Left Ventricular Myocardium Of Dogs Research Articles

Treatment of cardiomyocytes isolated from left ventricular myocardium of dogs with chronic heart failure with ALDA-1, an activator of aldehyde dehydrogenase-2, improves mitochondrial function

Abstract Background Reactive oxygen species (ROS) generated by dysfunctional mitochondria (MITO) of the failing heart plays an important role in lipid peroxidation of polyunsaturated fatty acids in cell and MITO membranes resulting in the production of aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). MITO aldehyde dehydrogenase-2 (mALDH2) is a key enzyme for the detoxification of endogenous aldehyde substrates through NAD dependent oxidation. ALDH2 catalyzes toxic 4HNE to nontoxic 4HNA in cardiac cells. We previously showed that mALDH2 expression and activity are markedly reduced in MITO of failing LV cardiomyocytes (CM), a maladaptation that can lead to considerable accumulation of 4-HNE in MITO and ultimately to progressive LV systolic and diastolic dysfunction. In this study, we tested the hypothesis that exposure of failing cardiomyocytes to ALDA-1, an activator of mALDH2, improves MITO function. Methods Freshly isolated CM from LV myocardium of dogs (n = 7) with heart failure (HF, LV ejection fraction ≤35%) and of normal (NL) dogs (n = 4) were incubated for 1 hour in absence (baseline) and presence of Alda-1 at concentration of 10 µM. After incubation, ADP-stimulated respiration (ADPresp) and maximal rate of ATP synthesis (ATPmax) were measured using Strathklein respirometer and bioluminescence ATP assay kit. Results Treatment of isolated CM from HF dogs with 10 µM Alda-1 significantly increased ADPresp (277 ± 18 vs. 189 ± 12 nAtom O/min/mg, p<0.05) and ATPmax (30 ± 3 vs. 16 ± 3 nmoles/min/mg, p<0.05) compared to untreated CM of HF dogs. Treatment of CM from NL dogs with 10 µM Alda-1 had no effect on ADPresp (488 ± 18 vs. 484 ± 19 nAtom O/min/mg) or ATPmax (54 ± 3 vs. 52 ± 2 nmoles/min/mg) compared to untreated NL CM. Conclusions Treatment of CM from HF dogs with Alda-1 improved overall MITO function. Activation of mALDH2 represents a potential viable therapeutic target for treating HF.

Abstract 278: Reduced HtrA2 Protein Levels in Mitochondria but Elevated Levels in Cytosol of Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: The high-temperature requirement serine peptidase 2 (HtrA2) is a pro-apoptotic mitochondrial serine protease involved in caspase-dependent as well as caspase-independent programmed cell death. When HtrA2 protein is tightly associated with mitochondria (MITO), its protease activity is masked. In contrast, once the protein is released from MITO into the cytosol (CS), its protease activity is exposed. Once active HtrA2 activates caspase-3, a pro-apoptotic enzyme, by degrading the caspase-3 inhibitor XIAP (x-linked inhibitor of apoptosis protein. Recent studies have shown that translocation of HtrA2 protein from the MITO to the CS occurs in mice hearts during experimental myocardial ischemic and reperfusion. Hypothesis: In this study we examined if translocation of HtrA2 from MITO to CS occurs in LV myocardium of dogs with chronic stable heart failure (HF). Methods: LV tissue from 7 dogs with chronic HF produced by multiple sequential intracoronary micro-embolizations (LV ejection fraction <35%) and 7 normal (NL) dogs was used to prepare crude homogenate (HG) and to isolate MITO and CS fractions. Using primary antibodies, HtrA2 protein was determined in HG, MITO and CS fractions by Western blotting coupled with chemiluminescence. GAPDH was used as an internal loading control for both HG and CS fractions. Porin was used as an internal loading control for MITO fractions. HtrA2 band intensity was normalized to GAPDH for HG and CS and to porin for MITO. Results: GAPDH and porin protein levels were unchanged between NL and HF dogs. Compared to NL, HtrA2 protein levels in dogs with HF were unchanged in HG (0.76 ± 0.04 vs. 0.73 ± 0.08), significantly reduced in MITO (0.17 ± 0.02 vs. 0.40 ± 0.05, p<0.05) and significantly increased in CS (0.90 ± 0.07 vs. 0.36 ± 0.05, p<0.05). Conclusions: Compared to NL dogs, HtrA2 protein levels are significantly reduced in MITO but increased in cytosol fractions from LV myocardium of dogs with chronic stable HF; a maladaptations that promotes cardiomyocyte apoptosis and progressive LV dysfunction. Inhibiting the translocation of HtrA2 from MITO to CS may have therapeutic potential for the preventing progressive dysfunction of the failing heart.

