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1 doi:1.138/nture11444 CMKIIN Gold prticles/ mitochondril re ( m ) CMKIIN mtcmkiin mtcmkiin SERCA ATP synthse HA mitoplsts mtcmkiin CMKIIN cytosolic mtcmkiin CMKIIN 97 kdl 64 kdl 51 kdl 14 kdl 6 kdl Supplementl Figure 1. CMKIIN is expressed in mitochondri of CMKIIN nd mtcmkiin mouse herts.. Representtive TEM imges of mitochondri isolted from herts showing HA-tgged CMKIIN expression in CMKIIN nd mtcmkiin trnsgenic mice, ut not in mice. Gold leling on mitochondri is mrked y lck circles. Summry dt (upper right) showing HA stining for n = 5 fields per condition, n = 6 herts/genotype, (p <.1). Dt represent men ± s.e.m.. Western nlysis of mitoplst lyste from, mtcmkiin nd CMKIIN herts. HA ntiody recognizes HA-CMKIIN in mitoplst nd cytosolic lystes from CMKIIN trnsgenic herts ut only in mitoplst lystes from mtcmkiin trnsgenic herts. HA ws not detected in lystes from herts. SERCA, n SR protein, is present in cytosol nd sent in mitochondril frctions. Inner mitochondril protein, ATP synthse (5), is present only in mitoplst frctions. 1
2 1 nd nd CMKIIN nd Reperfusion5 mn LVDP (mm hg) seline ischemi c Percent recovery in LVDP n = CMKIIN 1 1 d CMKIIN e f. g time (min) reperfusion CMKIIN CMKIIN. µm h Mitochondri injury score LVDP (mm hg) mm 1 Percent infrcted tissue 5 5 n = 3 CMKIIN CMKIIN Cspse 9 ctivity/ µg protein 1.1 n = n = CMKIIN CMKIIN Supplementl Figure. Hert function is eqully mintined fter ischemi nd reperfusion injury (I/R) y CMKII inhiition (CMKIIN) or cyclosporin A ().. Representtive left ventriculr developed pressure (LVDP) trces from herts nd CMKIIN-expressing herts perfused with or ( µm). The concentrtion of in the coronry perfuste is sed on lood serum levels of min fter injection in the study y Piot et l. 8.. Expnded LVDP trces efore nd fter I/R from time-points 1 nd (with rrows) in pnel. c. LVDP recovery following I/R s percentge of the seline vlue (p =.9). Numerls indicte numer of mesurements/group. d. Representtive triphenyl tetrzolium chloride (TTC) stined hert sections. The drk red stining represents living myocrdium, the solid lck outlines form oundries demrcting vile from ded tissue nd the dotted lines indicte totl re t risk. e. Summry dt from TTC stined herts with reltive re of infrct normlized to. Infrcted re ws mesured s percentge of totl t risk myocrdium (indicted y the dshed lines in pnel d, p <.1). f. Cspse 9 ctivity from herts treted s in pnel (p =.34). g. Representtive trnsmission electron microscopy (TEM) imges from herts treted s in pnel. h. Summry mitochondri injury scores for TEM studies y the following criteri: = no detectle disruption in ny mitochondri/field, 1 = criste disrupted in one mitochondrion/field, = disruption in >1 mitochondrion/field, 3 = 1 nd < 5% ruptured mitochondri/field nd 4 = 5% mitochondri ruptured/field (p =.8, out 5 mitochondri from 1 rndom fields were counted/genotype nd tretment). Dt represent men ± s.e.m.
