Actin dynamics is rapidly regulated by the PTEN and PIP2 signaling pathways leading to myocyte hypertrophy.

Mature cardiac myocytes are terminally differentiated, and the heart has limited capacity to replace lost myocytes. Thus adaptation of myocyte size plays an important role in the determination of cardiac function. The hypothesis tested is that regulation of the dynamic exchange of actin leads to cardiac hypertrophy. ANG II was used as a hypertrophic stimulant in mouse heart and neonatal rat ventricular myocytes (NRVMs) in culture for assessment of a mechanism for regulation of actin dynamics by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin dynamics in NRVMs rapidly increased in a PIP2-dependent manner, measured by imaging and fluorescence recovery after photobleaching (FRAP). A significant increase in PIP2 levels was found by immunoblotting in both adult mouse heart tissue and cultured NRVMs. Inhibition of phosphatase and tensin homolog (PTEN) in NRVMs markedly blunted ANG II-induced increases in actin dynamics, the PIP2 level, and cell size. Furthermore, PTEN activity was dramatically upregulated in ANG II-treated NRVMs but downregulated when PTEN inhibitors were used. The time course of the rise in the PIP2 level was inversely related to the fall in the PIP3 level, which was significant by 30 min in ANG II-treated NRVMs. However, significant translocation of PTEN to the plasma membrane occurred by 10 min, suggesting a crucial initial step for PTEN for the cellular responses to ANG II. In conclusion, PTEN and PIP2 signaling may play an important role in myocyte hypertrophy by the regulation of actin filament dynamics, which is induced by ANG II stimulation.

Long-term pharmacological activation of PPARgamma does not prevent left ventricular remodeling in dogs with advanced heart failure.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) activators affect the myocardium through inhibition of inflammatory cytokines and metabolic modulation but their effect in the progression of heart failure is unclear. In the present study, we examined the effects of the PPARgamma activator, GW347845 (GW), on the progression of heart failure. METHODS AND RESULTS: Heart failure was produced in 21 dogs by intracoronary microembolizations to LV ejection fraction (EF) less than 30% and randomized to 3 months of therapy with high-dose GW (10 mg/Kg daily, n = 7), low-dose GW (3 mg/Kg daily, n = 7), or no therapy (control, n = 7). In control dogs, EF significantly decreased (28 +/- 1 vs. 22 +/- 1%, p < 0.001) and end-diastolic volume (EDV) and end-systolic volume (ESV) increased during the 3 months of the follow-up period (64 +/- 4 vs. 76 +/- 5; p = 0.003, 46 +/- 3 vs. 59 +/- 4 ml, p = 0.002, respectively). In dogs treated with low-dose GW, EDV increased significantly (69 +/- 4 vs.81 +/- 5 ml, p = 0.01), whereas ESV remained statistically unchanged (50 +/- 3 vs. 54 +/- 3 ml, p = 0.10) resulting in modestly increased ejection fraction (27 +/- 1 vs. 32 +/- 3%, p = 0.05). In dogs treated with high-dose GW, both EDV and ESV increased (72 +/- 4 vs. 79 +/- 5 ml, p = 0.04; 53 +/- 3 vs. 62 +/- 5 ml, p = 0.04) and EF decreased (26 +/- 1 vs. 23 +/- 1%, p = 0.04) as with control dogs. There was significantly increased myocardial hypertrophy as evidenced by increased LV weight to body weight ratio and myocyte cross-section area in the GW treated animals compared to controls. Compared to control, treatment with GW had no effect on mRNA expression of PPARgamma, inflammatory cytokines, stretch response proteins, or transcription factors that may induce hypertrophy. CONCLUSIONS: Long-term PPARgamma activation with GW did not prevent progressive LV remodeling in dogs with advanced heart failure.

Long-term therapy with the acorn cardiac support device normalizes gene expression of growth factors and gelatinases in dogs with heart failure.

