Knockout Hearts Research Articles

Abstract 269: RBM20 Knockout Ameliorates Angiotensin II Induced Hypertension and Cardiac Hypertrophy

Introduction: RNA binding motif 20 (RBM20) is a muscle specific splicing factor that has been implicated in heart failure (HF). RBM20 regulates mRNA splicing of titin, a giant muscle protein that is expressed in cardiac and smooth muscles and plays a major role in passive stiffness of them. RBM20 knockout (KO) results in less myocardial stiffness by inducing large titin isoform expression. Hence, we hypothesize that RBM20 decreases arterial stiffness through large titin isoform expression, and thus reduces blood pressure (BP) in hypertension. The aim of this study was to investigate whether RBM20 could be a therapeutic target to reduce BP in patients with hypertension and prevent HF progression. Methods: Male wild type (WT) (Sprague-Dawley х Brown Norway) and Rbm20 KO rats (n=10 each) were subcutaneously implanted with osmotic mini-pump to continuously release angiotensin II (Ang II) at a rate of 400 ng/kg/min for 28 days. BP was measured by noninvasive tail-cuff system weekly. Pulse wave velocity (PWV) was measured by Doppler system. Cardiac remodeling was evaluated by echocardiography. Biochemistry and histological analyses were performed on aorta and heart tissues. Results: Larger titin isoform was increased and no difference of elastin and collagen expression was observed in RBM20 KO blood vessels when compared to WT. Basal BPs were the same in WT and KO rats (diastolic BP, KO 91.75 ± 1.83 vs. WT 94.33±1.88 mmHg; systolic BP, KO 127.6 ± 0.97 vs. WT 128.0 ± 1.35 mmHg). Ang II increased BP in both WT and KO rats, but KO had a significantly lower BP than WT (diastolic BP, KO 141.7 ± 5.00 vs. WT 182.0±4.84 mmHg; systolic BP, KO 195.4 ± 3.671 vs. WT 226.4 ± 3.093 mmHg, P<0.0001). PWV is lower in KO rats than in WT (KO 4.035 ± 0.21 mm/s vs. WT 5.128±0.37 mm/s, P<0.05). Cardiac hypertrophy was developed in both WT and KO hearts treated with Ang II, but it was alleviated in the KO heart (left ventricular wall, KO 0.324 ± 0.01 cm vs. WT 0.368 ± 0.013 cm, P<0.05). Conclusions: RBM20 KO reduces arterial stiffness through increased larger titin isoform expression, decreases Ang II-induced hypertension, and prevents HF progression. These findings suggest that RBM20 could be a potential novel target for the treatment of hypertension and hypertension-induced cardiac hypertrophy.

Abstract 103: Cytochrome b5 Reductase 3 Regulates Myoglobin Redox State and Controls Cardiac Function

Oxidative stress contributes to the adverse pathophysiology of cardiac remodeling in heart failure. During oxidative stress, hemoprotein heme-iron can be oxidized, often altering the function of these proteins. We previously reported that cytochrome b5 reductase 3 (Cyb5R3) regulates heme iron redox in the vascular wall. While conducting vasoreactivity studies with a small-molecule Cyb5R3 inhibitor, we unexpectedly observed inhibition resulted in dilated cardiomyopathy (DCM) with reduced ejection fraction. Based on this study, we generated the first cardiomyocyte Cyb5R3 knockout mouse. Heart specific Cyb5R3 knockout resulted in DCM with >50% lethality in 15 days. H&E staining, wheat germ agglutinin immunofluorescence staining, transmission electron microscopy and diffusion tensor MRI shows that loss of Cyb5R3 causes myocardial atrophy and fibrosis. Due to the known heme iron reducing function of Cyb5R3, electron paramagnetic resonance (EPR) was used to assess the abundance of reduced and oxidized myoglobin ex vivo. Cyb5R3 knockout hearts had approximately 30% more oxidation relative to controls. Hypoxyprobe staining, which senses tissue pO2 below 5mmHg, showed Cyb5R3 expression is required for maintaining intra-cardiomyocyte pO 2 , likely through myoglobin heme-iron reduction. Decreased pO 2 was supported by altered mitochondrial dynamics. Complex IV activity was significantly reduced, along with decreased mitochondrial size and total ATP. Heme degradation protein, heme oxygenase-1 (HO-1) was significantly upregulated, suggesting that Cyb5R3 knockout may result in increased free-heme toxicity leading to intracellular damage. To translate these findings to humans, we conducted a retrospective study using a high-frequency T117S variant in African Americans. T117S carriers had significantly accelerated mortality post-first acute cardiac event. A T117S mouse was generated and received trans-aortic constriction (TAC) for 2 weeks. Preliminary results suggest T117S TAC mice have accelerated cardiac remodeling and time to death after pressure overload relative to C57B6/J TAC controls. Together, these data support Cyb5R3 expression may have an important role as a heme-iron reductase in cardiomyocyte function.

