In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

Regulation of cardiomyocyte intracellular trafficking and signal transduction by protein palmitoylation.

Despite the well-established functions of protein palmitoylation in fundamental cellular processes, the roles of this reversible post-translational lipid modification in cardiomyocyte biology remain poorly studied. Palmitoylation is catalyzed by a family of 23 zinc finger and Asp-His-His-Cys domain-containing S-acyltransferases (zDHHC enzymes) and removed by select thioesterases of the lysophospholipase and α/ß-hydroxylase domain (ABHD)-containing families of serine hydrolases. Recently, studies utilizing genetic manipulation of zDHHC enzymes in cardiomyocytes have begun to unveil essential functions for these enzymes in regulating cardiac development, homeostasis, and pathogenesis. Palmitoylation co-ordinates cardiac electrophysiology through direct modulation of ion channels and transporters to impact their trafficking or gating properties as well as indirectly through modification of regulators of channels, transporters, and calcium handling machinery. Not surprisingly, palmitoylation has roles in orchestrating the intracellular trafficking of proteins in cardiomyocytes, but also dynamically fine-tunes cardiomyocyte exocytosis and natriuretic peptide secretion. Palmitoylation has emerged as a potent regulator of intracellular signaling in cardiomyocytes, with recent studies uncovering palmitoylation-dependent regulation of small GTPases through direct modification and sarcolemmal targeting of the small GTPases themselves or by modification of regulators of the GTPase cycle. In addition to dynamic control of G protein signaling, cytosolic DNA is sensed and transduced into an inflammatory transcriptional output through palmitoylation-dependent activation of the cGAS-STING pathway, which has been targeted pharmacologically in preclinical models of heart disease. Further research is needed to fully understand the complex regulatory mechanisms governed by protein palmitoylation in cardiomyocytes and potential emerging therapeutic targets.

Palmitoylation-dependent regulation of cardiomyocyte Rac1 signaling activity and minor effects on cardiac hypertrophy.

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2 weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.

Cardiomyocyte NOX4 regulates resident macrophage-mediated inflammation and diastolic dysfunction in stress cardiomyopathy.

In acute sympathetic stress, catecholamine overload can lead to stress cardiomyopathy. We tested the hypothesis that cardiomyocyte NOX4 (NADPH oxidase 4)-dependent mitochondrial oxidative stress mediates inflammation and diastolic dysfunction in stress cardiomyopathy. Isoproterenol (ISO; 5 mg/kg) injection induced sympathetic stress in wild-type and cardiomyocyte (CM)-specific Nox4 knockout (Nox4CM-/-) mice. Wild-type mice treated with ISO showed higher CM NOX4 expression, H2O2 levels, inflammasome activation, and IL18, IL6, CCL2, and TNFα levels than Nox4CM-/- mice. Spectral flow cytometry and t-SNE analysis of cardiac cell suspensions showed significant increases in pro-inflammatory and pro-fibrotic embryonic-derived resident (CCR2-MHCIIhiCX3CR1hi) macrophages in wild-type mice 3 days after ISO treatment, whereas Nox4CM-/- mice had a higher proportion of embryonic-derived resident tissue-repair (CCR2-MHCIIloCX3CR1lo) macrophages. A significant increase in cardiac fibroblast activation and interstitial collagen deposition and a restrictive pattern of diastolic dysfunction with increased filling pressure was observed in wild-type hearts compared with Nox4CM-/- 7 days post-ISO. A selective NOX4 inhibitor, GKT137831, reduced myocardial mitochondrial ROS, macrophage infiltration, and fibrosis in ISO-injected wild-type mice, and preserved diastolic function. Our data suggest sympathetic overstimulation induces resident macrophage (CCR2-MHCII+) activation and myocardial inflammation, resulting in fibrosis and impaired diastolic function mediated by CM NOX4-dependent ROS.

zDHHC9 Regulates Cardiomyocyte Rab3a Activity and Atrial Natriuretic Peptide Secretion Through Palmitoylation of Rab3gap1.

Production and release of natriuretic peptides by the stressed heart reduce cardiac workload by promoting vasodilation, natriuresis, and diuresis, which has been leveraged in the recent development of novel heart-failure pharmacotherapies, yet the mechanisms regulating cardiomyocyte exocytosis and natriuretic peptide release remain ill defined. We found that the Golgi S-acyltransferase zDHHC9 palmitoylates Rab3gap1 resulting in its spatial segregation from Rab3a, elevation of Rab3a-GTP levels, formation of Rab3a-positive peripheral vesicles, and impairment of exocytosis that limits atrial natriuretic peptide release. This novel pathway potentially can be exploited for targeting natriuretic peptide signaling in the treatment of heart failure.