Abstract 538: Reduced Protein and mRNA Levels of Velosin Containing Protein in Left Ventricular Myocardium of Dogs With Advanced Heart Failure Are Restored Following Chronic Therapy With Elamipretide

Introduction: The failing heart manifests abnormalities of mitochondrial function characterized by reduced respiration, reduced membrane potential and opening of permeability transition pore (mPTP). Recent studies have shown that velosin containing protein (VCP), an ATPase associate protein, preserves mitochondrial respiration and prevents opening of the mPTP. Long-term therapy with elamipretide (ELAM), a novel mitochondria-targeting tetra-peptide, was previously shown improve mitochondrial respiration, membrane potential and prevent opening of the mPTP in dogs with intracoronary microembolization-induced heart failure (HF). Hypothesis: This study tested the hypothesis that mRNA and protein levels of VCP are decreased in left ventricular (LV) myocardium of dogs with HF and that chronic therapy with ELAM reverses this dysregulation. Methods: Studies were performed using LV tissue from 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELAM (HF+ELAM, 0.5 mg/kg once daily, n=7) or no therapy at all (control, HF-CON, n=7). LV tissue from 6 normal (NL) dogs was used for comparisons. Protein levels of VCP and GAPDH, an internal loading control, were determined in LV SDS-extracts using Western blotting coupled with chemiluminescence detection and band intensity was expressed in densitometric units (du). Using isolated RNA and specific primers, mRNA expression of VCP normalized to GAPDH. Results: mRNA and protein levels of GAPDH in LV tissue remained unchanged among the 3 study groups. Compared to NL, mRNA expression of VCP was reduced by 3.67 fold in HF-CON. Compared to NL, protein level of VCP was significantly reduced compared to HF-CON (0.30 ± 0.04 vs. 1.77 ± 0.31 du, p<0.05). In HF+ELAM treated dogs, mRNA expression of VCP was reduced by only 1.07 fold compared to HF-CON (p<0.05). Similarly, in HF+ELAM, protein levels of VCP were significantly higher than in HF-CON (1.03 ± 0.16 du, p<0.05). Conclusions: VCP mRNA and protein levels are significantly reduced in LV myocardium of dogs with chronic HF. Long-term therapy with ELAM reverses this dysregulation. The findings support the observations of improved mitochondrial respiration and reduced mPTP opening in dogs with advanced HF following chronic therapy with ELAM.

Elamipretide Normalizes Protein and mRNA Expression Levels of Mitofilin in Left Ventricular Myocardium of Dogs with Advanced Heart Failure

Mitofilin (MF) is a protein of the inner mitochondrial (MITO) membrane and has critical functions in MITO morphology and MITO fusion and fission, specifically in the formation of tubular cristae and cristae junctions. MF also regulates cytochrome c release during apoptosis. Down-regulation of MF results in increased apoptosis and disorganization of the MITO inner membrane; abnormalities that are also manifested in the heart failure (HF). We showed that MF levels are markedly reduced in LV myocardium of dogs with HF as well as in explanted failed human heart. Elamipretide (ELAM), a novel MITO-targeting peptide, has been shown to improve MITO function and morphology in animals with experimental HF. This study tested the hypothesis that chronic therapy with ELAM can reverse the dysregulation of MF in LV myocardium of dogs with coronary microembolization-induced HF (LV ejection fraction ~30%).

