Animal preparation.

Adult male Sprague-Dawley rats (200–250 g) at 12 wk of age were subjected to either sham or ACF surgery in our laboratory as previously described (9, 21, 45, 50, 54, 56). Briefly, rats were anesthetized under 2.5% isoflurane, and, under sterile conditions, an incision was made into the abdominal cavity whereby the descending aorta and vena cava were revealed. Blood flow was restricted through sufficient pressure to the main vessels in this region. A needle was first inserted into the aorta, and, while still inside the aorta, it was then pierced through into the vena cava. The needle was then retracted while the external hole from the initial aorta puncture was sealed with sterile gel adhesive. With the hole created between the major vessels, the pressure of aortic blood caused back flow into the vena cava leaving the fistula open, resulting in VO in the heart due to excess venous return. In control animals, a sham surgery procedure was conducted whereby all aspects of the surgery were performed except for the needle puncture to form the fistula. Time points of 24 h, 4 wk, and 12 wk were selected based on the development of heart failure from acute, compensated, and decompensated stages (9, 22). Rats that underwent sham or ACF surgery were studied at 4 or 12 wk after surgery using echocardiography before being euthanized, and whole LV tissue was used for protein analysis, TEM, and immunohistochemistry. Rats at 24 h or 4 or 12 wk after surgery were euthanized, and whole hearts used were to isolate LV cardiomyocytes. The animal use in these experiments was approved by the University of Alabama at Birmingham Animal Resource Program (protocol 130409070).

Echocardiography.

Echocardiography was performed before the animals were euthanized using the Vivo 2100 imaging system (Visualsonics, Toronto, ON, Canada) as previously described by our laboratory (22, 45, 54, 56). LV volume was calculated from traced M-mode LV dimensions using the following Teicholz formula: V = [7/(2.4 + LVID)] × [LVID]³, where V is volume and LVID is LV internal dimension.

Protein extraction from frozen tissue for desmin analysis.

For direct protein analysis, rats that underwent ACF surgery were dissected at 24 h, 4 wk, and 12 wk, and LV tissue was snap frozen in liquid nitrogen. Frozen tissue was pulverized using a liquid nitrogen-cooled mixer mill MM 400 (Retsch) and 0.5-mm cooled beads (2 × 1 min 30 s at 25 Hz). The resulting powder was resuspended in 25 mM HEPES buffer to separate myofilament- and cytosolic-enriched fractions as previously described (1).

Isolation of LV cardiomyocytes.

Cardiomyocytes were isolated from rats in both surgical groups as previously described by our laboratory (22, 45, 56). Briefly, hearts were perfused with perfusion buffer (120 mmol/l NaCl, 15 mmol/l KCl, 0.5 mmol/l KH₂PO₄, 5 mmol/l NaHCO₃, 10 mmol/l HEPES, and 5 mmol/l glucose at pH 7.0) for 5 min and digested with perfusion buffer containing 1 mg/ml collagenase type II (Invitrogen, Carlsbad, CA) for 30 min at 37°C. The right ventricle, atria, and apex were removed before the perfused heart was minced. The digestion was filtered and washed, and cells were pelleted. Only samples with >80% viability (indicated by healthy rod-shaped cells) were used.

TEM of rat tissue.

Tissue was fixed in 2.5% glutaraldehyde-Sorensen’s phosphate buffer (no. 15980, Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C as previously described by our laboratory (22, 50, 56). After postfixation with 1% osmium tetroxide in 0.1 M cacodylate buffer, tissue was dehydrated in a graded series of ethanol and embedded in Epon resin. Semithin (0.5 μm) and ultrathin (90 nm) sections were cut, mounted on copper grids, and poststained with uranyl acetate and lead citrate. Sections were viewed with a Philips 201 transmission electron microscope (FEI, Hillsboro, OR) for qualitative changes in ultrastructure by EMLabs (Birmingham, AL).

Immunohistochemistry.