NADPH Oxidases in Diastolic Dysfunction and Heart Failure with Preserved Ejection Fraction.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases regulate production of reactive oxygen species (ROS) that cause oxidative damage to cellular components but also regulate redox signaling in many cell types with essential functions in the cardiovascular system. Research over the past couple of decades has uncovered mechanisms by which NADPH oxidase (NOX) enzymes regulate oxidative stress and compartmentalize intracellular signaling in endothelial cells, smooth muscle cells, macrophages, cardiomyocytes, fibroblasts, and other cell types. NOX2 and NOX4, for example, regulate distinct redox signaling mechanisms in cardiac myocytes pertinent to the onset and progression of cardiac hypertrophy and heart failure. Heart failure with preserved ejection fraction (HFpEF), which accounts for at least half of all heart failure cases and has few effective treatments to date, is classically associated with ventricular diastolic dysfunction, i.e., defects in ventricular relaxation and/or filling. However, HFpEF afflicts multiple organ systems and is associated with systemic pathologies including inflammation, oxidative stress, arterial stiffening, cardiac fibrosis, and renal, adipose tissue, and skeletal muscle dysfunction. Basic science studies and clinical data suggest a role for systemic and myocardial oxidative stress in HFpEF, and evidence from animal models demonstrates the critical functions of NOX enzymes in diastolic function and several HFpEF-associated comorbidities. Here, we discuss the roles of NOX enzymes in cardiovascular cells that are pertinent to the development and progression of diastolic dysfunction and HFpEF and outline potential clinical implications.

Sirtuin 5 levels are limiting in preserving cardiac function and suppressing fibrosis in response to pressure overload.

Heart failure (HF) is the inability of the heart to pump blood sufficiently to meet the metabolic demands of the body. HF with reduced systolic function is characterized by cardiac hypertrophy, ventricular fibrosis and remodeling, and decreased cardiac contractility, leading to cardiac functional impairment and death. Transverse aortic constriction (TAC) is a well-established model for inducing hypertrophy and HF in rodents. Mice globally deficient in sirtuin 5 (SIRT5), a NAD+-dependent deacylase, are hypersensitive to cardiac stress and display increased mortality after TAC. Prior studies assessing SIRT5 functions in the heart have all employed loss-of-function approaches. In this study, we generated SIRT5 overexpressing (SIRT5OE) mice, and evaluated their response to chronic pressure overload using TAC. Compared to littermate controls, SIRT5OE mice were protected against adverse functional consequences of TAC, left ventricular dilation and impaired ejection fraction. Transcriptomic analysis revealed that SIRT5 suppresses key HF sequelae, including the metabolic switch from fatty acid oxidation to glycolysis, immune activation, and fibrotic signaling pathways. We conclude that SIRT5 is a limiting factor in the preservation of cardiac function in response to experimental pressure overload.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy.

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1-/- mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.

Palmitoylation: A Fatty Regulator of Myocardial Electrophysiology.

Regulation of cardiac physiology is well known to occur through the action of kinases that reversibly phosphorylate ion channels, calcium handling machinery, and signaling effectors. However, it is becoming increasingly apparent that palmitoylation or S-acylation, the post-translational modification of cysteines with saturated fatty acids, plays instrumental roles in regulating the localization, activity, stability, sorting, and function of numerous proteins, including proteins known to have essential functions in cardiomyocytes. However, the impact of this modification on cardiac physiology requires further investigation. S-acylation is catalyzed by the zDHHC family of S-acyl transferases that localize to intracellular organelle membranes or the sarcolemma. Recent work has begun to uncover functions of S-acylation in the heart, particularly in the regulation of cardiac electrophysiology, including modification of the sodium-calcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel. Elucidating the regulatory functions of zDHHC enzymes in cardiomyocytes and determination of how S-acylation is altered in the diseased heart will shed light on how these modifications participate in cardiac pathogenesis and potentially identify novel targets for the treatment of cardiovascular disease. Indeed, proteins with critical signaling roles in the heart are also S-acylated, including receptors and G-proteins, yet the dynamics and functions of these modifications in myocardial physiology have not been interrogated. Here, we will review what is known about zDHHC enzymes and substrate S-acylation in myocardial physiology and highlight future areas of investigation that will uncover novel functions of S-acylation in cardiac homeostasis and pathophysiology.