P6170Elamipretide normalizes protein and mRNA expression levels of mitofilin in left ventricular myocardium of dogs with advanced heart failure

P6170Elamipretide normalizes protein and mRNA expression levels of mitofilin in left ventricular myocardium of dogs with advanced heart failure

Long-Term Therapy with Elamipretide Reverses Abnormalities of Cardiolipin Synthesis and Remodeling in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Long-Term Therapy with Elamipretide Reverses Abnormalities of Cardiolipin Synthesis and Remodeling in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Abstract 12984: Long-term Therapy With Elamipretide Normalizes Protein Levels of the Signal Transducer and Activator of Transcription 3 (STAT3) in Mitochondria of Left Ventricular Myocardium of Dogs With Chronic Heart Failure

Introduction: The signal transducer and activator of transcription 3 (STAT3) has recently been identified in mitochondria (MITO) of cardiomyocytes (mSTAT3). In STAT3-/- cells, the activities of MITO complexes I and II of the electron transport chain (ETC) were significantly reduced suggesting that mSTAT3 is required for optimal ETC function. In the heart, mSTAT3 activity is modulated by the heat shock protein H11 kinase (H11K) previously shown to be reduced in MITO of dogs with HF. We showed that long-term (3 months) therapy with elamipretide (ELA, Bendavia TM ), a novel MITO-targeting peptide, improves LV systolic function and to normalize MITO respiration, rate of ATP synthesis and H11K levels in MITO of LV cardiomyocytes of dogs with heart failure (HF). Hypothesis: This study tested the hypothesis that the level of mSTAT3 is reduced in LV myocardium of dogs with HF and that long-term therapy with ELA restores levels of mSTAT3. Methods: LV tissue was obtained from 14 dogs with microembolization-induced HF (LV ejection fraction ~30%) randomized to 3 months therapy with subcutaneous injections of ELA (0.5 mg/kg once daily, n=7) or saline (Control, n=7). LV tissue from 6 normal dogs was used for comparison. Protein levels of mSTAT3 and porin; the latter a MITO protein unchanged in HF (internal control), were determined in isolated MITO fractions from LV tissue using by Western blotting coupled with chemiluminescence. Protein band intensities were quantified in densitometric units (du). Results: Protein levels of porin was similar in all 3 study groups (NL:0.24±0.01; Control: 0.22±0.01; ELA: 0.23±0.01 du). Levels of mSTAT3 were 0.82±0.05 du in NL, decreased to 0.29±0.03 du in Controls (p<0.05 vs. NL) and were partially restored by ELA (0.53±0.05, p<0.05 vs. Control). Levels of mSTAT3 normalized to porin were 3.6±0.3 du in NL, decreased to 1.3±0.1 in Controls (p<0.05 vs. NL) and were restored to near normal levels following treatment with ELA (2.3±0.2, p<0.05 vs. Control). Conclusions: mSTAT3 level is reduced in MITO of from LV myocardium of HF dogs and restored after chronic therapy with ELA. Normalization of mSTAT3 by ELA likely contributed to the observed improvement in MITO function following therapy with ELA in dogs with chronic HF.

Abstract 432: Elamipretide (BendaviaTM) Restores Aldehyde Dehydrogenase-2 Protein Levels and 4-Hydroxy-2-Nonenal Protein Adducts in Left Ventricular Myocardium of Dogs with Heart Failure

