Mitochondrial morphology is not affected by bicarbonate.

<p>Mitochondrial morphology under control (A and B) or sI/R conditions (C and D) was measured by calculating their aspect ratio (A and C) (Major axis/Minor Axis) and format factor (B and D) (Perimeter²/(Pi*4*Area)). Representative images of mitochondrial morphology analyses using Keyence software; CoxIV is red, DAPI is blue and selected mitochondria are purple (E-H).</p

Proteaso me activity is not affected by bicarbonate.

<p>Proteasome activity was measured as described in Materials and Methods. Rat hearts were subjected to 30 min ischemia followed by 15 min reperfusion and the proteins were collected and proteasome activity was measured (A). HL-1 cells were subjected to 150 min ischemia followed by 5 min reperfusion; cellular extracts were obtained at the indicated times and proteasome activity was measured (B).</p

H₂O₂ production from isolated mitochondria is not increased by bicarbonate.

<p>Mitochondria isolated from rat hearts were subjected to 0 or 10% bicarbonate; hydrogen peroxide production as a function of oxygen consumption was measured in the presence of ADP (A), ADP + Oligomycin (B) or ADP + Oligomycin + CCCP (C). Respiratory control (RC) (D) was measured as described in Materials and Methods. To examine protein oxidative modifications, mitochondria were incubated in media with Succinate (E and G), or Succinate + Antimycin (F and H) for 15 min at 37°C; the mitochondria were then pelleted and protein carbonyl (E and F) and methionine sulfoxide (G and H) content was measured by western blot.</p

Autophagy markers accumulate in the mitochondrial fraction.

<p>HL-1 cells were subjected to 150 min ischemia followed by 5 min reperfusion, and cell extracts were obtained at the indicated times and probed for p62 by western blot (A). Isolated rat hearts were subjected to 30 min ischemia followed by 15 min reperfusion, and the heart homogenates were fractionated by differential centrifugation to yield cytosol (B, D, F) and mitochondria (C, E, G). The resulting fractions were probed for p62 (B and C), Beclin 1 (D and E) and Drp1 (F and G) by western blot.</p

Bicarbonate exacerbates ischemia/reperfusion injury.

<p>HL-1 cells were subjected to 150 min ischemia followed by 5 min of reperfusion (A); CK release to the supernatant was measured at the indicated times (B); protein carbonylation content was measured by western blot in the cell lysate before and after the 5 min reperfusion (C). Isolated rat hearts were subjected to ischemia for 30 min and 15 min reperfusion (D); CK release into the perfusate was measured during reperfusion (E); protein carbonylation content was measured by western blot at the end of reperfusion (15 min) (F).</p

Scheme representing the events that lead to increased damage in I/R and sI/R in the presence of bicarbonate.

<p>I/R causes mitochondrial oxidant production with resulting oxidative damage to macromolecules (orange glow). Outer mitochondrial membrane proteins such as TOM70 are degraded by the proteasome, and the damaged mitochondrion is separated from the network by Drp1 and marked for autophagic removal by p62. Bicarbonate interferes with mitophagy, resulting in the accumulation of oxidized proteins, functional impairment, and cell death. Bicarbonate does not affect mitochondrial oxidant production, morphology, proteasome activity, or general autophagy.</p

Bicarbonate Increases Ischemia-Reperfusion Damage by Inhibiting Mitophagy

<div><p>During an ischemic event, bicarbonate and CO₂ concentration increase as a consequence of O₂ consumption and lack of blood flow. This event is important as bicarbonate/CO₂ is determinant for several redox and enzymatic reactions, in addition to pH regulation. Until now, most work done on the role of bicarbonate in ischemia-reperfusion injury focused on pH changes; although reperfusion solutions have a fixed pH, cardiac resuscitation protocols commonly employ bicarbonate to correct the profound acidosis associated with respiratory arrest. However, we previously showed that bicarbonate can increase tissue damage and protein oxidative damage independent of pH. Here we show the molecular basis of bicarbonate-induced reperfusion damage: the presence of bicarbonate selectively impairs mitophagy, with no detectable effect on autophagy, proteasome activity, reactive oxygen species production or protein oxidation. We also show that inhibition of autophagy reproduces the effects of bicarbonate in reperfusion injury, providing additional evidence in support of this mechanism. This phenomenon is especially important because bicarbonate is widely used in resuscitation protocols after cardiac arrest, and while effective as a buffer, may also contribute to myocardial injury.</p></div

Bicarbonate promotes accumulation of mitophagy cargo and exacerbation of sI/R injury comparable to lysosomal blockade.

<p>TOM70 (A) (proteasome degraded) and COXIV (B) (autophagy degraded) were probed by western blot in total extracts from hearts subjected to 30 min ischemia followed by 5 min of reperfusion. HL-1 cells were subjected to 150 min ischemia followed by 5 min reperfusion in the presence or absence of bicarbonate and/or Bafilomycin A (autophagy inhibitor, Baf, red symbols); creatine kinase release was measured at the indicated times (C).</p

Bicarbonate does not change LC3 expression, but changes LC3 transcription.

<p>LC3 was measured by western blot or RT-PCR as described in the Material and Methods. Hearts were subjected to 30 min ischemia followed by 15 min reperfusion, after which protein was extracted to quantify LC3 expression (A-C). In the same treatment, mRNA was isolated and LC3 mRNA was quantified by RT-PCR (D). HL-1 cells were subjected to 150 min ischemia followed by 5 min reperfusion. Cell extracts prepared at the indicated times were probed for LC3 expression (E-G).</p

eNOS activity is increased in fasted DNP and CR tissues, and enhanced in the post-prandial period.

<p>eNOS and phospho-eNOS^Ser1177 expression were measured in the visceral adipose tissue (A) and skeletal muscle (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018433#s2" target="_blank">Materials and Methods</a>. Typical blots are shown above average densitometry results. Plasma nitrite (NO₂⁻) levels (C) were measured using an NO^• analyzer. AL, <i>ad libitum</i>; DNP, dinitrophenol-treated mice; CR, calorically-restricted mice. *<i>p</i><0.05 vs. fasting AL. ^#p<0.05 vs. group-matched fasting.</p