Bradshaw AD, Baicu CF, Rentz TJ, Van Laer AO, Bonnema DD, Zile MR. Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing. Am J Physiol Heart Circ Physiol 298: H614 -H622, 2010. First published December 11, 2009; doi:10.1152/ajpheart.00474.2009.-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 Ϯ 0.08 mg hydroxyproline/g dry wt and 0.04 Ϯ 0.005) than in old WT mice (1.70 Ϯ 0.10 mg hydroxyproline/g dry wt and 0.07 Ϯ 0.005, P Ͻ 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.secreted protein acidic and rich in cysteine; aging LEFT VENTRICULAR (LV) structural remodeling, such as changes in LV mass, volume, and geometry, are important predictors of functional and clinical outcomes (21,22,27,45). Advancing age, independent of any concurrent cardiovascular disease, can itself be associated with significant LV structural remodeling (16, 24). These age-dependent changes in LV structure may play an important role in the functional limitations that occur in advancing age (16,24). Previous studies have shown that with increasing age, the LV develops concentric remodeling (characterized by an increased LV mass-to-...

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60 -70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wildtype cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.Matricellular proteins are defined as proteins that are associated with the extracellular matrix (ECM) 2 but are not considered structural components of the ECM, in contrast to classical ECM proteins such as laminin and collagen I (1, 2). SPARC is a prototypic matricellular protein that exhibits counter-adhesive and anti-proliferative activity when added to cultured cells (3). SPARC has been shown to bind to a number of ECM proteins including collagens I, III, and IV (3).Expression of SPARC is elevated during development and decreases upon differentiation in a majority of tissues (4). However, expression of SPARC persists in tissues in which ECM remodeling is ongoing, such as bone and gut epithelia (4). Increased levels of SPARC are detected in response to injury where ECM remodeling is initiated, with fibroses in liver, lungs, and kidney, and in the skin of individuals with scleroderma (5-8). Hence, SPARC expression patterns implicate this protein as an important mediator of collagen I deposition and/or remodeling.The ␣1(I) and ␣2(I) subunits of procollagen I are synthesized with N-and C-propeptides that are enzymatically released by specific proteases to yield processed collagen I (9). Processing of procollagen I to collagen I is essential for correct assembly of collagen fibrils. Spatial and temporal regulation of procollagen processing has been proposed as a potential regulatory event in collagen fibril assembly (9). For example, antibodies against the N-propeptide of collagen I were immunolocalized exclusively to the surface of collagen fibrils that were 20 -40 nm in diameter (10). Retention of the prop...

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses; however, the molecular regulation of 2-AG synthesis is not well understood. Here we identify a novel functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacts with the C-terminal domain of DGLα, phosphorylates two serine residues, and inhibits DGLα activity. Moreover, CaMKII inhibition augments short-term retrograde eCB signaling at striatal glutamatergic synapses. Consistent with an inhibitory role for CaMKII in synaptic 2-AG synthesis, in vivo genetic inhibition of CaMKII increases striatal DGL activity and basal 2-AG levels. Moreover, blockade of 2-AG breakdown using concentrations of JZL-184 that have no significant effect in wild type mice produces a hypo-locomotor response in mice with reduced CaMKII activity. These findings provide novel mechanistic insight into the molecular regulation of striatal eCB signaling with implications for physiological control of motor function.

Background-Chronic pressure overload causes myocardial hypertrophy, increased fibrillar collagen content, and abnormal diastolic function. We hypothesized that one determinant of these pressure overload-induced changes is the extracellular processing of newly synthesized procollagen into mature collagen fibrils. We further hypothesized that secreted protein acidic and rich in cysteine (SPARC) plays a key role in post-synthetic procollagen processing in normal and pressure-overloaded myocardium. Methods and Results-To determine whether pressure overload-induced changes in collagen content and diastolic function are affected by the absence of SPARC, age-matched wild-type (WT) and SPARC-null mice underwent either transverse aortic constriction (TAC) for 4 weeks or served as nonoperated controls. Left ventricular (LV) collagen content was measured histologically by collagen volume fraction, collagen composition was measured by hydroxyproline assay as soluble collagen (1 mol/L NaCl extractable) versus insoluble collagen (mature cross-linked collagen), and collagen morphological structure was examined by scanning electron microscopy. SPARC expression was measured by immunoblot. LV, myocardial, and cardiomyocyte structure and function were assessed by echocardiographic, papillary muscle, and isolated cardiomyocyte studies. In WT mice, TAC increased LV mass, SPARC expression, myocardial diastolic stiffness, fibrillar collagen content, and soluble and insoluble collagen. In SPARC-null mice, TAC increased LV mass to an extent similar to WT mice. In addition, in SPARC-null mice, TAC increased fibrillar collagen content, albeit significantly less than that seen in WT TAC mice. Furthermore, the proportion of LV collagen that was insoluble was less in the SPARC-null TAC mice (86Ϯ2%) than in WT TAC mice (99Ϯ2%, PϽ0.05), and the proportion of collagen that was soluble was greater in the SPARC-null TAC mice (14Ϯ2%) than in WT TAC mice (1Ϯ2%, PϽ0.05) As a result, myocardial diastolic stiffness was lower in SPARC-null TAC mice (0.075Ϯ0.005) than in WT TAC mice (0.045Ϯ0.005, PϽ0.05). Conclusions-The absence of SPARC reduced pressure overload-induced alterations in extracellular matrix fibrillar collagen and diastolic function. These data support the hypothesis that SPARC plays a key role in post-synthetic procollagen processing and the development of mature cross-linked collagen fibrils in normal and pressure-overloaded myocardium. (Circulation. 2009;119:269-280.)