Caspase Cleavage of Gelsolin Is an Inductive Cue for Pathologic Cardiac Hypertrophy

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  Figure 1. Gelsolin is an essential caspase cleavage substrate during cardiomyocyte hypertrophy. A, Cardiomyocytes infected with p35‐adenovirus or green fluorescent protein (GFP)–adenovirus were treated with phenylephrine and subjected to immunoblot using a C‐terminal gelsolin antibody. Gelsolin cleavage was observed with GFP‐adenovirus, whereas these fragments were not present with p35‐adenovirus. Treatment with 2 μmol/L staurosporine served as a positive (+) control. B, Gelsolin in vitro cleavage assays for caspase alone, gelsolin alone, gelsolin with caspase 3 or 7, or gelsolin with caspase and caspase inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk) were probed with a C‐terminal gelsolin antibody. A smaller molecular weight gelsolin fragment (asterisk) is observed when gelsolin is incubated with caspase 3/7 and is reduced with z‐DEVD‐fmk. C, Recombinant gelsolin was subjected to an in vitro cleavage reaction, followed by SDS/PAGE and silver staining. Protein fragments were isolated and processed by liquid chromatography–tandem mass spectrometry. D, Red peptides represent those from the N‐terminal fragment (44 kDa plus 26‐kDa N‐terminal gelsolin Glutathione…

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  Figure 2. Expression of wild‐type or N‐terminal gelsolin can induce hypertrophy in primary cardiomyocytes. Primary cardiomyocytes were infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection [MOI]=1) during serum‐free or phenylephrine treatment (bar=40 μm). Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the GFP‐adenovirus–infected cardiomyocytes within each treatment. A significant increase in cell size was observed after wild‐type (n=3, **P<0.01) or N‐terminal gelsolin infection (n=3, *P<0.05) during 24 hours of serum‐free treatment, whereas the C‐terminal gelsolin or D401A gelsolin‐adenovirus did not significantly affect cell size. Similar results were observed after serum‐free treatment over 48 hours (wild‐type, n=3, ***P<0.001; N‐terminus, n=3, **P<0.01) and phenylephrine treatment over 3 hours (wild‐type, n=3, **P<0.01; N‐terminus, n=3, **P<0.01). ANP levels were also significantly increased after wild‐type and…

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  Figure 3. Expression of wild‐type or N‐terminal gelsolin can rescue the hypertrophy response reduced by caspase inhibition. Primary cardiomyocytes were treated with 20 μmol/L of the caspase 3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp fluoromethyl ketone (z‐DEVD‐fmk); infected with green fluorescent protein (GFP) control adenovirus, wild‐type gelsolin‐adenovirus, N‐terminal gelsolin‐adenovirus, C‐terminal gelsolin‐adenovirus, or D401A gelsolin‐adenovirus (multiplicity of infection=1); and treated with phenylephrine for 24 hours. Immunofluorescence analysis was completed (A), and cell size and atrial natriuretic peptide (ANP) levels were evaluated (B). All values were normalized to the caspase‐inhibited and GFP‐adenovirus–infected cardiomyocytes, which were reduced in size, and ANP levels were compared with those lacking caspase inhibitor treatment (n=3, **P<0.01; and n=3, **P<0.01, respectively). During caspase inhibition, a hypertrophy rescue was observed after wild‐type (n=3, **P<0.01) or N‐terminal gelsolin infection (n=3, **P<0.01) and identified by a significant increase in cell size and ANP levels compared with GFP‐adenovirus–infected cardiomyocytes (wild‐type, n=3, **P<0.01;…

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  Figure 4. Wild‐type or N‐terminal gelsolin overexpression can induce a hypertrophy response in the intact myocardium. A, Outline of in vivo study. Sprague‐Dawley rats were echo‐guided intramyocardial injected with either the control green fluorescent protein (GFP) adenovirus or one of the gelsolin adenoviruses. Echocardiography was performed at days 0, 14, 21, and 28. Rats were euthanized at day 35, and hearts and tibia were isolated. Three hearts per adenovirus group were optimal cutting temperature (OCT) compound embedded and cryosectioned. B, Echocardiographic data were taken on days 0, 14, 21, and 28 and represent the percentage change from the baseline measurement. Significant differences in these measurements were observed when comparing the gelsolin‐injected rats with those injected with the control GFP‐adenovirus. Significantly reduced left ventricular internal diameter (LVID) was observed in rats injected with N‐terminal gelsolin‐adenovirus compared with the GFP‐adenovirus (day 28) (n=7, *P<0.05). Although no significant increase in left ventricular posterior wall thickness (Figure S4) was observed, interventricular septum (IVS) measurements showed thickening in N‐terminal…

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  Figure 5. Gelsolin secreted by hypertrophic cardiomyocytes may be involved in the induction of hypertrophy. A, Primary cardiomyocytes were treated with conditioned hypertrophic media (Cond. Media), from phenylephrine‐ or procaspase activating compound (PAC‐1)–treated cardiomyocytes, supplemented with gelsolin antibody to block secreted gelsolin, gelsolin antibody plus gelsolin peptide, or MyoD antibody. Serum‐free medium treatment was used as a control. B, Cell size and atrial natriuretic peptide (ANP) levels were analyzed and are quantified. Phenylephrine (n=3, **P<0.01; ANP, n=3, **P<0.01) and PAC‐1 (n=3, **P<0.01; ANP, n=3, **P<0.01) treatment (regular controls) resulted in hypertrophy induction. Treatment of cardiomyocytes with phenylephrine‐ or PAC‐1–conditioned medium led to enlarged cardiomyocytes (phenylephrine, n=3, **P<0.01; PAC‐1, n=3, **P<0.01) and elevated ANP levels (phenylephrine, n=3, **P<0.01; PAC‐1, n=3, **P<0.01) compared with the serum‐free control. Conversely, cardiomyocytes treated with the conditioned phenylephrine (n=3, **P<0.01; ANP, n=3, **P<0.01) or PAC‐1 (n=3, **P<0.01; ANP, n=3, **P<0.01) hypertrophy medium plus a gelsolin antibody reduced the hypertrophy…