Background: Reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) are generated in the failing heart and contribute to cardiomyocyte injury and death and progressive left ventricular (LV) dysfunction. Aldehyde dehydrogenase-2 (ALDH2) plays a pivotal role in detoxifying mitochondrial (MITO) reactive aldehydes. We previously showed that long-term therapy with Elamipretide (ELA, Bendavia TM ), a novel MITO targeting peptide, improves LV systolic function and normalizes MITO function and rate of ATP synthesis in dogs with heart failure (HF). In this study, we examined the effects of long-term treatment with ELA on protein levels of 4-HNE adducts and ALDH2 in LV myocardium of dogs with coronary microembolization-induced HF (LV ejection fraction ~30%). Methods: LV tissue from 14 HF dogs randomized to 3 months monotherapy with subcutaneous injections of ELA (0.5 mg/kg once daily, n=7) or saline (control, CTR, n=7) and tissue from 6 normal (NL) dogs was used in the study. Using LV tissue extracts, 4-HNE protein adducts levels were quantified using a commercially available Elisa kit and expressed as ng/mg. In isolated MITO fractions prepared from LV tissue, protein levels of ALDH2 and porin, the latter used as an internal loading control, were determined by Western blotting coupled with Chemiluminescence. Band intensity was expressed in densitometric units (DU). Results: No changes in protein level of porin was observed among the 3 study groups (NL: 0.26±0.02; CTR: 0.24±0.01; BEN: 0.26±0.01 DU). 4-HNE protein adducts levels were increased in CTR dogs compared to NL dogs and treatment with ELA partially restored 4-HNE to near normal levels (NL: 185±21; CTR: 399±35*; ELA: 252 ± 18† ng/mg, *=p<0.05 vs. NL; †=p<0.05 vs. CTR). Compared to NL, dogs, ALDH2 protein levels were significantly reduced in HF CTR dogs (0.49±0.02 vs. 1.16±0.10; p<0.05). Treatment with ELA significantly increased ALDH2 protein compared to untreated CTR (0.74±0.02 DU, p<0.05). Conclusions: Long-term therapy with ELA normalizes ALDH2 and 4-HNE levels in LV myocardium of dogs with HF. Improved protein levels of ALDH2 in HF after therapy with ELA can account, in part, for the observed improvement of global LV function elicited by long-term therapy with ELA.

Chronic Therapy With Elamipretide (MTP-131), a Novel Mitochondria-Targeting Peptide, Improves Left Ventricular and Mitochondrial Function in Dogs With Advanced Heart Failure.

Elamipretide (MTP-131), a novel mitochondria-targeting peptide, was shown to reduce infarct size in animals with myocardial infarction and improve renal function in pigs with acute and chronic kidney injury. This study examined the effects of chronic therapy with elamipretide on left ventricular (LV) and mitochondrial function in dogs with heart failure (HF). Fourteen dogs with microembolization-induced HF were randomized to 3 months monotherapy with subcutaneous injections of elamipretide (0.5 mg/kg once daily, HF+ELA, n=7) or saline (control, HF-CON, n=7). LV ejection fraction, plasma n-terminal pro-brain natriuretic peptide, tumor necrosis factor-α, and C-reactive protein were measured before (pretreatment) and 3 months after initiating therapy (post-treatment). Mitochondrial respiration, membrane potential (Δψm), maximum rate of ATP synthesis, and ATP/ADP ratio were measured in isolated LV cardiomyocytes obtained at post-treatment. In HF-CON dogs, ejection fraction decreased at post-treatment compared with pretreatment (29 ± 1% versus 31 ± 2%), whereas in HF+ELA dogs, ejection fraction significantly increased at post-treatment compared with pretreatment (36 ± 2% versus 30 ± 2%; P<0.05). In HF-CON, n-terminal pro-brain natriuretic peptide increased by 88 ± 120 pg/mL during follow-up but decreased significantly by 774 ± 85 pg/mL in HF+ELA dogs (P<0.001). Treatment with elamipretide also normalized plasma tumor necrosis factor-α and C-reactive protein and restored mitochondrial state-3 respiration, Δψm, rate of ATP synthesis, and ATP/ADP ratio (ATP/ADP: 0.38 ± 0.04 HF-CON versus 1.16 ± 0.15 HF+ELA; P<0.001). Long-term therapy with elamipretide improves LV systolic function, normalizes plasma biomarkers, and reverses mitochondrial abnormalities in LV myocardium of dogs with advanced HF. The results support the development of elamipretide for the treatment of HF.

