DKO Hearts Research Articles

Reduced fatty acid uptake aggravates cardiac contractile dysfunction in streptozotocin-induced diabetic cardiomyopathy

Diabetes is an independent risk factor for the development of heart failure. Increased fatty acid (FA) uptake and deranged utilization leads to reduced cardiac efficiency and accumulation of cardiotoxic lipids, which is suggested to facilitate diabetic cardiomyopathy. We studied whether reduced FA uptake in the heart is protective against streptozotocin (STZ)-induced diabetic cardiomyopathy by using mice doubly deficient in fatty acid binding protein 4 (FABP4) and FABP5 (DKO mice). Cardiac contractile dysfunction was aggravated 8 weeks after STZ treatment in DKO mice. Although compensatory glucose uptake was not reduced in DKO-STZ hearts, total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. Tracer analysis with 13C6-glucose revealed that accelerated glycolysis in DKO hearts was strongly suppressed by STZ treatment. Levels of ceramides, cardiotoxic lipids, were similarly elevated by STZ treatment. These findings suggest that a reduction in total energy supply by reduced FA uptake and suppressed glycolysis could account for exacerbated contractile dysfunction in DKO-STZ hearts. Thus, enhanced FA uptake in diabetic hearts seems to be a compensatory response to reduced energy supply from glucose, and therefore, limited FA use could be detrimental to cardiac contractile dysfunction due to energy insufficiency.

JDP2 and ATF3 deficiencies dampen maladaptive cardiac remodeling and preserve cardiac function.

c-Jun dimerization protein (JDP2) and Activating Transcription Factor 3 (ATF3) are closely related basic leucine zipper proteins. Transgenic mice with cardiac expression of either JDP2 or ATF3 showed maladaptive remodeling and cardiac dysfunction. Surprisingly, JDP2 knockout (KO) did not protect the heart following transverse aortic constriction (TAC). Instead, the JDP2 KO mice performed worse than their wild type (WT) counterparts. To test whether the maladaptive cardiac remodeling observed in the JDP2 KO mice is due to ATF3, ATF3 was removed in the context of JDP2 deficiency, referred as double KO mice (dKO). Mice were challenged by TAC, and followed by detailed physiological, pathological and molecular analyses. dKO mice displayed no apparent differences from WT mice under unstressed condition, except a moderate better performance in dKO male mice. Importantly, following TAC the dKO hearts showed low fibrosis levels, reduced inflammatory and hypertrophic gene expression and a significantly preserved cardiac function as compared with their WT counterparts in both genders. Consistent with these data, removing ATF3 resumed p38 activation in the JDP2 KO mice which correlates with the beneficial cardiac function.Collectively, mice with JDP2 and ATF3 double deficiency had reduced maladaptive cardiac remodeling and lower hypertrophy following TAC. As such, the worsening of the cardiac outcome found in the JDP2 KO mice is due to the elevated ATF3 expression. Simultaneous suppression of both ATF3 and JDP2 activity is highly beneficial for cardiac function in health and disease.

ACE-inhibition induces a cardioprotective transcriptional response in the metabolic syndrome heart

Cardiovascular disease associated with metabolic syndrome has a high prevalence, but the mechanistic basis of metabolic cardiomyopathy remains poorly understood. We characterised the cardiac transcriptome in a murine metabolic syndrome (MetS) model (LDLR−/−; ob/ob, DKO) relative to the healthy, control heart (C57BL/6, WT) and the transcriptional changes induced by ACE-inhibition in those hearts. RNA-Seq, differential gene expression and transcription factor analysis identified 288 genes differentially expressed between DKO and WT hearts implicating 72 pathways. Hallmarks of metabolic cardiomyopathy were increased activity in integrin-linked kinase signalling, Rho signalling, dendritic cell maturation, production of nitric oxide and reactive oxygen species in macrophages, atherosclerosis, LXR-RXR signalling, cardiac hypertrophy, and acute phase response pathways. ACE-inhibition had a limited effect on gene expression in WT (55 genes, 23 pathways), and a prominent effect in DKO hearts (1143 genes, 104 pathways). In DKO hearts, ACE-I appears to counteract some of the MetS-specific pathways, while also activating cardioprotective mechanisms. We conclude that MetS and control murine hearts have unique transcriptional profiles and exhibit a partially specific transcriptional response to ACE-inhibition.

