Cardiomyocyte Expression of ZO-1 Is Essential for Normal Atrioventricular Conduction but Does Not Alter Ventricular Function.

RATIONALE: ZO-1 (Zonula occludens-1), a plasma membrane-associated scaffolding protein regulates signal transduction, transcription, and cellular communication. Global deletion of ZO-1 in the mouse is lethal by embryonic day 11.5. The function of ZO-1 in cardiac myocytes (CM) is largely unknown. OBJECTIVE: To determine the function of CM ZO-1 in the intact heart, given its binding to other CM proteins that have been shown instrumental in normal cardiac conduction and function. METHODS AND RESULTS: We generated ZO-1 CM-specific knockout (KO) mice using α-Myosin Heavy Chain-nuclear Cre (ZO-1cKO) and investigated physiological and electrophysiological function by echocardiography, surface ECG and conscious telemetry, intracardiac electrograms and pacing, and optical mapping studies. ZO-1cKO mice were viable, had normal Mendelian ratios, and had a normal lifespan. Ventricular morphometry and function were not significantly different between the ZO-1cKO versus control (CTL) mice, basally in young or aged mice, or even when hearts were subjected to hemodynamic loading. Atrial mass was increased in ZO-1cKO. Electrophysiological and optical mapping studies indicated high-grade atrioventricular (A-V) block in ZO-1cKO comparing to CTL hearts. While ZO-1-associated proteins such as vinculin, connexin 43, N-cadherin, and α-catenin showed no significant change with the loss of ZO-1, Connexin-45 and Coxsackie-adenovirus (CAR) proteins were reduced in atria of ZO-1cKO. Further, with loss of ZO-1, ZO-2 protein was increased significantly in ventricular CM in a presumed compensatory manner but was still not detected in the AV nodal myocytes. Importantly, the expression of the sodium channel protein NaV1.5 was altered in AV nodal cells of the ZO-1cKO versus CTL. CONCLUSIONS: ZO-1 protein has a unique physiological role in cardiac nodal tissue. This is in alignment with its known interaction with CAR and Cx45, and a new function in regulating the expression of NaV1.5 in AV node. Uniquely, ZO-1 is dispensable for function of the working myocardium.

Nexilin Is a New Component of Junctional Membrane Complexes Required for Cardiac T-Tubule Formation.

BACKGROUND: Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. The junctional membrane complexes in the cardiac dyads, where transverse (T) tubules are juxtaposed to the sarcoplasmic reticulum, are a prime example. T-tubule uncoupling and remodeling are well-known features of cardiac disease and heart failure. Even subtle alterations in the association between T-tubules and the junctional sarcoplasmic reticulum can cause serious cardiac disorders. NEXN (nexilin) has been identified as an actin-binding protein, and multiple mutations in the NEXN gene are associated with cardiac diseases, but the precise role of NEXN in heart function and disease is still unknown. METHODS: Nexn global and cardiomyocyte-specific knockout mice were generated. Comprehensive phenotypic and RNA sequencing and mass spectrometry analyses were performed. Heart tissue samples and isolated single cardiomyocytes were analyzed by electron and confocal microscopy. RESULTS: Global and cardiomyocyte-specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses revealed that NEXN interacted with junctional sarcoplasmic reticulum proteins, was essential for optimal calcium transients, and was required for initiation of T-tubule invagination and formation. CONCLUSIONS: These results demonstrated that NEXN is a pivotal component of the junctional membrane complex and is required for initiation and formation of T-tubules, thus providing insight into mechanisms underlying cardiomyopathy in patients with mutations in NEXN.

Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Statins, which reduce LDL-cholesterol by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin-induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity-dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin-1, dystrophin, epidermal growth factor receptor, and insulin receptor-ß; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte-equivalent HL-1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long-term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size-variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria- and endoplasmic reticulum-related genes; and a 21% increase in mortality in cardiac-specific vinculin-knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long-term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.-Godoy, J. C., Niesman, I. R., Busija, A. R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D. M., Kararigas, G., Patel, H. H., Zemljic-Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Infarct Fibroblasts Do Not Derive From Bone Marrow Lineages.

