Acute beta-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells

Abstract

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, "Takotsubo stress cardiomyopathy," is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of beta1/beta2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.

FIGURE 1. Representative EKG tracings monitoring the acute effects of subcutaneous administration of ISO (5 mg kg^-1)

EKG monitoring was continued for up to 6 h after ISO or placebo administration. After this time period, no deaths have been observed. From every representative tracing, an insert was created to highlight the specific morphologic EKG modifications (A-H). A, EKG output of CTRL animals (BASELINE). B-F, in ∼20% of the treated rats, the EKG showed ST ischemic changes (B and C) followed by rapid sustained ventricular tachycardia, which turned into ventricular fibrillation and death of the animal (D-F). G and H, in the majority of the rats treated with ISO, the same acute ST changes were evident, and periods of short bursts of ventricular tachycardia were registered during the EKG monitoring after ISO administration. I, the representative two-dimensional echocardiogram of a surviving animal shows a dramatic dilatation of the LV at day 1, as compared with the recording prior to ISO administration (CTRL). The chamber dimensions have returned to normal at day 6.

FIGURE 2. Isoproterenol caused extensive acute myocardial necrosis in the LV

A, low magnification (×2.5) representative hematoxylin and eosin cross-sections of the apical LV wall, epi- to endocardial, from CTRL and ISO-treated hearts. The higher magnification (×20) inset at 1 and 3 days after ISO shows myocytes with disrupted sarcolemmal membranes and pale cytoplasmic staining, implicating focal necrosis. B, representative picture of specific necrotic myocytes labeled in vivo with anti-myosin antibody (green). C, the fraction of necrotic myocytes were significantly increased at 1 day in the LV and decreased over time. *, p < 0.05 versus CTRL. D, plasma cTnI was significantly elevated at 1 day after ISO. *, p < 0.001 versus CTRL.

FIGURE 3. Isoproterenol caused acute myocyte apoptosis along with RyR2 dysfunction and reactive myocyte hypertrophy in the LV

A-D, representative picture of apoptotic myocytes stained by activated caspase-3 (A)(green) and TdT (B) (TdT (green), MHC (red), and DAPI (blue)). C and D, apoptotic (caspase-3- and TdT-positive) myocytes were significantly increased at 1 day in the LV and progressively decreased over time. *, p < 0.05 versus CTRL. E, representative Western blots of cleaved caspase-3 and PARP in isolated myocytes after ISO injection. Changes to caspase-3 and PARP progressively decreased over 6 days. F, distribution of myocyte sizes (open bars) at 1 day after ISO showing the fraction of dead myocytes (necrotic and apoptotic; solid bars) only in the largest myocytes. G, LV dry weight was slightly decreased at 1 day but significantly increased at 3 days, indicating net loss of myocytes followed by reactive cardiac hypertrophy at days 1-3 after ISO injection. *, p < 0.05 versus CTRL. H, quantitative real-time RT-PCR showed increased mRNA transcripts of β-MHC in ARVMs isolated from ISO-treated hearts at 3 days when compared with CTRL and 1 day. Data are presented as the ratio between numbers of β-MHC and GAPDH mRNA molecules per μg of RNA. *, p < 0.01. I and J, representative Western blots of phospho-PKA, PKAα, phospho-CaMKII, CaMKII, total RyR2, and PKA- and CaMKII-mediated RyR2 phosphorylation levels in ARVMs isolated from hearts of rats at 1, 3, and 6 days after ISO or saline vehicle (CTRL). K, the levels of calstabin were not affected by ISO injection, whereas calstabin was displaced from the RyR2 complex at 1 day as shown by the immunoprecipitation with RyR2 and the Western blot for calstabin.