Ablation of MMP9 Prevents Cardiac Pyroptosis of Ins2+/− T1DM Mice Heart

Pyroptosis, a caspase‐1‐dependent cell death mechanism, is upregulated in the diabetes mellitus (DM) heart. However, its underlying regulatory mechanisms are unclear. Here, we investigated the regulatory role of matrix metalloproteinase‐9 (MMP9), which is upregulated and promotes pathological remodeling, on pyroptosis of the DM heart. We hypothesized that upregulated MMP9 in the DM heart induces cardiac pyroptosis, which can be rescued by ablation of MMP9. To test the hypothesis, we created a novel DM mice strain where MMP9 gene is deleted (Ins2+/−/MMP9−/− or DKO), by crossbreeding Ins2+/− Akita (spontaneous, genetic T1DM model) male with MMP9−/− (MMP9KO) female mice. To assess the effect of DM on cardiac pyroptosis, we used Ins2+/− Akita and its sibling Ins2+/+ WT mice. For all experiments, we used fourteen‐week male mice. For in vitro studies, we used HL1 cells (cardiomyocyte cell line of murine origin) and generated MMP9−/− HL1 cells by CRISPER/Cas9 method. We treated HL1 and MMP9−/− HL1 cells with normal (NG= 5 mM) or high (HG= 25 mM) doses of D‐glucose for 24 hours. We measured the activity of MMP9 by in‐situ and in‐gel gelatin zymography. For pyroptosis, we measured the protein levels of NLRP3 (involved in the inflammasome complex formation that is required for pyroptosis initiation), caspase‐1 (the key enzyme for pyroptosis), and interleukine‐1β (IL‐1β) and interleukine‐18 (IL‐18) (the pro‐inflammatory cytokines that are cleaved by caspase‐1) by Western blotting. We corroborated cell death by pyroptosis using LDH release assay in vitro. We found absence of MMP9 activity in DKO hearts and MMP9−/− HL1 cardiomyocytes, and upregulation of MMP9 in Akita hearts and hyperglycemic HL1 cardiomyocytes, which supports our published reports and validate our model system. The densitometric analyses of Western blot bands revealed that pyroptosis markers are upregulated in Akita hearts but they are decreased in DKO hearts. The protein levels (mean± SE) of different pyroptosis markers in the heart are: NLRP3: WT 1.6 ± 0.2, Akita 2.5 ± 0.2, DKO 0.4 ± 0.0; cleaved/total caspase‐1: WT 0.2 ± 0.0, Akita 0.3 ± 0.0, DKO 0.2 ± 0.0; cleaved/total IL‐1β: WT 0.3 ± 0.0, Akita 0.5 ± 0.0, DKO 0.1 ± 0.0; cleaved IL‐18: WT 0.4 ± 0.0, Akita 1.0 ± 0.0, DKO 0.2 ± 0.0 (decimal values are rounded). The in vitro studies showed similar patterns of protein levels (mean± SE): NLRP3: HL1+NG 0.08 ± 0.02, HL1+HG 0.14 ± 0.01, MMP9−/− HL1+HG 0.08 ± 0.00; caspase‐1: HL1+NG 0.6 ± 0.1, HL1+HG 1.0 ± 0.08, MMP9−/− HL1+HG 0.40 ± 0.05; IL‐1β: HL‐1+NG 0.60 ± 0.03, HL‐1+HG 0.80 ± 0.04, MMP9−/− HL1+HG 0.10 ± 0.01; IL‐18: HL‐1+NG 1.60 ± 0.20, HL‐1+HG 2.40± 0.20, MMP9−/− HL1+HG 0.20 ± 0.03. The LDH release assay showed that high‐glucose treatment causes increased death of cardiomyocytes (absorbance at 490–690 nm for red formazan formation: HL1+NG 0.20 ± 0.01, HL1+HG 0.40 ± 0.00), which was decreased by ablation of MMP9 (MMP9−/− HL1+HG 0.10 ± 0.01). In conclusion, we have demonstrated for the first time that MMP9 inhibition could alleviate pyroptosis of the DM heart.Support or Funding InformationThis study is supported by the NIH grants HL‐113281 and HL‐116205 to P.K.M.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Robust suppression of cardiac energy catabolism with marked accumulation of energy substrates during lipopolysaccharide-induced cardiac dysfunction in mice