RATIONALE: Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar. OBJECTIVE: The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter. METHODS AND RESULTS: Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery. CONCLUSIONS: In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure.

Impaired mitophagy facilitates mitochondrial damage in Danon disease.

RATIONALE: Lysosomal associated membrane protein type-2 (LAMP-2) is a highly conserved, ubiquitous protein that is critical for autophagic flux. Loss of function mutations in the LAMP-2 gene cause Danon disease, a rare X-linked disorder characterized by developmental delay, skeletal muscle weakness, and severe cardiomyopathy. We previously found that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from Danon patients exhibited significant mitochondrial oxidative stress and apoptosis. Understanding how loss of LAMP-2 expression leads to cardiomyocyte dysfunction and heart failure has important implications for the treatment of Danon disease as well as a variety of other cardiac disorders associated with impaired autophagy. OBJECTIVE: Elucidate the pathophysiology of cardiac dysfunction in Danon disease. METHODS AND RESULTS: We created hiPSCs from two patients with Danon disease and differentiated those cells into hiPSC-CMs using well-established protocols. Danon hiPSC-CMs demonstrated an accumulation of damaged mitochondria, disrupted mitophagic flux, depressed mitochondrial respiratory capacity, and abnormal gene expression of key mitochondrial pathways. Restoring the expression of LAMP-2B, the most abundant LAMP-2 isoform in the heart, rescued mitophagic flux as well as mitochondrial health and bioenergetics. To confirm our findings in vivo, we evaluated Lamp-2 knockout (KO) mice. Impaired autophagic flux was noted in the Lamp-2 KO mice compared to WT reporter mice, as well as an increased number of abnormal mitochondria, evidence of incomplete mitophagy, and impaired mitochondrial respiration. Physiologically, Lamp-2 KO mice demonstrated early features of contractile dysfunction without overt heart failure, indicating that the metabolic abnormalities associated with Danon disease precede the development of end-stage disease and are not merely part of the secondary changes associated with heart failure. CONCLUSIONS: Incomplete mitophagic flux and mitochondrial dysfunction are noted in both in vitro and in vivo models of Danon disease, and proceed overt cardiac contractile dysfunction. This suggests that impaired mitochondrial clearance may be central to the pathogenesis of disease and a potential target for therapeutic intervention.

Mitochondrial reprogramming induced by CaMKIIδ mediates hypertrophy decompensation.

RATIONALE: Sustained activation of Gαq transgenic (Gq) signaling during pressure overload causes cardiac hypertrophy that ultimately progresses to dilated cardiomyopathy. The molecular events that drive hypertrophy decompensation are incompletely understood. Ca(2+)/calmodulin-dependent protein kinase II δ (CaMKIIδ) is activated downstream of Gq, and overexpression of Gq and CaMKIIδ recapitulates hypertrophy decompensation. OBJECTIVE: To determine whether CaMKIIδ contributes to hypertrophy decompensation provoked by Gq. METHODS AND RESULTS: Compared with Gq mice, compound Gq/CaMKIIδ knockout mice developed a similar degree of cardiac hypertrophy but exhibited significantly improved left ventricular function, less cardiac fibrosis and cardiomyocyte apoptosis, and fewer ventricular arrhythmias. Markers of oxidative stress were elevated in mitochondria from Gq versus wild-type mice and respiratory rates were lower; these changes in mitochondrial function were restored by CaMKIIδ deletion. Gq-mediated increases in mitochondrial oxidative stress, compromised membrane potential, and cell death were recapitulated in neonatal rat ventricular myocytes infected with constitutively active Gq and attenuated by CaMKII inhibition. Deep RNA sequencing revealed altered expression of 41 mitochondrial genes in Gq hearts, with normalization of ≈40% of these genes by CaMKIIδ deletion. Uncoupling protein 3 was markedly downregulated in Gq or by Gq expression in neonatal rat ventricular myocytes and reversed by CaMKIIδ deletion or inhibition, as was peroxisome proliferator-activated receptor α. The protective effects of CaMKIIδ inhibition on reactive oxygen species generation and cell death were abrogated by knock down of uncoupling protein 3. Conversely, restoration of uncoupling protein 3 expression attenuated reactive oxygen species generation and cell death induced by CaMKIIδ. Our in vivo studies further demonstrated that pressure overload induced decreases in peroxisome proliferator-activated receptor α and uncoupling protein 3, increases in mitochondrial protein oxidation, and hypertrophy decompensation, which were attenuated by CaMKIIδ deletion. CONCLUSIONS: Mitochondrial gene reprogramming induced by CaMKIIδ emerges as an important mechanism contributing to mitotoxicity in decompensating hypertrophy.

