ALDH2 Knockout Aggravates Pressure Overload Induced Cardiac Dysfunction And Energy Remodeling

Data from clinical and experimental research suggested that alteration of energy metabolism might be an important factor contributing to cardiac hypertrophy/heart failure pathogenesis. The most remarkable alteration is declination in cardiac fatty acid utilization, accompanied with ascend glucose oxidation, which could finally induce energy depletion and mitochondrial dysfunction. The inefficiency energy mode could further induce an impaired ATP production and block the conversion of chemical energy into mechanical power, which then eventually lead to irreversible heart failure. Recent experimental studies demonstrated that AMP-kinase (AMPK) modulators AICAR and metformin could attenuate cardiac remodeling via maintaining myocardial ATP level through activating AMPK, indicating that strategies of improving cardiac energy metabolic profile might be effective for heart failure treatment6. However, it is not always easy to modify/improve metabolic remolding at the end stage of various pathogenesis, and it is essential and more effective to find out and interfere a mediator, which plays a major role at the early disease stage in order to maintain near-normal cardiac metabolic profile and slow down the disease progress.Mitochondrial aldehyde dehydrogenase 2 (ALDH2), a key mitochondrial enzyme, was shown to be an important endogenous protector in a variety of cardiac injuries including endoplasmic reticulum (ER) stress, ischemia reperfusion (I/R) injury by our group and other researchers. Detoxification of toxic aldehydes-energy substrates oxidation by-products, such as 4-hydroxy 2-nonenol (4-HNE), was proven as a major working mechanism for ALDH2. Recently, Jin et al. reported that inactive ALDH2 enzyme enhanced glycolysis and induced metabolic remodeling. Meanwhile, Ren et al. reported elevated phosphorylation of AMPK in murine myocardium overexpressing ALDH2. Till now, the influence of ALDH2 on fatty acid and glucose metabolism, the primary energy sources of adult heart, has not been fully elucidated. We speculate that ALDH2 might also play an important role in the process of cardiac energy substrates utilization via modulating AMPK pathway, besides the known mechanism on detoxication of aldehyde.In the present study, we explored the role of ALDH2 deficiency on cardiac energy substrates utilization, fatty acid and glucose utilization and AMPK, PPARa signaling in a murine model of pressure overload-induced cardiac hypotrophy.PART ONEImpaction of ALDH2 on Pressure Overload induced Cardiac DysfuntionObjective:To explore the difference of cardiac function between ALDH2-/- and WT mice during TAC induced pressure overload.Methods:8-10weeks old WT and ALDH2-/-male mice were separated into TAC and Sham groups randomly. All the sugery mice were separated into four groups depending on different peried of pressure overload, including sham: 2weeks,4weeks and 8weeks. Echocardiography and invasive hemodynamics measurement were used to detect the cardiac function of mice from different groups.Results:There was no difference of basic charactrastics between ALDH2-/-and WT mice; the dysfunction of heart showed by EF and FS was worse in ALDH2-/-mice than WT mice, and the cardiac remodeling was more severe; the significant difference of dysfunction was abserved at 4week after pressure overload, IVS;d<. LVPW;d were thicker and LVESD was wider in ALDH2-/-mice; hemodynamics measurement showed that LVESP was elevated in ALDH2-/-TAC mice.Conclusions:Pressure overload models can be successfully builted in ALDH2-/-mice with TAC.4 weeks after TAC is the most sutible time for further experiment dule to the most significant changes of cardiac function. The impaction of ALDH2 on cardiac dysfunction during pressure overload has been proved by functional examination.PART TWOImpaction of ALDH2 on Cardiac Morphology during Pressure OverloadObjective:To explore the difference of cardiac remodeling, cardiomyocyte hypertrophy and structure of organelles between ALDH2-/-and WT mice during TAC induced pressure overload.Methods:Pressure overload was induced by TAC on 8-10 weeks old male WT and ALDH2-/-mice. Four weeks after TAC, heart were harvested, heart weight, body weight and weat lung weight were measured; HE and MASSON staning were performed on left ventrcal section for cell morphology and fibrosis; TUNEL were used to detect cell apoptosis; electron microscopy were used to explore mitochondria and myocardial fibers structure.Results:After 4 weeks pressure overload, heart from ALDH2-/-mice were significantly enlarged, heavier and accompanied with pulmonary edema; LV middle section area were increased, indicated cardiac remodeling; HE staining showed myocardial hypertrophy and decreased internal density of cardiomyocytes, suggesting more cell damage; fibrogenesis were decreased in ALDH2-/-mice and apoptosis were increased; mitochondria and myocardial fibers structure were