IRX2 activated by jumonji domain-containing protein 2A is crucial for cardiac hypertrophy and dysfunction in response to the hypertrophic stimuli.

BACKGROUND: Iroquois homeobox 2 (IRX2) is a member of the Iroquois family whose upregulation has been potentially correlated to cardiac hypertrophy. This work studied the function of IRX2 and its related molecules in hypertrophic cardiomyopathy (HCM). METHODS: A GEO dataset GSE32453 was analyzed to identify aberrantly expressed genes in HCM. Altered expression of IRX2 was induced in mice by lentivirus injection, followed by angiotensin II (Ang II) treatment to induce HCM. The function of IRX2 knockdown in ventricular dysfunction, heart volume and pathological changes in mice, and in surface area, oxidative stress and apoptosis of isolated cardiomyocytes were examined. Binding relationship between jumonji domain-containing protein 2A (JMJD2A) and IRX2 was predicted by online tools and validated. The interaction between JMJD2A and IRX2 in HCM development was examined by joint interventions. RESULTS: IRX2 was highly expressed in heart tissues with HCM. IRX2 knockdown prevented mice from Ang II-induced ventricular dysfunction, cardiac hypertrophy, inflammation and fibrosis in mouse heart, and it decreased the levels of cardiac hypertrophy-related markers, oxidative stress response, and apoptosis of Ang II-treated cardiomyocytes. JMJD2A catalyzed demethylation of H3K9me3 near the IRX2 promoter to activate its transcription. JMJD2A knockdown similarly exerted protective functions against cardiac hypertrophy in vivo and in vitro, but the protection was blocked upon further IRX2 upregulation. IRX2 was found to increase the Wnt/ß-catenin signaling activation. CONCLUSION: This work reports that JMJD2A activates IRX2 transcription and the Wnt/ß-catenin signaling to induce cardiac hypertrophy and dysfunction in HCM.

A Neuroligin Isoform Translated by circNlgn Contributes to Cardiac Remodeling.

[Figure: see text].

A directed network analysis of the cardiome identifies molecular pathways contributing to the development of HFpEF.

AIMS: The metabolic syndrome and associated comorbidities, like diabetes, hypertension and obesity, have been implicated in the development of heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms underlying the development of HFpEF remain to be elucidated. We developed a cardiome-directed network analysis and applied this to high throughput cardiac RNA-sequencing data from a well-established rat model of HFpEF, the obese and hypertensive ZSF1 rat. With this novel system biology approach, we explored the mechanisms underlying HFpEF. METHODS AND RESULTS: Unlike ZSF1-Lean, ZSF1-Obese and ZSF1-Obese rats fed with a high-fat diet (HFD) developed diastolic dysfunction and reduced exercise capacity. The number of differentially expressed genes amounted to 1591 and 1961 for the ZSF1-Obese vs. Lean and ZSF1-Obese+HFD vs. Lean comparison, respectively. For the cardiome-directed network analysis (CDNA) eleven biological processes related to cardiac disease were selected and used as input for the STRING protein-protein interaction database. The resulting STRING network comprised 3.460 genes and 186.653 edges. Subsequently differentially expressed genes were projected onto this network. The connectivity between the core processes within the network was assessed and important bottleneck and hub genes were identified based on their network topology. Classical gene enrichment analysis highlighted many processes related to mitochondrial oxidative metabolism. The CDNA indicated high interconnectivity between five core processes: endothelial function, inflammation, apoptosis/autophagy, sarcomere/cytoskeleton and extracellular matrix. The transcription factors Myc and Peroxisome Proliferator-Activated Receptor-α (Ppara) were identified as important bottlenecks in the overall network topology, with Ppara acting as important link between cardiac metabolism, inflammation and endothelial function. CONCLUSIONS: This study presents a novel systems biology approach, directly applicable to other cardiac disease-related transcriptome data sets. The CDNA approach enabled the identification of critical processes and genes, including Myc and Ppara, that are putatively involved in the development of HFpEF.

