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Background: Left ventricular pressure overload (LVPO) can develop from antecedent conditions, like aortic stenosis, and is associated with increased myocardial collagen content that can lead to increases in myocardial stiffness. Alleviation of LVPO in patients undergoing surgical aortic valve replacement (SAVR) resulted in reduced but persistent fibrosis evident in biopsies taken 5-yrs following SAVR. Little is known regarding cellular mechanisms that contribute to ECM turnover following hemodynamic unloading. Objective: Determine the time course of ECM remodeling after alleviation of LVPO. Methods: Transverse aortic constriction (TAC) was used to induce LVPO. Following 4wks of TAC, alleviation of LVPO was performed by removing the TAC band (unTAC). Five time points were assessed: Control no TAC, 4wk TAC, 4wk TAC+2wk unTAC, 4wk TAC+4wk unTAC, and 4wk TAC+6wk unTAC for cardiomyocyte cross-sectional area (CSA), collagen volume fraction (CVF), collagen degradation, and myocardial stiffness. Results: CSA increased by 47% at 4wk TAC vs control. Compared to 4wk TAC, CSA decreased by 20% at 2wk unTAC, but returned to control levels by 6wk unTAC. CVF increased by 204% at 4wk TAC vs control. Although CVF was similar between 4wk TAC and 2wk unTAC, 2wk unTAC demonstrated robust increases in markers of collagen degradation (cathepsin K, pro-matrix metalloproteinase 2 (pro-MMP2), MMP3, and MMP8). CVF significantly decreased at 4wk unTAC compared to 2wk unTAC. However, the markers of collagen degradation were reduced by 4wk- and 6wk unTAC likely contributing to persistent fibrosis. Furthermore, passive stiffness remained elevated at 6wk unTAC. Conclusion: In LVPO hearts, hypertrophy and fibrosis were present. Although cardiomyocyte hypertrophy fully regressed with normalization of hemodynamic load, increased myocardial stiffness and elevated interstitial fibrosis were persistent.

Left ventricular pressure overload (LVPO) can develop from antecedent diseases such as aortic valve stenosis and systemic hypertension and is characterized by accumulation of myocardial extracellular matrix (ECM). Evidence from patient and animal models supports limited reductions in ECM following alleviation of PO, however, mechanisms that control the extent and timing of ECM regression are undefined. LVPO, induced by 4 wk of transverse aortic constriction (TAC) in mice, was alleviated by removal of the band (unTAC). Cardiomyocyte cross-sectional area, collagen volume fraction (CVF), myocardial stiffness, and collagen degradation were assessed for: control, 2-wk TAC, 4-wk TAC, 4-wk TAC + 2-wk unTAC, 4-wk TAC + 4-wk unTAC, and 4-wk TAC + 6-wk unTAC. When compared with 4-wk TAC, 2-wk unTAC resulted in increased reactivity of collagen hybridizing peptide (CHP) (representing initiation of collagen degradation), increased levels of collagenases and gelatinases, decreased levels of collagen cross-linking enzymes, but no change in CVF. When compared with 2-wk unTAC, 4-wk unTAC demonstrated decreased CVF, which did not decline to control values. At 4-wk and 6-wk unTAC, CHP reactivity and mediators of ECM degradation were reduced versus 2-wk unTAC, whereas levels of tissue inhibitor of metalloproteinase (TIMP)-1 increased. ECM homeostasis changed in a time-dependent manner after removal of LVPO and is characterized by early increases in collagen degradation, followed by a later dampening of this process. Tempered ECM degradation with time is predicted to contribute to the finding that normalization of hemodynamic overload alone does not completely regress myocardial fibrosis. NEW & NOTEWORTHY In this study, a murine model demonstrated persistent interstitial fibrosis and myocardial stiffness following alleviation of pressure overload.