damaged more severe in ALDH2-/-mice.Conclusions:ALDH2 knockout promote the cardiac morphology changes induced by pressure overload, including cardiac remodeling, hypertrophy, apoptosis and organelles injury, which may lead to cardiac dysfuntion.PART TWOImpaction of ALDH2 on Cardiac Morphology during Pressure OverloadObjective:To explore the difference of cardiac remodeling, cardiomyocyte hypertrophy and structure of organelles between ALDH2-/-and WT mice during TAC induced pressure overload.Methods:Pressure overload was induced by TAC on 8-10 weeks old male WT and ALDH2-/-mice. Four weeks after TAC, heart were harvested, heart weight, body weight and weat lung weight were measured; HE and MASSON staning were performed on left ventrcal section for cell morphology and fibrosis; TUNEL were used to detect cell apoptosis; electron microscopy were used to explore mitochondria and myocardial fibers structure.Results:After 4 weeks pressure overload, heart from ALDH2-/-mice were significantly enlarged, heavier and accompanied with pulmonary edema; LV middle section area were increased, indicated cardiac remodeling; HE staining showed myocardial hypertrophy and decreased internal density of cardiomyocytes, suggesting more cell damage; fibrogenesis were decreased in ALDH2-/-mice and apoptosis were increased; mitochondria and myocardial fibers structure were damaged more severe in ALDH2-/-mice.Conclusions:ALDH2 knockout promote the cardiac morphology changes induced by pressure overload, including cardiac remodeling, hypertrophy, apoptosis and organelles injury, which may lead to cardiac dysfuntion.PART THREEImpaction of ALDH2 on Pressure Overload induced Cradiac Energy RemodelingObjective:To explore the difference of cardiac energy substrates utilization, including fatty acid, glucose and ATP, between ALDH2-/- and WT mice during TAC induced pressure overload.Methods:Pressure overload was induced by TAC on 8-10 weeks old male WT and ALDH2-/-mice. Cardiaomyocytes were cultured and stretched. Four weeks after TAC and 12 hours after stretch, heart and cells were harvested, acyl-CoA and acyl-carnitine were measured by LC-MS to indicate fatty acid utilization; glucose uptake were measured by 18F-FDG PET-CT and y counting; ATP level were measured by luciferase kit.Results:At basal condition, acyl-CoA of ALDH2-/-mice were increased and acyl-carnitine were decreased, which indicated the accumulation of acyl-CoA and inhibition of fatty acid transport into mitochondria; after 4weeks TAC acyl-CoA and acyl-carnitine were both decreased; glucose up take were increased in both WT and ALDH2-/-groups after pressure overload; ATP were on the contrary, decreased in ALDH2-/-TAC mice. Same results were observed at in-vitro experiments.Conclusions:ALDH2 knockout inhibit cardiac fatty acid transportation and increased glucose utilization, lead to cardiac energy remodeling and ATP scareity. weeks after TAC and 12 hours after stretch, heart and cells were harvested, acyl-CoA and acyl-carnitine were measured by LC-MS to indicate fatty acid utilization; glucose uptake were measured by 18F-FDG PET-CT and y counting; ATP level were measured by luciferase kit.Results:At basal condition, acyl-CoA of ALDH2-/-mice were increased and acyl-carnitine were decreased, which indicated the accumulation of acyl-CoA and inhibition of fatty acid transport into mitochondria; after 4weeks TAC acyl-CoA and acyl-carnitine were both decreased; glucose up take were increased in both WT and ALDH2-/-groups after pressure overload; ATP were on the contrary, decreased in ALDH2-/-TAC mice. Same results were observed at in-vitro experiments.Conclusions:ALDH2 knockout inhibit cardiac fatty acid transportation and increased glucose utilization, lead to cardiac energy remodeling and ATP scareity.PART FOURSignaling Pathways Involved in the Effects of ALDH2 on Cradiac Energy Remodeling during Pressure OverloadObjective:To explore the signaling pathways that ALDH2-/-involved in cardiac energy remodeling during TAC induced pressure overload.Methods:Pressure overload was induced by TAC on 8-10 weeks old male WT and ALDH2-/-mice. Cardiaomyocytes were cultured and stretched. Four weeks after TAC and 12 hours after stretch, heart and cells were harvested, p-AMPK, AMPK and PPARa protein expresstion were measured by western-blot; mRNA level of fatty acid transporter mCPT-I and glucose oxidation key enzyme PDH were were measured by real-time PCR.Results:p-AMPK and PPARa were significantly decreased in ALDH2-/-mice, but there was no different between TAC and Sham groups, which implayed that ALDH2 could regulate energy signal independently; downstream fatty acid pathway mCPT-I were decreased both by pressure overload and ALDH2 knockout, but PDH had no difference within four groups mice. Same results were observed at in-vitro experiments.Conclusions:Through the exploration of signaling pathways, p-AMPK/PPARa - CPT-I were proved as the most possible mechanism which involved in ALDH2 induced cardiac energy remodeling.