Acute NelfA knockdown restricts compensatory gene expression and precipitates ventricular dysfunction during cardiac hypertrophy.

Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.

Monitoring Cell-Type-Specific Gene Expression Using Ribosome Profiling In Vivo During Cardiac Hemodynamic Stress.

RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.

Loss of SRSF3 in Cardiomyocytes Leads to Decapping of Contraction-Related mRNAs and Severe Systolic Dysfunction.

RATIONALE: RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. OBJECTIVE: To investigate the role of SRSF3 in cardiac function. METHODS AND RESULTS: Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. CONCLUSIONS: We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools.

Loss of Apelin Augments Angiotensin II-Induced Cardiac Dysfunction and Pathological Remodeling.

Apelin is an inotropic and cardioprotective peptide that exhibits beneficial effects through activation of the APJ receptor in the pathology of cardiovascular diseases. Apelin induces the expression of angiotensin-converting enzyme 2 (ACE2) in failing hearts, thereby improving heart function in an angiotensin 1⁻7-dependent manner. Whether apelin antagonizes the over-activation of the renin⁻angiotensin system in the heart remains elusive. In this study we show that the detrimental effects of angiotensin II (Ang II) were exacerbated in the hearts of aged apelin-gene-deficient mice. Ang II-mediated cardiac dysfunction and hypertrophy were augmented in apelin knockout mice. The loss of apelin increased the ratio of angiotensin-converting enzyme (ACE) to ACE2 expression in the Ang II-stressed hearts, and Ang II-induced cardiac fibrosis was markedly enhanced in apelin knockout mice. mRNA expression of pro-fibrotic genes, such as transforming growth-factor beta (TGF-ß) signaling, were significantly upregulated in apelin knockout hearts. Consistently, treatment with the ACE-inhibitor Captopril decreased cardiac contractility in apelin knockout mice. In vitro, apelin ameliorated Ang II-induced TGF-ß expression in primary cardiomyocytes, accompanied with reduced hypertrophy. These results provide direct evidence that endogenous apelin plays a crucial role in suppressing Ang II-induced cardiac dysfunction and pathological remodeling.

Aryl Hydrocarbon Receptor Ablation in Cardiomyocytes Protects Male Mice From Heart Dysfunction Induced by NKX2.5 Haploinsufficiency.

Epidemiological studies in humans and research in vertebrates indicates that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous and biopersistent environmental toxicant, is associated with incidence of early congenital heart disease in the embryo and later in the adult. TCDD-mediated toxicity depends on the aryl hydrocarbon receptor (AHR) but the role of the TCDD-activated AHR in cardiac function is not well-defined. To characterize the mechanisms responsible for AHR-mediated disruption of heart function, we generated several mouse strains with cardiomyocyte-specific Ahr gene knockout. Here, we report results on one of these strains in which the Ahr gene was deleted by cre recombinase regulated by the promoter of the cardiomyocyte-specific Nkx2.5 gene. We crossed mice with loxP-targeted Ahrfx/fx alleles with Nkx2.5+/cre mice bearing a "knock-in" cre recombinase gene integrated into one of the Nkx2.5 alleles. In these mice, loss of one Nkx2.5 allele is associated with disrupted cardiac development. In males, Nkx2.5 hemizygosity resulted in cardiac haploinsufficiency characterized by hypertrophy, dilated cardiomyopathy, and impaired ejection fraction. Ahr ablation protected Nkx2.5+/cre haploinsufficient males from cardiac dysfunction while inducing a significant increase in body weight. These effects were absent or largely blunted in females. Starting at 3 months of age, mice were exposed by oral gavage to 1 µg/kg/week of TCDD or control vehicle for an additional 2 months. TCDD exposure restored cardiac physiology in aging males, appearing to compensate for the heart dysfunction caused by Nkx2.5 hemizygosity. Our findings underscore the conclusion that deletion of the Ahr gene in cardiomyocytes protects males from heart dysfunction due to NKX2.5 haploinsufficiency.