Selective Life-Long Skeletal Myofiber-Targeted VEGF Gene Ablation Impairs Exercise Capacity in Adult Mice.

Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF-/-) during embryogenesis (∼ E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (P < 0.0001, each muscle). VEGF levels were unchanged in the heart. Treadmill speed (WT 86 ± 4 cm/sec, skmVEGF-/- 70 ± 5 cm/sec, P = 0.006) and endurance (WT 78 ± 24 min, skmVEGF-/- 18 ± 4 min, P = 0.0004) were severely limited in skmVEGF-/- mice in contrast to minor effect of conditional skmVEGF gene deletion in the adult. Body weight was also reduced (WT 22.8 ± 1.6 g, skmVEGF-/-, 21.1 ± 1.5, P = 0.02), but the muscle mass/body weight ratio was unchanged. The capillary/fiber ratio was lower in skmVEGF-/- plantaris (WT 1.51 ± 0.12, skmVEGF-/- 1.16 ± 0.20, P = 0.01), gastrocnemius (WT 1.61 ± 0.08, skmVEGF-/- 1.39 ± 0.08, P = 0.01), EDL (WT 1.36 ± 0.07, skmVEGF-/- 1.14 ± 0.13, P = 0.03) and diaphragm (WT 1.39 ± 0.18, skmVEGF-/- 0.79 ± 0.16, P = 0.0001) but, not in soleus. Cardiac function (heart rate, maximal pressure, maximal dP/dt, minimal dP/dt,) in response to dobutamine was not impaired in anesthetized skmVEGF-/- mice. Isolated soleus and EDL fatigue times were 16% and 20% (P < 0.02) longer, respectively, in skmVEGF-/- mice than the WT group. These data suggest that skeletal myofiber VEGF expressed during development is necessary to establish capillary networks that allow maximal exercise capacity.

Lmo7 is dispensable for skeletal muscle and cardiac function.

Emery-Dreifuss muscular dystrophy (EDMD) is a degenerative disease primarily affecting skeletal muscles in early childhood as well as cardiac muscle at later stages. EDMD is caused by a number of mutations in genes encoding proteins associated with the nuclear envelope (e.g., Emerin, Lamin A/C, and Nesprin). Recently, a novel protein, Lim-domain only 7 (lmo7) has been reported to play a role in the molecular pathogenesis of EDMD. Prior in vitro and in vivo studies suggested the intriguing possibility that Lmo7 plays a role in skeletal or cardiac muscle pathophysiology. To further understand the in vivo role of Lmo7 in striated muscles, we generated a novel Lmo7-null (lmo7(-/-)) mouse line. Using this mouse line, we examined skeletal and cardiac muscle physiology, as well as the role of Lmo7 in a model of muscular dystrophy and regeneration using the dystrophin-deficient mdx mouse model. Our results demonstrated that lmo7(-/-) mice had no abnormalities in skeletal muscle morphology, physiological function, or regeneration. Cardiac function was also unaffected. Moreover, we found that ablation of lmo7 in mdx mice had no effect on the observed myopathy and muscular regeneration exhibited by mdx mice. Molecular analyses also showed no changes in dystrophin complex factors, MAPK pathway components, and Emerin levels in lmo7 knockout mice. Taken together, we conclude that Lmo7 is dispensable for skeletal muscle and cardiac physiology and pathophysiology.

Ca2+/Calmodulin-dependent protein kinase II δ mediates myocardial ischemia/reperfusion injury through nuclear factor-κB.

