Cardiomyocyte-restricted Knockout Research Articles

Phenotypic Differences Among Mice with Induced Cardiomyocyte‐Restricted Ablation of Cops5, Cops8, or Both

Background- Within a large subset of heart failure, the ubiquitin proteasome system has shown to be inadequate or even causative. The COP9 signalosome (CSN) holocomplex, a vital ubiquitination regulator, directly regulates Cullin-RING ligases (CRLs) via Cullin deneddylation. This holocomplex is formed by 8 unique subunits (COPS1~COPS8), where the deneddylase activity resides in COPS5. Our lab has shown in the past that cardiomyocyte-restricted knockout (CKO) of the Cops8 gene causes cardiomyocyte necroptosis, dilated cardiomyopathy, and shortened lifespan in mice. This study tests whether the protection against necroptosis by Cops8 is deneddylation-dependent or Cops8-specific, possibly alluding to subunit-specific functions independent of the holocomplex. Methods and Results- CKO of Cops8, Cops5, or both in adult mice was achieved using a tamoxifen-inducible Cre-LoxP system. Echocardiography performed 21 days after tamoxifen withdrawal showed no significant difference between MerCreMer (MCM) transgenic and Cops8-floxed/Cops5-floxed control groups. Compared with either control group, all CKO groups developed dilated cardiomyopathy, but the severity in the Cops5-CKO and the Cops8+Cops5 double CKO (dCKO) groups was greater than that in the Cops8-CKO group. Kaplan-Meier survival analyses revealed shortened lifespans for all CKO groups, compared with the MCM group. The post-tamoxifen lifespans of Cops5-CKO and dCKO mice were comparable (median 42 days vs. 42 days; p=0.66) but significantly shorter than that of the Cops8-CKO (81 days, p<0.0001). In Cops5-CKO and dCKO mice, we observed an increased number of cardiomyocytes positive for Evan's blue dye uptake and increased myocardial CD45 proteins compared to Cops8-CKO. All CKO myocardium showed increases in full-length caspase 8 but decreased cleaved caspase 8, indicating suppression of caspase 8 activation by defective CSN. Conclusions (1)Cops5-CKO and dCKO are similarly detrimental but are more so than Cops8-CKO; (2) the primary defect caused by Cops8-CKO results from impaired CSN holocomplex formation and thereby loss of Cullin deneddylation.

Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodeling during stress. Here we show that Mitochondrial Pyruvate Carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as the myocardium of mice induced to fail by angiotensin II or transverse-aortic constriction-induced. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure-overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-13C2-glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse-aortic constriction protected against cardiac hypertrophy and failure. Collectively, we demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

COP9 Signalosome Suppresses RIPK1-RIPK3-Mediated Cardiomyocyte Necroptosis in Mice.

Mechanisms governing the induction of heart failure by the impairment of autophagy and the ubiquitin-proteasome system and the molecular pathways to cardiomyocyte necrosis remain incompletely understood. COPS8 is an essential subunit of the COP9 (COnstitutive Photomorphogenesis 9) signalosome, a key regulator of ubiquitination. Mice with cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) show autophagic and ubiquitin-proteasome system malfunction and massive cardiomyocyte necrosis followed by acute heart failure and premature death, providing an excellent animal model to address the mechanistic gaps specified above. This study was conducted to determine the nature and underlying mechanisms of the cardiomyocyte necrosis in Cops8-cko mice. Compared with littermate control mice, myocardial protein levels of key factors in the necroptotic pathway (RIPK1 [receptor-interacting protein kinase 1], RIPK3, MLKL [mixed lineage kinase-like], the RIPK1-bound RIPK3), protein carbonyls, full-length Casp8 (caspase 8), and BCL2, as well as histochemical staining of superoxide anions were significantly higher but the cleaved Casp8 and the Casp8 activity were significantly lower in Cops8-cko mice. In vivo cardiomyocyte uptake of Evan's blue dye was used as an indicator of necrosis. Cops8-cko mice treated with a RIPK1 kinase inhibitor (Nec-1 [Necrostatin-1]) showed less Evans blue dye uptake (0.005% versus 0.20%; P<0.0001) and longer median lifespan (32.5 versus 27 days; P<0.01) than those treated with vehicle control. RIPK3 haploinsufficiency showed similar rescuing effects on Cops8-cko but Cyclophilin D deficiency did the opposite. Cardiac Cops8/COP9 signalosome malfunction causes RIPK1-RIPK3 dependent, but mitochondrial permeability transition pore independent, cardiomyocyte necroptosis in mice and the COP9 signalosome plays an indispensable role in suppressing cardiomyocyte necroptosis.