BackgroundMyocardial contractile dysfunction in sepsis has been attributed mainly to increased inflammatory cytokines, insulin resistance, and impaired oxidative phosphorylation of fatty acids (FAs). However, precise molecular mechanisms underlying the cardiac dysfunction in sepsis remain to be determined. We previously reported major shift in myocardial energy substrates from FAs to glucose, and increased hepatic ketogenesis in mice lacking fatty acid-binding protein 4 (FABP4) and FABP5 (DKO). PurposeWe sought to determine whether a shift of energy substrates from FAs to glucose and increased availability of ketone bodies are beneficial or detrimental to cardiac function under the septic condition. MethodsLipopolysaccharide (LPS, 10mg/kg) was intraperitoneally injected into wild-type (WT) and DKO mice. Twelve hours after injection, cardiac function was assessed by echocardiography and serum and hearts were collected for further analyses. ResultsCardiac contractile function was more deteriorated by LPS injection in DKO mice than WT mice despite comparable changes in pro-inflammatory cytokine production. LPS injection reduced myocardial uptake of FA tracer by 30% in both types of mice, while uptake of the glucose tracer did not significantly change in either group of mice in sepsis. Storage of glycogen and triacylglycerol in hearts was remarkably increased by LPS injection in both mice. Metabolome analysis revealed that LPS-induced suppression of pool size in the TCA cycle was more enhanced in DKO hearts. A tracing study with 13C6-glucose further revealed that LPS injection substantially reduced glucose-derived metabolites in the TCA cycle and related amino acids in DKO hearts. Consistent with these findings, glucose oxidation in vitro was similarly and markedly reduced in both mice. Serum concentration of β-hydroxybutyrate and cardiac expression of genes associated with ketolysis were reduced in septic mice. ConclusionsOur study demonstrated that LPS-induced cardiac contractile dysfunction is associated with the robust suppression of catabolism of energy substrates including FAs, glucose and ketone bodies and accumulation of glycogen and triacylglycerol in the heart. Thus, a fuel shift from FAs to glucose and/or ketone bodies may be detrimental rather than protective under septic conditions.

Abstract 269: Epicardial Deletion of Myocardin-related Transcription Factors Prevent Pathological Cardiac Remodeling After Ischemic Injury