In human heart failure and in murine hearts with left-ventricular pressure overload (LVPO), increases in fibrosis are associated with increases in myocardial stiffness. Secreted protein acidic and rich in cysteine (SPARC) is shown to be necessary for both cardiac fibrosis and increases in myocardial stiffness in response to LVPO; however, cellular sources of cardiac SPARC are incompletely defined. Irradiation and bone marrow transfer were undertaken to test the hypothesis that SPARC expression by bone marrow-derived cells is an important mediator of fibrosis in LVPO. In recipient SPARC-null mice transplanted with donor wild-type (WT) bone marrow and subjected to LVPO, levels of fibrosis similar to that of WT mice were found despite the lack of SPARC expression by resident cells. In recipient WT mice with donor SPARC-null bone marrow, significantly less fibrosis versus that of WT mice was found despite the expression of SPARC by resident cells. Increases in myocardial stiffness followed a similar pattern to that of collagen deposition. Myocardial macrophages were significantly reduced in SPARC-null mice with LVPO versus that of WT mice. Recipient SPARC-null mice transplanted with donor WT bone marrow exhibited an increase in cardiac macrophages versus that of SPARC-null LVPO and donor WT mice with recipient SPARC-null bone marrow. Expression of vascular cellular adhesion molecule (VCAM), a previously identified binding partner of SPARC, was assessed in all groups and with the exception of WT mice, increases in VCAM immunoreactivity with LVPO were observed. However, no differences in VCAM expression between bone marrow transplant groups were noted. In conclusion, SPARC expression by bone marrow-derived cells was critical for fibrotic deposition of collagen and influenced the expansion of myocardial macrophages in response to LVPO. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in LV and myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, a murine model of cardiac fibrosis induced by PO was used to demonstrate a critical function of SPARC in bone marrow-derived cells that drives cardiac fibrosis and increases in cardiac macrophages.

Mechanisms that contribute to myocardial fibrosis, particularly in response to left ventricular pressure overload (LVPO), remain poorly defined. To test the hypothesis that a myocardial-specific profile of secreted factors is produced in response to PO, levels of 44 factors implicated in immune cell recruitment and function were assessed in a murine model of cardiac hypertrophy and compared with levels produced in a model of pulmonary fibrosis (PF). Mice subjected to PO were assessed at 1 and 4 wk. Protein from plasma, LV, lungs, and kidneys were analyzed by specific protein array analysis in parallel with protein from mice subjected to silica-instilled PF. Of the 44 factors assessed, 13 proteins were elevated in 1-wk PO myocardium, whereas 18 proteins were found increased in fibrotic lung. Eight of those increased in 1-wk LVPO were not found to be increased in fibrotic lungs (CCL-11, CCL-12, CCL-17, CCL-19, CCL-21, CCL-22, IL-16, and VEGF). Additionally, six factors were increased in plasma of 1-wk LVPO in the absence of increases in myocardial levels. In contrast, in mice with PF, no factors were found increased in plasma that were not elevated in lung tissue. Of those factors increased at 1 wk, only TIMP-1 remained elevated at 4 wk of LVPO. Immunohistochemistry of myocardial vasculature at 1 and 4 wk revealed similar amounts of total vasculature; however, evidence of activated endothelium was observed at 1 wk and, to a lesser extent, at 4 wk LVPO. In conclusion, PO myocardium generated a unique signature of cytokine expression versus that of fibrotic lung.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, cytokine profiles produced in a murine model of cardiac fibrosis induced by PO were compared with those produced in response to silica-induced lung fibrosis. A unique profile of cardiac tissue-specific and plasma-derived factors generated in response to PO are reported.

Myocardial fibrosis and the resultant increases in left ventricular stiffness represent pivotal consequences of chronic pressure overload (PO) that impact both functional capacity and the rates of morbid and mortal events. However, the time course and cellular mechanisms that underlie PO-induced fibrosis have not been completely defined. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that has been shown to be required for insoluble collagen deposition and increased myocardial stiffness in response to PO in mice. As macrophages are associated with increases in fibrillar collagen, the hypothesis that macrophages represent a source of increased SPARC production in the PO myocardium was tested. The time course of changes in the myocardial macrophage population was compared with changes in procollagen type I mRNA, production of SPARC, fibrillar collagen accumulation, and diastolic stiffness. In PO hearts, mRNA encoding collagen type I was increased at 3 days, whereas increases in levels of total collagen protein did not occur until 1 wk and were followed by increases in insoluble collagen at 2 wk. Increases in muscle stiffness were not detected before increases in insoluble collagen content (>1 wk). Significant increases in myocardial macrophages that coincided with increased SPARC were found but did not coincide with increases in mRNA encoding collagen type I. Furthermore, immunohistochemistry and flow cytometry identified macrophages as a cellular source of SPARC. We conclude that myocardial macrophages play an important role in the time-dependent increases in SPARC that enhance postsynthetic collagen processing, insoluble collagen content, and myocardial stiffness and contribute to the development of fibrosis. NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in left ventricular and myocardial stiffness represent pivotal consequences of chronic pressure overload. In this study a murine model of cardiac fibrosis induced by pressure overload was used to establish a time course of collagen expression, collagen deposition, and cardiac macrophage expansion.