Inadequate ubiquitination-proteasome coupling contributes to myocardial ischemia-reperfusion injury.

The ubiquitin-proteasome system (UPS) degrades a protein molecule via 2 main steps: ubiquitination and proteasomal degradation. Extraproteasomal ubiquitin receptors are thought to couple the 2 steps, but this proposition has not been tested in vivo with vertebrates. More importantly, impaired UPS performance plays a major role in cardiac pathogenesis, including myocardial ischemia-reperfusion injury (IRI), but the molecular basis of UPS impairment remains poorly understood. Ubiquilin1 is a bona fide extraproteasomal ubiquitin receptor. Here, we report that mice with a cardiomyocyte-restricted knockout of Ubiquilin1 (Ubqln1-CKO mice) accumulated a surrogate UPS substrate (GFPdgn) and increased myocardial ubiquitinated proteins without altering proteasome activities, resulting in late-onset cardiomyopathy and a markedly shortened life span. When subject to regional myocardial ischemia-reperfusion, young Ubqln1-CKO mice showed substantially exacerbated cardiac malfunction and enlarged infarct size, and conversely, mice with transgenic Ubqln1 overexpression displayed attenuated IRI. Furthermore, Ubqln1 overexpression facilitated proteasomal degradation of oxidized proteins and the degradation of a UPS surrogate substrate in cultured cardiomyocytes without increasing autophagic flux. These findings demonstrate that Ubiquilin1 is essential to cardiac ubiquitination-proteasome coupling and that an inadequacy in the coupling represents a major pathogenic factor for myocardial IRI; therefore, strategies to strengthen coupling have the potential to reduce IRI.

Abstract 345: Inadequate Coupling between Ubiquitination and the Proteasome is a Major Pathogenic Factor of Myocardial Ischemia/Reperfusion Injury

Targeted removal of damaged/misfolded proteins is primarily done by the ubiquitin (Ub)-proteasome system (UPS). Generally, the UPS degrades a protein molecule via two main steps: ubiquitination of the protein and subsequent degradation of the ubiquitinated protein by the proteasome. The two steps are not always directly coupled. The proteasome can directly uptake nearby polyubiquitinated proteins via its Ub receptor subunits but extra-proteasomal Ub receptors may be required for shuttling the remote substrates to the proteasome for degradation. We have previously demonstrated that UPS function is impaired during acute myocardial ischemia/reperfusion (I/R) and this impairment plays a major role in I/R injury due to failure to timely remove damaged/misfolded proteins. The molecular underpinnings of UPS impairment in I/R injury remains poorly understood. This study shows an important role of Ubiquilin1, a bona fide extra-proteasomal Ub receptor, in cardiac ubiquitination-proteasome coupling during I/R. Mice with cardiomyocyte-restricted knockout of the Ubiquilin1 gene (CR-Ubqln1KO) or with Ubiquilin1 overexpression (Ubqln1OE) were subject to myocardial I/R created via left anterior descending artery ligation (for 30min) and subsequent release (for 24hr). The I/R induced decline of left ventricular maximum dP/dt, elevation of minimum dP/dt, and infarct size were significantly greater in CR-Ubqln1KO mice, and conversely, were markedly attenuated in Ubqln1OE mice, compared with their littermates with a respective control genotype. The accumulation of ubiquitinated proteins in I/R myocardium was increased by CR-Ubqln1KO and reduced by Ubqln1OE. Furthermore, in cultured cardiomyocytes, Ubqln1OE was able to enhance the degradation of a surrogate UPS substrate as well as a bona fide misfolded protein linked to human proteinopathy. These exciting new findings demonstrate for the first time that inadequate coupling between ubiquitination and proteasomal degradation hinders the degradation of damaged proteins in I/R myocardium and represents a major pathogenic factor to acute I/R injury. It is also suggested that facilitating the coupling is potentially a novel therapeutic strategy for reducing I/R injury.