RATIONALE: Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated as a maladaptive mediator of cardiac ischemic injury. We hypothesized that the inflammatory response associated with in vivo ischemia/reperfusion (I/R) is initiated through CaMKII signaling. OBJECTIVE: To assess the contribution of CaMKIIδ to the development of inflammation, infarct, and ventricular dysfunction after in vivo I/R and define early cardiomyocyte-autonomous events regulated by CaMKIIδ using cardiac-specific knockout mice. METHODS AND RESULTS: Wild-type and CaMKIIδ knockout mice were subjected to in vivo I/R by occlusion of the left anterior descending artery for 1 hour followed by reperfusion for various times. CaMKIIδ deletion protected the heart against I/R damage as evidenced by decreased infarct size, attenuated apoptosis, and improved functional recovery. CaMKIIδ deletion also attenuated I/R-induced inflammation and upregulation of nuclear factor-κB (NF-κB) target genes. Further studies demonstrated that I/R rapidly increases CaMKII activity, leading to NF-κB activation within minutes of reperfusion. Experiments using cyclosporine A and cardiac-specific CaMKIIδ knockout mice indicate that NF-κB activation is initiated independent of necrosis and within cardiomyocytes. Expression of activated CaMKII in cardiomyocytes leads to IκB kinase phosphorylation and concomitant increases in nuclear p65. Experiments using an IκB kinase inhibitor support the conclusion that this is a proximal site of CaMKII-mediated NF-κB activation. CONCLUSIONS: This is the first study demonstrating that CaMKIIδ mediates NF-κB activation in cardiomyocytes after in vivo I/R and suggests that CaMKIIδ serves to trigger, as well as to sustain subsequent changes in inflammatory gene expression that contribute to myocardial I/R damage.

Discoidin domain receptor 2 germline gene deletion leads to altered heart structure and function in the mouse.

Discoidin domain receptor 2 (DDR2) is a fibrillar collagen receptor that is expressed in mesenchymal cells throughout the body. In the heart, DDR2 is selectively expressed on cardiac fibroblasts. We generated a germline DDR2 knockout mouse and used this mouse to examine the role of DDR2 deletion on heart structure and function. Echocardiographic measurements from null mice were consistent with those from a smaller heart, with reduced left ventricular chamber dimensions and little change in wall thickness. Fractional shortening appeared normal. Left ventricular pressure measurements revealed mild inotropic and lusitropic abnormalities that were accentuated by dobutamine infusion. Both body and heart weights from 10-wk-old male mice were ~20% smaller in null mice. The reduced heart size was not simply due to reduced body weight, since cardiomyocyte lengths were atypically shorter in null mice. Although normalized cardiac collagen mass (assayed by hydroxyproline content) was not different in null mice, the collagen area fraction was statistically higher, suggesting a reduced collagen density from altered collagen deposition and cross-linking. Cultured cardiac fibroblasts from null mice deposited collagen at a slower rate than wild-type littermates, possibly due to the expression of lower prolyl 4-hydroxylase α-isoform 1 enzyme levels. We conclude that genetic deletion of the DDR2 collagen receptor alters cardiac fibroblast function. The resulting perturbations in collagen deposition can influence the structure and function of mature cardiomyocytes.

Thymosin beta 4 is dispensable for murine cardiac development and function.

RATIONALE: Thymosin beta 4 (Tß4) is a 43-amino acid factor encoded by an X-linked gene. Recent studies have suggested that Tß4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of tß4 has been reported to result in embryonic lethality at E14.5-16.5, with severe cardiac and angiogenic defects. However, this shRNA tß4-knockdown model did not completely abrogate Tß4 expression. To completely ablate Tß4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical. OBJECTIVE: We examined the role of Tß4 in developing and adult heart through global and cardiac specific tß4-knockout mouse models. METHODS AND RESULTS: Global tß4-knockout mice were born at mendelian ratios and exhibited normal heart and blood vessel formation. Furthermore, in adult global tß4-knockout mice, cardiac function, capillary density, expression of key cardiac fetal and angiogenic genes, epicardial marker expression, and extracellular matrix deposition were indistinguishable from that of controls. Tissue-specific tß4-deficient mice, generated by crossing tß4-floxed mice to Nkx2.5-Cre and αMHC-Cre, were also found to have no phenotype. CONCLUSIONS: We conclude that Tß4 is dispensable for embryonic viability, heart development, coronary vessel development, and adult myocardial function.