Epicardium-derived progenitor cell (EPDC) activation after injury is a cardio-restorative mechanism in the zebrafish heart, but this response is limited in adult mammals. Identification of mechanisms that regulate adult EPDC function is critical to enhance epicardium-mediated cardiac repair. We previously found that myocardin-related transcription factors (MRTFs) regulate EPDC mobilization and coronary vessel maturation during development. Adult mice lacking both MRTF-A and -B in the epicardium (DKO) were evaluated at baseline and after myocardial infarction (MI) in this study. In non-injured DKO hearts, MRTF expression is reduced in epicardium-derived lineages such as fibroblasts and pericytes, but is unchanged in cardiomyocytes and endothelial cells. DKO mice display increased cellular retention in the epicardium as compared to wildtype mice (WT), consistent with observations during embryonic development. While WT mice demonstrate a decline in cardiac function after MI, ejection fraction is preserved in DKO mice, which also display reduced fibrotic scarring and cardiomyocyte hypertrophy. In order to determine the expression levels of pro-fibrotic genes in epicardial-derived lineages, we isolated CD31-NG2+PDGFRβ+ (pericytes), CD31-NG2-PDGFRβ- (fibroblasts) and CD31+ (endothelial cells) populations from sham and 7-day post-MI hearts. Postn (11-fold), Col1a1 (4-fold), Col3a1 (4.8-fold) and Acta2 (5-fold) are similarly activated in pericyte and fibroblast populations after MI. Of note, pericytes express abundant levels of fibronectin (38-fold vs. 16-fold in fibroblasts) and pro-inflammatory interleukin-1β (41-fold vs 12-fold in fibroblasts) suggesting that pericytes may also contribute to inflammation and fibrosis after cardiac injury. Indeed, NG2+ pericytes are dramatically reduced in remote (5-fold) and damaged regions (3.3-fold) of DKO hearts compared to WT. From this data, we hypothesize that MRTF deletion alters pro-migratory and fibrotic gene programs and reduces inflammatory signals in EPDCs decreasing the presence of scar and improving cardiac function after ischemic injury.

Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning.

Anesthetic preconditioning (APC) is a clinically important phenomenon in which volatile anesthetics (VAs) protect tissues such as heart against ischemic injury. The mechanism of APC is thought to involve K channels encoded by the Slo gene family, and the authors showed previously that slo-2 is required for APC in Caenorhabditis elegans. Thus, the authors hypothesized that a slo-2 ortholog may mediate APC-induced cardioprotection in mammals. A perfused heart model of ischemia-reperfusion injury, a fluorescent assay for K flux, and mice lacking Slo2.1 (Slick), Slo2.2 (Slack), or both (double knockouts, Slo2.x dKO) were used to test whether these channels are required for APC-induced cardioprotection and for cardiomyocyte or mitochondrial K transport. In wild-type (WT) hearts, APC improved post-ischemia-reperfusion functional recovery (APC = 39.5 ± 3.7% of preischemic rate × pressure product vs. 20.3 ± 2.3% in controls, means ± SEM, P = 0.00051, unpaired two-tailed t test, n = 8) and lowered infarct size (APC = 29.0 ± 4.8% of LV area vs. 51.4 ± 4.5% in controls, P = 0.0043, n = 8). Protection by APC was absent in hearts from Slo2.1 mice (% recovery APC = 14.6 ± 2.6% vs. 16.5 ± 2.1% in controls, P = 0.569, n = 8 to 9, infarct APC = 52.2 ± 5.4% vs. 53.5 ± 4.7% in controls, P = 0.865, n = 8 to 9). APC protection was also absent in Slo2.x dKO hearts (% recovery APC = 11.0 ± 1.7% vs. 11.9 ± 2.2% in controls, P = 0.725, n = 8, infarct APC = 51.6 ± 4.4% vs. 50.5 ± 3.9% in controls, P = 0.855, n = 8). Meanwhile, Slo2.2 hearts responded similar to WT (% recovery APC = 41.9 ± 4.0% vs. 18.0 ± 2.5% in controls, P = 0.00016, n = 8, infarct APC = 25.2 ± 1.3% vs. 50.8 ± 3.3% in controls, P < 0.000005, n = 8). Furthermore, VA-stimulated K transport seen in cardiomyocytes or mitochondria from WT or Slo2.2 mice was absent in Slo2.1 or Slo2.x dKO. Slick (Slo2.1) is required for both VA-stimulated K flux and for the APC-induced cardioprotection.

Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4–6 weeks, leading to knockout of both these proteins in 8–10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca2+-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca2+ and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Ablation of Matrix Metalloproteinase-9 Prevents Cardiomyocytes Contractile Dysfunction in Diabetics.