Cell-specific ablation of Hsp47 defines the collagen-producing cells in the injured heart

Collagen production in the adult heart is thought to be regulated by the fibroblast, although cardiomyocytes and endothelial cells also express multiple collagen mRNAs. Molecular chaperones are required for procollagen biosynthesis, including heat shock protein 47 (Hsp47). To determine the cell types critically involved in cardiac injury­induced fibrosis theHsp47 gene was deleted in cardiomyocytes, endothelial cells, or myofibroblasts. Deletion ofHsp47 from cardiomyocytes during embryonic development or adult stages, or deletion from adult endothelial cells, did not affect cardiac fibrosis after pressure overload injury. However, myofibroblast-specific ablation of Hsp47; blocked fibrosis and deposition of collagens type I, III, and V following pressure overload as well as significantly reduced cardiac hypertrophy. Fibroblast-specific Hsp47-deleted mice showed lethality after myocardial infarction injury, with ineffective scar formation and ventricular wall rupture. Similarly, only myofibroblast-specific deletion of Hsp47reduced fibrosis and disease in skeletal muscle in a mouse model of muscular dystrophy. Mechanistically, deletion of Hsp47 from myofibroblasts reduced mRNA expression of fibrillar collagens and attenuated their proliferation in the heart without affecting paracrine secretory activity of these cells. The results show that myofibroblasts are the primary mediators of tissue fibrosis and scar formation in the injured adult heart, which unexpectedly affects cardiomyocyte hypertrophy.

Disruption of valosin-containing protein activity causes cardiomyopathy and reveals pleiotropic functions in cardiac homeostasis.

Valosin-containing protein (VCP), also known as p97, is an ATPase with diverse cellular functions, although the most highly characterized is targeting of misfolded or aggregated proteins to degradation pathways, including the endoplasmic reticulum-associated degradation (ERAD) pathway. However, how VCP functions in the heart has not been carefully examined despite the fact that human mutations in VCP cause Paget disease of bone and frontotemporal dementia, an autosomal dominant multisystem proteinopathy that includes disease in the heart, skeletal muscle, brain, and bone. Here we generated heart-specific transgenic mice overexpressing WT VCP or a VCPK524A mutant with deficient ATPase activity. Transgenic mice overexpressing WT VCP exhibit normal cardiac structure and function, whereas mutant VCP-overexpressing mice develop cardiomyopathy. Mechanistically, mutant VCP-overexpressing hearts up-regulate ERAD complex components and have elevated levels of ubiquitinated proteins prior to manifestation of cardiomyopathy, suggesting dysregulation of ERAD and inefficient clearance of proteins targeted for proteasomal degradation. The hearts of mutant VCP transgenic mice also exhibit profound defects in cardiomyocyte nuclear morphology with increased nuclear envelope proteins and nuclear lamins. Proteomics revealed overwhelming interactions of endogenous VCP with ribosomal, ribosome-associated, and RNA-binding proteins in the heart, and impairment of cardiac VCP activity resulted in aggregation of large ribosomal subunit proteins. These data identify multifactorial functions and diverse mechanisms whereby VCP regulates cardiomyocyte protein and RNA quality control that are critical for cardiac homeostasis, suggesting how human VCP mutations negatively affect the heart.

Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability.

Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7ß1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability.

Defective Flux of Thrombospondin-4 through the Secretory Pathway Impairs Cardiomyocyte Membrane Stability and Causes Cardiomyopathy.

Thrombospondins are stress-inducible secreted glycoproteins with critical functions in tissue injury and healing. Thrombospondin-4 (Thbs4) is protective in cardiac and skeletal muscle, where it activates an adaptive endoplasmic reticulum (ER) stress response, induces expansion of the ER, and enhances sarcolemmal stability. However, it is unclear if Thbs4 has these protective functions from within the cell, from the extracellular matrix, or from the secretion process itself. In this study, we generated transgenic mice with cardiac cell-specific overexpression of a secretion-defective mutant of Thbs4 to evaluate its exclusive intracellular and secretion-dependent functions. Like wild-type Thbs4, the secretion-defective mutant upregulates the adaptive ER stress response and expands the ER and intracellular vesicles in cardiomyocytes. However, only the secretion-defective Thbs4 mutant produces cardiomyopathy with sarcolemmal weakness and rupture that is associated with reduced adhesion-forming glycoproteins in the membrane. Similarly, deletion of Thbs4 in the mdx mouse model of Duchenne muscular dystrophy enhances cardiomyocyte membrane instability and cardiomyopathy. Finally, overexpression of the secretion-defective Thbs4 mutant in Drosophila, but not wild-type Thbs4, impaired muscle function and sarcomere alignment. These results suggest that transit through the secretory pathway is required for Thbs4 to augment sarcolemmal stability, while ER stress induction and vesicular expansion mediated by Thbs4 are exclusively intracellular processes.

