STAT3 as a common signal of ischemic conditioning: a lesson on "rigor and reproducibility" in preclinical studies on cardioprotection.

Ischemic conditioning before (ischemic preconditioning, IPC) or after (ischemic postconditioning, POCO) sustained myocardial ischemia/reperfusion (I/R), induced locally or remotely from the heart (remote IPC, RIPC), reduces infarct size. However, none of the identified signaling steps of ischemic conditioning was robust across models and species to be successfully translated to humans. In prior separate studies in pigs, activation of signal transducer and activator of transcription 3 (STAT3) was causal for infarct size reduction by IPC, POCO, and RIPC but it remains unclear whether or not STAT3 is truly a common denominator of cardioprotective signaling. We therefore, now analyzed the phosphorylation of STAT3 and other signaling proteins in left ventricular biopsies from our prior studies on IPC, POCO and RIPC in one approach. We developed a strategy for the quantification of protein phosphorylation in multiple samples from many experiments on different gels/membranes by Western blot. Along with reduced infarct size, the ratio of STAT3tyr705 phosphorylation/total STAT3 protein at early reperfusion was significantly increased by IPC (IPC 2.0 ± 0.3 vs. I/R 1.2 ± 0.2 arbitrary units), but only trendwise by POCO and RIPC (1.3 ± 0.2; 1.4 ± 0.2 arbitrary units); storage time for IPC samples was shorter than for POCO and RIPC samples. No other signaling protein phosphorylation was associated with reduced infarct size. We confirmed STAT3 phosphorylation with IPC. For POCO and RIPC we could not reproduce the findings from our earlier more focused studies. At this point, we can not distinguish between lack of robustness of the biological signal and methodological issues of our retrospective approach.

lschemic preconditioning in pigs: a causal role for signal transducer and activator of transcription 3.

Ischemic preconditioning (IPC), i.e., brief episodes of nonlethal myocardial ischemia-reperfusion (I/R) before sustained ischemia with subsequent reperfusion, reduces infarct size in all species tested so far, including humans. In rodents, the cardioprotective signal transduction causally involves an activation of Akt, ERK1/2, and STAT3. However, there are apparent species differences in the signal transduction between rodents and larger mammals such as pigs, where data on IPC's signal transduction are inconsistent for Akt and ERK1/2. The role of STAT3 has not yet been analyzed. Pigs were subjected to 60 min of left anterior descending coronary artery occlusion and 180 min of reperfusion without or with IPC (2 cycles of 3-min occlusion separated by 2 min of reperfusion 15 min before sustained I/R). Infarct size was analyzed by triphenyl tetrazolium chloride staining, and Akt, ERK1/2, and STAT3 phosphorylation was quantified in myocardial biopsies taken at baseline and early reperfusion. AG490 was used to block the STAT3 signaling pathway. IPC reduced infarct size (%area at risk; mean ± SE, I/R, 45 ± 3 vs. IPC, 18 ± 3, P < 0.05). Akt and ERK1/2 phosphorylation was increased at early reperfusion without and with IPC. In contrast, STAT3 phosphorylation at early reperfusion was only increased with IPC (%baseline; mean ± SE, I/R, 126 ± 29 vs. IPC, 408 ± 147, P < 0.05). AG490 prevented the IPC-related increase of STAT3 phosphorylation at reperfusion (%baseline; mean ± SE, 82 ± 12) and abolished IPC's cardioprotection (%area at risk; mean ± SE, 35 ± 4). In pigs, increased phosphorylation of STAT3 is causally involved, whereas Akt and ERK1/2 seem to play no role in IPC's cardioprotection.NEW & NOTEWORTHY In pig hearts in situ, ischemic preconditioning (IPC) causally involves increased phosphorylation of STAT3, whereas Akt and ERK1/2 play no role for cardioprotection. The cardioprotective signal transduction of IPC is similar to that of ischemic postconditioning and remote IPC in pigs.

Across-Species Transfer of Protection by Remote Ischemic Preconditioning With Species-Specific Myocardial Signal Transduction by Reperfusion Injury Salvage Kinase and Survival Activating Factor Enhancement Pathways.

RATIONALE: Reduction of myocardial infarct size by remote ischemic preconditioning (RIPC), that is, cycles of ischemia/reperfusion in an organ remote from the heart before sustained myocardial ischemia/reperfusion, was confirmed in all species so far, including humans. OBJECTIVE: To identify myocardial signal transduction of cardioprotection by RIPC. METHODS AND RESULTS: Anesthetized pigs were subjected to RIPC (4×5/5 minutes hindlimb ischemia/reperfusion) or placebo (PLA) before 60/180 minutes coronary occlusion/reperfusion. Phosphorylation of protein kinase B, extracellular signal-regulated kinase 1/2 (reperfusion injury salvage kinase [RISK] pathway), and signal transducer and activator of transcription 3 (survival activating factor enhancement [SAFE] pathway) in the area at risk was determined by Western blot. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Plasma sampled after RIPC or PLA, respectively, was transferred to isolated bioassay rat hearts subjected to 30/120 minutes global ischemia/reperfusion. RIPC reduced infarct size in pigs to 16±11% versus 43±11% in PLA (% area at risk; mean±SD; P<0.05). RIPC increased the phosphorylation of signal transducer and activator of transcription 3 at early reperfusion, and AG490 abolished the protection, whereas RISK blockade did not. Signal transducer and activator of transcription 5 phosphorylation was decreased at early reperfusion in both RIPC and PLA. In isolated rat hearts, pig plasma taken after RIPC reduced infarct size (25±5% of ventricular mass versus 38±5% in PLA; P<0.05) and activated both RISK and SAFE. RISK or SAFE blockade abrogated this protection. CONCLUSIONS: Cardioprotection by RIPC in pigs causally involves activation of signal transducer and activator of transcription 3 but not of RISK. Protection can be transferred with plasma from pigs to isolated rat hearts where activation of both RISK and SAFE is causally involved. The myocardial signal transduction of RIPC is the same as that of ischemic postconditioning.