Abstract 110: Numb Family Proteins Modulate Cardiac Morphogenesis by Regulating N-cadherin Trafficking to Plasma Membrane

Numb, an endocytic adaptor protein, is essential for multiple biologic processes. We previously reported that the deletion of Numb and its homologue Numblike (collectively known as, Numb Family Proteins or NFPs) specifically in heart causes a variety of morphogenetic defects, however, the key downstream target or signaling pathway that is disrupted and is responsible for each defect in the knockout was not determined. In this study, we investigated the cellular and molecular mechanisms of the trabeculation defect in the NFPs knockout. We found that during trabecular initiation, the cellular and mitotic spindle orientations of cardiomyocytes in the knockouts were significantly different from the control, which was further confirmed by the sparse cell-labeling / confetti clonal analysis. We then determined that N-Cadherin, which is required for trabecular formation in previous studies, is significantly reduced in the NFPs knockout. Genetic and biochemical tools were applied to demonstrate that Numb functions upstream of N-Cadherin and modulates its protein level to regulate trabeculation. Using chicken N-Cadherin driven by the aMHC promoter in transgenic mice, we found that cardiomyocytes that express chicken N-Cadherin in the NFPs knockout hearts preferentially localized to trabeculae compared to cardiomyocytes that do not express chicken N-Cadherin, thus partially rescuing the trabeculation defect of the knockout. This suggests that NFPs regulate trabeculation via maintaining the N-Cadherin protein level. To determine the mechanism of how NFPs regulate N-Cadherin protein, we generated a mouse line in which the endocytic motifs of Numb are deleted, and found the N-Cadherin protein level was not changed, indicating that Numb does not regulate N-Cadherin through endocytosis. Instead, we found that Numb localizes to the recycling endosome and that less N-Cadherin was localized to the membrane in the knockout, suggesting that NFPs are required for N-Cadherin recycling to the membrane. In summary, NFPs regulate N-Cadherin recycling to the plasma membrane during trabecular morphogenesis, and NFP deletion reduces N-Cadherin protein level, resulting in the trabeculation defect.

OP-19 - The dys-regulation of anti-oxidant defense via an impairment of Nrf2 response in the pathology of Friedreich’s ataxia

The transcription factor, nuclear factor-erythroid 2-related factor-2 (Nrf2), is the master regulator of antioxidant response. Decreased Nrf2 expression was previously reported in studies with models of the neuro-and cardio-degenerative disease, Friedreich’s ataxia (FA), where oxidative stress is a key contributor to its pathology. Using a mouse conditional frataxin knockout (KO) model, we examined the Nrf2 pathway in the frataxin-deficient heart and skeletal muscle. In the KO heart, our studies have demonstrated increased protein and GSH oxidation, decreased total and nuclear Nrf2 levels, and increased expression of its inhibitor, Keap1. However, the opposite was found in skeletal muscle. The activation of nuclear Nrf2 export/degradation machinery via Gsk3f3-signaling was demonstrated in the KO heart through the process of: (i) increased Gsk3f3 activation; (ii) f3-TrCP nuclear accumulation; and (iii) Fyn phosphorylation. This corresponded with decreased Nrf2-DNA-binding activity and a general decrease in Nrf2-target mRNA in KO hearts. Overall, increased levels of cytosolic Keap1, and activation of Gsk3f3-signaling are potential mechanisms for the decreased Nrf2 levels in the frataxin-deficient Heart, in contrast to skeletal muscle, where Nrf2 was not decreased.

