IRE1 phosphatase PP2Ce regulates adaptive ER stress response in the postpartum mammary gland.

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium

Catabolism of Branched-Chain Amino Acids in Heart Failure: Insights from Genetic Models

Genetic defects in amino acid metabolism are major causes of newborn diseases that often lead to abnormal development and function of the central nervous system. Their direct impact on cardiac development and function has rarely been investigated. Recently, the authors have established that a mitochondrial targeted 2C-type ser/thr protein phosphatase, PP2Cm, is the endogenous phosphatase of the branched-chain alpha keto acid-dehydrogenase complex (BCKD) and functions as a key regulator in branched-chain amino acid catabolism and homeostasis. Genetic inactivation of PP2Cm in mice leads to significant elevation in plasma concentrations of branched-chain amino acids and branched-chain keto acids at levels similar to those associated with intermediate mild forms of maple syrup urine disease. In addition to neuronal tissues, PP2Cm is highly expressed in cardiac muscle, and its expression is diminished in a heart under pathologic stresses. Whereas phenotypic features of heart failure are seen in PP2Cm-deficient zebra fish embryos, cardiac function in PP2Cm-null mice is compromised at a young age and deteriorates faster by mechanical overload. These observations suggest that the catabolism of branched-chain amino acids also has physiologic significance in maintaining normal cardiac function. Defects in PP2Cm-mediated catabolism of branched-chain amino acids can be a potential novel mechanism not only for maple syrup urine disease but also for congenital heart diseases and heart failure

Therapeutic Effect of Targeting Branched-Chain Amino Acid Catabolic Flux in Pressure-Overload Induced Heart Failure.

Background Branched-chain amino acid (BCAA) catabolic defect is an emerging metabolic hallmark in failing hearts in human and animal models. The therapeutic impact of targeting BCAA catabolic flux under pathological conditions remains understudied. Methods and Results BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), a small-molecule inhibitor of branched-chain ketoacid dehydrogenase kinase, was used to enhance BCAA catabolism. After 2 weeks of transaortic constriction, mice with significant cardiac dysfunctions were treated with vehicle or BT2. Serial echocardiograms showed continuing pathological deterioration in left ventricle of the vehicle-treated mice, whereas the BT2-treated mice showed significantly preserved cardiac function and structure. Moreover, BT2 treatment improved systolic contractility and diastolic mechanics. These therapeutic benefits appeared to be independent of impacts on left ventricle hypertrophy but associated with increased gene expression involved in fatty acid utilization. The BT2 administration showed no signs of apparent toxicity. Conclusions Our data provide the first proof-of-concept evidence for the therapeutic efficacy of restoring BCAA catabolic flux in hearts with preexisting dysfunctions. The BCAA catabolic pathway represents a novel and potentially efficacious target for treatment of heart failure

Glucagon Receptor Antagonism Ameliorates Progression of Heart Failure.

Mice were treated with a fully human monoclonal glucagon receptor antagonistic antibody REMD2.59 following myocardial infarction or pressure overload. REMD2.59 treatment blunted cardiac hypertrophy and fibrotic remodeling, and attenuated contractile dysfunction at 4 weeks after myocardial infarction. In addition, REMD2.59 treatment at the onset of pressure overload significantly suppressed cardiac hypertrophy and chamber dilation with marked preservation of cardiac systolic and diastolic function. Initiation of REMD2.59 treatment 2 weeks after pressure overload significantly blunted the progression of cardiac pathology. These results provide the first in vivo proof-of-concept evidence that glucagon receptor antagonism is a potentially efficacious therapy to ameliorate both onset and progression of heart failure

Low-volume jet injection for intradermal immunization in rabbits

BACKGROUND: This study tested a low-volume (20–30 μl/20–30 μg DNA) jet injection method for intradermal delivery of a DNA vaccine. Jet injection offers the advantages of a needle-less system, low-cost, rapid preparation of the injected DNA solution, and a simple delivery system. More than one construct can be injected simultaneously and the method may be combined with adjuvants. RESULTS: Low-volume jet injection targeted delivery of a DNA solution exclusively to the dermis and epidermis of rabbits. A three injection series of plasmid DNA, encoding the Hepatitis B Surface Antigen stimulated a humoral immune response in 2/5 rabbits. One rabbit developed a significant rise in antibody titer after 1 injection and one following 2 injections. There were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10–100 times higher than jet injection. CONCLUSION: Low-volume jet injection is a simple, effective methodology for intradermal DNA immunization

Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.