BACKGROUND: Passive mechanical containment of the failing left ventricle with the Acorn Cardiac Support Device (CSD) was shown to prevent progressive left ventricular dilation in dogs with heart failure and increase left ventricular ejection fraction. To examine possible mechanisms for improved cardiac function with a CSD, we examined the effect of CSD therapy on the mRNA gene expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP) 2 and 9, and tissue inhibitors of metalloproteinases (TIMP) 1 and 2. METHODS: Heart failure was produced in 12 dogs by multiple sequential intracoronary microembolizations. Six dogs were implanted with the CSD and 6 served as concurrent controls. Left ventricular tissue from 6 normal dogs was used for comparison. RESULTS: Compared with normal dogs, dogs with untreated heart failure showed downregulation of mRNA expression for VEGF and bFGF, whereas upregulation of mRNA expression for MMP-2 and MMP-9 was observed. Normalization of mRNA expression for all these genes was seen after treatment with the CSD. CONCLUSIONS: This study shows that preventing left ventricular dilation and myocardial stretch with the CSD promotes normalization of growth factor and MMP gene expression. These results suggest that modulation of gene activity may, in part, contribute to the improvement of left ventricular function observed with CSD therapy.

Long-term effects of non-excitatory cardiac contractility modulation electric signals on the progression of heart failure in dogs.

OBJECTIVE: We previously showed that acute delivery of non-excitatory cardiac contractility modulation (CCM) electric signal during the absolute refractory period improved LV function in dogs with chronic heart failure (HF). In the present study we examined the long-term effects of CCM signal delivery on the progression of LV dysfunction and remodeling in dogs with chronic HF. METHODS: Chronic HF was produced in 12 dogs by multiple sequential intracoronary microembolizations. The CCM signal was delivered using a lead implanted in the distal anterior coronary vein. A right ventricular and a right atrial lead were implanted and used for timing of CCM signal delivery. In six dogs, CCM signals were delivered continuously for 6 h daily with an average amplitude of 3.3 V for 3 months. Six HF dogs did not have leads implanted and served as controls. RESULTS: In control dogs, LV end-diastolic volume (EDV) and LV end-systolic volume (ESV) increased (64+/-5 ml vs. 75+/-6 ml, P=0.003; 46+/-4 ml vs. 57+/-4 ml, P=0.003; respectively), and ejection fraction (EF) decreased (28+/-1% vs. 23+/-1%, P=0.001) over the course of 3 months of follow-up. In contrast, CCM-treated dogs showed a smaller increase in EDV (66+/-4 vs. 73+/-5 ml, P=0.01), no change in ESV, and an increase in EF from 31+/-1 to 34+/-2% (P=0.04) after 3 months of therapy. CONCLUSIONS: In dogs with HF, long-term CCM therapy prevents progressive LV dysfunction and attenuates global LV remodeling. These findings provide compelling rationale for exploring the use of CCM for the treatment of patients with chronic HF.

Reversal of chronic molecular and cellular abnormalities due to heart failure by passive mechanical ventricular containment.

Passive mechanical containment of failing left ventricle (LV) with the Acorn Cardiac Support Device (CSD) was shown to prevent progressive LV dilation in dogs with heart failure (HF) and increase ejection fraction. To examine possible mechanisms for improved LV function with the CSD, we examined the effect of CSD therapy on the expression of cardiac stretch response proteins, myocyte hypertrophy, sarcoplasmic reticulum Ca2+-ATPase activity and uptake, and mRNA gene expression for myosin heavy chain (MHC) isoforms. HF was produced in 12 dogs by intracoronary microembolization. Six dogs were implanted with the CSD and 6 served as concurrent controls. LV tissue from 6 normal dogs was used for comparison. Compared with normal dogs, untreated HF dogs showed reduced cardiomyocyte contraction and relaxation, upregulation of stretch response proteins (p21ras, c-fos, and p38 alpha/beta mitogen-activated protein kinase), increased myocyte hypertrophy, reduced SERCA2a activity with unchanged affinity for calcium, reduced proportion of mRNA gene expression for alpha-MHC, and increased proportion of beta-MHC. Therapy with the CSD was associated with improved cardiomyocyte contraction and relaxation, downregulation of stretch response proteins, attenuation of cardiomyocyte hypertrophy, increased affinity of the pump for calcium, and restoration of alpha- and beta-MHC isoforms ratio. The results suggest that preventing LV dilation and stretch with the CSD promotes downregulation of stretch response proteins, attenuates myocyte hypertrophy and improves SR calcium cycling. These data offer possible mechanisms for improvement of LV function after CSD therapy.