Abstract 350: Brown Adipose Tissue, a Novel Mechanism to Induce both Angiogenesis and Arteriogenesis

Brown adipose tissue (BAT) is known to protect metabolism, but the extent to which it mediates angiogenesis and cardioprotection, the goal of this investigation, is not known. The Regulator of G Protein Signaling 14 (RGS14) knockout (KO) mouse is unique in that it not only extends longevity, but also enhances several aspects of healthful aging, i.e., protects against diabetes, obesity, exercise intolerance and increased brown adipose tissue by 47% compared with wild type littermates (WT). However, less is known about its role in cardioprotection. Accordingly we examined the effects of chronic myocardial ischemia after 1 and 3 weeks of permanent coronary artery occlusion (CAO) in 3-4 month old RGS14 KO and their WT. Scar size after 3 weeks CAO, determined by histology, was decreased by 33±5% in RGS14 KO, which was only possible in the face of permanent CAO if angiogenesis/arteriogenesis developed in the RGS14 KO mice hearts. Indeed, both angiogenesis assessed by Ki 67 endothelial cell staining and arteriogenesis, assessed by Ki 67 arteriole staining, more than doubled compared with WT. This was associated with a 206±25.5% increase in VEGF in the RGS14 KO hearts compared with WT. We then examined the role of BAT by using a simulated BAT KO, i.e., BAT was transplanted from the RGS14 KO to WT. In the WT with BAT transplants VEGF was increased by 90%. Infarct size, assessed as the fraction of area at risk, was lower, p<0.05, in the BAT transplants (27±2.8%), compared with infarct size in RGS14 KO donors (44±1.9%), a value similar to that observed in WT without the BAT transplant (46±1.0%). Thus, brown adipose tissue appears to be a novel mechanism to induce both angiogenesis and arteriogenesis, resulting in vascular protection from ischemia.

Interleukin 6 Knockout Inhibits Aging-Related Accumulation of p53 in the Mouse Myocardium.

Interleukin 6 (IL6) and p53 are linked by mutual regulatory mechanisms and are both upregulated in aging. The aim of this study was to evaluate the effects of aging and IL6 on expression of p53 in the mouse heart. Male C57BL6/J wild-type and IL6 knockout mice at the age of 4-5 months (young adult) and 24-30 months (old) were used. Myocardial expression of proteins such as p53, p21, Mdm2, and phospho-Akt/Akt was estimated using Western blotting and expression of p53 and p21 mRNA using real-time polymerase chain reaction. Expression of p53 protein was lower in IL6 knockout hearts than in wild-type hearts. Aging caused significant upregulation of p53 protein level; however, it was significantly higher in old wild-type hearts than in old IL6 knockout hearts (p < .05). Similar p53 mRNA levels in all groups implied IL6 influence on age-related proteasomal degradation of p53. Localization of p53 mainly in the extranuclear compartment and lack of p21 upregulation in aged hearts may suggest quenched transcriptional activity of p53 despite increased abundance of p53. We conclude that lack of IL6 attenuates expression of p53 protein in the hearts of young mice and diminishes its accumulation with aging by post-transcriptional mechanisms; however, this is not related to altered phenotype of aging heart.

Splicing Variation of TTN Novex Isoforms Across Species and RBM20 Does Not Regulate Splicing of Novex Isoforms in Cardiac Muscle.

TTN is a major disease‐causing gene in cardiac muscle. TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by alternative exon usages of exon 45 (Novex 1), exon 46 (Novex 2) and exon 48 (Novex 3). It is unknown how these exons are alternatively used in Novex isoforms across species and whether Novex isoforms are associated with heart disease. In this study, we compared alternative exon usage between species with the mVISTA online tool, and found that exon 45 is conserved across all species, but exons 46 and 48 only found in some species. Interestingly, we found a conserved region between exons 47 and 48 across species which has never been characterized before. RT‐PCR and DNA sequencing confirmed that this is a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, we found two new splicing forms, which one has intron 44 retention and the other has intron 45 retention. With Novex 2 primer pairs, we found that Novex 2 is lack of expression in pig, mouse and rat. A new truncated isoform with intron 46 retention is mainly expressed in human and frog heart, and another new truncated isoform with exon 45 and 46 are co‐expressed predominantly in chicken and frog heart. Using RBM20 knockout heart, we revealed that RBM20 doesn't regulate splicing of Novex isoforms. Finally, we examined the expression levels of Novex isoforms in human heart with cardiomyopathies, and we didn't find any difference. These results suggest that exon usage of Novex isoforms varies across species which is not associated with cardiomyopathies. Hence, their functional role in the heart need be further studied in the future, which is beyond the scope of this study.Support or Funding InformationThis work was supported by the NIFA‐USDA 1009266, National Institutes of Health NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Critical role of phosphodiesterase 2A in mouse congenital heart defects.