Elevated expression and activity of matrix metalloproteinase-9 (MMP9) and decreased contractility of cardiomyocytes are documented in diabetic hearts. However, it is unclear whether MMP is involved in the regulation of contractility of cardiomyocytes in diabetic hearts. In the present study, we tested the hypothesis that MMP9 regulates contractility of cardiomyocytes in diabetic hearts, and ablation of MMP9 prevents impaired contractility of cardiomyocytes in diabetic hearts. To determine the specific role of MMP9 in cardiomyocyte contractility, we used 12–14 week male WT (normoglycemic sibling of Akita), Akita, and Ins2+∕−/MMP9−∕− (DKO) mice. DKO mice were generated by cross-breeding male Ins2+∕− Akita (T1D) with female MMP9 knockout (MMP9−∕−) mice. We isolated cardiomyocytes from the heart of the above three groups of mice and measured their contractility and calcium transients. Moreover, we determined mRNA and protein levels of sarco-endoplasmic reticulum calcium ATPase-2a (SERCA-2a), which is involved in calcium handling during contractility of cardiomyocytes in WT, Akita, and DKO hearts using QPCR, Western blotting and immunoprecipitation, respectively. Our results revealed that in Akita hearts where increased expression and activity of MMP9 is reported, the rates of shortening and re-lengthening (±dL/dt) of cardiomyocytes were decreased, time to 90% peak height and baseline during contractility was increased, rate of calcium decay was increased, and calcium transient was decreased as compared to WT cardiomyocytes. However, these changes in Akita were blunted in DKO cardiomyocytes. The molecular analyses of SERCA-2a in the hearts showed that it was downregulated in Akita as compared to WT but was comparatively upregulated in DKO. These results suggest that abrogation of MMP9 gene prevents contractility of cardiomyocytes, possibly by increasing SERCA-2a and calcium transients. We conclude that MMP9 plays a crucial role in the regulation of contractility of cardiomyocytes in diabetic hearts.

Phospholamban-Independent Adrenergic Reserve in SERCA2 Ablated Hearts

Normal cardiac contraction and relaxation requires efficient operation of the SR Ca2+ ATPase, SERCA2a. In failing hearts, decreased SERCA2a expression and slowed SR Ca2+ reuptake are thought to significantly underlie contractile dysfunction. We have studied the relationship between decreased SERCA2a expression and cardiac function using a mouse model of conditional cardiac Serca2 ablation, the Serca2fl/fl mouse. In this model, Cre activation by tamoxifen injection efficiently deletes Serca2 from the heart; four weeks post-knockout, cardiac SERCA2a protein content is below 5% of normal levels. Serca2 KO mice survive 7-10 weeks post knockout with only mild in vivo impairment prior to this time, suggesting that this loss of SERCA2a protein can be temporarily compensated. We found that isolated SERCA2a KO hearts retained the ability to respond to s-adrenergic stimulation ex vivo despite the loss of most SERCA2a protein. Because the protein regulator of SERCA2a, phospholamban (PLN), is thought to be a major component of the cardiac s-adrenergic response, this finding was unexpected and warranted further detailed investigation. To identify the mechanism underlying this preserved s-adrenergic response in SERCA2-depleted hearts, we bred Serca2fl/fl and PLN-/- mice to generate an inducible Serca2 knockout mouse line lacking PLN. Serca2fl/fl;PLN-/- mice were injected with tamoxifen to induce Serca2 gene disruption, and isolated hearts from Serca2KO;PLN-/- (“DKO”) mice were evaluated by Langendorff perfusion 4-5 weeks after Serca2 knockout. Control PLN-/- hearts show little response to s-adrenergic stimulation, and DKO hearts have impaired contractility under baseline conditions. DKO hearts, however, show improved systolic and diastolic function when perfused with 50 nM isoproterenol, indicating that targets of s-adrenergic signaling beyond PLN are able to support a significant inotropic and lusitropic response when cardiac contractility is impaired. We will discuss how these findings support new mechanisms underlying heart performance during stress and disease.