Abstract 269: Duo-impairment of the Ubiquitin-Proteasome System and Autophagy by Ablation of COP9 Signalosome Subunit 8 Activates a Programmed Necrosis Pathway Mediated by RIP1-RIP3 Kinases but not Cyclophilin D-regulated Mitochondrial Membrane Permeability

Programmed cell death includes apoptosis and programmed necrosis (AKA, necroptosis). A well-recognized feature of necrosis is the loss of membrane integrity of the dying cell. Cells undergoing apoptosis, however, maintain their membrane integrity. We previously reported massive cardiomyocyte (CM) necrosis, before increased apoptosis is discernible, in mouse hearts with cardiomyocyte-restricted knockout of the COP9 signalosome subunit 8 (CR-Csn8KO). This is associated with defective autophagosome maturation and ubiquitin-proteasome system (UPS) dysfunction. Consequently, mice with perinatal CR-Csn8KO develop rapidly dilated cardiomyopathy (DCM) and die prematurely. The present study sought to search for the mechanisms underlying the CM necrosis. Here our immunoprecipitation revealed significant increases of RIP1-interacted RIP3 in CR-Csn8KO myocardium, indicative of activation of the RIP1-RIP3 pathway. The RIP1-RIP3 pathway is known to mediate necroptosis. To further test whether RIP1 activation plays an essential role in CM necrosis in CR-Csn8KO mice, we treated the mice with a RIP1 kinase specific inhibitor necrostatin-1 (Nec-1) or vehicle control via osmotic mini-pump implanted in the peritoneal cavity at 2 weeks of age. Nec-1 significantly suppressed CM necrosis as measured by the positivity of Evan’s blue dye (EBD) uptake (p&lt;0.00001), prevented left ventricle dilatation (p&lt;0.05) at 3 weeks, and delayed premature death of the CR-Csn8KO mice (p=0.0072). These results demonstrate that CM necrosis in CR-Csn8KO mice is necroptosis in which the RIP1-RIP3 kinases-mediated pathway plays a major pathogenic role, and that CM necroptosis is the primary cause of DCM and mouse premature death. Increased mitochondrial membrane permeability, which can be suppressed by inhibition of cyclophilin D, has been shown to play a critical mediating role in myocytes necrosis. Hence, we cross-bred CR-Csn8KO mice with cyclophilin D knockout mice and obtained the Kaplan-Meier survival curve. Unexpectedly, cyclophilin D deficiency exacerbated the premature death of CR-Csn8KO mice (p=0.007), indicating that cyclophilin D regulated mitochondrial membrane permeability does not play an important role in CM necrosis in CR-Csn8KO mouse hearts.

Protective Function of STAT3 in CVB3-Induced Myocarditis

The transcription factor signal transducer and activator of transcription 3 (STAT3) is an important mediator of the inflammatory process. We investigated the role of STAT3 in viral myocarditis and its possible role in the development to dilated cardiomyopathy. We used STAT3-deficent mice with a cardiomyocyte-restricted knockout and induced a viral myocarditis using Coxsackievirus B3 (CVB3) which induced a severe inflammation during the acute phase of the viral myocarditis. A complete virus clearance and an attenuated inflammation were examined in both groups WT and STAT3 KO mice 4 weeks after infection, but the cardiac function in STAT3 KO mice was significantly decreased in contrast to the infected WT mice. Interestingly, an increased expression of collagen I was detected in STAT3 KO mice compared to WT mice 4 weeks after CVB3 infection. Furthermore, the matrix degradation was reduced in STAT3 KO mice which might be an explanation for the observed matrix deposition. Consequently, we here demonstrate the protective function of STAT3 in CVB3-induced myocarditis. Since the cardiomyocyte-restricted knockout leads to an increased fibrosis, it can be assumed that STAT3 signalling in cardiomyocytes protects the heart against increased fibrosis through paracrine effects.

COP9 Signalosome Regulates Autophagosome Maturation

Autophagy is essential to intracellular homeostasis and is involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8), and is known to regulate the ubiquitin-proteasome system. However, it is unknown whether CSN plays a role in autophagy. Marked increases in the LC3-II and p62 proteins were observed on Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with green fluorescent protein-LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred before a global ubiquitin-proteasome system impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Downregulation of Rab7 was found to colocalize strikingly with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Long-term lysosomal inhibition with chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of nonselective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes. Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway, but also selective autophagy. Likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation. Impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.