Phosphodiesterase 2 A (Pde2A), a cAMP-hydrolysing enzyme, is essential for mouse development; however, the cause of Pde2A knockout embryonic lethality is unknown. To understand whether Pde2A plays a role in cardiac development, hearts of Pde2A deficient embryos were analysed at different stage of development. At the stage of four chambers, Pde2A deficient hearts were enlarged compared to the hearts of Pde2A heterozygous and wild-type. Pde2A knockout embryos revealed cardiac defects such as absence of atrial trabeculation, interventricular septum (IVS) defects, hypertrabeculation and thinning of the myocardial wall and in rare cases they had overriding aorta and valves defects. E14.5 Pde2A knockouts showed reduced cardiomyocyte proliferation and increased apoptosis in the IVS and increased proliferation in the ventricular trabeculae. Analyses of E9.5 Pde2A knockout embryos revealed defects in cardiac progenitor and neural crest markers, increase of Islet1 positive and AP2 positive apoptotic cells. The expression of early cTnI and late Mef2c cardiomyocyte differentiation markers was strongly reduced in Pde2A knockout hearts. The master transcription factors of cardiac development, Tbx, were down-regulated in E14.5 Pde2A knockout hearts. Absence of Pde2A caused an increase of intracellular cAMP level, followed by an up-regulation of the inducible cAMP early repressor, Icer in fetal hearts. In vitro experiments on wild-type fetal cardiomyocytes showed that Tbx gene expression is down-regulated by cAMP inducers. Furthermore, Pde2A inhibition in vivo recapitulated the heart defects observed in Pde2A knockout embryos, affecting cardiac progenitor cells. Interestingly, the expression of Pde2A itself was dramatically affected by Pde2A inhibition, suggesting a potential autoregulatory loop. We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

Connexin43 and zonula occludens-1 are targets of Akt in cardiomyocytes that correlate with cardiac contractile dysfunction in Akt deficient hearts

While deletion of Akt1 results in a smaller heart size and Akt2−/− mice are mildly insulin resistant, Akt1−/−/Akt2−/− mice exhibit perinatal lethality, indicating a large degree of functional overlap between the isoforms of the serine/threonine kinase Akt. The present study aimed to determine the cooperative contribution of Akt1 and Akt2 on the structure and contractile function of adult hearts. To generate an inducible, cardiomyocyte-restricted Akt2 knockout (KO) model, Akt2flox/flox mice were crossed with tamoxifen-inducible MerCreMer transgenic (MCM) mice and germline Akt1−/− mice to generate the following genotypes:Akt1+/+; Akt2flox/flox (WT), Akt2flox/flox; α-MHC-MCM (iAkt2 KO), Akt1−/−, and Akt1−/−; Akt2flox/flox; α-MHC-MCM mice (Akt1−/−/iAkt2 KO). At 28 days after the first tamoxifen injection, Akt1−/−/iAkt2 KO mice developed contractile dysfunction paralleling increased atrial and brain natriuretic peptide (ANP and BNP) levels, and repressed mitochondrial gene expression. Neither cardiac fibrosis nor apoptosis were detected in Akt1−/−/iAkt2 KO hearts. To explore potential molecular mechanisms for contractile dysfunction, we investigated myocardial microstructure before the onset of heart failure. At 3 days after the first tamoxifen injection, Akt1−/−/iAkt2 KO hearts showed decreased expression of connexin43 (Cx43) and connexin-interacting protein zonula occludens-1 (ZO-1). Furthermore, Akt1/2 silencing significantly decreased both Cx43 and ZO-1 expression in cultured neonatal rat cardiomyocytes in concert with reduced beating frequency. Akt1 and Akt2 are required to maintain cardiac contraction. Loss of Akt signaling disrupts gap junction protein, which might precipitate early contractile dysfunction prior to heart failure in the absence of myocardial remodeling, such as hypertrophy, fibrosis, or cell death.

Increasing T-type calcium channel activity by β-adrenergic stimulation contributes to β-adrenergic regulation of heart rates.