Autophagic cell death is dependent on lysosomal membrane permeability through Bax and Bak.

Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and previous we have shown that these two effectors are also needed for mitochondrial-dependent cellular necrosis (Karch et al., 2013). Here we show that mouse embryonic fibroblasts deficient in Bax/Bak1 are resistant to the third major form of cell death associated with autophagy through a mechanism involving lysosome permeability. Indeed, specifically targeting Bax only to the lysosome restores autophagic cell death in Bax/Bak1 null cells. Moreover, a monomeric-only mutant form of Bax is sufficient to increase lysosomal membrane permeability and restore autophagic cell death in Bax/Bak1 double-deleted mouse embryonic fibroblasts. Finally, increasing lysosomal permeability through a lysomotropic detergent in cells devoid of Bax/Bak1 restores autophagic cell death, collectively indicting that Bax/Bak integrate all major forms of cell death through direct effects on membrane permeability of multiple intracellular organelles.

Thrombospondin expression in myofibers stabilizes muscle membranes.

Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of ßPS integrin. This functional conservation emphasizes the fundamental importance of Thbs' as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.

The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy.

Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease.

Genetic lineage tracing defines myofibroblast origin and function in the injured heart.

Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction (MI) and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell type in terms of their origins and functional effects in vivo. Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen-inducible Cre for cellular lineage-tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Lineage tracing with four additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin(+) myofibroblasts reduces collagen production and scar formation after MI. Periostin-traced myofibroblasts also revert back to a less-activated state upon injury resolution. Our results define the myofibroblast as a periostin-expressing cell type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21(+) tissue-resident fibroblasts.

Dissection of Thrombospondin-4 Domains Involved in Intracellular Adaptive Endoplasmic Reticulum Stress-Responsive Signaling.

Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function, regulating the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6α (Atf6α). In the present study, we dissected the domains of Thbs4 that mediate interactions with ER proteins, such as BiP (Grp78) and Atf6α, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted from cardiomyocytes, as were the type III repeat (T3R) and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wild-type Thbs4 and the calcium-binding mutant interacted with Atf6α, induced an adaptive ER stress response, and caused expansion of intracellular vesicles. In contrast, overexpression of a related secreted oligomeric glycoprotein, Nell2, which lacks only the T3R and TSP-C domains, did not cause these effects. Finally, deletion of Atf6α abrogated Thbs4-induced vesicular expansion. Taken together, these data identify the critical intracellular functional domains of Thbs4, which was formerly thought to have only extracellular functions.

LRRC10 is required to maintain cardiac function in response to pressure overload.

We previously reported that the cardiomyocyte-specific leucine-rich repeat containing protein (LRRC)10 has critical functions in the mammalian heart. In the present study, we tested the role of LRRC10 in the response of the heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10(-/-)) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in Lrrc10(-/-) mice compared with control mice. In addition to dilation and cardiomyopathy, Lrrc10(-/-) mice showed a pronounced increase in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting that the absence of LRRC10 renders the heart more disease prone with greater hypertrophy and structural remodeling, although rates of cardiac fibrosis and myocyte dropout were not different from control mice. Lrrc10(-/-) cardiomyocytes also exhibited reduced contractility in response to ß-adrenergic stimulation, consistent with loss of cardiac ventricular performance after pressure overload. We have previously shown that LRRC10 interacts with actin in the heart. Here, we show that His(150) of LRRC10 was required for an interaction with actin, and this interaction was reduced after pressure overload, suggesting an integral role for LRRC10 in the response of the heart to mechanical stress. Importantly, these experiments demonstrated that LRRC10 is required to maintain cardiac performance in response to pressure overload and suggest that dysregulated expression or mutation of LRRC10 may greatly sensitize human patients to more severe cardiac disease in conditions such as chronic hypertension or aortic stenosis.