Rat hearts were immersion fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (5 μm) were mounted on slides, deparaffinized in xylene, and rehydrated in a gradient series of ethanol. Sections used for fluorescent imaging were blocked with 5% goat serum and 1% bovine serum in PBS and incubated overnight at 4°C with either desmin antibody (1:100, ab6322, Abcam, Cambridge, UK) or voltage-dependent anion channel (VDAC; 1:100, ab15895, Abcam). Alexa fluor-conjugated secondary antibodies (1:500 each, Molecular Probes, Eugene, OR) were applied to visualize desmin (green) and VDAC (red) in the tissue. Nuclei (blue) were stained with DAPI (1.5 μg/ml, Vector Laboratories, Burlingame, CA). Image acquisition (×100 objective, ×4,000 video-screen magnification) was performed on a Leica DM6000 epifluorescence microscope with Simple-PCI software (Compix, Cranberry Township, PA). Images were adjusted appropriately to remove background fluorescence.

TUNEL assay.

A TUNEL assay was performed on paraffin-embedded sections using the DeadEnd fluorometric TUNEL kit (Promega, Madison, WI) as previously described by our laboratory (9).

Western blot analysis.

M-PER Mammalian Protein Extraction Reagent with Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail (all from Life Technologies, Grand Island, NY) at 1:100 concentrations were used for our sample preparation. Cell lysates prepared from isolated cardiomyocytes (30–50 μg protein) were separated on a 4–12% bis-Tris gradient gel (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membranes, which were incubated overnight at 4°C with the following antibodies: light chain (LC)3A/B (1:1,000, catalog no. 2741), p62/SQSTM1 (1:1,000, catalog no. 5114), beclin-1 (1:1,000, catalog no.3495), dynamin-related protein 1 (DRP1; 1:1,000, catalog no. 8570), phospho-DRP1 (Ser⁶¹⁶, 1:1,000, catalog no. 3455), PTEN-induced putative kinase 1 (PINK1; 1:1,000, catalog no. 6946), parkin (1:1,000, catalog no. 4211), heat shock protein (HSP)60 (1:1,000, catalog no. 12165), Bad (1:1,000, catalog no. 9239), phospho-Bad (Ser¹⁵⁵, 1:1,000, catalog no. 9297), Bcl-x_L (1:1,000, catalog no. 2764), Akt (1:1,000, catalog no. 4691), and phospho-Akt (Ser⁴⁷³, 1:1,000, catalog no. 4060) (all from Cell Signaling Technology, Danvers, MA) as well as cyclophilin F (also known as CypD, 1:1,000, ab110324, Abcam) and GAPDH (1:1,000, GT239, GeneTex, Irvine, CA).

Membranes were then washed and incubated for 1 h with the appropriate Amersham enhanced chemiluminescence horseradish peroxidase-linked secondary antibody (GE Healthcare Bio-Sciences, Pittsburgh, PA). Membranes were saturated with chemiluminescent substrate (Pierce, Rockford, IL), and images were taken using a charge-coupled device camera and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD). For desmin Western blot analysis on tissue, myofilament-enriched fractions were assayed for protein concentration using BCA (Pierce) diluted in lithium dodecyl sulfate buffer (Life Technologies) completed with 1% DTT and boiled at 95°C for 10 min. The resulting denatured samples were loaded onto 4–12% NuPAGE precast gels (Life Technologies), separated, and blotted on PVDF (Millipore) as previously described (1). Immunostaining using anti-desmin antibody (1:10,000 in Tris-buffered solution with 5% milk, DE-U-10; Sigma) was detected using a secondary red goat anti-mouse antibody (1:10,000, Li-Cor). After detection, membranes were stripped for 15 min (Restore, Pierce) and stained with Direct Blue 71 (Sigma) to confirm equal loading as previously described (1). For densitometry, the signal intensity of intact desmin was normalized to the average of sham-operated control animals at 24 h (equal to 100) to account for gel-to-gel variability. The signal intensity of the desmin fragment was expressed as a percentage of intact desmin within each lane, thus expressing the relative contribution of cleavage.

Mass spectrometry.

For mass spectrometry (MS) analysis, tissue protein extracts were separated on precast SDS-PAGE gels (bis-Tris 4–12%, Life Technologies) using MES buffer (Life Technologies). After separation, the gel slab was fixed overnight in 10% acetic acid-40% methanol (Fisher Scientific) followed by staining with blue silver staining (7). The electronic images of the desmin blot and the resulting Coomassie-stained gel were aligned using the molecular weight markers (SeeBlue; Life Technologies), and the corresponding bands were hand-picked using a clean scalpel and forceps. The excised bands were reduced to 1-mm³ cubes and subjected to in-gel digestion as previously described (1). Extracted peptides were analyzed using an Orbitrap Q Exactive plus MS (Thermo Scientific) with a 90-min gradient and 70K-35K resolution. The resulting spectra were searched against the Rattus norvegicus RefSeq2015 database with fixed carbamidomethyl, variable oxidation, deamidation, and phosphorylation with no enzyme (de novo sequencing) using Peaks (BSI).