Cav3.1 T-type Ca2+ channel current (ICa-T ) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β-adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β-adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β-adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. ICa-T in sinoatrial nodal cells can be upregulated by β-adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T-type Ca2+ channel can be a target for heart disease treatments that aim to slow down the heart rate ABSTRACT: Cav3.1 (α1G ) T-type Ca2+ channel (TTCC) is expressed in mouse sinoatrial node cells (SANCs) and atrioventricular (AV) nodal cells and contributes to heart rate (HR) genesis and AV conduction. However, its role in HR regulation and AV conduction acceleration by the β-adrenergic system (SAS) is unclear. In the present study, L- (ICa-L ) and T-type (ICa-T ) Ca2+ currents were recorded in SANCs from Cav3.1 transgenic (TG) and knockout (KO), and control mice. ICa-T was absent in KO SANCs but enhanced in TG SANCs. In anaesthetized animals, different doses of isoproterenol (ISO) were infused via the jugular vein and the HR was recorded. The EC50 of the HR response to ISO was lower in TG mice but higher in KO mice, and the maximal percentage of HR increase by ISO was greater in TG mice but less in KO mice. In Langendorff-perfused hearts, ISO increased HR and shortened PR intervals to a greater extent in TG but to a less extent in KO hearts. KO SANCs had significantly slower spontaneous beating rates than control SANCs before and after ISO; TG SANCs had similar basal beating rates as control SANCs probably as a result of decreased ICa-L but a greater response to ISO than control SANCs. ICa-T in SANCs was significantly increased by ISO. ICa-T upregulation by β-adrenergic stimulation contributes to HR and conduction regulation by the SAS. TTCC can be a target for slowing the HR.

The transcription factor MEF2A fine-tunes gene expression in the atrial and ventricular chambers of the adult heart

The distinct morphological and functional properties of the cardiac chambers arise from an elaborate developmental program involving cell lineage determination, morphogenesis, and dynamic spatiotemporal gene expression patterns. Although a number of transcription factors have been identified for proper gene regulation in the chambers, the complete transcriptional network that controls these patterns remains poorly defined. Previous studies have implicated the MEF2C transcription factor in the regulation of chamber-restricted enhancers. To better understand the mechanisms of MEF2-mediated regional gene regulation in the heart, we took advantage of MEF2A knock-out (KO) mice, a model that displays a predominantly ventricular chamber phenotype. Transcriptomic analysis of atrial and ventricular tissue from adult MEF2A KO hearts revealed a striking difference in chamber gene expression, with a larger proportion of dysregulated genes in the atrial chambers. Canonical pathway analysis of genes preferentially dysregulated in the atria and ventricles revealed distinct MEF2A-dependent cellular processes in each cardiac chamber. In the atria, MEF2A regulated genes involved in fibrosis and adhesion, whereas in the ventricles, it controlled inflammation and endocytosis. Finally, analysis of transcription factor-binding site motifs of differentially dysregulated genes uncovered distinct MEF2A co-regulators for the atrial and ventricular gene sets, and a subset of these was found to cooperate with MEF2A. In conclusion, our results suggest a mechanism in which MEF2 transcriptional activity is differentially recruited to fine-tune gene expression levels in each cardiac chamber. This regulatory mechanism ensures optimal output of these gene products for proper physiological function of the atrial and ventricular chambers.

Sorcin ablation plus β-adrenergic stimulation generate an arrhythmogenic substrate in mouse ventricular myocytes

Sorcin, a penta-EF hand Ca2+-binding protein expressed in cardiomyocytes, is known to interact with ryanodine receptors and other Ca2+ regulatory proteins. To investigate sorcin's influence on cardiac excitation-contraction coupling and its role in the development of cardiac malfunctions, we generated a sorcin knockout (KO) mouse model. Sorcin KO mice presented ventricular arrhythmia and sudden death when challenged by acute stress induced by isoproterenol plus caffeine. Chronic stress, which was induced by transverse aortic constriction, significantly decreased the survival rate of sorcin KO mice. Under isoproterenol stimulation, spontaneous Ca2+ release events were frequently observed in sorcin KO cardiomyocytes. Sorcin KO hearts of adult, but not young mice developed overexpression of L-type Ca2+ channel and Na+-Ca2+ exchanger, which enhanced ICa and INCX. Consequently, spontaneous Ca2+ release events in sorcin KO cardiomyocytes were more likely to induce arrhythmogenic delayed afterdepolarizations. Our study demonstrates sorcin deficiency may trigger cardiac ventricular arrhythmias due to Ca2+ disturbances, and evidences the critical role of sorcin in maintaining Ca2+ homeostasis, especially during the adrenergic response of the heart.

