Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

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.

Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease.

Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.

Aortic pathology from protein kinase G activation is prevented by an antioxidant vitamin B12 analog.

People heterozygous for an activating mutation in protein kinase G1 (PRKG1, p.Arg177Gln) develop thoracic aortic aneurysms and dissections (TAAD) as young adults. Here we report that mice heterozygous for the mutation have a three-fold increase in basal protein kinase G (PKG) activity, and develop age-dependent aortic dilation. Prkg1R177Q/+ aortas show increased smooth muscle cell apoptosis, elastin fiber breaks, and oxidative stress compared to aortas from wild type littermates. Transverse aortic constriction (TAC)-to increase wall stress in the ascending aorta-induces severe aortic pathology and mortality from aortic rupture in young mutant mice. The free radical-neutralizing vitamin B12-analog cobinamide completely prevents age-related aortic wall degeneration, and the unrelated anti-oxidant N-acetylcysteine ameliorates TAC-induced pathology. Thus, increased basal PKG activity induces oxidative stress in the aorta, raising concern about the widespread clinical use of PKG-activating drugs. Cobinamide could be a treatment for aortic aneurysms where oxidative stress contributes to the disease, including Marfan syndrome.

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.

Luma is not essential for murine cardiac development and function.

AIMS: Luma is a recently discovered, evolutionarily conserved protein expressed in mammalian heart, which is associated with the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex. The LINC complex structurally integrates the nucleus and the cytoplasm and plays a critical role in mechanotransduction across the nuclear envelope. Mutations in several LINC components in both humans and mice result in various cardiomyopathies, implying they play essential, non-redundant roles. A single amino acid substitution of serine 358 to leucine (S358L) in Luma is the unequivocal cause of a distinct form of arrhythmogenic cardiomyopathy. However, the role of Luma in heart has remained obscure. In addition, it also remains to be determined how the S358L mutation in Luma leads to cardiomyopathy. METHODS AND RESULTS: To determine the role of Luma in the heart, we first determined the expression pattern of Luma in mouse heart. Luma was sporadically expressed in cardiomyocytes throughout the heart, but was highly and uniformly expressed in cardiac fibroblasts and vascular smooth muscle cells. We also generated germline null Luma mice and discovered that germline null mutants were viable and exhibited normal cardiac function. Luma null mice also responded normally to pressure overload induced by transverse aortic constriction. In addition, localization and expression of other LINC complex components in both cardiac myocytes and fibroblasts was unaffected by global loss of Luma. Furthermore, we also generated and characterized Luma S358L knock-in mice, which displayed normal cardiac function and morphology. CONCLUSION: Our data suggest that Luma is dispensable for murine cardiac development and function and that the Luma S358L mutation alone may not cause cardiomyopathy in mice.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo.

Adipocyte-specific loss of PPARγ attenuates cardiac hypertrophy.

Adipose tissue is a key endocrine organ that governs systemic homeostasis. PPARγ is a master regulator of adipose tissue signaling that plays an essential role in insulin sensitivity, making it an important therapeutic target. The selective PPARγ agonist rosiglitazone (RSG) has been used to treat diabetes. However, adverse cardiovascular effects have seriously hindered its clinical application. Experimental models have revealed that PPARγ activation increases cardiac hypertrophy. RSG stimulates cardiac hypertrophy and oxidative stress in cardiomyocyte-specific PPARγ knockout mice, implying that RSG might stimulate cardiac hypertrophy independently of cardiomyocyte PPARγ. However, candidate cell types responsible for RSG-induced cardiomyocyte hypertrophy remain unexplored. Utilizing cocultures of adipocytes and cardiomyocytes, we found that stimulation of PPARγ signaling in adipocytes increased miR-200a expression and secretion. Delivery of miR-200a in adipocyte-derived exosomes to cardiomyocytes resulted in decreased TSC1 and subsequent mTOR activation, leading to cardiomyocyte hypertrophy. Treatment with an antagomir to miR-200a blunted this hypertrophic response in cardiomyocytes. In vivo, specific ablation of PPARγ in adipocytes was sufficient to blunt hypertrophy induced by RSG treatment. By delineating mechanisms by which RSG elicits cardiac hypertrophy, we have identified pathways that mediate the crosstalk between adipocytes and cardiomyocytes to regulate cardiac remodeling.

