A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

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.

Overexpression of the Na+/K+ ATPase α2 but not α1 isoform attenuates pathological cardiac hypertrophy and remodeling.

RATIONALE: The Na+ / K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+ / Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance. OBJECTIVE: To determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart. METHODS AND RESULTS: Cardiac-specific transgenic mice overexpressing either NKA-α1 or NKA-α2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-α1 had no protective effect, overexpression of NKA-α2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-α2 overexpression but not NKA-α1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-α2 showed greater affinity for Na+ compared with NKA-α1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-α2 but not NKA-α1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal. CONCLUSIONS: Our results suggest that the protective effect produced by increased expression of NKA-α2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-α1.

Transient receptor potential channels contribute to pathological structural and functional remodeling after myocardial infarction.

RATIONALE: The cellular and molecular basis for post-myocardial infarction (MI) structural and functional remodeling is not well understood. OBJECTIVE: Our aim was to determine if Ca2+ influx through transient receptor potential canonical (TRPC) channels contributes to post-MI structural and functional remodeling. METHODS AND RESULTS: TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte-specific expression of a dominant-negative (loss-of-function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival in mice. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 overexpression in adult feline myocytes induced calcineurin (Cn)-nuclear factor of activated T-cells (NFAT)-mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in adult feline myocytes increased rested state contractions and increased spontaneous sarcoplasmic reticulum Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady-state pacing, likely because of enhanced sarcoplasmic reticulum Ca2+ leak. CONCLUSIONS: Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function.

Cardiac-specific deletion of protein phosphatase 1ß promotes increased myofilament protein phosphorylation and contractile alterations.

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, ß and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with the loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-loxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1ß) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca(2+) handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, the loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1ß isoform in affecting cardiac contractile function.

STIM1 elevation in the heart results in aberrant Ca²âº handling and cardiomyopathy.

Stromal interaction molecule 1 (STIM1) is a Ca(2+) sensor that partners with Orai1 to elicit Ca(2+) entry in response to endoplasmic reticulum (ER) Ca(2+) store depletion. While store-operated Ca(2+) entry (SOCE) is important for maintaining ER Ca(2+) homeostasis in non-excitable cells, it is unclear what role it plays in the heart, although STIM1 is expressed in the heart and upregulated during disease. Here we analyzed transgenic mice with STIM1 overexpression in the heart to model the known increase of this protein in response to disease. As expected, STIM1 transgenic myocytes showed enhanced Ca(2+) entry following store depletion and partial co-localization with the type 2 ryanodine receptor (RyR2) within the sarcoplasmic reticulum (SR), as well as enrichment around the sarcolemma. STIM1 transgenic mice exhibited sudden cardiac death as early as 6weeks of age, while mice surviving past 12weeks of age developed heart failure with hypertrophy, induction of the fetal gene program, histopathology and mitochondrial structural alterations, loss of ventricular functional performance and pulmonary edema. Younger, pre-symptomatic STIM1 transgenic mice exhibited enhanced pathology following pressure overload stimulation or neurohumoral agonist infusion, compared to controls. Mechanistically, cardiac myocytes isolated from STIM1 transgenic mice displayed spontaneous Ca(2+) transients that were prevented by the SOCE blocker SKF-96365, increased L-type Ca(2+) channel (LTCC) current, and enhanced Ca(2+) spark frequency. Moreover, adult cardiac myocytes from STIM1 transgenic mice showed both increased diastolic Ca(2+) and maximal transient amplitude but no increase in total SR Ca(2+) load. Associated with this enhanced Ca(2+) profile was an increase in cardiac nuclear factor of activated T-cells (NFAT) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) activity. We conclude that STIM1 has an unexpected function in the heart where it alters communication between the sarcolemma and SR resulting in greater Ca(2+) flux and a leaky SR compartment.

A caveolae-targeted L-type Ca²+ channel antagonist inhibits hypertrophic signaling without reducing cardiac contractility.

RATIONALE: The source of Ca(2+) to activate pathological cardiac hypertrophy is not clearly defined. Ca(2+) influx through the L-type Ca(2+) channels (LTCCs) determines "contractile" Ca(2+), which is not thought to be the source of "hypertrophic" Ca(2+). However, some LTCCs are housed in caveolin-3 (Cav-3)-enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. OBJECTIVE: To test the idea that LTCCs in Cav-3-containing signaling domains are a source of Ca(2+) to activate the calcineurin-nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. METHODS AND RESULTS: We developed reagents that targeted Ca(2+) channel-blocking Rem proteins to Cav-3-containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca(2+) influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. CONCLUSIONS: We provide proof of concept that Ca(2+) influx through LTCCs within caveolae signaling domains can activate "hypertrophic" signaling, and this Ca(2+) influx can be selectively blocked without reducing cardiac contractility.

Inhibition of PKCα/ß with ruboxistaurin antagonizes heart failure in pigs after myocardial infarction injury.