Abstract 20950: An Intrinsic Mechanism for a Gain in Proteasome Activity Preserving Cardiac Function

Cardiac remodeling and heart failure are characterized by misaligned adaptations in protein synthesis and degradation, harboring the therapeutic potential for interventional harmonization. Partial inhibition of protein degradation via the ubiquitin-proteasome system was reported to reduce acute hypertrophic remodeling as well as recover systolic function. The studies are suggestive of an inadequate gain in proteasome activity, contributing to cardiac remodeling and heart failure. Aim was to identify a common mechanism promoting proteasome activity during cardiac remodeling and heart failure and to test whether it contributes to the pathogenesis. Incorporation of the inducible proteasome subunit Lmp2 has been described to increase proteasome activity. In fact, it is increasingly expressed and incorporated in proteasome complexes during isoproterenol-induced hypertrophic remodeling, which is associated with a gain in proteasome activity. We found that Lmp2 is also increasingly expressed upon transverse aortic constriction and in a murine model of familial hypertrophic cardiomyopathy. Knock-out (KO) of Lmp2 in mice increased the caspase-like proteasome activity by 19% (n=5, p&lt;0.05), but had no apparent influence on cardiac morphology or function. As predicted, lack of Lmp2 confined the regulatory capacity of proteasomes to gain activity during hypertrophic remodeling induced by isoproterenol. Unexpectedly, the restriction in additional activity did not reduce cardiac remodeling as previously described for proteasome inhibition. In contrast, lack of Lmp2 exacerbated hypertrophic remodeling and caused loss of systolic function (fractional shortening: -32% vs. wildtype, n≥8, p&lt;0.01). Cardiomyocyte-specific expression of Lmp2 via gene transfer into adult Lmp2 KO mice completely rescued the isoproterenol-induced deterioration of cardiac remodeling and function. Interestingly, the deprived gain in proteasome activity in Lmp2 KO hearts rather affected a specific subset than the general pool of ubiquitinated proteins. In conclusion, a gain in proteasome activity during acute cardiac remodeling is not entirely maladaptive. At least in part, it is dependent on Lmp2, controlling remodeling and preserving cardiac function.

Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

Mitochondrial electron transport is essential for oxidative phosphorylation (OXPHOS). Electron transport chain (ETC) activity generates an electrochemical gradient that is used by the ATP synthase to make ATP. ATP synthase is organized into supramolecular units called synthasomes that increase the efficiency of ATP production, while within ATP synthase is the cyclophilin D (CypD) regulated mitochondrial permeability transition pore (PTP). We investigated whether synthasomes are dynamic structures that respond to metabolic demands and whether CypD regulates this dynamic. Isolated heart mitochondria from wild-type (WT) and CypD knockout (KO) mice were treated to either stimulate OXPHOS or open the PTP. The presence and dynamics of mitochondrial synthasomes were investigated by native electrophoresis, immunoprecipitation, and sucrose density centrifugation. We show that stimulation of OXPHOS, inhibition of the PTP, or deletion of CypD increased high order synthasome assembly. In contrast, OXPHOS inhibition or PTP opening increased synthasome disassembly in WT, but not in CypD KO heart mitochondria. CypD activity also correlated with synthasome assembly in other tissues, such as liver and brain. We conclude that CypD not only regulates the PTP, but also regulates the dynamics of synthasome assembly depending on the bioenergetic state of the mitochondria.

Pacsin 2 is required for the maintenance of a normal cardiac function in the developing mouse heart

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (If), as well as L-type Ca2+ channel (ICaL), and sodium channel (INa). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases.

Loss of Protein Kinase Novel 1 (PKN1) is associated with mild systolic and diastolic contractile dysfunction, increased phospholamban Thr17 phosphorylation, and exacerbated ischaemia-reperfusion injury.