Knockout of insulin receptors in cardiomyocytes attenuates coronary arterial dysfunction induced by pressure overload

Ablating insulin receptors in cardiomyocytes causes subendocardial fibrosis and left ventricular (LV) dysfunction after 4 wk of transverse aortic constriction (TAC). To determine whether these maladaptive responses are precipitated by coronary vascular dysfunction, we studied mice with cardiomyocyte-restricted knock out of insulin receptors (CIRKO) and wild-type (WT) TAC mice before the onset of overt LV dysfunction. Two weeks of TAC produced comparable increases (P < 0.05 vs. respective sham) in heart weight/body weight (mg/g) in WT-TAC (8.03 ± 1.14, P < 0.05 vs. respective sham) and CIRKO-TAC (7.76 ± 1.25, P < 0.05 vs. respective sham) vs. WT-sham (5.64 ± 0.11) and CIRKO-sham (4.64 ± 0.10) mice. In addition, 2 wk of TAC were associated with similar LV geometry and function (echocardiography) and interstitial fibrosis (picrosirius red staining) in CIRKO and WT mice. Responses to acetylcholine (ACh), N(G)-monomethyl-L-arginine (l-NMMA), and sodium nitroprusside (SNP) were measured in coronary arteries that were precontracted to achieve ∼70% of maximal tension development using the thromboxane A(2) receptor mimetic U-46619 (∼3 × 10(-6) M). ACh-evoked vasorelaxation was absent in WT-TAC but was present in CIRKO-TAC albeit reduced relative to sham-operated animals. l-NMMA-evoked tension development was similar in vessels from CIRKO-TAC mice but was lower (P < 0.05) in WT-TAC animals vs. the respective sham-operated groups, and SNP-evoked vasorelaxation was similar among all mice. Thus estimates of stimulated and basal endothelial nitric oxide release were better preserved in CIRKO vs. WT mice in response to 2 wk of TAC. These findings indicate that maladaptive LV remodeling previously observed in CIRKO-TAC mice is not precipitated by coronary artery dysfunction, because CIRKO mice exhibit compensatory mechanisms (e.g., increased eNOS transcript and protein) to maintain coronary endothelial function in the setting of pressure overload.

Controlling cardiomyocyte length: the role of renin and PPAR-γ

Renin and peroxisome proliferator-activated receptor (PPAR-γ) interact directly with cardiomyocytes and influence protein synthesis. We investigated their effects and interaction on the size of cardiomyocytes. Effects of renin and PPAR-γ activation were studied in cultured adult rat ventricular cardiomyocytes, transgenic mice with a cardiomyocyte-restricted knockout of PPAR-γ, and transgenic rats overexpressing renin, TGR(mRen2)27. The length and width of cardiomyocytes were analysed 24 h after administration of factors. Renin caused an unexpected effect on the length of cardiomyocytes that was inhibited by mannose-6-phosphate and monensin, but not by administration of glucose-6-phosphate. Endothelin-1 used as a classical pro-hypertrophic agonist increased cell width but not cell length. Renin caused an activation of p38 and p42/44 mitogen-activated protein (MAP) kinases. The latter activation was impaired by mannose-6-phosphate. Inhibition of p42/44 but not of p38 MAP kinase activation attenuated the effect of renin on cell length. In contrast, activation of PPAR-γ reduced cell length. Feeding wild-type mice with pioglitazone, a PPAR-γ agonist, reduced cell length. Cardiomyocytes isolated from PPAR-γ knockout mice were longer, and their length was not affected by pioglitazone. Cardiomyocytes isolated from TGR(mRen2)27 rats were longer than those of non-transgenic littermates. Cell length was reduced by feeding these mice with pioglitazone. Pioglitazone affected cell length independent of blood pressure. The length of cardiomyocytes is controlled by the activation of cardiac-specific mannose-6-phosphate/insulin-like growth factor II receptors and activation of PPAR-γ. This type of cell size modification differs from that of any other known pro-hypertrophic agonists.

Impaired insulin signaling accelerates cardiac mitochondrial dysfunction after myocardial infarction