Activation of the Nkx2.5-Calr-p53 signaling pathway by hyperglycemia induces cardiac remodeling and dysfunction in adult zebrafish.

Hyperglycemia is an independent risk factor for diabetic cardiomyopathy in humans; however, the underlying mechanisms have not been thoroughly elucidated. Zebrafish (Danio rerio) was used in this study as a novel vertebrate model to explore the signaling pathways of human adult cardiomyopathy. Hyperglycemia was induced by alternately immersing adult zebrafish in a glucose solution or water. The hyperglycemic fish gradually exhibited some hallmarks of cardiomyopathy such as myocardial hypertrophy and apoptosis, myofibril loss, fetal gene reactivation, and severe arrhythmia. Echocardiography of the glucose-treated fish demonstrated diastolic dysfunction at an early stage and systolic dysfunction at a later stage, consistent with what is observed in diabetic patients. Enlarged hearts with decreased myocardial density, accompanied by decompensated cardiac function, indicated that apoptosis was critical in the pathological process. Significant upregulation of the expression of Nkx2.5 and its downstream targets calreticulin (Calr) and p53 was noted in the glucose-treated fish. High-glucose stimulation in vitro evoked marked apoptosis of primary cardiomyocytes, which was rescued by the p53 inhibitor pifithrin-µ. In vitro experiments were performed using compound treatment and genetically via cell infection. Genetically, knockout of Nkx2.5 induced decreased expression of Nkx2.5, Calr and p53 Upregulation of Calr resulted in increased p53 expression, whereas the level of Nkx2.5 remained unchanged. An adult zebrafish model of hyperglycemia-induced cardiomyopathy was successfully established. Hyperglycemia-induced myocardial apoptosis was mediated, at least in part, by activation of the Nkx2.5-Calr-p53 pathway in vivo, resulting in cardiac dysfunction and hyperglycemia-induced cardiomyopathy.

Plin5 alleviates myocardial ischaemia/reperfusion injury by reducing oxidative stress through inhibiting the lipolysis of lipid droplets.

Myocardial ischaemia-reperfusion (I/R) injury is a complex pathophysiological process. Current research has suggested that energy metabolism disorders, of which the abnormal consumption of fatty acids is closely related, compose the main pathological basis for myocardial I/R injury. Lipid droplets (LD) are critical regulators of lipid metabolism by LD-associated proteins. Among the lipid droplet proteins, the perilipin family members regulate lipolysis and lipogenesis through different mechanisms. Plin5, an important perilipin protein, promotes LD generation and lowers fatty acid oxidation, thus protecting the myocardium from lipotoxicity. This study investigated the protective effects of Plin5 in I/R myocardium. Our results indicated that Plin5 deficiency exacerbated the myocardial infarct area, aggravated left ventricular systolic dysfunction, reduced lipid storage, and elevated free fatty acids. Plin5-deficient myocardium exhibited severely damaged mitochondria, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity. Furthermore, the decreased phosphorylation of PI3K/Akt in Plin5-null cardiomyocytes might contribute to I/R injury aggravation. In conclusion, Plin5, a new regulator of myocardial lipid metabolism, decreases free fatty acid peroxidation by inhibiting the lipolysis of intracellular lipid droplets, thus providing cardioprotection against I/R injury and shedding new light on therapeutic solutions for I/R diseases.

Blood Gene Signatures of Chagas Cardiomyopathy With or Without Ventricular Dysfunction.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 7 million people in Latin American areas of endemicity. About 30% of infected patients will develop chronic Chagas cardiomyopathy (CCC), an inflammatory cardiomyopathy characterized by hypertrophy, fibrosis, and myocarditis. Further studies are necessary to understand the molecular mechanisms of disease progression. Transcriptome analysis has been increasingly used to identify molecular changes associated with disease outcomes. We thus assessed the whole-blood transcriptome of patients with Chagas disease. Microarray analysis was performed on blood samples from 150 subjects, of whom 30 were uninfected control patients and 120 had Chagas disease (1 group had asymptomatic disease, and 2 groups had CCC with either a preserved or reduced left ventricular ejection fraction [LVEF]). Each Chagas disease group displayed distinct gene expression and functional pathway profiles. The most different expression patterns were between CCC groups with a preserved or reduced LVEF. A more stringent analysis indicated that 27 differentially expressed genes, particularly those related to natural killer (NK)/CD8+ T-cell cytotoxicity, separated the 2 groups. NK/CD8+ T-cell cytotoxicity could play a role in determining Chagas disease progression. Understanding genes associated with disease may lead to improved insight into CCC pathogenesis and the identification of prognostic factors for CCC progression.

