Cardiac extracellular matrix remodeling: Fibrillar collagens and Secreted Protein Acidic and Rich in Cysteine (SPARC)

The cardiac interstitium is a unique and adaptable extracellular matrix (ECM) that provides a milieu in which myocytes, fibroblasts, and endothelial cells communicate and function. The composition of the ECM in the heart includes structural proteins such as fibrillar collagens and matricellular proteins that modulate cell:ECM interaction. Secreted Protein Acidic and Rich in Cysteine (SPARC), a collagen-binding matricellular protein, serves a key role in collagen assembly into the ECM. Recent results demonstrated increased cardiac rupture, dysfunction and mortality in SPARC-null mice in response to myocardial infarction that was associated with a decreased capacity to generate organized, mature collagen fibers. In response to pressure overload induced-hypertrophy, the decrease in insoluble collagen incorporation in the left ventricle of SPARC-null hearts was coincident with diminished ventricular stiffness in comparison to WT mice with pressure overload. This review will focus on the role of SPARC in the regulation of interstitial collagen during cardiac remodeling following myocardial infarction and pressure overload with a discussion of potential cellular mechanisms that control SPARC-dependent collagen assembly in the heart

426 A Beat Away from Precision Medicine: Characterizing Human Cardiac Fibroblast Responsiveness to Hemodynamic Unloading in Heart Failure with Reduced Ejection Fraction

OBJECTIVES/GOALS: Myocardial interstitial fibrosis leads to high hemodynamic load resulting in heart failure (HFrEF). Previous studies show that treatment with a left ventricular assist device (LVAD) does not reduce fibrosis. We hypothesize that human cardiac fibroblasts are highly activated in HFrEF and remain unresponsive to hemodynamic unloading by LVAD. METHODS/STUDY POPULATION: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). RESULTS/ANTICIPATED RESULTS: Quantification of PSR-stained sections reveals a significant increase in collagen content in the HFrEF tissue (CVF = 2.8) compared to control tissues (CVF = 0.9) that remained elevated in LVEX hearts (CVF = 3.1). HCFs from LV biopsies were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs were plated on substrates with stiffnesses reflective of normal myocardium (2kPa) or HFrEF myocardium (8kPa). Cells were collected at 4- and 7-day time points and levels of collagen I and alpha-smooth muscle actin were quantified by western blot analysis. Control HCFs were responsive to changes in substrate stiffness producing more Col I and a-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in protein production on 2 vs. 8kPa. DISCUSSION/SIGNIFICANCE: Our data suggests that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and might contribute to persistent increases in fibrosis. These findings bring us one step closer to elucidating mechanisms behind fibrosis in HFrEF which could lead to targeted therapies to improve patient outcomes from LVAD support

β3 Integrin-mediated ubiquitination activates survival signaling during myocardial hypertrophy

Identifying the molecular mechanisms activated in compensatory hypertrophy and absent during decompensation will provide molecular targets for prevention of heart failure. We have previously shown enhanced ubiquitination (Ub) during the early growth period of pressure overload (PO) hypertrophy near intercalated discs of cardiomyocytes, where integrins are important for mechanotransduction. In this study, we tested the role of integrins upstream of Ub, whether enhanced Ub contributes to survival signaling in early PO, and if loss of this mechanism could lead to decreased ventricular function. The study used a β3 integrin (−/−) mouse and a wild-type mouse as a control for in vivo PO by transverse aortic constriction (TAC) and for cultured cardiomyocytes in vitro, stimulated with the integrin-activating peptide RGD. We demonstrate β3 integrin mediates transient Ub of targeted proteins during PO hypertrophy, which is necessary for cardiomyocyte survival and to maintain ventricular function. Prosurvival signaling proceeds by initiation of NF-κB transcription of the E3 ligase, cIAP1. In PO β3−/− mice, absence of this mechanism correlates with increased TUNEL staining and decreased ventricular mass and function by 4 wk. This is the first study to show that a β3 integrin/Ub/NF-κB pathway contributes to compensatory hypertrophic growth.—Johnston, R. K., Balasubramanian, S., Kasiganesan, H., Baicu, C. F., Zile, M. R., Kuppuswamy, D. β3 Integrin-mediated ubiquitination activates survival signaling during myocardial hypertrophy

Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing

Advanced age, independent of concurrent cardiovascular disease, can be associated with increased extracellular matrix (ECM) fibrillar collagen content and abnormal diastolic function. However, the mechanisms causing this left ventricular (LV) remodeling remain incompletely defined. We hypothesized that one determinant of age-dependent remodeling is a change in the extent to which newly synthesized procollagen is processed into mature collagen fibrils. We further hypothesized that secreted protein acidic and rich in cysteine (SPARC) plays a key role in the changes in post-synthetic procollagen processing that occur in the aged myocardium. Young (3 mo old) and old (18–24 mo old) wild-type (WT) and SPARC-null mice were studied. LV collagen content was measured histologically by collagen volume fraction, collagen composition was measured by hydroxyproline assay as soluble collagen (1 M NaCl extractable) versus insoluble collagen (mature cross-linked), and collagen morphological structure was examined by scanning electron microscopy. SPARC expression was measured by immunoblot analysis. LV and myocardial structure and function were assessed using echocardiographic and papillary muscle experiments. In WT mice, advanced age increased SPARC expression, myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen. In SPARC-null mice, advanced age also increased myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen but significantly less than those seen in WT old mice. As a result, insoluble collagen and myocardial diastolic stiffness were lower in old SPARC-null mice (1.36 ± 0.08 mg hydroxyproline/g dry wt and 0.04 ± 0.005) than in old WT mice (1.70 ± 0.10 mg hydroxyproline/g dry wt and 0.07 ± 0.005, P < 0.05). In conclusion, the absence of SPARC reduced age-dependent alterations in ECM fibrillar collagen and diastolic function. These data support the hypothesis that SPARC plays a key role in post-synthetic procollagen processing and contributes to the increase in collagen content found in the aged myocardium