Kinome analysis.

M-PER Mammalian Protein Extraction Reagent with Halt Phosphatase Inhibitor Cocktail and Halt Protease Inhibitor Cocktail (all from Life Technologies) at 1:100 concentrations were used for our sample preparation. Kinome analysis was performed at the University of Alabama Birmingham Kinome Core (Birmingham, AL). Before samples were loaded onto the four-well tyrosine (PTK) chips or four-well serine/threonine (STK) chips, lysates were quantified using a standard BCA assay. Lysates were loaded at 15 μg/well (PTK) or 2 μg/well (STK) onto the PamChips after preparation. The in vitro kinase assay was performed using PamGene Evolve software on the PamStation12. Images were then imported to BioNavigator for raw data analysis. As applicable upstream kinase prediction was performed with data from Kinexus Phosphonet, and pathway analysis was performed with GeneGo MetaCore.

Statistical analysis.

Values are presented as means ± SE. An unpaired t-test was used to compare age-matched rats in the sham-operated and ACF groups at each time point of 24 h, 4 wk, and 12 wk for Western blots, and a Mann-Whitney U-test using SPSS 24 software was used for echocardiography measures. P values of <0.05 were considered statistically significant.

Echocardiography.

A dimensional and volumetric echocardiographic analysis was performed to determine the extent of LV remodeling in response to the pure VO of ACF. As shown in Table 1, there were subtle increases in LV end-diastolic dimension and LV end-diastolic volume at 24 h that resulted in a significant decrease in the LV end-diastolic mass-to-volume ratio. As expected, there was an increase in LVEF and LV fractional shortening at this early stage of a pure stretch and ejection into the low-pressure fistula. At 4 and 12 wk of ACF, there was a significant increase in LV end-diastolic dimension (40% and 38%, respectively, P < 0.05) compared with age-matched sham-operated rats. However, there was a concomitant 140% and 130% increase in LV end-diastolic volume (P < 0.05). This reflects the change to a more spherical LV geometry manifested by a decrease in sphericity index at both 4 and 12 wk of ACF compared with age-matched sham-operated animals (1.75 ± 0.04 vs. 2.03 ± 0.07, P < 0.01, and 1.71 ± 0.02 vs. 1.87 ± 0.05, P < 0.05, respectively). Consistent with an eccentric and spherical remodeling pattern, there was a 48% and 56% increase in LV mass compared with the 2.5-fold increase in LV end-diastolic volume (both P < 0.05) and a decrease in the relative LV wall thickness at end diastole at 4 and 12 wk of ACF (P < 0.05). Indexes of systolic function, including LV fractional shortening, LVEF, and velocity of circumferential fractional shortening adjusted for heart rate (VCF_r), did not differ from age-matched sham-operated animals at 4 wk of ACF; however, all were decreased significantly compared with age-matched sham-operated animals at 12 wk of ACF (P < 0.05). These echocardiographic indexes demonstrate that an adverse LV spherical eccentric remodeling at 4 and 12 wk of ACF ultimately results in a decrease in LV shortening indexes at 12 wk.

Desmin and mitochondrial loss of organization and function with ACF.