Preservation of myocardial fatty acid oxidation prevents diastolic dysfunction in mice subjected to angiotensin II infusion.

RATIONALE: Diastolic dysfunction is a common feature in many heart failure patients with preserved ejection fraction and has been associated with altered myocardial metabolism in hypertensive and diabetic patients. Therefore, metabolic interventions to improve diastolic function are warranted. In mice with a germline cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2), systolic dysfunction induced by pressure-overload was prevented by maintaining cardiac fatty acid oxidation (FAO). However, it has not been evaluated whether this strategy would prevent the development of diastolic dysfunction in the adult heart. OBJECTIVE: To test the hypothesis that augmenting cardiac FAO is protective against angiotensin II (AngII)-induced diastolic dysfunction in an adult mouse heart. METHODS AND RESULTS: We generated a mouse model to induce cardiac-specific deletion of ACC2 in adult mice. Tamoxifen treatment (20mg/kg/day for 5days) was sufficient to delete ACC2 protein and increase cardiac FAO by 50% in ACC2 flox/flox-MerCreMer+ mice (iKO). After 4weeks of AngII (1.1mg/kg/day), delivered by osmotic mini-pumps, iKO mice showed normalized E/E' and E'/A' ratios compared to AngII treated controls (CON). The prevention of diastolic dysfunction in iKO-AngII was accompanied by maintained FAO and reduced glycolysis and anaplerosis. Furthermore, iKO-AngII hearts had a~50% attenuation of cardiac hypertrophy and fibrosis compared to CON. In addition, maintenance of FAO in iKO hearts suppressed AngII-associated increases in oxidative stress and sustained mitochondrial respiratory complex activities. CONCLUSION: These data demonstrate that impaired FAO is a contributor to the development of diastolic dysfunction induced by AngII. Maintenance of FAO in this model leads to an attenuation of hypertrophy, reduces fibrosis, suppresses increases in oxidative stress, and maintains mitochondrial function. Therefore, targeting mitochondrial FAO is a promising therapeutic strategy for the treatment of diastolic dysfunction.

Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution.

Matrix metalloproteinase-9 (MMP-9) is robustly elevated in the first week post-myocardial infarction (MI). Targeted deletion of the MMP-9 gene attenuates cardiac remodeling post-MI by reducing macrophage infiltration and collagen accumulation through increased apoptosis and reduced inflammation. In this study, we used a translational experimental design to determine whether selective MMP-9 inhibition early post-MI would be an effective therapeutic strategy in mice. We enrolled male C57BL/6J mice (3-6months old, n=116) for this study. Mice were subjected to coronary artery ligation. Saline or MMP-9 inhibitor (MMP-9i; 0.03µg/day) treatment was initiated at 3h post-MI and the mice were sacrificed at day (D) 1 or 7 post-MI. MMP-9i reduced MMP-9 activity by 31±1% at D1 post-MI (p<0.05 vs saline) and did not affect survival or infarct area. Surprisingly, MMP-9i treatment increased infarct wall thinning and worsened cardiac function at D7 post-MI. While MMP-9i enhanced neutrophil infiltration at D1 and macrophage infiltration at D7 post-MI, CD36 levels were lower in MMP-9i compared to saline, signifying reduced phagocytic potential per macrophage. Escalation and prolongation of the inflammatory response at D7 post-MI in the MMP-9i group was evident by increased expression of 18 pro-inflammatory cytokines (all p<0.05). MMP-9i reduced cleaved caspase 3 levels at D7 post-MI, consistent with reduced apoptosis and defective inflammation resolution. Because MMP-9i effects on inflammatory cells were significantly different from previously observed MMP-9 null mechanisms, we evaluated pre-MI (baseline) systemic differences between C57BL/6J and MMP-9 null plasma. By mass spectrometry, 34 plasma proteins were significantly different between groups, revealing a previously unappreciated altered baseline environment pre-MI when MMP-9 was deleted. In conclusion, early MMP-9 inhibition delayed inflammation resolution and exacerbated cardiac dysfunction, highlighting the importance of using translational approaches in mice.