Diabetes increases mortality and accelerates left ventricular (LV) dysfunction following myocardial infarction (MI). This study sought to determine the impact of impaired myocardial insulin signaling, in the absence of diabetes, on the development of LV dysfunction following MI. Mice with cardiomyocyte-restricted knock out of the insulin receptor (CIRKO) and wildtype (WT) mice were subjected to proximal left coronary artery ligation (MI) and followed for 14 days. Despite equivalent infarct size, mortality was increased in CIRKO-MI vs. WT-MI mice (68% vs. 40%, respectively). In surviving mice, LV ejection fraction and d P/d t were reduced by > 40% in CIRKO-MI vs. WT-MI. Relative to shams, isometric developed tension in LV papillary muscles increased in WT-MI but not in CIRKO-MI. Time to peak tension and relaxation times were prolonged in CIRKO-MI vs. WT-MI suggesting impaired, load-independent myocardial contractile function. To elucidate mechanisms for impaired LV contractility, mitochondrial function was examined in permeabilized cardiac fibers. Whereas maximal ADP-stimulated mitochondrial O 2 consumption rates ( V ADP) with palmitoyl carnitine were unchanged in WT-MI mice relative to sham-operated animals, V ADP was significantly reduced in CIRKO-MI (13.17 ± 0.94 vs. 9.14 ± 0.88 nmol O 2/min/mgdw, p < 0.05). Relative to WT-MI, expression levels of GLUT4, PPAR-α, SERCA2, and the FA-Oxidation genes MCAD, LCAD, CPT2 and the electron transfer flavoprotein ETFDH were repressed in CIRKO-MI. Thus reduced insulin action in cardiac myocytes accelerates post-MI LV dysfunction, due in part to a rapid decline in mitochondrial FA oxidative capacity, which combined with limited glucose transport capacity that may reduce substrate utilization and availability.

Abstract 5569: Paracrine Effects of Cardiomyocyte STAT3 Determine the Vasculogenic Differentiation Potential of Cardiac Progenitor Cells: Potential Role of Endogenous Erythropoietin

Mice with a cardiomyocyte-restricted knock out of STAT3 (αMHC-Cretg/+; STAT3flox/flox, STAT3-KO) show a continuous decrease of cardiac vascularization and develop heart failure beyond the age of 9 months. We investigated the role of cardiomyocyte STAT3-driven paracrine effects on Sca-1+ cardiac progenitor cells (CPC) in the mouse heart. CPC were immunomag-netically isolated from KO and wildtype (STAT3flox/flox, WT) hearts (age: 3 months). PCR and Western blot confirmed deletion of STAT3 in cardiomyocytes of KO mice, while CPC from KO showed normal expression of STAT3. The total number of CPC per heart was similar between WT and KO mice. FACS analysis revealed a reduced number of endothelial progenitor cells (defined by co-expression of Sca-1, CD31 and CD38, −25%, P&lt;0.05) in CPC from KO compared to CPC from WT. In vitro culture for 4 weeks on fibronectin-coated plates of CPC from KO revealed reduced proliferation (−33%, p&lt;0.01), impaired endothelial cell (EC) tubeformation (monitored with Tie2, eNOS and CD31 immunohistochemistry (IHC), p&lt;0.01) and enhanced adipocyte differentiation (oil red staining and RT-PCR, p&lt;0.05) compared with CPC from WT. Microarray of freshly isolated CPC reflected this differences in EC and adipocyte differentiation on the mRNA level (i.e. EC marker Prostaglandin E Rezeptor-3: 2.3-fold lower; adipocyte marker Lipocalin-2 2.7-fold higher in CPC from KO hearts). Microarray results from whole left ventricular tissue showed a decrease in gene expression of Erythropoietin (Epo) in KO hearts (-9,25-fold). ELISA, IHC and methylcellulose assay confirm expression of active EPO by cardiomyocytes. CPC express high levels of EPO receptor (IHC, RT-PCR). Epo enhanced tube formation and sprouting of EC and attenuated adipocyte differentiation of CPCs from KO. In vivo treatment with Epo rescued impaired proliferation, promoted EC differentiation and attenuated adipocyte differentiation of CPC from KO hearts. Conclusion: STAT3-dependent paracrine factors from cardiomyocytes regulate proliferation, differentiation and vasculogenic properties of CPCs. Cardiomyocyte derived EPO is an important paracrine mediator that promotes differentiation into EC and attenuates differentiation into adipocytes from CPCs in the adult heart.