We recently reported a loss of organization in the cardiomyocyte cytoskeletal-mitochondrial architecture in rats with pure VO and in rats with ACF (56) and in humans with isolated MR (4). In the present study, we now demonstrate that desmin breakdown and mitochondrial disorganization occur as early as 24 h after ACF induction and persist at 4 and 12 wk of ACF (Fig. 1). In the normal LV, immmunofluorescent staining for desmin demonstrated a normal striated registry at the Z disks and more intense staining at the intercalated disks with a linear pattern of VDAC staining between sarcomeres. In hearts with ACF, there was near complete loss of desmin staining in some cardiomyocytes with intense VDAC staining that was consistent with the mitochondrial clustering in TEM images that characterize ACF sections (Fig. 2). The normal interfibrillar position of mitochondria closely straddling the A band of the sarcomere and its proximity to the myofibrillar structure were found in age-matched sham-operated and ACF rats, as shown in Fig. 2. In rats with ACF, at all time points, there was mitochondrial clustering with numerous small mitochondria that disrupted the normal highly organized linear registry of mitochondria with the sarcomere between the Z disks (Fig. 2). This disorganization was accompanied by the loss of the I bands and blurring of the Z disk. The mitochondrial disorganization at 24 h and 4 wk became more severe at 12 wk in animals with ACF with mitochondrial matrix swelling and cristae fragmentation, as demonstrated in the TEM images from two different rats with ACF shown in Fig. 3. It is of interest that these changes in mitochondrial disorganization and cristae loss along with myofibrillar breakdown were similar to those described in des/des^−/− mice and in R120G-αβ-crystallin transgenic mice in very early stages before their early demise (37, 51, 52). These mitochondrial abnormalities were also consistent with our previous studies of ACF at 8 wk, in which there was a dissipation of mitochondrial membrane potential (ΔΨ_m), which was restored by the mitochondrial antioxidant MitoQ (56).

Desmin cleavage and ACF and translocation of αB-crystallin.

Due to the profound changes observed in the desmin cytoskeleton, we investigated the idea that desmin cleavage could represent an early sign of adverse remodeling as manifested by the decrease in the LV end-diastolic volume mass-to-volume ratio. We measured the desmin cleavage product in ACF as early as 24 h after surgery using Western blot analysis (Fig. 4). Desmin and a 49-kDa band were separated using conventional SDS-PAGE. The 49-kDa fragment was approximately twofold increased at 24 h (P = 0.016) versus age-matched sham-operated animals, whereas intact desmin was only 10% increased (P = 0.04). At 4 wk of ACF, the 49-kDa fragment was increased approximately threefold (P = 0.021) with respect to age-matched sham-operated animals, whereas intact desmin was elevated by only 19% (P = 0.036). At 12 wk of ACF, there was no difference in intact desmin, whereas the 49-kDa fragment was increased twofold (P = 0.055). A second desmin fragment of ~25 kDa was also detected, but it did not display any significant difference at the three time points in animals with ACF versus sham-operated animals (Fig. 5, top).

To rule out the possibility of nonspecific staining with the desmin antibody, we excised the corresponding bands from Coomassie-stained gels and subjected them to analysis by MS. The presence of desmin in all bands detected by the antibody was confirmed by de novo sequencing. Interestingly, the NH₂-terminus of desmin, which is important for its assembly, was missing in both fragments (Fig. 5, bottom right).

Desmin’s chaperone protein, αB-crystallin, moved from its normal location at the Z disk (6) to the perinuclear area in rats with ACF at 4 and 12 wk. αB-crystallin followed the normal striated pattern along the Z disk in age-matched sham-operated control animals at 4 and 12 wk. In contrast, this regular distribution was replaced by αB-crystallin accumulation in the perinuclear area in ACF at both 4 and 12 wk (Fig. 6). These findings are consistent with what was also reported in patients with isolated MR and preserved LVEF (4).

Oxidative stress in cardiomyocytes.

One of the major causes of desmin disruption and mitochondrial disarray is excessive oxidative stress. We have previously demonstrated increased oxidative stress in isolated cardiomyocytes with ACF at 24 h (50) and 8 wk (56). Here, we studied the distribution of hydroxynonenal (HNE; Fig. 7, red) (4), which is a strong indicator of lipid peroxidation. As we have previously reported in people with isolated MR (4), there was a marked increase in HNE intensity in cardiomyocytes with ACF at both the 4- and 12-wk time points compared with age-matched sham-operated control animals. HNE staining was especially prominent in the perinuclear region, which coincides with damaged mitochondria in the TEM images shown in Fig. 2 and VDAC staining shown in Fig. 1 in hearts with ACF. The colocalization of HNE and VDAC is consistent with lipid peroxidation of the membrane-rich mitochondria in the cardiomyocyte.

Autophagy and apoptosis.