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, normalizes Complex-I Activity and reduces the NADH/NAD ratio in left ventricular myocardium of dogs with advanced heart failure

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, normalizes Complex-I Activity and reduces the NADH/NAD ratio in left ventricular myocardium of dogs with advanced heart failure

Abstract 13767: Thymosin β4 and its Cleavage Product Ac-SDKP Are Down-regulated in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: Thymosin β4 (Tβ4) is a 43 amino acid peptide and has been shown to promote angiogenesis, suppress pro-inflammatory cytokines and protect cardiomyocytes from apoptosis and oxidative stress injury. The Tβ4 cleavage product Ac-SDKP is a tetrapeptide (Acetyl-Ser-Asp-Lys-Pro) that has been shown to inhibit collagen production by fibroblasts and collagen deposition in the LV of rats with hypertension or myocardial infarction. In the setting of chronic heart failure (HF), LV dysfunction and chamber remodeling are associated with interstitial fibrosis, reduced capillary density, cardiomyocyte apoptosis and increased production of reactive oxygen species (ROS). Hypothesis: This study tested the hypothesis that protein levels of both Tβ4 and its cleavage product Ac-SDKP are down-regulated in LV myocardium of dogs with advanced chronic HF. Methods: LV tissue was obtained from the LV free wall of 6 normal dogs and 6 dogs with chronic HF (LV ejection fraction ~30%) produced by multiple sequential intracoronary microembolizations. Protein levels of Tβ4 and β-actin, as internal control, were determined by Western blotting and bands expressed in densitometric units (du). Levels of Ac-SDKP were determined by ELISA and expressed in ng/mg protein. Results: Protein level of β-actin did not change significantly between normal dogs (1.34 ± 0.19 du) and dogs with chronic HF (1.22 ± 0.18 du). Protein level of Tβ4 was significantly lower in LV myocardium of dogs with HF compared to normal dogs (0.36 ± 0.06 vs. 1.64 ± 0.13 du, p&lt;0.05). Similarly, levels of Ac-SDKP were significantly lower in LV myocardium of HF dogs compared to normal dogs (93 ± 9 vs. 155 ± 4 ng/mg protein, p&lt;0.05). Conclusions: Protein levels of Tβ4 and its cleavage tetrapeptide Ac-SDKP are markedly down-regulated in LV myocardium dogs with chronic HF. These findings are in-line with the reported increase of pro-inflammatory cytokines, interstitial fibrosis, cardiomyocyte apoptosis, and ROS formation as well as reduced capillary density in the failing LV myocardium of dogs with microembolization-induced HF as well as patients with HF. The findings support the therapeutic targeting of Tβ4 and Ac-SDKP as potential treatment for chronic HF.

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, normalizes functional mitochondrial abnormalities in left ventricular myocardium of dogs with heart failure

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, normalizes functional mitochondrial abnormalities in left ventricular myocardium of dogs with heart failure

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, reverses mitochondrial abnormalities in left ventricular myocardium of dogs with advanced heart failure

Background: Mitochondria (MITO) of failed human hearts and hearts of dogs with experimental heart failure manifest functional abnormalities characterized by reduced state-3 respiration, reduced membrane potential (Δψm) and increased opening of membrane permeability transition pores (mPTP). These abnormalities lead to reduced ATP synthesis that adversely impacts LV function. Bendavia (MTP-131), a novel, first in class, MITO-targeting peptide, has been shown to improve LV systolic function in dogs with chronic heart failure and to improve ATP synthesis in multiple organs including heart, kidney and skeletal muscle in other animal models of disease. The present study tested the hypothesis that long-term therapy with Bendavia reverses functional MITO abnormalities in dogs with chronic heart failure. Methods: Studies were performed in cardiomyocytes isolated from LV myocardium of 14 HF dogs produced by intracoronary microembolizations (LV ejection fraction ∼30%). Dogs were randomized to 3 months monotherapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). MITO state-3 respiration was measured using a Clark electrode. Δψm was measured using the dye JC-1 and mPTP was measured based on the rate of calcein exit from MITO. MITO ATP synthesis was measured using the bioluminescent ApoSENSORTM assay kit. Results: Compared to Control, Bendavia significantly improved MITO state-3 respiration, increased Δψm, decreased the rate of calcein exit from MITO thus decreasing mPTP opening and increased in ATP synthesis (Table). View this table: Indexes of mitochondria function Conclusions: Therapy with Bendavia reverses abnormalities of MITO function in LV myocardium of dogs with chronic heart failure and increases ATP synthesis. The latter can explain the observed improvement of LV systolic function following long-term therapy with Bendavia in dogs with HF.