THE ADULT MURINE HEART IS PROTECTED AGAINST ISCHEMIA-REPERFUSION INJURY IN THE ABSENCE OF BOTH MITOFUSIN (MFN) PROTEINS

BackgroundCardiac-specific ablation of both Mfn1 and Mfn2 in the adult heart results in mitochondrial fragmentation and a lethal cardiomyopathy after about 6 weeks. The effect of combined Mfn1 and Mfn2...

Identification of apolipoprotein D as a cardioprotective gene using a mouse model of lethal atherosclerotic coronary artery disease

Mice with homozygous null mutations in the HDL receptor (scavenger receptor class B, type I, or SR-BI) and apolipoprotein E (apoE) genes [SR-BI/apoE double KO (SR-BI(-/-)/apoE(-/-) or dKO) mice] spontaneously develop occlusive, atherosclerotic coronary artery disease (CAD) and die prematurely (50% mortality at 42 d of age). Using microarray mRNA expression profiling, we identified genes whose expression in the hearts of dKO mice changed substantially during disease progression [at 21 d of age (no CAD), 31 d of age (small myocardial infarctions), and 43 d of age (extensive myocardial infarctions) vs. CAD-free SR-BI(+/-)/apoE(-/-) controls]. Expression of most genes that increased >sixfold in dKO hearts at 43 d also increased after coronary artery ligation. We examined the influence and potential mechanism of action of apolipoprotein D (apoD) whose expression in dKO hearts increased 80-fold by 43 d. Analysis of ischemia/reperfusion-induced myocardial infarction in both apoD KO mice and wild-type mice with abnormally high plasma levels of apoD (adenovirus-mediated hepatic overexpression) established that apoD reduces myocardial infarction. There was a correlation of apoD's ability to protect primary cultured rat cardiomyocytes from hypoxia/reoxygenation injury with its potent ability to inhibit oxidation in a standard antioxidation assay in vitro. We conclude that dKO mice represent a useful mouse model of CAD and apoD may be part of an intrinsic cardioprotective system, possibly as a consequence of its antioxidation activity.

Pocket proteins critically regulate cell cycle exit of the trabecular myocardium and the ventricular conduction system

SummaryDuring development, the ventricular conduction system (VCS) arises from the trabecular or spongy myocardium. VCS and trabecular myocytes proliferate at a significantly slower rate than compact zone myocardial cells, establishing a transmural cell cycle gradient. The molecular determinants of VCS/trabecular myocyte cell cycle arrest are not known. Given the importance of pocket proteins (Rb, p107 and p130) in mediating G0/G1 arrest in many cell types, we examined the role of this gene family in regulating cell cycle exit of the trabecular myocardium and ventricular conduction system. Using a combinatorial knockout strategy, we found that graded loss of pocket proteins results in a spectrum of heart and lung defects. p107/p130 double knockout (dKO) hearts manifest dysregulated proliferation within the compact myocardium and trabecular bases, while the remaining trabecular region cell cycle exits normally. Consequently, dKO hearts exhibit defective cardiac compaction, septal hyperplasia and biventricular outflow tract obstruction, while the VCS appears relatively normal. Loss of all three pocket proteins (3KO) is necessary to completely disrupt the transmural cell cycle gradient. 3KO hearts exhibit massive overgrowth of the trabecular myocardium and ventricular conduction system, which leads to fetal heart failure and death. Hearts carrying a single pocket protein allele are able to maintain the transmural cell cycle gradient. These results demonstrate the exquisite sensitivity of trabecular and conduction myocytes to pocket protein function during ventricular chamber development.