The marked loss of organization of desmin cytoskeleton and mitochondria as visualized with TEM has been associated with an arrest of autophagy in the des/des^−/− mouse. In the cardiomyocytes with ACF at 4 and 12 wk that we studied, there was a significant increase in several autophagy protein markers, including microtubule-associated protein 1A/1B-LC3 (LC3-II/LC3-I ratio), p62/SQSTM1, and beclin-1 (Fig. 8A). In addition, LC3-II/GAPDH was increased 2.24- and 1.98-fold compared with age-matched sham-operated rats and those with ACF at both 4 and 12 wk, respectively.

Markers of mitophagy were also increased with ACF at 4 and 12 wk, including total DRP1 and Ser⁶¹⁶-phosphorylated phospho-DRP1, PINK1, parkin, HSP60, and CypD (Fig. 8B). DRP1 affects mitochondrial morphology and is important in mitochondrial fission in mammalian cells. Specifically, DRP1 phosphorylation at Ser⁶¹⁶ promotes mitochondrial fission (17), which has been reported to precede mitophagy and make mitochondria more suitable for engulfment by the autophagic machinery (39). However, this increase in mitophagy protein markers was countered by a significant sixfold increase in p62/SQSTM1 (Fig. 8A), which accumulates when autophagy is inhibited and decreases when autophagy is induced (30). Increased mitophagy markers may be affected by differences in total mitochondrial abundance, but this was ruled out by measured citrate synthase activity, which was not significantly different between sham-operated control animals and those with ACF at 12 wk (data not shown). Nevertheless, these are static markers of mitophagy in the face of extensive desmin/mitochondrial damage. Thus, it is possible that autophagy/mitophagy is activated and thereby increases the autophagic/mitophagic flux, but this increase may still not be adequate to remove the damaged mitochondria in a timely fashion at these stages of ACF VO. In addition, apoptosis was not induced by ACF at either 4 or 12 wk, as indicated both by increased protein levels of the antiapoptotic markers including total Akt, phospho-Akt (Ser⁴⁷³), total Bad, phospho-Bad (Ser¹⁵⁵), and Bcl-x_L (Fig. 9) and by TUNEL staining (Fig. 10).

Kinome analysis.

To further interrogate and verify the mechanistic underpinnings of this antiapoptotic phenotype of the ACF cardiomyocyte by Western blot analysis, we performed a kinome analysis of LV cardiomyoctyes isolated from the LVs of rats with ACF at 4 and 12 wk and age-matched sham-operated rats. Kinomic profiling is a functional proteomic approach that can help elucidate the central intracellular signaling pathways driving particular biological processes or phenotypes (Tables 2 and 3) (16, 29).

The tyrosine kinome at 4 wk of ACF did not show any significant differences from that of control animals at 4 wk. The serine/threonine kinome identified activation of MSK1 (90-kDa ribosomal protein S6 kinase 5) involved in the stress-activated MAPK cascade in response to cellular stress and inflammation; MAPK-activated protein kinases 2 and 3 (MAPKAPK2 and MAPKAPK3, respectively) involved in cell growth, tissue-specific gene expression, and cell differentiation in response to cellular stress; 70-kDa ribosomal protein S6 kinase 2 (p70S6Kb) involved in translational initiation and cell growth; and Ca²⁺/calmodulin-dependent protein kinase IV (CaMK4) involved in transcriptional regulation and mitochondrial organization and biogenesis. There was an induction of PIM1, PIM2, and PIM3 protooncogene serine/threonine-protein kinases involved in cell survival and proliferation and negative regulation of apoptosis by inhibition of proapoptotic proteins.

The tyrosine kinome at 12 wk of ACF had activation of AXL, an antiapoptotic receptor tyrosine kinase involved in cell growth (5), and the serine/threonine kinome had sustained activation of PIM1 and PIM3 as well as PKB (Akt1 and Akt2), which play a critical role in regulating cell survival and metabolism via many different signaling pathways (20). The serine/threonine kinome also showed activation of PKG (PKG1 and PKG2), which is involved in actin cytoskeleton organization and biogenesis, and PKA (PKACa and PKACb), which is involved in actin dynamics, microtubule dynamics, and inhibition of apoptosis. With regard to potential posttranslation modifications of desmin (28, 49, 55), the serine/threonine kinome demonstrated activation of PKG1, PKG2, PKACa, and PKACb (Tables 2 and 3).