AimsPKN1 is a stress-responsive protein kinase acting downstream of small GTP-binding proteins of the Rho/Rac family. The aim was to determine its role in endogenous cardioprotection.Methods and resultsHearts from PKN1 knockout (KO) or wild type (WT) littermate control mice were perfused in Langendorff mode and subjected to global ischaemia and reperfusion (I/R). Myocardial infarct size was doubled in PKN1 KO hearts compared to WT hearts. PKN1 was basally phosphorylated on the activation loop Thr778 PDK1 target site which was unchanged during I/R. However, phosphorylation of p42/p44-MAPK was decreased in KO hearts at baseline and during I/R. In cultured neonatal rat ventricular cardiomyocytes (NRVM) and NRVM transduced with kinase dead (KD) PKN1 K644R mutant subjected to simulated ischaemia/reperfusion (sI/R), PhosTag® gel analysis showed net dephosphorylation of PKN1 during sI and early R despite Thr778 phosphorylation. siRNA knockdown of PKN1 in NRVM significantly decreased cell survival and increased cell injury by sI/R which was reversed by WT- or KD-PKN1 expression. Confocal immunofluorescence analysis of PKN1 in NRVM showed increased localization to the sarcoplasmic reticulum (SR) during sI. GC-MS/MS and immunoblot analysis of PKN1 immunoprecipitates following sI/R confirmed interaction with CamKIIδ. Co-translocation of PKN1 and CamKIIδ to the SR/membrane fraction during sI correlated with phospholamban (PLB) Thr17 phosphorylation. siRNA knockdown of PKN1 in NRVM resulted in increased basal CamKIIδ activation and increased PLB Thr17 phosphorylation only during sI. In vivo PLB Thr17 phosphorylation, Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA2) expression and Junctophilin-2 (Jph2) expression were also basally increased in PKN1 KO hearts. Furthermore, in vivo P-V loop analysis of the beat-to-beat relationship between rate of LV pressure development or relaxation and end diastolic P (EDP) showed mild but significant systolic and diastolic dysfunction with preserved ejection fraction in PKN1 KO hearts.ConclusionLoss of PKN1 in vivo significantly reduces endogenous cardioprotection and increases myocardial infarct size following I/R injury. Cardioprotection by PKN1 is associated with reduced CamKIIδ-dependent PLB Thr17 phosphorylation at the SR and therefore may stabilize the coupling of SR Ca2+ handling and contractile function, independent of its kinase activity.

C1q/Tumor Necrosis Factor-Related Protein-9 Regulates the Fate of Implanted Mesenchymal Stem Cells and Mobilizes Their Protective Effects Against Ischemic Heart Injury via Multiple Novel Signaling Pathways.

Cell therapy remains the most promising approach against ischemic heart injury. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair after myocardial infarction. CTRP9 (C1q/tumor necrosis factor-related protein-9) is a novel prosurvival cardiokine with significantly downregulated expression after myocardial infarction. Here we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting implanted stem cell survival and cardioprotection. Mice were subjected to myocardial infarction and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Survival, cardiac remodeling and function, cardiomyocytes apoptosis, and ADSCs engraftment were evaluated. Whether CTRP9 directly regulates ADSCs function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms of CTRP9. Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 (P<0.05 or P<0.01 versus CTRP9 alone), suggesting a synergistic effect. Administration of CTRP9 at a dose recovering physiological CTRP9 levels significantly prolonged ADSCs retention/survival after implantation. Conversely, the number of engrafted ADSCs was significantly reduced in the CTRP9 knockout heart. In vitro study demonstrated that CTRP9 promoted ADSCs proliferation and migration, and it protected ADSCs against hydrogen peroxide-induced cellular death. CTRP9 enhances ADSCs proliferation/migration by extracellular regulated protein kinases (ERK)1/2-matrix metallopeptidase 9 signaling and promotes antiapoptotic/cell survival via ERK-nuclear factor erythroid-derived 2-like 2/antioxidative protein expression. N-cadherin was identified as a novel CTRP9 receptor mediating ADSCs signaling. Blockade of either N-cadherin or ERK1/2 completely abolished the previously noted CTRP9 effects. Although CTRP9 failed to promote ADSCs cardiogenic differentiation, CTRP9 promotes superoxide dismutase 3 expression and secretion from ADSCs, protecting cardiomyocytes against oxidative stress-induced cell death. We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms. These include binding with N-cadherin, activation of ERK-matrix metallopeptidase 9 and ERK-nuclear factor erythroid-derived 2-like 2 signaling, and upregulation/secretion of antioxidative proteins. These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.

