Constitutive protein kinase G activation exacerbates stress-induced cardiomyopathy.

BACKGROUND AND PURPOSE: Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long-term PKG activation in the heart are unknown. EXPERIMENTAL APPROACH: We characterized the cardiac phenotype of mice carrying a heterozygous knock-in mutation of PKG1 (Prkg1R177Q/+ ), which causes constitutive, cGMP-independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. KEY RESULTS: Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. CONCLUSION AND IMPLICATIONS: Long-term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

PRDM16 Is a Compact Myocardium-Enriched Transcription Factor Required to Maintain Compact Myocardial Cardiomyocyte Identity in Left Ventricle.

BACKGROUND: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily has an effect on left ventricles (LVs) and is often associated with LV dilation and dysfunction. However, in part because of the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying the susceptibility of LVs to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 (PR domain-containing 16) cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. METHODS: Prdm16 cardiomyocyte-specific knockout (Prdm16cKO) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and chromatin immunoprecipitation deep sequencing were performed to identify direct transcriptional targets of PRDM16 in cardiomyocytes. Single-cell RNA sequencing in combination with spatial transcriptomics was used to determine cardiomyocyte identity at the single-cell level. RESULTS: Cardiomyocyte-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. PRDM16 functioned mechanistically as a compact myocardium-enriched transcription factor that activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16cKO LV compact myocardial cardiomyocytes shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial cardiomyocytes or neurons. Chamber-specific transcriptional regulation by PRDM16 was attributable in part to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. CONCLUSIONS: These results demonstrate that disruption of proper specification of compact cardiomyocytes may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of the LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.

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.

Suppression of Endothelial AGO1 Promotes Adipose Tissue Browning and Improves Metabolic Dysfunction.

BACKGROUND: Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor-vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. METHODS: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus. RESULTS: We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. CONCLUSIONS: Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.

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.

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.

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.

Combinatorial interactions of genetic variants in human cardiomyopathy.

Dilated cardiomyopathy (DCM) is a leading cause of morbidity and mortality worldwide; yet how genetic variation and environmental factors impact DCM heritability remains unclear. Here, we report that compound genetic interactions between DNA sequence variants contribute to the complex heritability of DCM. By using genetic data from a large family with a history of DCM, we discovered that heterozygous sequence variants in the TROPOMYOSIN 1 (TPM1) and VINCULIN (VCL) genes cose-gregate in individuals affected by DCM. In vitro studies of patient-derived and isogenic human-pluripotent-stem-cell-derived cardio-myocytes that were genome-edited via CRISPR to create an allelic series of TPM1 and VCL variants revealed that cardiomyocytes with both TPM1 and VCL variants display reduced contractility and sarcomeres that are less organized. Analyses of mice genetically engineered to harbour these human TPM1 and VCL variants show that stress on the heart may also influence the variable penetrance and expressivity of DCM-associated genetic variants in vivo. We conclude that compound genetic variants can interact combinatorially to induce DCM, particularly when influenced by other disease-provoking stressors.

Reduction of myocardial ischaemia-reperfusion injury by inactivating oxidized phospholipids.

Aims: Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results: OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023). Conclusions: OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

A secretory pathway kinase regulates sarcoplasmic reticulum Ca2+ homeostasis and protects against heart failure.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

Publisher Correction: Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

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.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

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.

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.

Increased Echogenicity and Radiodense Foci on Echocardiogram and MicroCT in Murine Myocarditis.

OBJECTIVES: To address the question as to whether echocardiographic and/or microcomputed tomography (microCT) analysis can be utilized to assess the extent of Coxsackie B virus (CVB) induced myocarditis in the absence of left ventricular dysfunction in the mouse. BACKGROUND: Viral myocarditis is a significant clinical problem with associated inflammation of the myocardium and myocardial injury. Murine models of myocarditis are commonly used to study the pathophysiology of the disease, but methods for imaging the mouse myocardium have been limited to echocardiographic assessment of ventricular dysfunction and, to a lesser extent, MRI imaging. METHODS: Using a murine model of myocarditis, we used both echocardiography and microCT to assess the extent of myocardial involvement in murine myocarditis using both wild-type mice and CVB cleavage-resistant dystrophin knock-in mice. RESULTS: Areas of increased echogenicity were only observed in the myocardium of Coxsackie B virus infected mice. These echocardiographic abnormalities correlated with the extent of von Kossa staining (a marker of membrane permeability), inflammation, and fibrosis. Given that calcium phosphate uptake as imaged by von Kossa staining might also be visualized using microCT, we utilized microCT imaging which allowed for high-resolution, 3-dimensional images of radiodensities that likely represent calcium phosphate uptake. As with echocardiography, only mice infected with Coxsackie B virus displayed abnormal accumulation of calcium within individual myocytes indicating increased membrane permeability only upon exposure to virus. CONCLUSIONS: These studies demonstrate new, quantitative, and semi-quantitative imaging approaches for the assessment of myocardial involvement in the setting of viral myocarditis in the commonly utilized mouse model of viral myocarditis.