Loss of myocardial retinoic acid receptor α induces diastolic dysfunction by promoting intracellular oxidative stress and calcium mishandling in adult mice.

Retinoic acid receptor (RAR) has been implicated in pathological stimuli-induced cardiac remodeling. To determine whether the impairment of RARα signaling directly contributes to the development of heart dysfunction and the involved mechanisms, tamoxifen-induced myocardial specific RARα deletion (RARαKO) mice were utilized. Echocardiographic and cardiac catheterization studies showed significant diastolic dysfunction after 16wks of gene deletion. However, no significant differences were observed in left ventricular ejection fraction (LVEF), between RARαKO and wild type (WT) control mice. DHE staining showed increased intracellular reactive oxygen species (ROS) generation in the hearts of RARαKO mice. Significantly increased NOX2 (NADPH oxidase 2) and NOX4 levels and decreased SOD1 and SOD2 levels were observed in RARαKO mouse hearts, which were rescued by overexpression of RARα in cardiomyocytes. Decreased SERCA2a expression and phosphorylation of phospholamban (PLB), along with decreased phosphorylation of Akt and Ca2+/calmodulin-dependent protein kinase II δ (CaMKII δ) was observed in RARαKO mouse hearts. Ca2+ reuptake and cardiomyocyte relaxation were delayed by RARα deletion. Overexpression of RARα or inhibition of ROS generation or NOX activation prevented RARα deletion-induced decrease in SERCA2a expression/activation and delayed Ca2+ reuptake. Moreover, the gene and protein expression of RARα was significantly decreased in aged or metabolic stressed mouse hearts. RARα deletion accelerated the development of diastolic dysfunction in streptozotocin (STZ)-induced type 1 diabetic mice or in high fat diet fed mice. In conclusion, myocardial RARα deletion promoted diastolic dysfunction, with a relative preserved LVEF. Increased oxidative stress have an important role in the decreased expression/activation of SERCA2a and Ca2+ mishandling in RARαKO mice, which are major contributing factors in the development of diastolic dysfunction. These data suggest that impairment of cardiac RARα signaling may be a novel mechanism that is directly linked to pathological stimuli-induced diastolic dysfunction.

New Cell Adhesion Molecules in Human Ischemic Cardiomyopathy. PCDHGA3 Implications in Decreased Stroke Volume and Ventricular Dysfunction.

BACKGROUND: Intercalated disks are unique structures in cardiac tissue, in which adherens junctions, desmosomes, and GAP junctions co-localize, thereby facilitating cardiac muscle contraction and function. Protocadherins are involved in these junctions; however, their role in heart physiology is poorly understood. We aimed to analyze the transcriptomic profile of adhesion molecules in patients with ischemic cardiomyopathy (ICM) and relate the changes uncovered with the hemodynamic alterations and functional depression observed in these patients. METHODS AND RESULTS: Twenty-three left ventricular tissue samples from patients diagnosed with ICM (n = 13) undergoing heart transplantation and control donors (CNT, n = 10) were analyzed using RNA sequencing. Forty-two cell adhesion genes involved in cellular junctions were differentially expressed in ICM myocardium. Notably, the levels of protocadherin PCDHGA3 were related with the stroke volume (r = -0.826, P = 0.003), ejection fraction (r = -0.793, P = 0.004) and left ventricular end systolic and diastolic diameters (r = 0.867, P = 0.001; r = 0.781, P = 0.005, respectively). CONCLUSIONS: Our results support the importance of intercalated disks molecular alterations, closely involved in the contractile function, highlighting its crucial significance and showing gene expression changes not previously described. Specifically, altered PCDHGA3 gene expression was strongly associated with reduced stroke volume and ventricular dysfunction in ICM, suggesting a relevant role in hemodynamic perturbations and cardiac performance for this unexplored protocadherin.