Ablation of MMP9 ameliorates epigenetic modifications and mitigates diabetic cardiomyopathy

Mutation of Insulin2 gene (orthologous to human Insulin) activates latent matrix metalloproteinase‐9 (MMP9) that fosters cardiomyopathy. Epigenetic modifications such as DNA methylation through DNA methyl transferases (DNMTs) and histone de‐acetylation by HDAC play crucial role in cardiac dysfunction. However, the epigenetic signature of MMP9 in diabetic hearts is unclear. We hypothesize that abrogation of MMP9 attenuates DNA methylation and histone de‐acetylation in diabetic hearts and ameliorates contractile dysfunction. To test the hypothesis, we created double knock out (DKO: Ins2+/−/MMP9−/−) mice by targeted deletion of MMP9 in Ins2+/− Akita, and determined the levels of DNMT‐1, HDAC, H3K9 and sarco‐endoplasmic reticulum ATPase‐2a (serca‐2a) in WT (C57BL/6J), Ins2+/− (Akita) and DKO hearts by RT‐QPCR, Western blot and confocal microscopy. We also measured contractility of cardiomyocytes using Ion‐optics device. The results revealed that DNMT‐1, HDAC and H3K9 are robust in Akita but alleviated in DKO hearts. Conversely, serca‐2a is down regulated in Akita but induced in DKO. The rate of contraction and relaxation (±dL/dt) is improved in DKO as compared to Akita. We conclude that targeted deletion of MMP9 ameliorates diabetic cardiomyopathy through epigenetic modifications.

Abstract 19599: Loss of Alpha-Catenins Increases Nuclear Yap and Cardiomyocyte Proliferation and Improves Cardiac Function Following Myocardial Infarction

Background: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional co-activator Yap is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators that control Yap activity in the heart remain largely unknown. Objective: To investigate the role of α-catenins in the heart using cardiac-specific αE- and αT-catenin double knockout (α-cat DKO) mice. Methods: We used two cardiac-specific Cre transgenes to delete both αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes either in the perinatal or in the adult heart, and performed a series of experiments to examine the role of α-catenins under normal and pathological conditions. Results: Postnatal depletion of α-catenins caused a decrease in cardiomyocyte size and an increase in cardiomyocyte number in the α-cat DKO mice. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the α-cat DKO hearts. In comparison, ablation of α-catenins in the adult heart did not result in a proliferative phenotype. However, inactivation of α-catenins improved cardiac function following myocardial infarction. Increased nuclear Yap along with increased number of cells in cytokinesis indicated that cardiomyocyte proliferation contributed, at least in part, to the improved contractile function in the α-cat DKO heart following injury. Conclusions: These studies demonstrate that α-catenins are critical regulators of Yap, a transcriptional co-activator essential for cardiomyocyte proliferation. Furthermore, we provide proof-of-concept that inhibiting α-catenins might be a useful strategy to promote myocardial regeneration following injury.

Loss of Cadherin-Binding Proteins β-Catenin and Plakoglobin in the Heart Leads to Gap Junction Remodeling and Arrhythmogenesis

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.

Abstract P123: β- and γ-Catenin Cooperate to Maintain Mechanoelectrical Coupling in the Heart

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart-muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome including plakoglobin (also known as γ-catenin). Plakoglobin (PG) is a unique cell adhesion molecule as it is located in both desmosomes and adherens junctions located at the intercalated disc (ID) where it functions to link cadherins to the cytoskeleton. Redistribution of PG away from ID is found in virtually all cases of ARVC, regardless of the specific mutation, suggesting that PG plays a fundamental role in the pathogenesis of ARVC. To investigate the role of PG in ARVC, we generated an inducible cardiac-restricted knockout (CKO) of the PG gene in mice. Despite gap junction remodelling, PG CKO mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin, showed increased association with the gap junction protein, connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin protects PG mutant animals from sudden arrhythmic death, we generated mice lacking both PG and β-catenin specifically in the heart (i.e. double knockout, DKO). The PG/β-catenin DKO mice exhibited acute cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD 3 – 5 months (124 day median survival) after deleting the genes. The PG/β-catenin DKO hearts were susceptible to induced arrhythmias (8/11 DKO versus 0/9 WT, p &lt; 0.01) consistent with the SCD phenotype. Furthermore, spontaneous lethal arrhythmias were captured in PG/β-catenin DKO mice with telemetric monitoring. In contrast to the PG and β-catenin single mutants, N-cadherin was significantly reduced at the ID in the PG/β-catenin DKO mice. Consistent with decreased desmoglein-2 and plakophilin-2 at the ID, quantitative analysis of TEM images demonstrated a significant reduction in the number of desmosomes (7.36 ± 3.68 DKO versus 36.37 ± 4.44 WT per 100-µm ID; n = 10 fields per heart; p &lt; 0.001). In conclusion, these studies suggest that increasing β-catenin interaction with Cx43 may enhance gap junction function in ARVC patients, thus improving electrical conduction in the heart.

