Cristine Joelle Reitz

Background: Myocardial infarction (MI) initiates ventricular remodeling that extends beyond the infarct into remote, viable myocardium, where diffuse extracellular matrix (ECM) remodeling and stiffening contribute to progressive dysfunction and heart failure. Latent transforming growth factor binding protein 2 (LTBP2) is an injury-induced, microfibril-associated ECM glycoprotein that, uniquely among the LTBP family, does not covalently bind latent TGF-β. We asked whether LTBP2 regulates remote remodeling after MI, and how a protein lacking latent TGF-β–binding capacity could influence TGF-β–dependent fibrosis. Methods: Wild-type and Ltbp2 -null mice underwent permanent coronary ligation or sham surgery and were followed for 28 days by echocardiography and speckle-tracking strain analysis. Remote myocardium was assessed by histology, atomic force microscopy, and quantitative proteomics. Cellular mechanism was interrogated in primary cardiac fibroblasts (CFs) using a TGF-β reporter assay and quantitative imaging of LTBP1 and LTBP2 matrix incorporation. Sufficiency was tested with a minimal fibrillin-1–binding module of LTBP2 delivered as purified protein or by lentiviral expression. Results: LTBP2 was induced in injury-activated CFs and localized to infarct and remote myocardium by 7 days post-MI. Ltbp2 deletion improved 28-day survival (P=0.0272) without altering infarct size or early post-infarct function. By 28 days, Ltbp2 -null hearts showed improved ejection fraction and faster diastolic strain rates, with protection concentrated in segments remote from the infarct. Remote myocardium contained less interstitial collagen and was significantly less stiff, while cardiomyocyte hypertrophy was unaffected. Proteomic profiling showed enrichment of ECM organization and TGF-β–responsive programs in wild-type hearts that was attenuated in knockouts. In fibroblasts, loss of LTBP2 increased LTBP1 matrix incorporation and shifted TGF-β from the soluble to the matrix-bound compartment; conversely, the minimal LTBP2 module bound fibrillin-1 directly and mobilized latent TGF-β from established matrix within 20 minutes. Conclusions: LTBP2 drives maladaptive remodeling of remote myocardium after MI by limiting matrix sequestration of latent TGF-β and expanding the activatable pool, resolving how an LTBP that does not bind latent TGF-β amplifies TGF-β–dependent fibrosis. LTBP2 and its fibrillin-1 interaction surface may be tractable targets. Novelty and Significance: What Is Known?: Remodeling of the remote, non-infarcted myocardium after myocardial infarction drives progressive fibrosis, ventricular stiffening, and heart failure, largely independent of infarct size. Transforming growth factor-β (TGF-β) is a central profibrotic signal, and its availability is controlled by latent TGF-β binding proteins (LTBPs) that tether latent complexes to fibrillin-rich microfibrils. LTBP2 is induced in fibrotic tissue but, unlike other family members, does not bind latent TGF-β, and how it influences TGF-β–driven fibrosis has been unresolved. What New Information Does This Article Contribute?: LTBP2 is induced in injury-activated cardiac fibroblasts, deposited into fibrillin-rich matrix of both infarct and remote myocardium, and elevated in failing human left ventricle across ischemic, non-ischemic, and hypertrophic cardiomyopathy. Deletion of Ltbp2 improves survival, reduces remote fibrosis and tissue stiffness, and preserves regional systolic and diastolic mechanics without altering infarct size or the cardiomyocyte hypertrophic response. A minimal fibrillin-1–binding module of LTBP2 is sufficient to drive latent TGF-β out of the matrix and into the activatable pool, explaining how a protein that cannot bind latent TGF-β nonetheless amplifies TGF-β–driven fibrosis. Summary: Most therapies after myocardial infarction target the infarct itself, yet much of the decline toward heart failure originates in the surviving muscle surrounding the scar, where diffuse fibrosis stiffens the ventricle and impairs filling. We identify LTBP2 as a driver of this process. LTBP2 is produced by activated fibroblasts and incorporated into the microfibrillar scaffold of the matrix, where it reduces the amount of latent TGF-β held in reserve and increases the pool available for activation. Mice lacking LTBP2 survive infarction better and develop less fibrosis and stiffening in remote myocardium, with preserved regional mechanics but an intact hypertrophic response, indicating that the effect is specific to the interstitium. Conversely, introducing a short fibrillin-1–binding fragment of LTBP2 is enough to release latent TGF-β from the matrix within minutes. Because LTBP2 alters where TGF-β resides rather than blocking the pathway outright, and appears dispensable in the uninjured heart, targeting this interaction may offer a way to limit pathologic fibrosis without the toxicity of global TGF-β inhibition. Graphical LTBP2 remodels microfibrillar composition to enhance TGF-β bioavailability and profibrotic signaling: Under homeostatic conditions (left), latent TGF-β is sequestered within the extracellular matrix (ECM) as part of the large latent complex (LLC), in which TGF-β is covalently associated with LTBP1 or LTBP4 and tethered to fibrillin-1 microfibrils. This matrix sequestration maintains a balanced distribution between bound and soluble pools of latent TGF-β, thereby limiting ligand activation and downstream signaling. During fibroblast activation (right), LTBP2 expression is upregulated and incorporated into newly assembling fibrillin-1 microfibrillar networks. Although LTBP2 does not covalently bind latent TGF-β, it competes with TGF-β–binding LTBP isoforms for fibrillin-1 binding sites, reducing LLC deposition within the matrix. This competitive remodeling shifts the equilibrium from matrix sequestration toward an expanded soluble LLC pool. Soluble latent complexes are more readily activated through integrin-mediated mechanical stress or proteolytic processing, increasing local active TGF-β levels. This model provides a mechanistic framework whereby LTBP2 indirectly amplifies TGF-β signaling by altering microfibrillar composition and competitive binding dynamics within the ECM, promoting enhanced TGF-β bioavailability and sustained profibrotic remodeling in the remote myocardium.