Background— MicroRNAs (miRNAs) and histone deacetylases (HDACs) serve a significant role in the pathogenesis of a variety of cardiovascular diseases. The transcriptional regulation of miRNAs is poorly understood in cardiac hypertrophy. We investigated whether the expression of miR-133a is epigenetically regulated by class I and IIb HDACs during hypertrophic remodeling. Methods and Results— Transverse aortic constriction (TAC) was performed in CD1 mice to induce pressure overload hypertrophy. Mice were treated with class I and IIb HDAC inhibitor (HDACi) via drinking water for 2 and 4 weeks post TAC. miRNA expression was determined by real-time polymerase chain reaction. Echocardiography was performed at baseline and post TAC end points for structural and functional assessment. Chromatin immunoprecipitation was used to identify HDACs and transcription factors associated with miR-133a promoter. miR-133a expression was downregulated by 0.7- and 0.5-fold at 2 and 4 weeks post TAC, respectively, when compared with vehicle control ( P Conclusions— The results reveal that HDACs play a role in the regulation of pressure overload–induced miR-133a downregulation. This work is the first to provide insight into an epigenetic-miRNA regulatory pathway in pressure overload–induced cardiac fibrosis.

Historically, the tissue inhibitors of matrix metalloproteinases (TIMPs) were considered monochromatic in function. However, differential TIMP profiles more recently observed with left ventricular (LV) dysfunction and matrix remodeling suggest more diverse biological roles for individual TIMPs. This study tested the hypothesis that cardiac-specific overexpression (TIMP-4OE) or deletion (knockout; TIMP-4KO) would differentially affect LV function and structure following pressure overload (LVPO). LVPO (transverse aortic constriction) was induced in mice (3.5 ± 0.1 mo of age, equal sex distribution) with TIMP-4OE ( n = 38), TIMP-4KO ( n = 24), as well as age/strain-matched wild type (WT, n = 25), whereby indexes of LV remodeling and function such as LV mass and ejection fraction (LVEF) were determined at 28 days following LVPO. Following LVPO, both early (7 days) and late (28 days) survival was ∼25% lower in the TIMP-4KO group ( P < 0.05). While LVPO increased LV mass in all groups, the relative hypertrophic response was attenuated with TIMP-4OE. With LVPO, LVEF was similar between WT and TIMP-4KO (48 ± 2% and 45 ± 3%, respectively) but was higher with TIMP-4OE (57 ± 2%, P < 0.05). With LVPO, LV myocardial collagen expression (type I, III) increased by threefold in all groups ( P < 0.05), but surprisingly this response was most robust in the TIMP-4KO group. These unique findings suggest that increased myocardial TIMP-4 in the context of a LVPO stimulus may actually provide protective effects with respect to survival, LV function, and extracellular matrix (ECM) remodeling. These findings challenge the canonical belief that increased levels of specific myocardial TIMPs, such as TIMP-4 in and of themselves, contribute to adverse ECM accumulation following a pathological stimulus, such as LVPO.

Background— Although matrix metalloproteinases (MMPs) were initially thought to result primarily in extracellular matrix degradation, certain MMP types, such as membrane type-1 (MT1) MMP, may also be involved in profibrotic cascades through hydrolysis of latency-associated transforming growth factor–binding protein (LTBP-1) and activation of transforming growth factor–dependent profibrotic signaling. The present study tested the hypothesis that MT1-MMP plays a direct role in the matrix remodeling response to a left ventricular (LV) pressure overload (PO) stimulus. Methods and Results— Wild-type (WT) and transgenic mice with cardiac-restricted MT1-MMP overexpression or MT1-MMP reduced expression underwent PO for 4 weeks. PO resulted in a 57% increase in LV mass (no change in LV end diastolic volume, resulting in an increase in the LV mass/volume ratio consistent with concentric remodeling), a 60% increase in MT1-MMP–mediated LTBP-1 hydrolysis and a 190% increase in collagen content in WT mice. Although LV mass was similar among WT, MT1-MMP overexpression, and MT1-MMP reduced expression after PO, significant differences in LV function, MT1-MMP–mediated LTBP-1 hydrolysis, and collagen content occurred. PO in MT1-MMP overexpression increased LTBP-1 hydrolysis (18%), collagen content (60%), and left atrial dimension (19%; indicative of LV diastolic dysfunction) when compared with WT. PO in MT1-MMP reduced expression reduced left atrial dimension (19%), LTBP-1 hydrolysis (40%), and collagen content (32%) when compared with both WT. Conclusions— Despite an equivalent PO stimulus and magnitude of LV myocardial growth, altering MT1-MMP levels caused specific matrix-dependent changes in remodeling, thereby demonstrating a mechanistic role in the development of the maladaptive remodeling and myocardial fibrotic response to PO.