Intracoronary Gene Transfer of Adenylyl Cyclase 6 in Patients With Heart Failure: A Randomized Clinical Trial.

IMPORTANCE: Gene transfer has rarely been tested in randomized clinical trials. OBJECTIVE: To evaluate the safety and efficacy of intracoronary delivery of adenovirus 5 encoding adenylyl cyclase 6 (Ad5.hAC6) in heart failure. DESIGN, SETTING, AND PARTICIPANTS: A randomized, double-blind, placebo-controlled, phase 2 clinical trial was conducted in US medical centers (randomization occurred from July 19, 2010, to October 30, 2014). Participants 18 to 80 years with symptomatic heart failure (ischemic and nonischemic) and an ejection fraction (EF) of 40% or less were screened; 86 individuals were enrolled, and 56 were randomized. Data analysis was of the intention-to-treat population. Participants underwent exercise testing and measurement of left ventricular EF (echocardiography) and then cardiac catheterization, where left ventricular pressure development (+dP/dt) and decline (-dP/dt) were recorded. Participants were randomized (3:1 ratio) to receive 1 of 5 doses of intracoronary Ad5.hAC6 or placebo. Participants underwent a second catheterization 4 weeks later for measurement of dP/dt. Exercise testing and EF were assessed 4 and 12 weeks after randomization. INTERVENTIONS: Intracoronary administration of Ad5.hAC6 (3.2 × 109 to 1012 virus particles) or placebo. MAIN OUTCOMES AND MEASURES: Primary end points included exercise duration and EF before and 4 and 12 weeks after randomization and peak rates of +dP/dt and -dP/dt before and 4 weeks after randomization. Fourteen placebo participants were compared (intention to treat) with 24 Ad5.hAC6 participants receiving the highest 2 doses (D4 + 5). RESULTS: Fifty-six individuals were randomized and monitored for up to 1 year. Forty-two participants (75%) received Ad5.hAC6 (mean [SE] age, 63 [1] years; EF, 30% [1%]), and 14 individuals (25%) received placebo (age, 62 [1] years; EF, 30% [2%]). Exercise duration showed no significant group differences (4 weeks, P = .27; 12 weeks, P = .47, respectively). The D4 + 5 participants had increased EF at 4 weeks (+6.0 [1.7] EF units; n = 21; P < .004), but not 12 weeks (+3.0 [2.4] EF units; n = 21; P = .16). Placebo participants showed no increase in EF at 4 weeks or 12 weeks. Exercise duration showed no between-group differences (4-week change from baseline: placebo, 27 [36] seconds; D4 + 5, 44 [25] seconds; P = .27; 12-week change from baseline: placebo, 44 [28] seconds; D4 + 5, 58 [29 seconds, P = .47). AC6 gene transfer increased basal left ventricular peak -dP/dt (4-week change from baseline: placebo, +93 [51] mm Hg/s; D4 + 5, -39 [33] mm Hg/s; placebo [n = 21]; P < .03); AC6 did not increase arrhythmias. The admission rate for patients with heart failure was 9.5% (4 of 42) in the AC6 group and 28.6% (4 of 14) in the placebo group (relative risk, 0.33 [95% CI, 0.08-1.36]; P = .10). CONCLUSIONS AND RELEVANCE: AC6 gene transfer safely increased LV function beyond standard heart failure therapy, attainable with one-time administration. Larger trials are warranted. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00787059.

MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy.

MLP (muscle LIM protein)-deficient mice count among the first mouse models for dilated cardiomyopathy (DCM), yet the exact role of MLP in cardiac signalling processes is still enigmatic. Elevated PKCα signalling activity is known to be an important contributor to heart failure. Here we show that MLP directly inhibits the activity of PKCα. In end-stage DCM, PKCα is concentrated at the intercalated disc of cardiomyocytes, where it is sequestered by the adaptor protein CARP in a multiprotein complex together with PLCß1. In mice deficient for both MLP and CARP the chronic PKCα signalling chain at the intercalated disc is broken and they remain healthy. Our results suggest that the main role of MLP in heart lies in the direct inhibition of PKCα and that chronic uninhibited PKCα activity at the intercalated disc in the absence of functional MLP leads to heart failure.