Molecular heterogeneity in heart failure (HF) is increasingly recognized as a major barrier to effective therapy development. Here we performed proteomic profiling of left ventricular tissues from 149 patients with advanced HF spanning nine clinical etiologies. Despite clinical diversity, etiology-stratified analyses revealed convergence toward a shared end-stage proteomic landscape. By contrast, unbiased proteome-based clustering stratified patients into two molecular subgroups characterized by divergent remodeling phenotypes, including differences in fibrosis and immune cell infiltration. Phosphoproteomic profiling was consistent with this stratification, and the analyses further uncovered selective activation of druggable signaling networks in a molecularly defined subgroup of patients. Moreover, our remodeling-associated subgrouping proteins also stratified patients and predicted survival trajectories in an independent plasma-based HF cohort from the UK Biobank, extending our findings to earlier disease stages. Together, our study provides a rich resource for molecular classification of patients with HF, supporting the development of precision-guided therapeutic strategies.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms, as well as testing drug safety and efficacy. However, current hiPSC-CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents an hiPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to existing high-performing reference formulations, our medium significantly matured morphology, Ca²⁺ handling, electrophysiology, and metabolism, which was further validated by multi-omic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional hiPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 μN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.

Heart disease remains a leading cause of death in North America and worldwide. Despite advances in therapies, the chronic nature of cardiovascular diseases ultimately results in frequent hospitalizations and steady rates of mortality. Systems biology approaches have provided a new frontier toward unraveling the underlying mechanisms of cell, tissue, and organ dysfunction in disease. Mapping the complex networks of molecular functions across the genome, transcriptome, proteome, and metabolome has enormous potential to advance our understanding of cardiovascular disease, discover new disease biomarkers, and develop novel therapies. Computational workflows to interpret these data‐intensive analyses as well as integration between different levels of interrogation remain important challenges in the advancement and application of systems biology‐based analyses in cardiovascular research. This review will focus on summarizing the recent developments in network biology‐level profiling in the heart, with particular emphasis on modeling of human heart failure. We will provide new perspectives on integration between different levels of large “omics” datasets, including integration of gene regulatory networks, protein–protein interactions, signaling networks, and metabolic networks in the heart.

The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell–cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM.

Rest has long been considered beneficial to patient healing; however, remarkably, there are no evidence-based experimental models determining how it benefits disease outcomes. Here, we created an experimental rest model in mice that briefly extends the morning rest period. We found in 2 major cardiovascular disease conditions (cardiac hypertrophy, myocardial infarction) that imposing a short, extended period of morning rest each day limited cardiac remodeling compared with controls. Mechanistically, rest mitigates autonomic-mediated hemodynamic stress on the cardiovascular system, relaxes myofilament contractility, and attenuates cardiac remodeling genes, consistent with the benefits on cardiac structure and function. These same rest-responsive gene pathways underlie the pathophysiology of many major human cardiovascular conditions, as demonstrated by interrogating open-source transcriptomic data; thus, patients with other conditions may also benefit from a morning rest period in a similar manner. Our findings implicate rest as a key driver of physiology, creating a potentially new field — as broad and important as diet, sleep, or exercise — and provide a strong rationale for investigation of rest-based therapy for major clinical diseases.

Alcohol use is a contributor in the premature deaths of approximately 3 million people annually. Among the risk factors for alcohol misuse is circadian rhythm disruption; however, this connection remains poorly understood. Inhibition of the circadian nuclear receptor REV-ERBα is known to disrupt molecular feedback loops integral to daily oscillations, and impact diurnal fluctuations in the expression of proteins required for reward-related neurotransmission. However, the role of REV-ERBα in alcohol and substance use-related phenotypes is unknown. Herein, we used a Rev-erbα knockout mouse line and ethanol two-bottle choice preference testing to show that disruption of Rev-erbα reduces ethanol preference in male and female mice. Rev-erbα null mice showed the lowest ethanol preference in a two-bottle choice test across all genotypes, whereas there were no ethanol preference differences between heterozygotes and wildtypes. In a separate experiment, alcohol-consuming wildtype C57Bl/6N mice were administered the REV-ERBα/β inhibitor SR8278 (25 mg/kg or 50 mg/kg) for 7 days and alcohol preference was evaluated daily. No differences in alcohol preference were observed between the treatment and vehicle groups. Our data provides evidence that genetic variation in REV-ERBα may contribute to differences in alcohol drinking.