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes.

Diabetic cardiomyopathy (DbCM), which consists of cardiac hypertrophy and failure in the absence of traditional risk factors, is a major contributor to increased heart failure risk in type 2 diabetes patients. In rodent models of DbCM, cardiac hypertrophy and dysfunction have been shown to depend upon saturated fatty acid (SFA) oversupply and de novo sphingolipid synthesis. However, it is not known whether these effects are mediated by bulk SFAs and sphingolipids or by individual lipid species. In this report, we demonstrate that a diet high in SFA induced cardiac hypertrophy, left ventricular systolic and diastolic dysfunction, and autophagy in mice. Furthermore, treatment with the SFA myristate, but not palmitate, induced hypertrophy and autophagy in adult primary cardiomyocytes. De novo sphingolipid synthesis was required for induction of all pathological features observed both in vitro and in vivo, and autophagy was required for induction of hypertrophy in vitro. Finally, we implicated a specific ceramide N-acyl chain length in this process and demonstrated a requirement for (dihydro)ceramide synthase 5 in cardiomyocyte autophagy and myristate-mediated hypertrophy. Thus, this report reveals a requirement for a specific sphingolipid metabolic route and dietary SFAs in the molecular pathogenesis of lipotoxic cardiomyopathy and hypertrophy.

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH2- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.

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.

Objectives to investigate the feasibility of placing stents across renal arteries.Design we have studied in pigs: (i) the feasibility of accurately placing a fenestrated stent in front of one renal ostium; (ii) the short-term effects on renal arteries and function after the placement of such a fenestrated stent. Materials and methods eight fenestrated Palmaz stents were placed over pigs» renal ostia under fluoroscopy. Five weeks later, angiograms were performed and the animals were sacrificed. Proliferation of the healing tissues over the ostia was measured and analysed by microscopy. Serum creatinine was measured prior to all angiograms and at 5 weeks. Results all eight stents were correctly placed. One stent later migrated and was excluded from the study. One pig died at day 1. Gross examination confirmed the correct placement of the fenestrations in four pigs out of seven (57%). In the six remaining pigs, at 5 weeks, there was no angiographic evidence of stent misplacement and all the kidneys were fully perfused. Nine renal ostia were covered by struts and neointima with a mean area of coverage of 38±5% altogether. No tissue proliferation was observed over the three renal ostia located in front of the fenestration. Serum creatinine did not significantly increase at 5 weeks. Conclusion creating a fenestration in a stent for renal arteries may be worth while in order to avoid neointimal covering of the renal ostia. However, accurate placement of such a fenestrated stent remains a difficult task.