Molecular Alterations in a Mouse Cardiac Model of Friedreich Ataxia: An Impaired Nrf2 Response Mediated via Upregulation of Keap1 and Activation of the Gsk3β Axis

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a master regulator of the antioxidant response. However, studies in models of Friedreich ataxia, a neurodegenerative and cardiodegenerative disease associated with oxidative stress, reported decreased Nrf2 expression attributable to unknown mechanisms. Using a mouse conditional frataxin knockout (KO) model in the heart and skeletal muscle, we examined the Nrf2 pathway in these tissues. Frataxin KO results in fatal cardiomyopathy, whereas skeletal muscle was asymptomatic. In the KO heart, protein oxidation and a decreased glutathione/oxidized glutathione ratio were observed, but the opposite was found in skeletal muscle. Decreased total and nuclear Nrf2 and increased levels of its inhibitor, Kelch-like ECH-associated protein 1, were evident in the KO heart, but not in skeletal muscle. Moreover, a mechanism involving activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3β (Gsk3β) signaling was demonstrated in the KO heart. This process involved the following: i) increased Gsk3β activation, ii) β-transducin repeat containing E3 ubiquitin protein ligase nuclear accumulation, and iii) Fyn phosphorylation. A corresponding decrease in Nrf2-DNA-binding activity and a general decrease in Nrf2-target mRNA were observed in KO hearts. Paradoxically, protein levels of some Nrf2 antioxidant targets were significantly increased in KO mice. Collectively, cardiac frataxin deficiency reduces Nrf2 levels via two potential mechanisms: increased levels of cytosolic Kelch-like ECH-associated protein 1 and activation of Gsk3β signaling, which decreases nuclear Nrf2. These findings are in contrast to the frataxin-deficient skeletal muscle, where Nrf2 was not decreased.

Knockout of the ATPase inhibitory factor 1 protects the heart from pressure overload-induced cardiac hypertrophy

Mitochondrial ATP synthase catalyzes the coupling of oxidative phosphorylation. Under pathological conditions, ATP synthase hydrolyzes ATP to replenish protons from the matrix into the intermembrane space, sustaining mitochondrial membrane potential. ATPase inhibitory factor 1 (IF1) is a nuclear-encoded, ATP synthase-interacting protein that selectively inhibits the hydrolysis activity of ATP synthase, which may render the protective role of IF1 in ischemic hearts. However, the in vivo cardiac function of IF1 and the potential therapeutic application targeting IF1 remain obscure. In the present study, we uncovered that IF1 is upregulated in mouse hearts with pressure overload-induced hypertrophy and in human hearts with dilated cardiomyopathy. IF1 knockout (KO) mice were protected against cardiac dysfunction and pathological development induced by transverse aortic constriction (TAC) or isoproterenol infusion. The reduced ATP hydrolysis activated AMPK activity in IF1 KO hearts, which together facilitated autophagy. These results suggest that IF1 upregulation in the failing heart may be a maladaptive response. Inhibiting IF1 in the hypertrophied heart not only prevents cell death from excessive mitochondrial depolarization but also activates AMPK signaling and increases autophagy. Therefore, IF1 inhibition may serve as a potential therapeutic target in treating pathological cardiac hypertrophy and heart failure.

How does pressure overload cause cardiac hypertrophy and dysfunction? High-ouabain affinity cardiac Na+ pumps are crucial.

Left ventricular hypertrophy is frequently observed in hypertensive patients and is believed to be due to the pressure overload and cardiomyocyte stretch. Three recent reports on mice with genetically engineered Na+ pumps, however, have demonstrated that cardiac ouabain-sensitive α2-Na+ pumps play a key role in the pathogenesis of transaortic constriction-induced hypertrophy. Hypertrophy was delayed/attenuated in mice with mutant, ouabain-resistant α2-Na+ pumps and in mice with cardiac-selective knockout or transgenic overexpression of α2-Na+ pumps. The latter, seemingly paradoxical, findings can be explained by comparing the numbers of available (ouabain-free) high-affinity (α2) ouabain-binding sites in wild-type, knockout, and transgenic hearts. Conversely, hypertrophy was accelerated in α2-ouabain-resistant (R) mice in which the normally ouabain-resistant α1-Na+ pumps were mutated to an ouabain-sensitive (S) form (α1S/Sα2R/R or "SWAP" vs. wild-type or α1R/R α2S/S mice). Furthermore, transaortic constriction-induced hypertrophy in SWAP mice was prevented/reversed by immunoneutralizing circulating endogenous ouabain (EO). These findings show that EO and its receptor, ouabain-sensitive α2, are critical factors in pressure overload-induced cardiac hypertrophy. This complements reports linking elevated plasma EO to hypertension, cardiac hypertrophy, and failure in humans and elucidates the underappreciated role of the EO-Na+ pump pathway in cardiovascular disease.