Abstract 090: Long-term Therapy with a Bendavia (MTP-131), a Novel Mitochondria-Targeting Peptide, Normalizes Cardiolipin Biosynthesis and Remodeling in Left Ventricular Myocardium of Dogs with Advanced Heart Failure

Background: Cardiolipin (CL) is a signature phospholipid of the mitochondrial inner membrane and responsible for modulation of activities of various enzymes essential for oxidative phosphorylation. Cardiac CL is biosynthesized in a series of steps from phosphatidic acid and remodeled into a form which contains four 18:2 fatty acid chains, tetralinoleol CL [(18:2) 4 CL]. Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, was previously shown to improve left ventricular (LV) function, increase ATP synthesis, and increase the activity and protein levels of mitochondria cytochrome c oxidase (complex IV) in LV myocardium of dogs with advanced heart failure (HF). This study examined the effects of Bendavia on total CL and (18:2) 4 CL in LV myocardium of dogs with HF. Methods: LV tissue was obtained from 12 dogs with microembolization-induced HF randomized to 3 months therapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). LV tissue from 7 normal dogs was used for comparison. Total CL and (18:2) 4 CL were measured using electrospray ionization mass spectroscopy and quantified in nmol/mg of non-collagen protein (NCP). Results: Total CL was significantly decreased in LV myocardium of Control dogs compared to normal dogs (19.1±1.1 vs. 26.7±1.4 nmol/mg, p&lt;0.05) as was (18:2) 4 CL (14.2±0.9 vs. 20.5±1.2 nmol/mg, p&lt;0.05). Long-term therapy with Bendavia significantly increased both total CL (23.6±1.1 nmol/mg) and (18:2) 4 CL (17.8±1.0 nmol/mg) to near normal levels in LV myocardium of treated HF dogs compared to untreated HF Controls (p&lt;0.05). Conclusions: Total CL and (18:2) 4 CL are decreased in LV myocardium of dogs with HF. Treatment with Bendavia normalizes total CL and (18:2) 4 CL. These findings are consistent with observed normalization of mitochondrial complex IV activity, normalization of rate of ATP synthesis and improvement of global LV performance in dogs with HF after long-term therapy with Bendavia.

Abstract 309: Long-Term Therapy with Ivabradine Increases ACE-2 Activity in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: Angiotensin converting enzyme 2 (ACE2) is a homolog of ACE. ACE2 removes the terminal amino-acid from angiotensin-II (Ang II) to form Ang(1-7) which antagonizes the effects of Ang-II by activating the Mas receptor. We showed that ivabradine (IVA), a novel inhibitor of the cardiac pacemaker current I f , improves left ventricular (LV) function and prevents progressive LV remodeling in dogs with microembolization-induced heart failure (HF). Because RAAS system overactivity contributes to the progression of HF, we assessed changes in Ang-II level and ACE and ACE2 activity in LV of dogs treated with IVA. Methods: Studies were performed in LV tissue from 24 HF dogs randomized to 3 months therapy with high dose (HD) IVA (30 mg twice daily, n=8), low dose (LD) IVA (15 mg twice daily, n=8) or to no therapy at all (Control, CON, n=8). LV tissue from 6 normal (NL) dogs was used for comparison. A fluorogenic peptide, captopril (ACE inhibitor) and DX600 (ACE2 inhibitor) were used to quantify the activity of ACE and ACE2 in LV homogenate. Activity was expressed as fluorescence unit (FU)/μ g protein/hour. Ang II level was determined by ELISA. Results: IVA reduced heart rate (HR) and significantly increased LV ejection fraction in a dose-dependent manner. Ang II level and ACE and ACE2 activities were up-regulated in HF CON compared to NL dogs. Compared to CON, treatment with IVA reduced ACE activity and Ang II level but increased ACE2 activity in a dose-dependent manner (Table). Conclusions: In dogs with HF, therapy with IVA reduces activation of ACE and Ang II level but increases activation of ACE2. The latter may contribute to the observed beneficial effects of IVA on LV function and remodeling in HF.