RATIONALE: Protein kinase Cα (PKCα) activity and protein level are induced during cardiac disease where it controls myocardial contractility and propensity to heart failure in mice and rats. For example, mice lacking the gene for PKCα have enhanced cardiac contractility and reduced susceptibility to heart failure after long-term pressure overload or after myocardial infarction injury. Pharmacological inhibition of PKCα/ß with Ro-32-0432, Ro-31-8220 or ruboxistaurin (LY333531) similarly enhances cardiac function and antagonizes heart failure in multiple models of disease in both mice and rats. OBJECTIVE: Large and small mammals differ in several key indexes of heart function and biochemistry, lending uncertainty as to how PKCα/ß inhibition might affect or protect a large animal model of heart failure. METHODS AND RESULTS: We demonstrate that ruboxistaurin administration to a pig model of myocardial infarction-induced heart failure was protective. Twenty-kilogram pigs underwent left anterior descending artery occlusion resulting in myocardial infarctions and were then divided into vehicle or ruboxistaurin feed groups, after which they were monitored monthly for the next 3 months. Ruboxistaurin administered pigs showed significantly better recovery of myocardial contractility 3 months after infarction injury, greater ejection fraction, and greater cardiac output compared with vehicle-treated pigs. CONCLUSIONS: These results provide additional evidence in a large animal model of disease that PKCα/ß inhibition (with ruboxistaurin) represents a tenable and novel therapeutic approach for treating human heart failure.

Regulation of cardiac fibroblast cell death by unfolded protein response signaling.

The endoplasmic reticulum (ER) is a tightly regulated organelle that requires specific environmental properties to efficiently carry out its function as a major site of protein synthesis and folding. Embedded in the ER membrane, ER stress sensors inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) serve as a sensitive quality control system collectively known as the unfolded protein response (UPR). In response to an accumulation of misfolded proteins, the UPR signals for protective mechanisms to cope with the cellular stress. Under prolonged unstable conditions and an inability to regain homeostasis, the UPR can shift from its original adaptive response to mechanisms leading to UPR-induced apoptosis. These UPR signaling pathways have been implicated as an important feature in the development of cardiac fibrosis, but identifying effective treatments has been difficult. Therefore, the apoptotic mechanisms of UPR signaling in cardiac fibroblasts (CFs) are important to our understanding of chronic fibrosis in the heart. Here, we summarize the maladaptive side of the UPR, activated downstream pathways associated with cell death, and agents that have been used to modify UPR-induced apoptosis in CFs.

Assessing wound closure in mice using skin-punch biopsy.

Defects in myofibroblast function may cause wound healing defects in a variety of tissue types. Here we describe a simple skin-punch biopsy approach to screen mouse models for defects in wound closure that does not require extensive surgical training or expensive equipment. Experimental results may serve as an initial proof of concept to determine whether further investigation is necessary or if defects in myofibroblast function observed in other systems also result in reduced skin wound healing.

Analysis of the Rem2 - voltage dependant calcium channel beta subunit interaction and Rem2 interaction with phosphorylated phosphatidylinositide lipids.

Voltage dependant calcium channels (VDCC) play a critical role in coupling electrical excitability to important physiological events such as secretion by neuronal and endocrine cells. Rem2, a GTPase restricted to neuroendocrine cell types, regulates VDCC activity by a mechanism that involves interaction with the VDCC beta subunit (Ca(V)beta). Mapping studies reveal that Rem2 binds to the guanylate kinase domain (GK) of the Ca(V)beta subunit that also contains the high affinity binding site for the pore forming and voltage sensing VDCC alpha subunit (Ca(V)alpha) interaction domain (AID). Moreover, fine mapping indicates that Rem2 binds to the GK domain in a region distinct from the AID interaction site, and competitive inhibition studies reveal that Rem2 does not disrupt Ca(V)alpha - Ca(V)beta binding. Instead, the Ca(V)beta subunit appears to serve a scaffolding function, simultaneously binding both Rem2 and AID. Previous studies have found that in addition to Ca(V)beta binding, Rem2 must be localized to the plasma membrane to inhibit VDCC function. Plasma membrane localization requires the C-terminus of Rem2 and binding studies indicate that this domain directs phosphorylated phosphatidylinositide (PIP) lipids association. Plasma membrane localization may provide a unique point of regulation since the ability of Rem2 to bind PIP lipids is inhibited by the phosphoserine dependant binding of 14-3-3 proteins. Thus, in addition to Ca(V)beta binding, VDCC blockade by Rem2 is likely to be controlled by both the localized concentration of membrane PIP lipids and direct 14-3-3 binding to the Rem2 C-terminus.

The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling.

