A mechanistic role for cardiac myocyte apoptosis in heart failure (original) (raw)
Generation of transgenic mice with inducible cardiac myocyte apoptosis. To explore whether cardiac myocyte apoptosis may be a mechanism of heart failure, we created transgenic mice in which heart muscle cell death could be activated at will. This was accomplished by the cardiac-specific expression of a fusion protein (Figure 1a) consisting of three modules of human FKBP-12 (pk mutant) attached to the p20 and p10 catalytic domains of human procaspase-8 (13). This FKBP–caspase-8 fusion protein would be predicted to be catalytically inactive unless forced into close proximity. Forced approximation can be stimulated by systemic administration to mice of FK1012H2, a small molecule that can simultaneously bind two FKBP modules and, thereby, induce the oligomerization of the transgene protein (17).
Southern blot analysis demonstrated the transgene in ten independent lines, two of which, lines 7 and 169 (Figure 1b), represent the highest and lowest expressers, respectively (Figure 1c). As a control for potential effects of overexpression, an additional transgenic line was generated that expresses comparable levels of the identical transgene protein except for a point mutation (C360A) that ablates caspase activity (Figure 1c). Three-week-old pups from each of these transgenic lines appeared grossly normal and exhibited no histological or echocardiographic abnormalities (not shown).
Effects of acute induction of apoptosis. To determine the effects of caspase activation and induction of cardiac myocyte apoptosis in these apparently normal animals, mice were injected with FK1012H2 or vehicle control. No gross or histological abnormalities developed following administration of vehicle to any of the animals or FK1012H2 to WT animals. In contrast, following FK1012H2 administration, all line 7 (n > 100) and line 169 (n = 25) transgenic mice died (Figure 2a). The time to death varied inversely with the dose of FK1012H2, ranging from 4 to 18 hours (Figure 2b). The lethal effect of FK1012H2 was dependent on caspase function, as the C360A transgenic line, which expresses comparable levels of the point-mutated, catalytically inactive caspase, was completely resistant to even the highest doses of this drug (Figure 2a). In addition, FK1012H2-induced death in line 7 mice was markedly delayed by coadministration of _N_-[(1,3-dimethylindole-2-carbonyl)valinyl]-3-amino-4-oxo-5-fluoropentanoic acid (IDN 1965; Idun Pharmaceuticals), a dipeptide fluoromethylketone irreversible pseudosubstrate polycaspase inhibitor (Figure 2c). As expected, FK1012H2 triggered proteolytic cleavage of the transgene protein in the hearts of transgenic lines 7 and 169 mice, consistent with caspase activation; in contrast, the catalytically inactive mutant protein in C360A hearts remained intact (Figure 2d). Analysis of cardiac DNA from line 7 transgenic mice treated with FK1012H2 revealed strong internucleosomal laddering consistent with apoptosis (Figure 2e). Similarly, TUNEL of heart sections demonstrated abundant DNA-strand breaks in line 7 transgenic mice that received FK1012H2 (Figure 2f). Echocardiography of these mice revealed marked increases in wall thickness, which, upon histological examination, were found to represent edema (not shown). Thus, activation of exogenous caspase-8 in the heart results in massive cardiac myocyte apoptosis and death of the mouse.