Renin overexpression leads to increased titin-based stiffness contributing to diastolic dysfunction in hypertensive mRen2 rats.

Hypertension (HTN) is a major risk factor for heart failure. We investigated the influence of HTN on cardiac contraction and relaxation in transgenic renin overexpressing rats (carrying mouse Ren-2 renin gene, mRen2, n = 6). Blood pressure (BP) was measured. Cardiac contractility was characterized by echocardiography, cellular force measurements, and biochemical assays were applied to reveal molecular mechanisms. Sprague-Dawley (SD) rats (n = 6) were used as controls. Transgenic rats had higher circulating renin activity and lower cardiac angiotensin-converting enzyme two levels. Systolic BP was elevated in mRen2 rats (235.11 ± 5.32 vs. 127.03 ± 7.56 mmHg in SD, P < 0.05), resulting in increased left ventricular (LV) weight/body weight ratio (4.05 ± 0.09 vs. 2.77 ± 0.08 mg/g in SD, P < 0.05). Transgenic renin expression had no effect on the systolic parameters, such as LV ejection fraction, cardiomyocyte Ca(2+)-activated force, and Ca(2+) sensitivity of force production. In contrast, diastolic dysfunction was observed in mRen2 compared with SD rats: early and late LV diastolic filling ratio (E/A) was lower (1.14 ± 0.04 vs. 1.87 ± 0.08, P < 0.05), LV isovolumetric relaxation time was longer (43.85 ± 0.89 vs. 28.55 ± 1.33 ms, P < 0.05), cardiomyocyte passive tension was higher (1.74 ± 0.06 vs. 1.28 ± 0.18 kN/m(2), P < 0.05), and lung weight/body weight ratio was increased (6.47 ± 0.24 vs. 5.78 ± 0.19 mg/g, P < 0.05), as was left atrial weight/body weight ratio (0.21 ± 0.03 vs. 0.14 ± 0.03 mg/g, P < 0.05). Hyperphosphorylation of titin at Ser-12742 within the PEVK domain and a twofold overexpression of protein kinase C-α in mRen2 rats were detected. Our data suggest a link between the activation of renin-angiotensin-aldosterone system and increased titin-based stiffness through phosphorylation of titin's PEVK element, contributing to diastolic dysfunction.

Inhibition of let-7 augments the recruitment of epicardial cells and improves cardiac function after myocardial infarction.

Heart failure due to myocardial infarction is a major cause of mortality. The microRNA (miR) family let-7 is expressed during embryonic development and is up-regulated in differentiated cells. The aim of this study was to study the role of let-7 after acute myocardial infarction (AMI). We designed an antimiR to inhibit the highest expressed members of the let-7 family, let-7 a, b and c. Administration at day 0 and day 2 after AMI resulted in sustained knockdown of let-7 after 28days. Let-7 inhibition prevented deterioration of cardiac functions compared to control treatment which was especially due to improvements in the infarcted, apical cardiac segments. We observed higher contents of fibrosis in the border zone as well as increased numbers of cells positive for TCF21, which is also expressed in epicardial cells. Markers were augmented after let-7 inhibition and let-7 blocked EMT in epicardial cells in vitro. Lineage tracing in TCF21(iCre/+):R26R(tdT) mice showed abundant tomato positive cells in the infarct and border zone. In conclusion, let-7 inhibition resulted in functional benefits due to an increase in recruitment of epicardial cells and EMT.

Heterozygous caveolin-3 mice show increased susceptibility to palmitate-induced insulin resistance.