Cardiomyocyte‐restricted PPARδ deletion in PPARα null mice leads to cardiac hypertrophy and heart failure

Mice with cardiac PPARδ deficiency exhibit basal fatty acid oxidation (FAO) and bioenergetic deficits, leading to cardiac hypertrophy and heart failure. While PPARα also regulates cardiac FAO gene expression, PPARα null mice display no basal cardiac phenotype. We crossed cardiomyocyte‐restricted the PPARδ−/− (CR‐PPARδ−/−) mice with the PPARα null mice to generate a mouse line that PPARδ is eliminated specifically from cardiomyocytes of the PPARα mice (DKO). QPCR revealed that transcript expression of FAO genes in PPARδ null hearts was depressed similarly to those in PPARα null hearts. Double knockout of both PPARδ and PPARα did not lead to further depression of selected FAO gene expression compared with those from PPARδ and PPARα single knockout hearts. The protein abundance of these FAO enzymes was similarly depressed among these mice. There was no overt basal pathophysiological change in PPARα null hearts. In contrast, pathophysiological changes in the DKO hearts were similar to those of CR‐PPARδ−/− hearts. DKO mice exhibited cardiac dysfunction, mitochondria abnormality and cardiac hypertrophy after 4–5 month of their ages. However, it is interesting that DKO mice exhibited generally delayed dilated cardiomyopathy with lipid overload when compared with the CR‐PPARδ−/− hearts. We conclude that PPARδ plays a more essential role than PPARα does in the heart at basal condition.

Unmasking different mechanical and energetic roles for the small heat shock proteins CryAB and HSPB2 using genetically modified mouse hearts

CryAB and HSPB2 are small heat shock proteins constitutively expressed in the heart. CryAB protects cytoskeletal organization and intermediate filament assembly; the functions of HSPB2 are unknown. The promoters of CryAB and HSPB2 share regulatory elements, making identifying their separate functions difficult. Here, using a genetic approach, we report distinct roles for these sHSPs, with CryAB protecting mechanical properties and HSPB2 protecting energy reserve. Isolated hearts of wild type mice (WT), mice lacking both sHSPs (DKO), WT mice overexpressing mouse CryAB protein (mCryAB(Tg)), and mice with no HSPB2 made by crossing DKO with mCryAB(Tg) (DKO/mCryAB(Tg)) were stressed with either ischemia/reperfusion or inotropic stimulation. Contractile performance and energetics were measured using 31P NMR spectroscopy. Ischemia/reperfusion caused severe diastolic dysfunction in DKO hearts. Recovery of [ATP] and [PCr] during reperfusion was impaired only in DKO/mCryAB(Tg). During inotropic stimulation, DKO/mCryAB(Tg) showed blunted systolic and diastolic function and revealed massive energy wasting on acute stress: |deltaG(-ATP)| decreased in DKO by 6.4 +/- 0.7 and in DKO/mCryAB(Tg) by 5.5 +/- 0.8 kJ/mol compared with only approximately 3.3 kJ/mol in WT and mCryAB(Tg). Thus, CryAB and HSPB2 proteins play nonredundant roles in the heart, CryAB in structural remodeling and HSPB2 in maintaining energetic balance.

Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling

Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.