We aimed to understand the genetic control of cardiac remodeling using an isoproterenol-induced heart failure model in mice, which allowed control of confounding factors in an experimental setting. We characterized the changes in cardiac structure and function in response to chronic isoproterenol infusion using echocardiography in a panel of 104 inbred mouse strains. We showed that cardiac structure and function, whether under normal or stress conditions, has a strong genetic component, with heritability estimates of left ventricular mass between 61% and 81%. Association analyses of cardiac remodeling traits, corrected for population structure, body size and heart rate, revealed 17 genome-wide significant loci, including several loci containing previously implicated genes. Cardiac tissue gene expression profiling, expression quantitative trait loci, expression-phenotype correlation, and coding sequence variation analyses were performed to prioritize candidate genes and to generate hypotheses for downstream mechanistic studies. Using this approach, we have validated a novel gene, Myh14, as a negative regulator of ISO-induced left ventricular mass hypertrophy in an in vivo mouse model and demonstrated the up-regulation of immediate early gene Myc, fetal gene Nppb, and fibrosis gene Lgals3 in ISO-treated Myh14 deficient hearts compared to controls

Rescue of Pressure Overload-Induced Heart Failure by Estrogen Therapy.

BackgroundEstrogen pretreatment has been shown to attenuate the development of heart hypertrophy, but it is not known whether estrogen could also rescue heart failure (HF). Furthermore, the heart has all the machinery to locally biosynthesize estrogen via aromatase, but the role of local cardiac estrogen synthesis in HF has not yet been studied. Here we hypothesized that cardiac estrogen is reduced in HF and examined whether exogenous estrogen therapy can rescue HF.Methods and resultsHF was induced by transaortic constriction in mice, and once mice reached an ejection fraction (EF) of ≈35%, they were treated with estrogen for 10 days. Cardiac structure and function, angiogenesis, and fibrosis were assessed, and estrogen was measured in plasma and in heart. Cardiac estrogen concentrations (6.18±1.12 pg/160 mg heart in HF versus 17.79±1.28 pg/mL in control) and aromatase transcripts (0.19±0.04, normalized to control, P<0.05) were significantly reduced in HF. Estrogen therapy increased cardiac estrogen 3-fold and restored aromatase transcripts. Estrogen also rescued HF by restoring ejection fraction to 53.1±1.3% (P<0.001) and improving cardiac hemodynamics both in male and female mice. Estrogen therapy stimulated angiogenesis as capillary density increased from 0.66±0.07 in HF to 2.83±0.14 (P<0.001, normalized to control) and reversed the fibrotic scarring observed in HF (45.5±2.8% in HF versus 5.3±1.0%, P<0.001). Stimulation of angiogenesis by estrogen seems to be one of the key mechanisms, since in the presence of an angiogenesis inhibitor estrogen failed to rescue HF (ejection fraction=29.3±2.1%, P<0.001 versus E2).ConclusionsEstrogen rescues pre-existing HF by restoring cardiac estrogen and aromatase, stimulating angiogenesis, and suppressing fibrosis

Metabolic status differentiates Trp53inp2 function in pressure-overload induced heart failure

Cardiometabolic disorders encompass a broad range of cardiovascular complications associated with metabolic dysfunction. These conditions have an increasing share in the health burden worldwide due to worsening endemic of hypertension, obesity, and diabetes. Previous studies have identified Tumor Protein p53-inducible Nuclear Protein 2 (Trp53inp2) as a molecular link between hyperglycemia and cardiac hypertrophy. However, its role in cardiac pathology has never been determined in vivo. In this study, we generated a cardiac specific knockout model of Trp53inp2 (Trp53inp2-cKO) and investigated the impact of Trp53inp2 inactivation on the pathogenesis of heart failure under mechanic or/and metabolic stresses. Based on echocardiography assessment, inactivation of Trp53inp2 in heart led to accelerated onset of HFrEF in response to pressure-overload, with significantly reduced ejection fraction and elevated heart failure marker genes comparing to the control mice. In contrast, inactivation of Trp53inp2 ameliorated cardiac dysfunction induced by combined stresses of high fat diet and moderate pressure overload (Cardiometabolic Disorder Model). Moreover, Trp53inp2 inactivation led to reduced expression of glucose metabolism genes in lean, pressure-overloaded hearts. However, the same set of genes were significantly induced in the Trp53inp2-cKO hearts under both mechanical and metabolic stresses. In summary, we have demonstrated for the first time that cardiomyocyte Trp53inp2 has diametrically differential roles in the pathogenesis of heart failure and glucose regulation under mechanical vs. mechanical plus metabolic stresses. This insight suggests that Trp53inp2 may exacerbate the cardiac dysfunction during pressure overload injury but have a protective effect in cardiac diastolic function in cardiometabolic disease

Glucagon Receptor Antagonist for Heart Failure With Preserved Ejection Fraction.