MicroRNA-133 controls cardiac hypertrophy.

Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.

Selective deletion of long but not short Cypher isoforms leads to late-onset dilated cardiomyopathy.

Cypher long (CypherL) and short (CypherS) isoforms are distinguished from each other by the presence and absence of three C-terminal LIM domains, respectively. Cypher isoforms are developmentally regulated, and mutations affecting both long and short isoforms are linked to muscle disease in humans. Given these data, we hypothesized that various Cypher isoforms play overlapping and unique roles in striated muscle. To determine the specific role of Cypher isoforms in striated muscle, we generated two mouse lines in which either CypherS or CypherL isoforms were specifically deleted. Mice specifically, deficient in CypherS isoforms had no detectable muscle phenotype. In contrast, selective loss of CypherL isoforms resulted in partial neonatal lethality. Surviving mutants exhibited growth retardation and late-onset dilated cardiomyopathy, which was associated with cardiac fibrosis and calcification, leading to premature adult mortality. At a young age, preceding development of cardiomyopathy, hearts from these mutants exhibited defects in both Z-line ultrastructure and specific aberrations in calcineurin-NFAT and protein kinase C pathways. Earlier onset of cardiac dilation relative to control wild-type mice was observed in young CypherL isoform knockout mice consequent to pressure overload, suggesting a greater susceptibility to the disease. In summary, we have identified unique roles for CypherL isoforms in maintaining Z-line ultrastructure and signaling that are distinct from the roles of CypherS isoforms, while highlighting the contribution of mutations in the long isoforms to the development of dilated cardiomyopathy.

ß1 integrin gene excision in the adult murine cardiac myocyte causes defective mechanical and signaling responses.

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which ß1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of ß1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte ß1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of ß1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that ß1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

Absence of SOCS3 in the cardiomyocyte increases mortality in a gp130-dependent manner accompanied by contractile dysfunction and ventricular arrhythmias.

BACKGROUND: Suppressor of cytokine signaling-3 (SOCS3) is a key negative-feedback regulator of the gp130 receptor that provides crucial signaling for cardiac hypertrophy and survival; however, an in vivo role of SOCS3 regulation on cardiac gp130 signaling remains obscure. METHODS AND RESULTS: We generated cardiac-specific SOCS3 knockout (SOCS3 cKO) mice. These mice showed increased activation of gp130 downstream signaling targets (STAT3, ERK1/2, AKT, and p38) from 15 weeks of age and developed cardiac dysfunction from approximately 25 weeks of age with signs of heart failure. Surprisingly, SOCS3 cKO failing hearts had minimal histological abnormalities with intact myofibril ultrastructure. In addition, Ca(2+) transients were significantly increased in SOCS3 cKO failing hearts compared with wild-type hearts. We also found that Ser23/24 residues of troponin I were hypophosphorylated in SOCS3 cKO hearts before the manifestation of cardiac dysfunction. These data suggested the presence of abnormalities in myofilament Ca(2+) sensitivity in SOCS3 cKO mice. In addition to the contractile dysfunction, we found various ventricular arrhythmias in SOCS3 cKO nonfailing hearts accompanied by a sarcoplasmic reticulum Ca(2+) overload. To determine the contribution of gp130 signaling to the cardiac phenotype that occurs with SOCS3 deficiency, we generated cardiac-specific gp130 and SOCS3 double KO mice. Double KO mice lived significantly longer and had different histological abnormalities when compared with SOCS3 cKO mice, thus demonstrating the importance of gp130 signaling in the SOCS3 cKO cardiac phenotype. CONCLUSIONS: Our results demonstrate an important role of SOCS3 regulation on cardiac gp130 signaling in the pathogenesis of contractile dysfunction and ventricular arrhythmias.

Pressure overload-induced cardiac remodeling and dysfunction in the absence of interleukin 6 in mice.