Insulin resistance and diabetes are comorbidities of obesity and affect one in 10 adults in the United States. Despite the high prevalence, the mechanisms of cardiac insulin resistance in obesity are still unclear. We test the hypothesis that the insulin receptor localizes to caveolae and is regulated through binding to caveolin-3 (CAV3). We further test whether haploinsufficiency forCAV3 increases the susceptibility to high-fat-induced insulin resistance. We used in vivo and in vitro studies to determine the effect of palmitate exposure on global insulin resistance, contractile performance of the heart in vivo, glucose uptake in the heart, and on cellular signaling downstream of theIR We show that haploinsufficiency forCAV3 increases susceptibility to palmitate-induced global insulin resistance and causes cardiomyopathy. On the basis of fluorescence energy transfer (FRET) experiments, we show thatCAV3 andIRdirectly interact in cardiomyocytes. Palmitate impairs insulin signaling by a decrease in insulin-stimulated phosphorylation of Akt that corresponds to an 87% decrease in insulin-stimulated glucose uptake inHL-1 cardiomyocytes. Despite loss of Akt phosphorylation and lower glucose uptake, palmitate increased insulin-independent serine phosphorylation ofIRS-1 by 35%. In addition, we found lipid induced downregulation ofCD36, the fatty acid transporter associated with caveolae. This may explain the problem the diabetic heart is facing with the simultaneous impairment of glucose uptake and lipid transport. Thus, these findings suggest that loss ofCAV3 interferes with downstream insulin signaling and lipid uptake, implicatingCAV3 as a regulator of theIRand regulator of lipid uptake in the heart.

Thioredoxin-1 Attenuates Ventricular and Mitochondrial Postischemic Dysfunction in the Stunned Myocardium of Transgenic Mice.

AIM: We evaluated the effect of thioredoxin1 (Trx1) system on postischemic ventricular and mitochondrial dysfunction using transgenic mice overexpressing cardiac Trx1 and a dominant negative (DN-Trx1) mutant (C32S/C35S) of Trx1. Langendorff-perfused hearts were subjected to 15 min of ischemia followed by 30 min of reperfusion (R). We measured left ventricular developed pressure (LVDP, mmHg), left ventricular end diastolic pressure (LVEDP, mmHg), and t63 (relaxation index, msec). Mitochondrial respiration, SERCA2a, phospholamban (PLB), and phospholamban phosphorylation (p-PLB) Thr17 expression (Western blot) were also evaluated. RESULTS: At 30 min of reperfusion, Trx1 improved contractile state (LVDP: Trx1: 57.4 ± 4.9 vs. Wt: 27.1 ± 6.3 and DN-Trx1: 29.2 ± 7.1, p < 0.05); decreased myocardial stiffness (LVEDP: Wt: 24.5 ± 4.8 vs. Trx1: 11.8 ± 2.9, p < 0.05); and improved the isovolumic relaxation (t63: Wt: 63.3 ± 3.2 vs. Trx1: 51.4 ± 1.9, p < 0.05). DN-Trx1 mice aggravated the myocardial stiffness and isovolumic relaxation. Only the expression of p-PLB Thr17 increased at 1.5 min R in Wt and DN-Trx1 groups. At 30 min of reperfusion, state 3 mitochondrial O2 consumption was impaired by 13% in Wt and by 33% in DN-Trx1. ADP/O ratios for Wt and DN-Trx1 decrease by 25% and 28%, respectively; whereas the Trx1 does not change after ischemia and reperfusion (I/R). Interestingly, baseline values of complex I activity were increased in Trx1 mice; they were 24% and 47% higher than in Wt and DN-Trx1 mice, respectively (p < 0.01). INNOVATION AND CONCLUSION: These results strongly suggest that Trx1 ameliorates the myocardial effects of I/R by improving the free radical-mediated damage in cardiac and mitochondrial function, opening the possibility of new therapeutic strategies in coronary artery disease. Antioxid. Redox Signal. 25, 78-88.