Increased myocardial extracellular matrix collagen represents an important structural milestone during the development of left ventricular (LV) pressure overload (PO); however, the proteolytic pathways that contribute to this process are not fully understood. This study tested the hypothesis that membrane type 1-matrix metalloproteinase (MT1-MMP) is directly induced at the transcriptional level in vivo during PO and is related to changes in LV collagen content. PO was induced in vivo by transverse aortic constriction in transgenic mice containing the full length human MT1-MMP promoter region ligated to luciferase (MT1-MMP Prom mice). MT1-MMP promoter activation (luciferase expression), expression, and activity; collagen volume fraction (CVF); and left atrial dimension were measured at 1 ( n = 8), 2 ( n = 12), and 4 ( n = 17) wk following PO. Non-PO mice ( n = 10) served as controls. Luciferase expression increased by fivefold at 1 wk, fell at 2 wk, and increased again by ninefold at 4 wk of PO ( P < 0.05). MT1-MMP expression and activity increased at 1 wk, fell at 2 wk, and increased again at 4 wk after PO. CVF increased at 1 wk, remained unchanged at 2 wk, and increased by threefold at 4 wk of PO ( P < 0.05). There was a strong positive correlation between CVF and MT1-MMP activity ( r = 0.80, P < 0.05). Left atrial dimension remained unchanged at 1 and 2 wk but increased by 25% at 4 wk of PO. When a mechanical load was applied in vitro to LV papillary muscles isolated from MT1-MMP Prom mice, increased load caused MT1-MMP promoter activation to increase by twofold and MT1-MMP expression to increase by fivefold ( P < 0.05). These findings challenge the canonical belief that PO suppresses overall matrix proteolytic activity, but rather supports the concept that certain proteases, such as MT1-MMP, play a pivotal role in PO-induced matrix remodeling and fibrosis.

Cardiac hypertrophy is generated in response to hemodynamic overload by altering steady-state protein metabolism such that the rate of protein synthesis exceeds the rate of protein degradation. To determine the relative contributions of protein synthesis and degradation in regulating cardiac hypertrophy in mice, a continuous infusion strategy was developed to measure myocardial protein synthesis rates in vivo. Osmotic mini-pumps were implanted in the abdominal cavity to infuse radiolabeled leucine in mice that are conscious and ambulatory. Protein synthesis rates were calculated by measuring incorporation of leucine into myocardial protein over 24 h prior to each time point and dividing by the specific radioactivity of plasma leucine. Compared to sham-operated controls, fractional rates of protein synthesis (K(s)) increased significantly at days 1 and 3 of TAC, but was lower on day 7 and returned to control values by day 14. These changes coincided with the curvilinear increase in LV mass that characterizes the hypertrophic response. Fractional rates of protein degradation (K(d)) were calculated by subtracting the rate of myocardial growth from the corresponding K(s) value. K(d) fell at days 1 and 3 of TAC, increased at day 7 and returned to control on day 14. Thus, the increase in LV mass generated in response to pressure overload is caused by acceleration of K(s) and suppression of K(d). As the growth rate slows, a new steady-state is achieved once the hypertrophic response is completed.

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

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 (86±2%) than in WT TAC mice (99±2%, P P P 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.

Introduction: Both pressure overload hypertrophy (POH) and aging cause increased diastolic chamber stiffness. Whether and to what extent these changes are dependent on increased LV mass, increased myocardial viscoelastic stiffness, changes in the extracellular matrix (ECM) and/or changes in the constitutive properties of the cardiac muscle cells is not completely defined. Methods: Three groups of mice (C57Bl) were studied: young adults (controls; n 7, age 16 5 wks), POH (n 6, age 16 1 wks) and senescent (n 7, age 96 10 wks). POH was created by transverse aortic constriction (TAC) for 4 weeks. Left ventricular (LV) structure and function were determined by echocardiography. LV papillary muscles were isolated and viscoelastic properties studied. Cardiomyocytes were isolated (collagenase), embedded in a gel matrix and viscoelastic properties studied. In both papillary muscles and cardiomyocytes a stress vs strain hysteresis loop was derived, which was fit by a constitutive equation to derive the elastic stiffness constant β. Additionally, normalized area of the hysteresis loop was used to derive the viscosity constant η. Results: In the POH mice echocardiography showed a significant increase in LV/ Body Weight ratio (3.04 0.16 before vs 5.69 1.09* mg/gm after TAC) with no change in end diastolic volume or ejection fraction. Stiffness and viscosity were increased in papillary muscle and cardiomyocytes in both POH and senescent mice (Table). POH caused a 118% increase in myocardial stiffness and a 21% increase in cardiomyocyte stiffness. Aging caused a 29% increase in myocardial stiffness and a 31% increase in cardiomyocyte stiffness. Therefore, both the ECM and cardiomyocyte properties play a role in increased myocardial stiffness. Conclusion: Data suggest that the observed increase in myocardial stiffness may be more dependent on changes in the ECM in POH, but more dependent on changes in the cardiomyocyte in aging. Therefore, treatment developed to decrease myocardial stiffness may target either ECM or cardiomyocyte but must affect both to completely normalize the increased stiffness that occurs in POH and aging.