3 Percent +dp/dt mx CMKIIN Percent -dp/dt mx CMKIIN dp/dt mx (mmhg/s) CMKIIN -dp/dt mx (mmhg/s) CMKIIN LVDP (mmhg) CMKIIN Supplementl Figure 3. Mechnicl function in isolted herts is mintined fter ischemi nd reperfusion y CMKIIN expression in trnsgenic herts or y in herts.. Percent recovery of mximum contrctility (+dp/dtmx) nd relxtion (-dp/dtmx, from time points 1 nd, see Supplementry Fig. ) in mouse herts sujected to I/R injury.. Bseline recordings for ech of the prmeters. p <.1. Numerls in rs indicte the numer of herts studied. Dt represent men ± s.e.m. 3
4 CMKII coxiv R-IgG c SERCA Clnexin Totl Mitoplst Mitochondril Cytosol ] 97 SR 97 proteins d.5 µm. µm Totl Mitoplst Mitochondril Cytosol e.15 Gold prticles/mitochondril re ( m ) CMKIICoxIV R IgG CMKII nucleolr mfn1 VDAC CoxIV 51 nucler 8] protein 97 mitochondril 8]proteins 14 pmol/min/µg.1.5. mtcmkiin Supplementl Figure 4. CMKII ssocites with mitochondri.. Representtive immuno-em imges of isolted mitochondri with ntiodies ginst CMKII or CoxIV (rrows).. Summry dt of gold leling for CMKII, using CoxIV nd rit IgG primry ntiodies s positive nd negtive controls, respectively. n = 5 fields per ntiody. c. Immunodetection of CMKII in mitochondri. Western lot shows srcoplsmic reticulum (SR) proteins in totl nd cytosol cell frctions, nucler proteins in totl nd cytosol cell frctions nd outer mitochondril proteins (mfn1 nd VDAC)predominntly in whole mitochondril frction while inner memrne spce nd inner memrne protein (CoxIV) re in oth mitochondril frctions. d. Coomssie stin of gel used in c. e. CMKII ctivity ssy mesuring C + nd clmodulin-dependent ctivity in isolted mitochondri (n = 4 mesurements/ genotype, p =.4). Dt represent men ± s.e.m. 4
5 Stte 4 (succinte) Stte 3 (succinte) Stte 3/Stte 4 (succinte) Respirtion mtcmkiin Respirtion mtcmkiin RCR mtcmkiin Stte 4 (glut/ml) Stte 3 (glut/ml) Stte 3/Stte 4 (glut/ml) 3. Respirtion 5 5 Respirtion Respirtory Control Rtio mtcmkiin mtcmkiin. mtcmkiin ADP:O rtio Respirtion mtcmkiin.5 5. mtcmkiin potentil (mv) Supplementl Figure 5. Mitochondril respirtion nd memrne potentil re not different etween nd mtcmkiin mitochondri. Both memrne potentil nd respirtion rte showed no significnt differences under stte 3 or 4 conditions or in coupling of inner mitochondril memrne potentil nd respirtion in mtcmkiin compred with control mice using succinte or glutmte nd mlte (glut/ml) s mitochondril sustrtes. The ADP:O nd the reltionship etween respirtion nd memrne potentil were determined in succinte. Mitochondril smples were prepred from nd mtcmkiin herts (n = 5 or 7 smples/group for mitochondri respiring in glut/ml or succinte, respectively) from -month old mice. Dt represent men ± s.e.m. 5
6 3 3 Normlized CG5N Normlized CG5N 1 1 mtcmkiin mtcmkiin mtcmkiin mtcmkiin Ru36 mtcmkiin Ru36 mtcmkiin Ru C + cpcity (mm) C + cpcity (mm) mtcmkiin - - mtcmkiin Supplementl Figure 6. Mitochondirl clcium retention nd cpcity in permeilized crdiomyocytes.. CG5N fluorescence trces, normlized to the first pek, show mitochondril C + uptke kinetics in sponin-permeilized Supplementl Figure nd 6. leisttin- Mitochondirl (1 clcium µm) nd thpsigrgin- (5 µm) treted crdiomyocytes isolted from or mtcmkiin trnsgenic herts. Ech