BackgroundHeart failure with preserved ejection fraction (HFpEF) is an emerging major unmet need and one of the most significant clinic challenges in cardiology. The pathogenesis of HFpEF is associated with multiple risk factors. Hypertension and metabolic disorders associated with obesity are the 2 most prominent comorbidities observed in patients with HFpEF. Although hypertension-induced mechanical overload has long been recognized as a potent contributor to heart failure with reduced ejection fraction, the synergistic interaction between mechanical overload and metabolic disorders in the pathogenesis of HFpEF remains poorly characterized.MethodWe investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction in lean C57Bl/6J or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice, followed by single-nuclei RNA-seq and targeted manipulation of a top-ranked signaling pathway differentially affected in the 2 experimental cohorts.ResultsIn contrast to the post-trans-aortic constriction C57Bl/6J lean mice, which developed pathological features of heart failure with reduced ejection fraction over time, the post-trans-aortic constriction ob/ob mice showed no significant changes in ejection fraction but developed characteristic pathological features of HFpEF, including diastolic dysfunction, worsened cardiac hypertrophy, and pathological remodeling, along with further deterioration of exercise intolerance. Single-nuclei RNA-seq analysis revealed significant transcriptome reprogramming in the cardiomyocytes stressed by both pressure overload and obesity/diabetes, markedly distinct from the cardiomyocytes singularly stressed by pressure overload or obesity/diabetes. Furthermore, glucagon signaling was identified as the top-ranked signaling pathway affected in the cardiomyocytes associated with HFpEF. Treatment with a glucagon receptor antagonist significantly ameliorated the progression of HFpEF-related pathological features in 2 independent preclinical models. Importantly, cardiomyocyte-specific genetic deletion of the glucagon receptor also significantly improved cardiac function in response to pressure overload and metabolic stress.ConclusionsThese findings identify glucagon receptor signaling in cardiomyocytes as a critical determinant of HFpEF progression and provide proof-of-concept support for glucagon receptor antagonism as a potential therapy for the disease

Metabolic status differentiates Trp53inp2 function in pressure-overload induced heart failure

Cardiometabolic disorders encompass a broad range of cardiovascular complications associated with metabolic dysfunction. These conditions have an increasing share in the health burden worldwide due to worsening endemic of hypertension, obesity, and diabetes. Previous studies have identified Tumor Protein p53-inducible Nuclear Protein 2 (Trp53inp2) as a molecular link between hyperglycemia and cardiac hypertrophy. However, its role in cardiac pathology has never been determined in vivo. In this study, we generated a cardiac specific knockout model of Trp53inp2 (Trp53inp2-cKO) and investigated the impact of Trp53inp2 inactivation on the pathogenesis of heart failure under mechanic or/and metabolic stresses. Based on echocardiography assessment, inactivation of Trp53inp2 in heart led to accelerated onset of HFrEF in response to pressure-overload, with significantly reduced ejection fraction and elevated heart failure marker genes comparing to the control mice. In contrast, inactivation of Trp53inp2 ameliorated cardiac dysfunction induced by combined stresses of high fat diet and moderate pressure overload (Cardiometabolic Disorder Model). Moreover, Trp53inp2 inactivation led to reduced expression of glucose metabolism genes in lean, pressure-overloaded hearts. However, the same set of genes were significantly induced in the Trp53inp2-cKO hearts under both mechanical and metabolic stresses. In summary, we have demonstrated for the first time that cardiomyocyte Trp53inp2 has diametrically differential roles in the pathogenesis of heart failure and glucose regulation under mechanical vs. mechanical plus metabolic stresses. This insight suggests that Trp53inp2 may exacerbate the cardiac dysfunction during pressure overload injury but have a protective effect in cardiac diastolic function in cardiometabolic disease