D. Dirk Bonnema

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.

A Direct Test of the Hypothesis That Increased Microtubule Network Density Contributes to Contractile Dysfunction of the Hypertrophied Heart

Contractile dysfunction in pressure overload-hypertrophied myocardium has been attributed in part to the increased density of a stabilized cardiocyte microtubule network. The present study, the first to employ wild-type and mutant tubulin transgenes in a living animal, directly addresses this microtubule hypothesis by defining the contractile mechanics of the normal and hypertrophied left ventricle (LV) and its constituent cardiocytes from transgenic mice having cardiac-restricted replacement of native beta(4)-tubulin with beta(1)-tubulin mutants that had been selected for their effects on microtubule stability and thus microtubule network density. In each case, the replacement of cardiac beta(4)-tubulin with mutant hemagglutinin-tagged beta(1)-tubulin was well tolerated in vivo. When LVs in intact mice and cardiocytes from these same LVs were examined in terms of contractile mechanics, baseline function was reduced in mice with genetically hyperstabilized microtubules, and hypertrophy-related contractile dysfunction was exacerbated. However, in mice with genetically hypostabilized cardiac microtubules, hypertrophy-related contractile dysfunction was ameliorated. Thus, in direct support of the microtubule hypothesis, we show here that cardiocyte microtubule network density, as an isolated variable, is inversely related to contractile function in vivo and in vitro, and microtubule instability rescues most of the contractile dysfunction seen in pressure overload-hypertrophied myocardium.

Regulation of Ncx1 expression: Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.

Comparison of percutaneous coronary intervention safety before and during the establishment of a transradial program at a teaching hospital.

This study sought to examine the safety of percutaneous coronary intervention (PCI) before and during de novo establishment of a transradial (TR) program at a teaching hospital. TR access remains underused in the United States, where cardiology fellowship programs continue to produce cardiologists with little TR experience. Establishment of TR programs at teaching hospitals may affect PCI safety. Starting in July 2009 a TR program was established at a teaching hospital. PCI-related data for academic years 2008 to 2009 (Y1) and 2009 to 2010 (Y2) were prospectively collected and retrospectively analyzed. Of 1,366 PCIs performed over 2 years, 0.1% in Y1 and 28.7% in Y2 were performed by TR access. No major complications were identified in 194 consecutive patients undergoing TR PCI, and combined bleeding and vascular complication rates were lower in Y2 versus Y1 (0.7% vs 2.0%, p = 0.05). Patients treated in Y2 versus Y1 and by TR versus transfemoral approach required slightly more fluoroscopy but similar contrast volumes and had similar procedural durations, lengths of stay, and predischarge mortality rates. PCI success rates were 97% in Y1, 97% in Y2, and 98% in TR cases. TR PCIs were performed by 13 cardiology fellows and 9 attending physicians, none of whom routinely performed TR PCI previously. In conclusion, de novo establishment of a TR program improved PCI safety at a teaching hospital. TR programs are likely to improve PCI safety at other teaching hospitals and should be established in all cardiology fellowship training programs.