Heart rate reduction and longevity in mice.

Heart rate correlates inversely with life span across all species, including humans. In patients with cardiovascular disease, higher heart rate is associated with increased mortality, and such patients benefit from pharmacological heart rate reduction. However, cause-and-effect relationships between heart rate and longevity, notably in healthy individuals, are not established. We therefore prospectively studied the effects of a life-long pharmacological heart rate reduction on longevity in mice. We hypothesized, that the total number of cardiac cycles is constant, and that a 15% heart rate reduction might translate into a 15% increase in life span. C57BL6/J mice received either placebo or ivabradine at a dose of 50 mg/kg/day in drinking water from 12 weeks to death. Heart rate and body weight were monitored. Autopsy was performed on all non-autolytic cadavers, and parenchymal organs were evaluated macroscopically. Ivabradine reduced heart rate by 14% (median, interquartile range 12-15%) throughout life, and median life span was increased by 6.2% (p = 0.01). Body weight and macroscopic findings were not different between placebo and ivabradine. Life span was not increased to the same extent as heart rate was reduced, but nevertheless significantly prolonged by 6.2%.

Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption.

Connexin 43 (Cx43) is present at the sarcolemma and the inner membrane of cardiomyocyte subsarcolemmal mitochondria (SSM). Lack or inhibition of mitochondrial Cx43 is associated with reduced mitochondrial potassium influx, which might affect mitochondrial respiration. Therefore, we analysed the importance of mitochondrial Cx43 for oxygen consumption. Acute inhibition of Cx43 in rat left ventricular (LV) SSM by 18α glycyrrhetinic acid (GA) or Cx43 mimetic peptides (Cx43-MP) reduced ADP-stimulated complex I respiration and ATP generation. Chronic reduction of Cx43 in conditional knockout mice (Cx43(Cre-ER(T)/fl) + 4-OHT, 5-10% of Cx43 protein compared with control Cx43(fl/fl) mitochondria) reduced ADP-stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O(2)/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O(2)/min.*mg protein in Cx43(fl/fl) mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43(Cre-ER(T)/fl) +4-OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43(fl/fl) mice (19.8 ± 1.3%, n = 8, P < 0.05); complex II activities were similar between genotypes. Supporting the importance of Cx43 for respiration, in Cx43-overexpressing HL-1 cardiomyocytes complex I respiration was increased, whereas complex II respiration remained unaffected. Taken together, mitochondrial Cx43 is required for optimal complex I activity and respiration and thus mitochondrial ATP-production.

Kinetics and Signal Activation Properties of Circulating Factor(s) From Healthy Volunteers Undergoing Remote Ischemic Pre-Conditioning.

Although remote ischemic pre-conditioning (RIPC) reduced infarct size in animal experiments and proof-of-concept clinical trials, recent phase III trials failed to confirm cardioprotection during cardiac surgery. Here, we characterized the kinetic properties of humoral factors that are released after RIPC, as well as the signal transduction pathways that were responsible for cardioprotection in an ex vivo model of global ischemia reperfusion injury. Venous blood from 20 healthy volunteers was collected at baseline and 5 min, 30 min, 1 h, 6 h, and daily from 1 to 7 days after RIPC (3 × 5/5 min upper-limb ischemia/reperfusion). Plasma-dialysates (cut-off: 12 to 14 kDa; dilution: 1:20) were infused into Langendorff-perfused mouse hearts subjected to 20/120 min global ischemia/reperfusion. Infarct size and phosphorylation of signal transducer and activator of transcription (STAT)3, STAT5, extracellular-regulated kinase 1/2 and protein kinase B were determined. In a subgroup of plasma-dialysates, an inhibitor of STAT3 (Stattic) was used in mouse hearts. Perfusion with baseline-dialysate resulted in an infarct size of 39% of ventricular mass (interquartile range: 36% to 42%). Perfusion with dialysates obtained 5 min to 6 days after RIPC significantly reduced infarct size by ∼50% and increased STAT3 phosphorylation beyond that with baseline-dialysate. Inhibition of STAT3 abrogated these effects. These results suggest that RIPC induces the release of cardioprotective, dialyzable factor(s) within 5 min, and that circulate for up to 6 days. STAT3 is activated in murine myocardium by RIPC-induced human humoral factors and is causally involved in cardioprotection.