Abstract 1115: Impaired Angiogenic Potential of Cardiac Progenitor Cells in Mice with a Cardiomyocyte-Restricted Knock Out of STAT3

Mice with a cardiomyocyte-restricted knock out of STAT3 (KO: alpha-MHC-Cre tg/+; STAT3 flox/flox ) show a continuous decrease of the cardiac capillary density and develop heart failure beyond the age of 9 months. We sought to determine the paracrine influence of cardiomyocyte STAT3 on the endothelial differentiation potential of cardiac progenitor cells (CPC) of the adult mouse heart. Sca-1 + CPC were isolated from male mice hearts by MACS separation. STAT3 was entirely deleted in cardiomyocytes of KO mice, while CPC from KO showed normal expression of STAT3 (confirmed by PCR and Western blot). No difference in the total number of CPC per heart was observed between wildtype (WT: STAT3 flox/flox ) and KO mice. FACS analysis revealed a reduced number of endothelial progenitor cells (as defined by coexpression of Sca-1, CD31 and CD38, −25%, P&lt;0.05) among CPC from KO compared to CPC from WT. The differentiation potential of CPC from WT and KO was analyzed during in vitro culture on fibronectin-coated plates. After 4 weeks of culture RT-PCR for CD31 and immunohistochemistry (IHC) for endothelial cell (EC) marker tie2 and isolectin B4 was performed. CPC from WT showed markedly more efficient EC differentiation and tube formation compared to CPC from KO (p&lt;0.01). In contrast, adipocyte differentiation was enhanced in CPC from KO (p&lt;0.05, oil red staining and RT-PCR). Proliferation capacity of CPC from KO was reduced by 33% (p&lt;0.01) as compared to CPC from WT. Microarray results of freshly isolated CPC were consistent with the differences in EC and adipocyte differentiation (i.e. prostaglandin E receptor 3 up 2.3-fold in CPC from WT, Lipocalin-2 up 2.7-fold in CPC from KO). We did not observe cardiomyocyte differentiation (IHC for alpha-sarcomeric actinin; RT-PCR for Nkx 2.5, alpha-MHC, or alpha-skeletal actin) of CPC from both genotypes, neither in vitro by addition of oxytocin, 5-AZA, DMSO, nor following intramyocardial injection of CPC in vivo. Conclusion: STAT3-dependent paracrine mediators released from cardiomyocytes are determinants of differentiation and vasculogenic properties of new EC derived from cardiac progenitor cells. The identification of these factors may offer new approaches to enforce the endogenous vasculogenic repair potential of the adult heart.

Abstract 542: Mice with Cardiomyocyte-Restricted IGF-1 Receptor Deletion Exhibit Diminished Cardiomyocyte Size and Resistance to Exercise-Induced Cardiac Hypertrophy

Insulin-like growth factor 1 (IGF-1) and IGF-1 receptors are expressed in murine hearts, and IGF-1 receptor signaling in cardiac muscle has been proposed to play a role in growth, differentiation, and cell survival, but mechanisms by which IGF-1 modulates myocardial structure and function are only partially understood. To investigate the role of IGF-1 signaling on cardiac development and physiology, we generated mice with cardiomyocyte-restricted knockout of the IGF-1receptor (IGF-1R −/−) by crossing α-MHC-Cre mice with mice containing a floxed exon 3 of the IGF-1R gene. Ablation of IGF-1 receptors in cardiomyocytes did not alter baseline heart weight to tibia length (HW/TL) ratios at 8 weeks or 12 weeks of age. However, wheat germ agglutinin (WGA-FITC) staining revealed that myocyte cross-sectional area was reduced by 18.8% (P &lt; 0.05). To define the contribution of IGF-1 receptor signaling in the development of physiological hypertrophy; mice [WT (n = 7) or IGF-1R −/− (n = 9)] were subjected to 4 weeks swim (Sw) training and compared with Sedentary (Sed) wild type (WT) (n = 5) or IGF-1R −/− (n = 7) mice. HW/TL ratios increased by 19.2% in WT animals after swim training (5.19 ± 0.26 vs. 6.18 ± 0.2, P &lt; 0.05), but only by 5.5% in Sw IGF-1R −/− mice (5.58 ± 0.18 vs. 5.89 ± 0.22, P = 0.32) and the fold increase in HW/TL was significantly greater in Sw WT vs. Sw IGF-1R −/− (P &lt; 0.05). Despite resistance to hypertrophy, cardiac systolic function assessed by ejection fraction was preserved in Sw IGF-1R −/− (before Sw 0.71 ± 0.02 vs. after Sw 0.67 ± 0.02, P = 0.12). Phosphorylation of Akt was significantly increased in both trained WT and IGF-1R−/− mice at Ser473 [fold-change 1.61 ± 0.09 (P &lt; 0.05) and 2.11 ± 0.19 (P &lt; 0.01), respectively] and Thr308 [2.42 ± 0.24 and 2.61 ± 0.59 (P &lt; 0.01), respectively] vs. Sed. Surprisingly Ser (473) Akt phosphorylation was greater in Sw IGF-1R−/− vs. Sw WT (P &lt; 0.05). These data define an essential role for IGF-1 signaling in mediating physiologic cardiomyocyte hypertrophy, and indicate that although Akt signaling might be necessary for mediating physiological cardiac hypertrophy it is not sufficient in the absence of myocardial IGF-1R signaling.

