one-dimensional slices through the Asp C3 are shown to display significant differences in peak intensity between the WT and DN-MCU heart extracts.
Fig. S7.
Fig. S7.
Reversal of elevated diastolic cytosolic [Ca2+] in voltage clamp-stimulated DN-MCU ventricular myocytes by ATP dialysis. (A) Representative [Ca2+] transients from isolated ventricular WT (black tracings) and DN-MCU (red tracings) myocytes stimulated at 1 and 3 Hz in the absence or presence of ATP (5 mM), which was added to the pipette (intracellular) solution. Summary data for diastolic (B) and systolic (C) cytosolic [Ca2+] in WT and DN-MCU ventricular myocytes in the presence and absence of pipette solution with 5 mM ATP. Cell number in each group is indicated in parentheses: (B) 1 Hz WT (7), WT + ATP (8), DN-MCU (16), DN-MCU + ATP (11); 3 Hz WT (8), WT + ATP (7), DN-MCU (13), DN-MCU + ATP (9). (C) 1 Hz WT (6), WT + ATP (6), DN-MCU (9), DN-MCU + ATP (6); 3 Hz WT (8), WT + ATP (7), DN-MCU (9), DN-MCU + ATP (10). ***P < 0.001, one-way ANOVA analysis of means. Error bars show SEM.
Fig. S8.
Fig. S8.
ICa and INCX recordings and SR Ca2+ content. (A) ICa recording in ventricular myocytes. Sample size (n) noted in parentheses. Error bars represent SEM. (B) INCX recording in patched ventricular myocytes. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. Sample size (n) noted in parentheses. Error bars represent SEM. (C) Summary SR Ca2+ content data from isolated ventricular myocytes loaded with Fluo-4 AM at baseline before and after addition of isoproterenol (ISO) (100 nM). Maximum SR Ca2+ release was triggered by a caffeine spritz (SI Materials and Methods). Sample size (n) is indicated for each group. **P < 0.01 and ****P < 0.0001, Student’s t test. Error bars show SEM.
Fig. 4.
Fig. 4.
DN-MCU mitochondria have normal OCR. (A) Representative electron paramagnetic resonance spectra of the DMPO-OH spin adduct produced by isolated cardiac mitochondria. (B) Summary showing the signal intensity of DMPO-OH baseline (P = 0.07, Student’s t test) and after addition of antimycin A (1 μM) or SOD (100 U/0.5 mL). (C) OCR in permeabilized myocardial fibers with 156 nM Ca2+ and 10 mM succinate in different states V0 (no secondary substrate), VADP (1 mM ADP), and VOligo (1 mM oligomycin). Units are pmol oxygen/min/mg of dry fiber weight. (*P < 0.05 from WT in same state, Student’s t test). (D) Bioluminescent quantification of ATP (*P < 0.05 Student’s t test). (E) Calculated ATP:O ratios in permeabilized fibers. (F) Baseline OCR from isolated mitochondria without added ADP. (G) OCR normalized to baseline after addition of ADP (4 mM), oligomycin (2.5 μg/mL), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (4 μM), and antimycin A (4 μM). All error bars represent SEM. Sample size (n) indicated for each group in parentheses.
Fig. S9.
Fig. S9.
DN-MCU hearts are not protected from ischemia-reperfusion injury. (A) Percentage change in TMRM signal from baseline in heart tissue during ischemia reperfusion. Two-way ANOVA analysis shows that the genotype was not a significant source of variation. (B) Percentage change in DCF signal from baseline in heart tissue during ischemia reperfusion, (*P < 0.05, **P < 0.01) compared with WT at same time point, Student’s t test; (#P < 0.05) compared with baseline value within genotype, Dunnett’s multiple comparison post test; (P < 0.05) Two-way ANOVA testing genotype as a source of variation. (C) Glutathione peroxidase activity. (D) Catalase activity. (E) Copper/zinc SOD-specific activity. (F) Manganese SOD-specific activity. (G) Total SOD activity. Sample size (n) is indicated for each group in parentheses. *P < 0.05, Student’s t test. Error bars show SEM. (H) Representative TTC-stained heart sections after ischemia reperfusion. The black line outlines viable tissue. (I) Quantification of viable myocardium from TTC staining. All error bars represent SEM. Sample size (n) is indicated for each group in parentheses.
Fig. 5.
Fig. 5.
Broad transcriptional reprogramming in DN-MCU hearts. (A) Hierarchical clustering of 700 differentially expressed genes (WT, black; DN-MCU, gray). (B) Graph shows P values for 10 functional terms enriched in the DN-MCU gene set. (C) qRT-PCR data showing validation of selected functional annotation terms listed in B. Superscript numbers indicate the functional annotation cluster being queried from B. (D) Western blot detecting Bax protein in whole-heart homogenates. Coomassie-stained blot shows gel loading for each sample. (E) Quantification of Bax Western blot (*P < 0.05, **P < 0.01, Student’s t test). All error bars represent SEM. Sample size (n) indicated for each group in parentheses.
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