RGK proteins constitute a novel subfamily of small Ras-related proteins that function as potent inhibitors of voltage-dependent (VDCC) Ca(2+) channels and regulators of actin cytoskeletal dynamics. Within the larger Ras superfamily, RGK proteins have distinct regulatory and structural characteristics, including nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains conserved regulatory sites which control both subcellular localization and function. RGK GTPases interact with the VDCC beta-subunit (Ca(V)beta) and inhibit Rho/Rho kinase signaling to regulate VDCC activity and the cytoskeleton respectively. Binding of both calmodulin and 14-3-3 to RGK proteins, and regulation by phosphorylation controls cellular trafficking and the downstream signaling of RGK proteins, suggesting that a complex interplay between interacting protein factors and trafficking contribute to their regulation.

Overlapping and differential functions of ATF6α versus ATF6ß in the mouse heart.

Hemodynamic stress on the mammalian heart results in compensatory hypertrophy and activation of the unfolded protein response through activating transcription factor 6α (ATF6α) in cardiac myocytes, but the roles of ATF6α or the related transcription factor ATF6ß in regulating this hypertrophic response are not well-understood. Here we examined the effects of loss of ATF6α or ATF6ß on the cardiac response to pressure overload. Mice gene-deleted for Atf6 or Atf6b were subjected to 2 weeks of transverse aortic constriction, and each showed a significant reduction in hypertrophy with reduced expression of endoplasmic reticulum (ER) stress-associated proteins compared with controls. However, with long-term pressure overload both Atf6 and Atf6b null mice showed enhanced decompensation typified by increased heart weight, pulmonary edema and reduced function compared to control mice. Our subsequent studies using cardiac-specific transgenic mice expressing the transcriptionally active N-terminus of ATF6α or ATF6ß revealed that these factors control overlapping gene expression networks that include numerous ER protein chaperones and ER associated degradation components. This work reveals previously unappreciated roles for ATF6α and ATF6ß in regulating the pressure overload induced cardiac hypertrophic response and in controlling the expression of genes that condition the ER during hemodynamic stress.

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.

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.

Mitsugumin 29 regulates t-tubule architecture in the failing heart.

Transverse tubules (t-tubules) are uniquely-adapted membrane invaginations in cardiac myocytes that facilitate the synchronous release of Ca2+ from internal stores and subsequent myofilament contraction, although these structures become disorganized and rarefied in heart failure. We previously observed that mitsugumin 29 (Mg29), an important t-tubule organizing protein in skeletal muscle, was induced in the mouse heart for the first time during dilated cardiomyopathy with heart failure. Here we generated cardiac-specific transgenic mice expressing Mg29 to model this observed induction in the failing heart. Interestingly, expression of Mg29 in the hearts of Csrp3 null mice (encoding muscle LIM protein, MLP) partially restored t-tubule structure and preserved cardiac function as measured by invasive hemodynamics, without altering Ca2+ spark frequency. Conversely, gene-deleted mice lacking both Mg29 and MLP protein showed a further reduction in t-tubule organization and accelerated heart failure. Thus, induction of Mg29 in the failing heart is a compensatory response that directly counteracts the well-characterized loss of t-tubule complexity and reduced expression of anchoring proteins such as junctophilin-2 (Jph2) that normally occur in this disease. Moreover, preservation of t-tubule structure by Mg29 induction significantly increases the function of the failing heart.

Caveolae-localized L-type Ca2+ channels do not contribute to function or hypertrophic signalling in the mouse heart.

AIMS: L-type Ca2+ channels (LTCCs) in adult cardiomyocytes are localized to t-tubules where they initiate excitation-contraction coupling. Our recent work has shown that a subpopulation of LTCCs found at the surface sarcolemma in caveolae of adult feline cardiomyocytes can also generate a Ca2+ microdomain that activates nuclear factor of activated T-cells signaling and cardiac hypertrophy, although the relevance of this paradigm to hypertrophy regulation in vivo has not been examined. METHODS AND RESULTS: Here we generated heart-specific transgenic mice with a putative caveolae-targeted LTCC activator protein that was ineffective in initiating or enhancing cardiac hypertrophy in vivo. We also generated transgenic mice with cardiac-specific overexpression of a putative caveolae-targeted inhibitor of LTCCs, and while this protein inhibited caveolae-localized LTCCs without effects on global Ca2+ handling, it similarly had no effect on cardiac hypertrophy in vivo. Cardiac hypertrophy was elicited by pressure overload for 2 or 12 weeks or with neurohumoral agonist infusion. Caveolae-specific LTCC activator or inhibitor transgenic mice showed no greater change in nuclear factor of activated T-cells activity after 2 weeks of pressure overload stimulation compared with control mice. CONCLUSION: Our results indicate that LTCCs in the caveolae microdomain do not affect cardiac function and are not necessary for the regulation of hypertrophic signaling in the adult mouse heart.

Fibroblast-specific TGF-ß-Smad2/3 signaling underlies cardiac fibrosis.

The master cytokine TGF-ß mediates tissue fibrosis associated with inflammation and tissue injury. TGF-ß induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-ß signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-ß-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-ß receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-ß mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-ß-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.