Abstract 281: Long-Term Therapy with a Partial Adenosine A1-Receptor Agonist Upregulates mRNA and Protein Expression of Glucose Transporters in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: Recruitment of glucose transport proteins GLUT-1 and -4 is a mechanism by which the heart increases glucose transport for metabolism in response to increased energy demands. In the failing LV, mRNA and protein levels of GLUT-1 and -4 are down-regulated and the response to increased demands is met by increased dependence on fatty acid oxidation. We previously showed that treatment with the partial adenosine A1-receptor agonist capadenoson (CAP) improves LV systolic function in dogs with advanced heart failure (HF). This study examined the effects of CAP on the regulation of GLUT-1 and -4 in LV myocardium of dogs with chronic HF. Methods: Studies were performed in LV tissue from 12 HF dogs randomized to 3 months oral therapy with the CAP (7.5 mg twice daily, n=6) or to no therapy at all (Control, n=6). LV tissue from 6 normal (NL) dogs was used for comparison. mRNA expression of GLUT-1 and -4 was measured in LV tissue homogenate using real time polymerase chain reaction (RT-PCR). Protein levels of GLUT-1 and -4 and calsequestrin (CSQ), a sarcoplasmic reticulum protein that does not change in HF (internal control), were measured in SDS-extract using Western blotting coupled with chemiluminescent method. Band intensity was quantified in densitometric units (du). Results: Compared to NL dogs, Control HF dogs showed a 3.6 fold decrease in mRNA expression of GLUT-1 and a 2.7 fold decrease in the expression of GLUT-4. CAP-treatment in HF dogs improved mRNA expression of GLUT-1 (1.38 fold decrease) and GLUT-4 (0.8 fold decrease). Similarly, compared to NL dogs, Control HF dogs showed a significant decrease of protein levels of GLUT-1 (0.64 ± 0.04 vs. 0.26 ± 0.04 du, p&lt;0.05) and GLUT-4 (1.42 ± 0.06 vs. 0.40 ± 0.05 du, p&lt;0.05). CAP treatment significantly increased protein levels of both GLUT-1 (0.49 ± 0.03 du) and GLUT-4 (0.82 ± 0.03) compared to Control HF dogs (p&lt;0.05). CSQ levels were similar in all study groups. Conclusions: In dogs with chronic HF, long-term therapy with CAP improves mRNA and protein expression of GLUT-1 and -4. This improvement in expression of glucose transport proteins is likely to restore partial dependency of the failing heart to more efficient glucose metabolism, and therefore, lead to improved myocardial energetics and pump function.

