Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivate

Supplemental table 2 with references

proteomic analysis in HFpEF </p

Mitochondrial Dysfunction Contributes to Alveolar Developmental Arrest in Hyperoxia-Exposed Mice

This study investigated whether mitochondrial dysfunction contributes to alveolar developmental arrest in a mouse model of bronchopulmonary dysplasia (BPD). To induce BPD, 3-day-old mice were exposed to 75% O2. Mice were studied at two time points of hyperoxia (72 h or 2 wk) and after 3 weeks of recovery in room air (RA). A separate cohort of mice was exposed to pyridaben, a complex-I (C-I) inhibitor, for 72 hours or 2 weeks. Alveolarization was quantified by radial alveolar count and mean linear intercept methods. Pulmonary mitochondrial function was defined by respiration rates, ATP-production rate, and C-I activity. At 72 hours, hyperoxic mice demonstrated significant inhibition of C-I activity, reduced respiration and ATP production rates, and significantly decreased radial alveolar count compared with controls. Exposure to pyridaben for 72 hours, as expected, caused significant inhibition of C-I and ADP-phosphorylating respiration. Similar to hyperoxic littermates, these pyridaben-exposed mice exhibited significantly delayed alveolarization compared with controls. At 2 weeks of exposure to hyperoxia or pyridaben, mitochondrial respiration was inhibited and associated with alveolar developmental arrest. However, after 3 weeks of recovery from hyperoxia or 2 weeks after 72 hours of exposure to pyridaben alveolarization significantly improved. In addition, there was marked normalization of C-I and mitochondrial respiration. The degree of hyperoxia-induced pulmonary simplification and recovery strongly (r2 = 0.76) correlated with C-I activity in lung mitochondria. Thus, the arrest of alveolar development induced by either hyperoxia or direct inhibition of mitochondrial oxidative phosphorylation indicates that bioenergetic failure to maintain normal alveolar development is one of the fundamental mechanisms responsible for BPD

Supplemental Table 3-mouse LV DEGs.xlsx

Differentially expressed gene in the left ventricle of the mouse following 3 days AngII/PE infusion. This is the mouse model of HFpEF.</p

Image_1_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.JPEG

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

Video_2_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.MOV

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

Supplemental Table 4 mouse-human comp.xlsx

Comparison between mouse LV Day 3 AngII/PE with human HFpEF</p

Video_1_Fibroblast growth factor receptor signaling in cardiomyocytes is protective in the acute phase following ischemia-reperfusion injury.MOV

Fibroblast growth factor receptors (FGFRs) are expressed in multiple cell types in the adult heart. Previous studies have shown a cardioprotective effect of some FGF ligands in cardiac ischemia-reperfusion (I/R) injury and a protective role for endothelial FGFRs in post-ischemic vascular remodeling. To determine the direct role FGFR signaling in cardiomyocytes in acute cardiac I/R injury, we inactivated Fgfr1 and Fgfr2 (CM-DCKO) or activated FGFR1 (CM-caFGFR1) in cardiomyocytes in adult mice prior to I/R injury. In the absence of injury, inactivation of Fgfr1 and Fgfr2 in adult cardiomyocytes had no effect on cardiac morphometry or function. When subjected to I/R injury, compared to controls, CM-DCKO mice had significantly increased myocyte death 1 day after reperfusion, and increased infarct size, cardiac dysfunction, and myocyte hypertrophy 7 days after reperfusion. No genotype-dependent effect was observed on post-ischemic cardiomyocyte cross-sectional area and vessel density in areas remote to the infarct. By contrast, transient activation of FGFR1 signaling in cardiomyocytes just prior to the onset of ischemia did not affect outcomes after cardiac I/R injury at 1 day and 7 days after reperfusion. These data demonstrate that endogenous cell-autonomous cardiomyocyte FGFR signaling supports the survival of cardiomyocytes in the acute phase following cardiac I/R injury and that this cardioprotection results in continued improved outcomes during cardiac remodeling. Combined with the established protective role of some FGF ligands and endothelial FGFR signaling in I/R injury, this study supports the development of therapeutic strategies that promote cardiomyocyte FGF signaling after I/R injury.</p

Nelfinavir inhibits intra-mitochondrial calcium influx and protects brain against hypoxic-ischemic injury in neonatal mice.

Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca(2+)-induced membrane permeabilization, greater ADP-phosphorylating activity and reduced cytochrome C release during reperfusion. Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca(2+) content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca(2+) release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca(2+) content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca(2+) uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca(2+) influx, and did not prevent Ca(2+) induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca(2+) uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca(2+) flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca(2+) overload → mitochondrial membrane permeabilization → secondary energy failure

Nelfinavir limits post-ischemic mitochondrial cytochrome C release, brain infarct volume and cellular mortality.

<p><b>A, B</b> - Western blot analysis for the presence of Cytochrome C (Cyt C) in cytosolic (Cytosol) and mitochondrial (Mitos) fractions obtained from the ipsilateral hemisphere at five hrs after HI insult. β-actin was used as a loading control and cytochrome C oxidase (COX IV) was used as a purity control of cytosolic fraction and loading control for mitochondrial fraction. Quantitative data presented in (B) are expressed in arbitrary OD units normalized to β-actin. Vehicle –treated mice, n = 3, NLF-treated mice, n = 4. <b>C, D</b> - Cerebral infarct volume and representative TTC-stained brain slices obtained at 24 hrs of reperfusion after HI in the vehicle and NLF-treated mice. <b>E, F</b> – Cell mortality at 6 hours of reperfusion following 12 hours of OGD in HT-22 cells treated with vehicle (n = 7), NLF (n = 7) or RuR (n = 7) compared to naives (n = 4). <b>E</b> - Representative images of HT-22 cells in different experimental conditions stained with Propidium Iodide (red) and Hoechst (blue). Note that amount of red cells predominates in OGD-vehicle group. Confocal microscopy. Scale bar = 50 µm. <b>F</b> - Quantitative evaluation of cell mortality. One-way Anova, only significant difference is shown.</p