Cardiac Hypertrophy Caused by Peroxisome Proliferator- Activated Receptor-γ Agonist Treatment Occurs Independently of Changes in Myocardial Insulin Signaling

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands are insulin sensitizers, widely used in the treatment of type 2 diabetes. A consistent observation in preclinical species is the development of cardiac hypertrophy after short-term treatment with these agents. The mechanisms for this hypertrophy are incompletely understood. Given the important role of insulin signaling in the regulation of myocardial size, we tested the hypothesis that augmentation of myocardial insulin signaling may play a role in PPAR-gamma ligand-induced cardiac hypertrophy. We treated mice with cardiomyocyte-restricted knockout of insulin receptors (CIRKO) and littermate controls (wild type) with 2-(2-(4-phenoxy-2-propylphenoxy) ethyl) indole-5-acetic acid (COOH), which is a non-thiazolidinedione PPAR-gamma agonist for 2 wk. Two weeks of COOH treatment increased heart weights by 22% in CIRKO mice and 16% in wild type, and induced similar fold increase in the expression of hypertrophic markers such as alpha-skeletal actin, brain natriuretic peptide, and atrial natriuretic peptide in CIRKO and wild-type (WT) hearts. COOH treatment increased plasma volume by 10% in COOH-treated WT and CIRKO mice but did not increase systolic or diastolic blood pressure. Echocardiographic analysis was also consistent with volume overload, as evidenced by increased left ventricular diastolic diameters and cardiac output in COOH-treated CIRKO and WT mice. These data indicate that cardiac hypertrophy after PPAR-gamma agonist treatment can occur in the absence of myocardial insulin signaling and is likely secondary to the hemodynamic consequences of plasma volume expansion.

Genetic inhibition of cullin‐based ubiquitin ligase dynamics in adult mouse hearts suffices to cause heart failure (HF)

BACKGROUND: The COP9 signalosome (CSN) is an evolutionarily conserved protein complex formed by 8 subunits (CSN1 ~ CSN8). By deneddylating cullins (Cul), CSN regulates the dynamics of Cul-based E3s, a large family of ubiquitin ligases. The ubiquitin-proteasome system (UPS) degrades most cellular proteins. UPS malfunction in the heart is implicated in HF genesis but a causative relationship has not been established yet. METHODS & RESULTS: We achieved cardiomyocyte-restricted knockout of the Csn8 gene (CR-KO) in adult mice, using the Cre-loxP system with a tamoxifen-inducible modified Cre. Cardiac CSN8 protein nearly disappeared by 5 days after CR-KO induction. CR-KO increased the protein levels of neddylated Cul1 and Cul4a, p62/sequestosome 1, and the lipidated form of LC3 (LC3-II). Electron microscopy reveals marked ultrastructure changes: myofibril alignment disruption, mitochondrial degeneration, and massive autophagic vacuoles in CR-KO myocytes. Echocardiography shows progressive left ventricular (LV) wall thinning and marked declines in LV ejection fraction and fraction shortening. Significantly reduced cardiac contractility and relaxation were also detected by LV hemodynamics. All CR-KO mice died within 8 days after CR-KO induction. CONCLUSIONS: CSN8 is required for CSN activities and for maintaining cardiac structure and function in adult mice. UPS impairment suffices to cause HF.