Congestive heart failure is associated with increased expression of pro-inflammatory cytokines, and the levels of these cytokines correlate with heart failure severity and prognosis. Chronic interleukin 6 (IL-6) stimulation leads to left ventricular (LV) hypertrophy and dysfunction, and deletion of IL-6 reduces LV hypertrophy after angiotensin II infusion. In this study, we tested the hypothesis that IL-6 deletion has favorable effects on pressure-overloaded hearts. We performed transverse aortic constriction on IL-6-deleted (IL6KO) mice and C57BL/6J mice (CON) to induce pressure overload. Pressure overload was associated with similar LV hypertrophy, dilation, and dysfunction in CON and IL6KO mice. Re-activation of the fetal gene program was also similar in pressure-overloaded CON and IL6KO mice. There were no differences between CON and IL6KO mice in LV fibrosis or expression of extracellular matrix proteins after pressure overload. In addition, no group differences in apoptosis or autophagy were seen. These data indicate that IL-6 deletion does not block LV remodeling and dysfunction induced by pressure overload. Attenuated content of IL-11 appears to be a compensatory mechanism for IL-6 deletion in pressure-overloaded hearts. We infer from these data that limiting availability of IL-6 alone is not sufficient to attenuate LV remodeling and dysfunction in failing hearts.

Nesprin 1 is critical for nuclear positioning and anchorage.

Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.

Phospholamban ablation rescues sarcoplasmic reticulum Ca(2+) handling but exacerbates cardiac dysfunction in CaMKIIdelta(C) transgenic mice.

RATIONALE: We previously showed that transgenic mice expressing Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca(2+) leak and lowering of SR Ca(2+) load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca(2+) load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG. OBJECTIVE: Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIdelta(C) expression were corrected, and establish the mechanistic basis for these changes. METHODS AND RESULTS: CaMKII-TG were crossed with PLN knockout (PLN-KO) mice to generate KO/TG mice. Myocytes from wild type (WT), CaMKII-TG, PLN-KO and KO/TG were compared. The decreased SR Ca(2+) load and twitch Ca(2+) transients seen in CaMKII-TG were normalized in KO/TG. Surprisingly the heart failure phenotype was exacerbated, as indicated by increased left ventricular dilation, decreased ventricular function, increased apoptosis and greater mortality. In KO/TG myocytes SR Ca(2+) sparks and leak were significantly increased, presumably because of the combined effects of restored SR Ca(2+) load and RyR2 phosphorylation. Mitochondrial Ca(2+) loading was increased in cardiomyocytes from KO/TG versus WT or CaMKII-TG mice and this was dependent on elevated SR Ca(2+) sparks. Cardiomyocytes from KO/TG showed poor viability, improved by inhibiting SR Ca(2+) release and mitochondrial Ca(2+) loading. CONCLUSIONS: Normalizing cardiomyocyte SR Ca(2+) loading in the face of elevated CaMKII and RyR2 phosphorylation leads to enhanced SR Ca(2+) leak and mitochondrial Ca(2+) elevation, associated with exacerbated cell death, heart failure and mortality.

Loss of enigma homolog protein results in dilated cardiomyopathy.

RATIONALE: The Z-line, alternatively termed the Z-band or Z-disc, is a highly ordered structure at the border between 2 sarcomeres. Enigma subfamily proteins (Enigma, Enigma homolog protein, and Cypher) of the PDZ-LIM domain protein family are Z-line proteins. Among the Enigma subfamily, Cypher has been demonstrated to play a pivotal role in the structure and function of striated muscle, whereas the role of Enigma homolog protein (ENH) in muscle remains largely unknown. OBJECTIVE: We studied the role of Enigma homolog protein in the heart using global and cardiac-specific ENH knockout mouse models. METHODS AND RESULTS: We identified new exons and splice isoforms for ENH in the mouse heart. Impaired cardiac contraction and dilated cardiomyopathy were observed in ENH null mice. Mice with cardiac specific ENH deletion developed a similar dilated cardiomyopathy. Like Cypher, ENH interacted with Calsarcin-1, another Z-line protein. Moreover, biochemical studies showed that ENH, Cypher short isoform and Calsarcin-1 are within the same protein complex at the Z-line. Cypher short isoform and Calsarcin-1 proteins are specifically downregulated in ENH null hearts. CONCLUSIONS: We have identified an ENH-CypherS-Calsarcin protein complex at the Z-line. Ablation of ENH leads to destabilization of this protein complex and dilated cardiomyopathy.

CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.