FIGURE 4. Isoproterenol induces myocyte death through RyR-2 hyperphosphorylation and calstabin complex dissociation

A, ARVMs were isolated and plated in culture dishes before ISO was administered in vitro, and cell apoptosis was measured by TdT labeling (B, TdT (green), MHC (red), and DAPI (blue)). C, ISO caused increased apoptosis of ARVMs in a dose-dependent manner (all dishes were supplemented with 0.1 mm ascorbic acid (AA); hatched bars). *, p < 0.001 versus CTRL and ascorbic acid alone. D, the presence of JTV519 (1.0 μm) prevented ISO-induced myocyte apoptosis. E and F, on the other hand, verapamil (20 μm) and diltiazem (20 μm) had no effect on attenuating ISO-induced apoptosis. *, p < 0.001 versus CTRL and ascorbic acid alone. G, representative Western blots of phospho-PKA, PKAα, phospho-CaMKII, CaMKII, total RyR2, and PKA- and CaMKII-mediated RyR2 phosphorylation levels and calstabin in in vitro ARVMs cultured with ISO for 0, 5, 10, and 20 min in the absence or presence of JTV. Also, representative immunoprecipitation with RyR2 antibody and the Western blot for calstabin is shown.

FIGURE 5. Adult c-Kit^pos CSCs exhibit decreased and inverted β₁- and β₂-AR expression

A, fraction of apoptotic c-Kit^pos CSCs in the hearts of CTRL and ISO-treated (1 day) rats (p = not significant). B, c-Kit^pos CSCs sorted by immunomagnetic beads showed a purity of ∼90% when analyzed by flow cytometry. C and D, representative real time RT-PCR and Western blot results showing decreased mRNA transcripts and protein expression of β₁- and β₂-ARs in isolated c-Kit^pos CSCs compared with ARVMs. c-Kit^pos CSCs exhibited an inverted expression of β₁- and β₂-ARs. RT-PCR data are presented as the ratio between numbers of β₁- or β₂-ARs and GAPDH mRNA molecules per μg of RNA. *, p < 0.01 versus β₁-AR; ‡, p < 0.01 versus ARVMs. E, β₁- and β₂-ARs were also detected on isolated c-Kit^pos CSCs using immunocytochemistry. Left, β₁-ARs (green), c-Kit (red), and DAPI (blue). Right, β₂-ARs (green), c-Kit (red), and DAPI (blue). Merged images (yellow) are shown at the bottom. Insets, magnified images.

FIGURE 6. Adult c-Kit^pos CSCs are resistant to the damaging effects of ISO

A and B, representative immunostaining showing apoptotic CSCs (c-Kit (red), TdT (green), and DAPI (blue)) in CTRL (A) and ISO-treated (B) CSCs in vitro. C, isolated c-Kit^pos CSCs exposed to the same doses of ISO as ARVMs (Fig. 4C) were 2 orders of magnitude more resistant. Subsequently, only the highest dose of ISO (10 μm) caused significant CSC apoptosis. *, p < 0.01 versus CTRL and AA alone. D, the specific β₁-AR blocker, CGP20172A (0.3 μm), attenuated CSC apoptosis. E, in contrast, in the presence of a specific β₂-AR blocker (ICI 118551; 0.1 μm) CSC apoptosis increased with ISO dose. F and G, representative Western blot and quantitative real time RT-PCR results showing the absence of RyR2 in c-Kit^pos CSCs when compared with ARVMs. Data are presented as the ratio between numbers of RyR2 and GAPDH mRNA molecules per μg of RNA. *, p < 0.01 versus ARVMs.

FIGURE 7. Adult c-Kit^pos CSCs are activated in response to ISO-induced damage

A, c-Kit^pos CSC number was elevated in the myocardium after ISO administration. *, p < 0.05 versus CTRL (B). The percentage of c-Kit^pos CSCs positive for the cell cycle marker, Ki67, increased after ISO injection, peaking at 1 day. *, p < 0.05 versus CTRL. C, representative immunohistochemistry staining of c-Kit^pos/Ki67^pos CSCs in ISO-treated rat myocardium (c-Kit (green), Ki67 (white), α-sarcomeric actin (red), and DAPI (blue)). D, ISO exposure had no effect on CSC proliferation, measured by bromodeoxyuridine incorporation, in vitro (p = not significant).