5 research outputs found
TAT-Ndi1 overcomes effects of complex I dysfunction.
<p><b>A</b>. ATP levels in rat heart tissue +/− TAT-Ndi1
following 30 min ischemia and 15min reperfusion or without treatment
(veh). TAT-Ndi1 prevents depletion of ATP stores in I/R hearts.
(n = 4, *p<0.05,
***p<0.0005). <b>B</b>. Dihydroethidium stained 1mm
rat heart sections +/−TAT-Ndi1 following 30 min no-flow
ischemia and 15 min reperfusion. TAT-Ndi1 reduces superoxide production
following I/R (representative image, n = 3).
<b>C</b>. Total free MDA levels normalized to total protein in
hearts perfused 20 min with or without TAT-Ndi1 and subjected to 30 min
ischemia and 15 min reperfusion or perfused constantly with vehicle
(n = 3, **p<0.005). <b>D</b>.
NAD<sup>+</sup>/NADH ratios from rat hearts perfused 15 min
+/− TAT-Ndi1 then subjected to 30 min ischemia and 15 min
reperfusion or perfused continuously with vehicle
(n = 4, *p<0.05, **p<0.005).</p
Mitochondrial integrity and function is preserved by TAT-Ndi1.
<p><b>A</b>. The absorbance of cardiac mitochondrial suspension from
rat heart tissue was measured in the presence or absence of TAT-Ndi1.
Hearts were perfused +/− TAT-Ndi1 for 20 min prior to
isolating mitochondria. TAT-Ndi1 protects against calcium-induced
mitochondrial swelling and this inhibition is abolished by Ndi1
inhibitor, flavone (representative trace, n = 4).
<b>B</b>. Slope and V<sub>max</sub> of mitochondrial swelling
are reduced in mitochondria with TAT-Ndi1 (n = 4,
p<0.05). <b>C</b>. Oxygen consumption of mitochondria isolated
from rat hearts with or without TAT-Ndi1 and subjected to I/R
(I/R+TAT-Ndi1:double line, I/R alone: dashed line) or constantly
perfused (Con:thick line, +TAT-Ndi1:thin line). Oxygen levels were
continuously monitored using a platinum Clark-type oxygen electrode.
Changes of O<b><sub>2</sub></b> concentration in chamber are shown
with administration of treatments indicated (n = 4,
representative trace). <b>D</b>. Rate of oxygen consumption
following addition of complex I substrates palmitoyl-L-carnitine/malate
and ADP (1mM final) prior to (black bars) and following (grey bars)
addition of rotenone (*p<0.05). Mitochondria were isolated from
hearts +/− TAT-Ndi1 subjected to I/R or constantly perfused
(control and Ndi1 alone).</p
TAT-Ndi1 is cardioprotective in the Langendorff-perfused rat heart model of ischemia/reperfusion.
<p><b>A</b>. Rat hearts were perfused with or without TAT-Ndi1 for 20
min prior to 30 min ischemia and 2 hour reperfusion. Frozen sections
were stained with TTC. TAT-Ndi1 reduced infarct size
61.5%±8.01. Mean and S.D. from at least 5 hearts per
condition. (*p<0.05). <b>B</b>. Perfusate collected prior
to ischemia (baseline) and 15 min following onset of reperfusion.
Creatine kinase release was reduced 51.6%±9.8 following
ischemia/reperfusion in hearts perfused with TAT-Ndi1. Mean and S.D.
from at least 4 hearts per condition. (**p<0.01).
<b>C</b>. Hearts were subjected to 30 min ischemia and
perfused with or without TAT-Ndi1 at the onset of reperfusion. Hearts
were reperfused for 2 hours. Sections were stained with TTC
(representative image, n = 5). TAT-Ndi1 reduced
infarct size 67.1%±17.1. Mean and S.D. from at least 5
hearts per condition (*p<0.05).</p
Generation of TAT-Ndi1 and expression <i>in vitro</i>.
<p><b>A</b>. Map of TAT-Ndi1 construct generated from inserting full
length NDI1 gene (1,539bp) from pHook(NDI1) into the 6xHis-TAT-HA
cloning vector. <b>B</b>. Lysates of adult rat ventricular
myocytes were transduced with TAT-Ndi1 at 500nM in complete maintenance
media for 20 min. Cell lysates were probed with anti-HA antibody to
detect TAT-Ndi1. <b>C</b>. Adult cardiac myocytes (first and
second rows) and HL-1 cells (third row) were transduced with TAT-Ndi1 at
500nM for 1 or 15 min as indicated, fixed and double-labeled with
affinity-purified rabbit antibody to <i>S. cerevisiae</i> Ndi1
and mouse monoclonal cytochrome <i>c</i> antibody.</p
Ndi1-mediated cytoprotection following simulated ischemia reperfusion is specific.
<p><b>A</b>. pHook(Ndi1) and pDsRed2-mito were transiently transfected
into HL-1 cells. Ndi1 (stained with anti-HA antibody) co-localized with
mitochondria (DsRed-mito) (merged). <b>B</b>. Ndi1-transfected and
empty pHook-transfected HL-1 cells and neonatal rat cardiomyocytes were
subjected to 2 hours simulated ischemia and 24 hours reperfusion. Cell
death was scored by permeability to Yo-Pro-1 stain and only transfected
cells were scored. (≥250 cells scored per experiment,
n = 3, *p<0.05). <b>C</b>. Neonatal
rat cardiomyocytes transfected with empty pHook or pHook(Ndi1) were
subjected to 2 hours simulated ischemia and 24 hours reperfusion.
Pretreatment with Ndi1-inhibitor flavone abolished the cytoprotective
effect of Ndi1 expression. Cell death was scored by permeability to
Yo-Pro-1 stain and only transfected cells were scored. (≥250 cells
scored per experiment, n = 3,
**p<0.005).</p