C + retention nd cpcity in permeilized crdiomyocytes. trnsient ws detected with CG5N in response. CG5N fluorescence trces, to n ddition of 1 µm C + normlized to the first pek, show mitochondril C + uptke kinetics in (t times indicted). n = 4 experiments for nd mtcmkiin. sponin-permeilized nd leisttin- (1 µm) nd thpsigrgin- (5 µm) treted crdiomyocytes isolted Representtive trces shown for crdiomyocytes from nd mtcmkiin herts dditionlly treted with from or mtcmkiin trnsgenic herts. Ech C (5 µm) or Ru36 (.1 µm).. Totl extr-mitochondril + trnsient C + ws detected with CG5N in response clered y mitochondri for nd mtcmkiin to n ddition crdiomyocytes, of 1 µm C with + nd (t times without indicted)., n = 7 n (), = 4 experiments 4 ( with ), for 6 (mtcmkiin) nd mtcmkiin. nd 4 Representtive (mtcmkiin with trces ), shown p <.1 for crdiomyocytes p<.1. Dt from represent nd men mtcmkiin ± s.e.m. herts dditionlly treted with (5 µm) or Ru36 (.1 µm).. Totl extr-mitochondril C + clered y mitochondri for nd mtcmkiin crdiomyocytes, with nd without, n = 7 (), 4 ( with ), 6 (mtcmkiin) nd 4 (mtcmkiin with ), p <.1 p<.1. Dt represent men ± s.e.m. 6
7 control mtcmkiin CFP YFP CFP YFP Fluorescence (.u.) Before After Before After ATP Time [ s] 1 µm CFP YFP Fluorescence (.u.) ATP Time [ s] 1 µm Supplementl Figure 7. Mitochondril-trgeted cmeleon monitored C + uptke in HeL cells.. Trgeting of cmeleon 4mtD3cpv nd mtcmkiin or control vector (myc-pcdna) to mitochondri fter 4 h of trnsfection in HeL cells. The CFP fluorescence nd YFP FRET signls re differentilly responsive to ddition of ATP.. Representtive trcings of sl FRET (YFP, F535) nd the relted CFP (F48) fluorescence in cells trnsfected with 4mtD3cpv long with mtcmkiin or control. As indicted, cells were stimulted with 1 µm ATP. Individul fluorescent responses of 4mtD3cpv tht corresponded to the experiments shown in. Summry dt shown in Fig. 1f re tken from reltive fluorescent intensities t rrow nd rrowhed. 7
8 c I MCU (-16 mv), pa/pf 15 n.s. 1 5 Control Ru36 Voltge (mv) 1 Voltge (mv) Control K/M -1 - T/D -3 5 d Voltge (mv) I MCU (pa) Control -1 - I MCU (pa) mtcmkiin CMKIIN -5-1 I MCU (pa) c-a c-a T/D e 1 Voltge (mv) SS/AA SS/AA + T/D + T/D I MCU (pa) Supplementl Figure 8. CMKII increses I MCU.. I MCU is sensitive to Ru36 (1 nm) ut not (5 µm). n = 7 (control), 6 () nd 5 (Ru36), p <.1. Dt represent men ± s.e.m.. Representtive trces showing I MCU is incresed y constitutively ctivted CMKII mutnt (T/D), with. mm C + nd ATP, ut not with kinse inctive CMKII (K/M), CM nd ATP. c. Representtive trces showing I MCU is incresed y clyculin A (c-a) nd no dditionl increse in I MCU occurred with ddition of ctivted CMKII. d. Representtive trces showing reduced I MCU recorded with. mm C + in mitoplsts from CMKIIN nd mtcmkiin compred to herts. e. Constitutively ctive CMKII, T/D increses I MCU on mitoplsts isolted from HEK cells overexpressing MCU, ut not mutted (serines 57 nd 9 to lnines, SS/AA) MCU. 8
9 15 p =.8 Bseline LVDP (mm Hg) 1 5 mtcmkiin Supplementl Figure 9. Left ventriculr developed pressure (LVDP) recorded from nd mtcmkiin Lngendorff-perfused herts. n = 14 herts/genotype, dt represent men ± s.e.m. 9
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