Contractile dysfunction in hypertrophied hearts with deficient insulin receptor signaling: possible role of reduced capillary density

Diabetics have worse outcomes than nondiabetics after a variety of cardiac insults. We tested the hypothesis that impaired insulin receptor signaling in myocytes worsens cardiac remodeling and function following injury, even in the absence of hyperglycemia. Mice with cardiomyocyte-restricted knock out of the insulin receptor (CIRKO) and wild type (WT) mice were treated with isoproterenol (ISO) for 2 or 5 days. Heart rates and cardiac mass increased comparably following ISO in WT and CIRKO mice. After 5 days, WT hearts were hyperdynamic by echocardiographic and left ventricular pressure measurements. However, CIRKO hearts had a blunted increase in contractility and relaxation following ISO. Interestingly, single myocytes isolated from both CIRKO ISO and WT ISO hearts had increased cellular shortening with prolonged time to peak shortening vs. respective shams. Thus, loss of myocytes or extramyocyte factors, rather than intrinsic dysfunction of surviving myocytes, caused the blunted inotropic response in ISO treated CIRKO hearts. Indeed, CIRKO ISO mice had increased troponin release after 2 days and greater interstitial and sub-endocardial fibrosis at 5 days than did ISO WT. Apoptosis assessed by TUNEL and caspase staining was increased in CIRKO ISO compared to WT ISO hearts; however, very few of the apoptotic nuclei were clearly in cardiac myocytes. After 5 days of ISO treatment, VEGF expression was increased in WT but not in CIRKO hearts. In keeping with this finding, capillary density was reduced in CIRKO ISO relative to WT ISO. Basal expression of hypoxia-inducible factor-1α was lower in CIRKO vs. WT hearts and may explain the blunted VEGF response. Thus, absence of insulin receptor signaling in the cardiac myocyte worsens catecholamine-mediated myocardial injury, at least in part, via mechanisms that tend to impair myocardial blood flow and increase ischemic injury.

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein.

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.

Optical mapping of propagation changes induced by elevated extracellular potassium ion concentration in genetically altered mouse hearts

Diabetes is associated with high rates of cardiovascular disease and sudden death. Therefore, dissecting specific mechanisms, such as the effects of impaired insulin signaling on cardiac electrophysiology may lead to better diagnosis and treatment. Lack of insulin receptors in mouse myocytes has been shown to reduce repolarizing potassium currents and prolong action potential duration. We hypothesized that these changes would manifest as rate-related effects on electrical propagation in the intact heart. This study employed optical mapping to characterize propagation changes in intact mouse hearts with cardiomyocyte-restricted knock out of insulin receptors (CIRKO). Methods: Fluorescent signals emitted from excited Di-4-ANEPPS in isolated Langendorff perfused mouse hearts were recorded from the left ventricular epicardium using an 8 by 8 photo diode array. The study included hearts from 8 CIRKO mice and 8 wild type (WT) littermate controls. Hearts were stimulated from the right atrium or the left ventricle at basic cycle lengths ranging from 160 to 280 ms under normal conditions and then after 5 minutes of perfusion with elevated potassium ion concentration (9.4 mM). Results: None of the 8 CIRKO hearts maintained regular responses to atrial stimulation at the 160 ms cycle length under normal conditions; however, all of the WT hearts were captured at this rate. Total activation time for a 4 mm by 4 mm area was longer for CIRKO hearts when compared with WT. Average epicardial conduction velocity was slower for the CIRKO when compared to WT. Propagation delay due to the presence of high [K+] e was significant in both CIRKO and WT mice, but significantly longer for the CIRKO hearts. Conclusions: These results show that in addition to reducing repolarization currents, impaired myocardial insulin signaling leads to impaired electrical impulse propagation particularly at increased heart rates. These data suggest a link between impaired myocardial insulin signaling and the increased risk of arrhythmia and sudden death in patients with diabetes.

Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury.

The transcription factor signal transducer and activator of transcription 3 (STAT3) participates in a wide variety of physiological processes and directs seemingly contradictory responses such as proliferation and apoptosis. To elucidate its role in the heart, we generated mice harboring a cardiomyocyte-restricted knockout of STAT3 using Cre/loxP-mediated recombination. STAT3-deficient mice developed reduced myocardial capillary density and increased interstitial fibrosis within the first 4 postnatal months, followed by dilated cardiomyopathy with impaired cardiac function and premature death. Conditioned medium from STAT3-deficient cardiomyocytes inhibited endothelial cell proliferation and increased fibroblast proliferation, suggesting the presence of paracrine factors attenuating angiogenesis and promoting fibrosis in vitro. STAT3-deficient mice showed enhanced susceptibility to myocardial ischemia/reperfusion injury and infarction with increased cardiac apoptosis, increased infarct sizes, and reduced cardiac function and survival. Our study establishes a novel role for STAT3 in controlling paracrine circuits in the heart essential for postnatal capillary vasculature maintenance, interstitial matrix deposition balance, and protection from ischemic injury and heart failure.