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.

Normalization of Naxos plakoglobin levels restores cardiac function in mice.

Arrhythmogenic cardiomyopathy (AC) is associated with mutations in genes encoding intercalated disc proteins and ultimately results in sudden cardiac death. A subset of patients with AC have the autosomal recessive cardiocutaneous disorder Naxos disease, which is caused by a 2-base pair deletion in the plakoglobin-encoding gene JUP that results in a truncated protein with reduced expression. In mice, cardiomyocyte-specific plakoglobin deficiency recapitulates many aspects of human AC, and overexpression of the truncated Naxos-associated plakoglobin also results in an AC-like phenotype; therefore, it is unclear whether Naxos disease results from loss or gain of function consequent to the plakoglobin mutation. Here, we generated 2 knockin mouse models in which endogenous Jup was engineered to express the Naxos-associated form of plakoglobin. In one model, Naxos plakoglobin bypassed the nonsense-mediated mRNA decay pathway, resulting in normal levels of the truncated plakoglobin. Moreover, restoration of Naxos plakoglobin to WT levels resulted in normal heart function. Together, these data indicate that a gain of function in the truncated form of the protein does not underlie the clinical phenotype of patients with Naxos disease and instead suggest that insufficiency of the truncated Naxos plakoglobin accounts for disease manifestation. Moreover, these results suggest that increasing levels of truncated or WT plakoglobin has potential as a therapeutic approach to Naxos disease.

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.

Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition. Instead, pressure overload promoted comparable proliferation and activation of two resident fibroblast lineages, including a previously described epicardial population and a population of endothelial origin. Together, these data present a paradigm for the origins of cardiac fibroblasts during development and in fibrosis. Furthermore, these data indicate that therapeutic strategies for reducing pathogenic cardiac fibroblasts should shift from targeting presumptive EndoMT or infiltrating hematopoietically derived fibroblasts, toward common pathways upregulated in two endogenous fibroblast populations.

Inhibition of Coxsackievirus-associated dystrophin cleavage prevents cardiomyopathy.

Heart failure in children and adults is often the consequence of myocarditis associated with Coxsackievirus (CV) infection. Upon CV infection, enteroviral protease 2A cleaves a small number of host proteins including dystrophin, which links actin filaments to the plasma membrane of muscle fiber cells (sarcolemma). It is unknown whether protease 2A-mediated cleavage of dystrophin and subsequent disruption of the sarcolemma play a role in CV-mediated myocarditis. We generated knockin mice harboring a mutation at the protease 2A cleavage site of the dystrophin gene, which prevents dystrophin cleavage following CV infection. Compared with wild-type mice, we found that mice expressing cleavage-resistant dystrophin had a decrease in sarcolemmal disruption and cardiac virus titer following CV infection. In addition, cleavage-resistant dystrophin inhibited the cardiomyopathy induced by cardiomyocyte-restricted expression of the CV protease 2A transgene. These findings indicate that protease 2A-mediated cleavage of dystrophin is critical for viral propagation, enteroviral-mediated cytopathic effects, and the development of cardiomyopathy.

Intravenous adeno-associated virus serotype 8 encoding urocortin-2 provides sustained augmentation of left ventricular function in mice.

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. Here we ask whether a single intravenous injection of adeno-associated virus type 8 encoding murine urocortin-2 (AAV8.UCn2) could provide long-term elevation in plasma UCn2 levels and increased left ventricular (LV) function. Normal mice received AAV8.UCn2 (5×10¹¹ genome copies, intravenous). Plasma UCn2 increased 15-fold 6 weeks and >11-fold 7 months after delivery. AAV8 DNA and UCn2 mRNA expression was persistent in LV and liver up to 7 months after a single intravenous injection of AAV8.UCn2. Physiological studies conducted both in situ and ex vivo showed increases in LV +dP/dt and in LV -dP/dt, findings that endured unchanged for 7 months. SERCA2a mRNA and protein expression was increased in LV samples and Ca²âº transient studies showed an increased rate of Ca²âº decline in cardiac myocytes from mice that had received UCn2 gene transfer. We conclude that a single intravenous injection of AAV8.UCn2 increases plasma UCn2 and increases LV systolic and diastolic function for at least 7 months. The simplicity of intravenous injection of a long-term expression vector encoding a gene with paracrine activity to increase cardiac function is a potentially attractive strategy in clinical settings. Future studies will determine the usefulness of this approach in the treatment of heart failure.

Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac fibrosis in mice.

BACKGROUND: Circulating subclinical lipopolysaccharide (LPS) occurs in health and disease. Ingesting high fatty meals increases LPS that cause metabolic endotoxemia. Subclinical LPS in periodontal disease may impair endothelial function. The heart may be targeted as cardiac cells express TLR4, the LPS receptor. It was hypothesized that recurrent exposure to subclinical LPS increases mortality and causes cardiac fibrosis. METHODS: C57Bl/6 mice were injected with intraperitoneal saline (control), low dose LPS (0.1 or 1 mg/kg), or moderate dose LPS (10 or 20 mg/kg), once a week for 3 months. Left ventricular (LV) function (echocardiography), hemodynamics (tail cuff pressure) and electrocardiograms (telemetry) were measured. Cardiac fibrosis was assessed by picrosirius red staining and LV expression of fibrosis related genes (QRT-PCR). Adult cardiac fibroblasts were isolated and exposed to LPS. RESULTS: LPS injections transiently increased heart rate and blood pressure (<6 hours) and mildly decreased LV function with full recovery by 24 hours. Mice tolerated weekly LPS for 2-3 months with no change in activity, appearance, appetite, weight, blood pressure, LV function, oximetry, or blood chemistries. Mortality increased after 60-90 days with moderate, but not low dose LPS. Arrhythmias occurred a few hours before death. LV collagen fraction area increased dose-dependently from 3.0±0.5% (SEM) in the saline control group, to 5.6±0.5% with low dose LPS and 9.7±0.9% with moderate dose LPS (P<0.05 moderate vs low dose LPS, and each LPS dose vs control). LPS increased LV expression of collagen Iα1, collagen IIIα1, MMP2, MMP9, TIMP1, periostin and IL-6 (P<0.05 moderate vs low dose LPS and vs control). LPS increased α-SMA immunostaining of myofibroblasts. LPS dose-dependently increased IL-6 in isolated adult cardiac fibroblasts. CONCLUSIONS: Recurrent exposure to subclinical LPS increases mortality and induces cardiac fibrosis.

Impaired exercise capacity and skeletal muscle function in a mouse model of pulmonary inflammation.

Pulmonary TNFα has been linked to reduced exercise capacity in a subset of patients with moderate to severe chronic obstructive pulmonary disease (COPD). We hypothesized that prolonged, high expression of pulmonary TNFα impairs cardiac and skeletal muscle function, and both contribute to exercise limitation. Using a surfactant protein C promoter-TNFα construct, TNFα was overexpressed throughout life in mouse lungs (SP-C/TNFα+). TNFα levels in wild-type (WT) female serum and lung were two- and threefold higher than in WT male mice. In SP-C/TNFα+ mice, TNFα increased similarly in both sexes. Treadmill exercise was impaired only in male SP-C/TNFα+ mice. While increases in lung volume and airspace size induced by TNFα were comparable in both sexes, pulmonary hypertension along with lower body and muscle mass were evident only in male mice. Left ventricular (LV) function (cardiac output, stroke volume, LV maximal pressure, and LV maximal pressure dP/dt) was not altered by TNFα overexpression. Fatigue measured in isolated soleus and EDL was more rapid only in soleus of male SP-C/TNFα+ mice and accompanied by a loss of oxidative IIa fibers, citrate synthase activity, and PGC-1α mRNA and increase in atrogin-1 and MuRF1 expression also only in male mice. In situ gastrocnemius fatigue resistance, reflecting both oxygen availability and contractility, was decreased similarly in female and male SP-C/TNFα+ mice. These data indicate that male, but not female, mice overexpressing pulmonary TNFα are susceptible to exercise limitation, possibly due to muscle wasting and loss of the oxidative muscle phenotype, with protection in females possibly due to estrogen.

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.