Activation of the caspase transgene in vivo results in massive cardiac myocyte apoptosis and death of the animals. (a) Mortality following intraperitoneal administration of vehicle alone (81.9% polyethylene glycol 400, 9.1% Tween-80, 9% dimethylacetate) or FK1012H2 (30 mg/kg in the vehicle) to 3-week-old WT mice and transgenic line 7, 169, and C360A mice. Animals were observed for death for 7 days following FK1012H2 administration. (b) Inverse dose-dependence of the time to death following intraperitoneal administration of the indicated doses of FK1012H2 to WT or transgenic line 7 (TG) mice. Animals were observed for death for 7 days following FK1012H2 administration, with no deaths occurring in the WT group. (c) Caspase inhibition delays time to death following transgene activation. Vehicle (0.9% saline) or the polycaspase inhibitor IDN 1965 (12 mg/kg in vehicle) was administered intraperitoneally to line 7 transgenic mice 45 minutes before FK1012H2 (30 mg/kg intraperitoneally) and every 4 hours thereafter as indicated by the triangles. Bars represent the survival times of individual animals that received vehicle or IDN 1965. Animals were observed for death until all had died. *P < 0.0001. (d) Activation of the transgene caspase by FK1012H2. Immunoblot of cardiac homogenates from WT and transgenic line 7, 169, and C360A mice treated 1.5 hours earlier with vehicle or FK1012H2 (30 mg/kg intraperitoneally). Processing of procaspase-8 is indicated by disappearance of the uncleaved moiety; cleavage fragments are not reliably detected in tissue homogenates, presumably because of rapid degradation. The lower portion of the blot was reacted with an antibody against mouse tubulin as a loading control. (e) Induction of cardiac apoptosis by FK1012H2. Genomic DNA from the hearts of WT and transgenic line 7 mice 1 hour after the administration of vehicle or FK1012H2 (30 mg/kg i.v.) was size-fractionated on an agarose gel containing ethidium bromide. (f) TUNEL analysis of FK1012H2-induced apoptosis. Paraffin-embedded sections of hearts from WT and line 7 transgenic mice 1 hour after administration of vehicle or FK1012H2 (30 mg/kg i.v.). TUNEL-positive cells were primarily myocytes, but additional unidentified cells were also present that may represent degenerating myocytes or infiltrating inflammatory cells due to magnitude and rapidity of the death. Bar, 20 μM.
Effects of chronically low levels of cardiac myocyte apoptosis. Our intention in generating the FKBP–caspase-8 mice was to create a model in which the effects of low levels of myocyte apoptosis on cardiac structure and function could be evaluated. Clearly, the rapidity and magnitude of cell death following FK1012H2-induced caspase activation did not accurately model the very low levels of cardiac myocyte loss observed during heart failure in humans and rodents. Unexpectedly, however, even in the absence of FK1012H2, transgenic mice from the highly expressing transgenic line 7 were noted to exhibit increased mortality beginning at approximately 8–9 weeks of age (Figure 3a). In contrast, normal longevity was observed in the low-expressing transgenic line 169 mice (despite the rapid death exhibited by these animals when given FK1012H2) and in mice expressing the C360A mutant. We hypothesized that the decreased survival in line 7 mice resulted from cardiomyopathy due to low, but abnormal, levels of myocyte apoptosis. To test this hypothesis, cardiac structure and function were first evaluated at several time points. At 3 weeks of age, echocardiography and cardiac histology were normal (not shown). In contrast, by 9 weeks of age, line 7 transgenic mice showed left ventricular dilation and marked depression of fractional shortening (Figure 3, b and c). This was accompanied by histological myocyte dropout, interstitial fibrosis, thinning of the myocardium, and dilation of all four cardiac chambers (Figure 3e), indicative of dilated cardiomyopathy. Cardiac catheterization measurements showed elevated left ventricular end-diastolic pressures and depressed basal and isoproterenol-stimulated +dP/dt and –dP/dt (Figure 3d), consistent with combined systolic and diastolic dysfunction. The lower-expressing line 169, which had normal longevity, exhibited an intermediate level of left ventricular dilation and contractile dysfunction (Figure 3c). In contrast, mice expressing the C360A mutant transgene had normal cardiac dimensions, function, and histology (Figure 3c and data not shown). These data demonstrate that the FKBP–caspase-8 transgenic mice spontaneously develop a dilated cardiomyopathy between 3 and 9 weeks of age. This phenotype requires a catalytically active caspase, and the severity and mortality of this syndrome are related to the dose of the transgene protein.