Cardiac contractility modulation electrical signals normalize expression of the excitation-contraction coupling proteins in left ventricular myocardium of dogs with heart failure

Cardiac contractility modulation electrical signals normalize expression of the excitation-contraction coupling proteins in left ventricular myocardium of dogs with heart failure

Effects of Chronic Therapy with Cardiac Contractility Modulation Electrical Signals on Cytoskeletal Proteins and Matrix Metalloproteinases in Dogs with Heart Failure

Objectives: Therapy with cardiac contractility modulation (CCM) electrical signals delivered to left ventricular (LV) muscle during the absolute refractory period improves LV systolic and diastolic function in dogs with heart failure (HF). This study examined the effects of CCM therapy on mRNA and protein expression of cytoskeletal proteins, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in the LV myocardium of dogs with HF. Methods: HF was produced in 14 dogs by coronary microembolizations. Dogs were randomized to 3 months of CCM therapy (n = 7) or to sham-operated controls (n = 7). LV tissue from 6 normal (NL) dogs was used for comparison. mRNA expression was measured using reverse-transcriptase polymerase chain reaction and protein expression using Western blots. Results: Compared with NL dogs, controls showed upregulation of mRNA and protein expression of the cytoskeletal proteins tubulin and fibronectin and MMP-1, MMP-2 and MMP-9, and downregulation of the cytoskeletal protein titin. Normalized expression of all these genes and proteins was seen after CCM therapy. No differences in expression of TIMP-1 and TIMP-2 were observed among groups. Conclusions: CCM therapy normalizes expression of key cytoskeletal proteins and MMPs and may partly explain the improvement in LV function seen in HF following CCM therapy.

Abstract 3295: De-induction of the Maladaptive Fetal Gene Program During Chronic Monotherapy with Metoprolol Succinate is Retained Following Withdrawal of Therapy in Dogs with Chronic Heart Failure

Background: Chronic therapy with extended release metoprolol succinate (MET), a selective β1 adrenergic receptor blocker, improves left ventricular (LV) function and attenuates global LV remodeling in dogs with chronic heart failure (HF). We previously showed that chronic therapy with β-blockade results in de-induction of the fetal gene program (FGP) in LV myocardium of dogs with HF. In this study, we tested the hypothesis that in dogs with HF withdrawal of chronic MET does not lead to re-induction of FGP. Methods: Studies were performed in 17 intracoronary microembolization-induced HF dogs randomized to 3 months oral therapy with MET (100 mg, once daily, n=11) or no therapy at all (Controls, n=6). In dogs randomized to MET, 6 were sacrificed after 3 months of therapy and in the remaining 5, MET was withdrawn after 3 months of therapy and dogs were observed for 6 weeks after withdrawal of MET (MET-W) and then sacrificed. LV tissue was also obtained from 6 normal (NL) dogs for comparison. mRNA expression of the FGP genes namely, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), sarcoplasmic reticulum calcium ATPase (CAA), cardiac β-1 adren-ergic receptor (AR) and α-myosin heavy chain isoform (α-MHC) was measured using reverse transcriptase polymerase chain reaction (RT-PCR) and bands were quantified in densitometric units (du). Results: In Controls, mRNA expression of ANP and BNP increased and expression of CAA, β 1-AR and α-MHC decreased. Treatment with MET decreased expression of ANP and BNP and increased expression of CAA, β 1-AR and α-MHC. Except for α-MHC, the improvement in FGP seen during MET treatment was preserved in MET-W dogs. Conclusions: Withdrawal of MET is associated with sustained de-induction of the FGP in LV myocardium of dogs with HF. This observation supports the concept that chronic β-blockade therapy in HF confers lasting reversal of LV remodeling and molecular recovery of the failing myocardium.

Cardiac Contractility Modulation Electrical Signals Normalize Expression of the Excitation-Contraction Coupling Proteins Presenillin-2 and Sorcin in Left Ventricular Myocardium of Dogs with Chronic Heart Failure

Background: In the normal heart, during membrane depolarization, presenillin-2 (PS2) activates the sarcoplasmic reticulum (SR) ryanodine Ca2+ release channel (RyR2) to release large amounts of Ca2+ from the SR. At the end of systole (high Ca2+), sorcin (SOR) interacts with RyR2 to stop the SR Ca2+ release. In heart failure (HF), the RyR2 is “leaky” causing continuous release of Ca2+ into the cytosol and leading to Ca2+ overload as well as to depletion of calcium from the SR a maladaptation deemed detrimental in HF.