Very low levels of myocyte apoptosis are sufficient to cause a lethal, dilated cardiomyopathy. (a) Kaplan-Meier survival curve of WT mice, and transgenic line 7, 169, and C360A mice that have never been treated with FK1012H2. P < 0.0001 for line 7 vs. WT, line C360A, or line 169. (b) Representative two-dimensionally directed M-mode echocardiograms through the interventricular septum (IVS) and left ventricular posterior wall (PW) from 9-week-old WT and transgenic line 7 mice in the absence of FK1012H2. The electrocardiogram is shown at the bottom of each echocardiogram. (c) Quantitation of M-mode echocardiographic parameters in conscious WT and transgenic line 7, 169, and C360A mice in the absence of FK1012H2. EDD, left ventricular end-diastolic dimension; FS, fractional shortening. *P < 0.01, **P < 0.001. (d) Left ventricular hemodynamics by cardiac catheterization in 9-week-old WT and transgenic line 7 mice under basal conditions or in response to isoproterenol (500 pg i.v.), in the absence of FK1012H2. LVEDP, left ventricular end-diastolic pressure. *P < 0.02, **P < 0.002. (e) Histological analysis of 9-week-old WT and transgenic line 7 mouse hearts in the absence of FK1012H2. Coronal sections stained with H&E (bar, 1 mm), and sections from the indicated area of the left ventricular free wall stained with Masson’s trichrome (bar, 25 μm). (f) Apoptotic cardiac myocytes in WT and transgenic mice in the absence of FK1012H2. Left panels: Double staining for TUNEL (green) and desmin (red, to identify myocytes) in paraffin sections from the hearts of 9-week-old WT and line 7 transgenic mice in the absence of FK1012H2. Bar, 10 μm. Right panel: Number of TUNEL-positive cardiac myocytes per 105 nuclei in 9-week-old WT and transgenic line 7 and C360A mice in the absence of FK1012H2. *P < 0.002, **P < 0.0003.
To investigate the potential role of myocyte apoptosis in the dilated cardiomyopathy of the FKBP–caspase-8 mice, TUNEL staining was performed on heart sections from 7.5- to 8.0-week-old animals that had never received FK1012H2 (Figure 3f). The frequency of spontaneous myocyte apoptosis in WT murine hearts was 1.59 ± 0.7 myocytes per 105 cardiac nuclei, similar to that noted previously in healthy human hearts (8–10). Mice expressing the C360A inactive caspase exhibited similar basal rates. In contrast, the frequency of myocyte apoptosis in line 7 mice was 23.2 ± 2.8 myocytes per 105 cardiac nuclei, 15 times higher than that in WT (P < 0.001). Despite being abnormally elevated, however, this frequency is still quite low. In fact, it is four to ten times lower than the most conservative estimates of myocyte death in failing human hearts (Table 1). These data demonstrate that induction of a very low level of myocyte apoptosis, lower than that observed in human heart failure, is sufficient to induce a lethal, dilated cardiomyopathy.
Frequency of cardiac myocyte apoptosis in human heart failure vs. FKBP–caspase-8 mice
Prevention of cardiomyopathy by caspase inhibition. If the low levels of myocyte apoptosis in the FKBP–caspase-8 transgenic mice really play a causal role in the resulting heart failure phenotype, then inhibition of this cell death should ameliorate the pathology. To test this, the polycaspase inhibitor IDN 1965 was administered to line 7 animals by continuous subcutaneous infusion via osmotic minipump. The infusion was started at 3.5–4.0 weeks of age, when the hearts were still normal, and continued to 7.5–8.0 weeks of age, when the cardiomyopathy is florid in untreated animals. As expected, caspase inhibition markedly reduced the rates of myocyte apoptosis (Figure 4a). Strikingly, inhibition of myocyte death was accompanied by complete abrogation of left ventricular dilation (Figure 4, b and c), dramatic improvement in left ventricular systolic function (Figure 4b), and lessening of histological features of cardiomyopathy such as fibrosis (Figure 4c). These data provide direct evidence that, in this model, myocyte apoptosis plays a critical role in the development of dilated cardiomyopathy.
Abrogation of dilated cardiomyopathy by caspase inhibition. Vehicle or the polycaspase inhibitor IDN 1965 (12.5 μg/h) was administered to line 7 transgenic mice by continuous subcutaneous infusion using osmotic minipumps (model 1002; ALZET Corp., Cupertino, California, USA), beginning at 3.5–4.0 weeks of age, when cardiac dimensions, function, and histology are normal, and continuing until sacrifice at 7.5–8.0 weeks of age, when these transgenic mice uniformly exhibit a severe dilated cardiomyopathy. At 7.5–8.0 weeks of age, echocardiography, TUNEL, and histological examination of cardiac tissue were performed. (a) Number of TUNEL-positive cardiac myocytes per 105 nuclei in vehicle- and IDN 1965–treated line 7 mice. *P < 0.03. (b) M-mode echocardiographic parameters from vehicle- and IDN 1965–treated line 7 mice. *P < 0.0003. (c) Coronal sections from vehicle- and IDN 1965–treated line 7 mice stained with H&E (bar, 1 mm), and sections from the indicated area of the left ventricular free wall stained with Masson’s trichrome (bar, 20 μm).