Hypoxia, angiotensin-II, and norepinephrine mediated apoptosis is stimulus specific in canine failed cardiomyocytes: a role for p38 MAPK, Fas-L and cyclin D1.

BACKGROUND: Apoptosis may contribute to the myocardial dysfunction associated with heart failure (HF). Activation of the p38 MAPK cascade can induce apoptosis in non-cardiac cells through increased expression of Fas-L, or through decreased expression of cyclin D(1). AIMS: We tested the hypothesis that hypoxia (HX), angiotensin-II (A-II) and norepinephrine (NEPI) can mediate apoptosis by activating p38 MAPK, and thus initiating stimulus specific changes in Fas-L and cyclin D(1) expression in failing cardiomyocytes. METHODS AND RESULTS: Cardiomyocytes isolated from ten dogs with HF induced by coronary microembolizations were subjected to HX or A-II or NEPI with and without a p38 MAPK inhibitor (SB 203580). TUNEL staining for DNA fragmentation and Western blots for p38 MAPK, Fas-L and cyclin D(1) detection were performed. HX-induced apoptosis was associated with increased Fas-L expression, A-II-induced apoptosis was associated with increased Fas-L and decreased cyclin D(1) expression, and NEPI-induced apoptosis was associated with decreased cyclin D(1) expression. Inhibition of p38 MAPK activity attenuated stress-induced apoptosis in all experiments and reversed changes in Fas-L and cyclin D(1) expression. CONCLUSIONS: HX, A-II and NEPI mediate apoptosis in failing cardiomyocytes via different effects on Fas-L and cyclin D(1) expression. Inhibition of p38 MAPK reversed these effects, suggesting that apoptosis induced by HX, A-II and NEPI involves activation of p38 MAPK upstream from Fas-L and cyclin D(1).

Cardiac contractility modulation with nonexcitatory electric signals improves left ventricular function in dogs with chronic heart failure.

BACKGROUND: Nonexcitatory electrical, signals termed cardiac contractility modulation (CCM) have been shown to improve contractile force of isolated papillary muscles. In this study, we examined the effects of CCM signal delivery on left ventricular function in dogs with chronic heart failure (HF). METHODS AND RESULTS: Chronic HF (ejection fraction

Effects of the AT1-receptor antagonist eprosartan on the progression of left ventricular dysfunction in dogs with heart failure.

1. We examined the effects of eprosartan, an AT(1) receptor antagonist, on the progression of left ventricular (LV) dysfunction and remodelling in dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction, EF 30 to 40%). 2. Dogs were randomized to 3 months of oral therapy with low-dose eprosartan (600 mg once daily, n=8), high-dose eprosartan (1200 mg once daily, n=8), or placebo (n=8). 3. In the placebo group, LV end-diastolic (EDV) and end-systolic (ESV) volumes increased after 3 months (68+/-7 vs 82+/-9 ml, P<0.004, 43+/-1 vs 58+/-7 ml, P<0.003, respectively), and EF decreased (37+/-1 vs 29+/-1%, P<0.001). In dogs treated with low-dose eprosartan, EF, EDV, and ESV remained unchanged over the course of therapy, whereas in dogs treated with high-dose eprosartan, EF increased (38+/-1 vs 42+/-1%, P<0.004) and ESV decreased (41+/-1 vs 37+/-1 ml, P<0.006), Eprosartan also decreased interstitial fibrosis and cardiomyocyte hypertrophy. 4. We conclude that eprosartan prevents progressive LV dysfunction and attenuates progressive LV remodelling in dogs with moderate HF and may be useful in treating patients with chronic HF.

Effects of long-term monotherapy with eplerenone, a novel aldosterone blocker, on progression of left ventricular dysfunction and remodeling in dogs with heart failure.

BACKGROUND: In heart failure (HF), aldosterone has been implicated in the formation of reactive interstitial fibrosis, a maladaptation that contributes to left ventricular (LV) remodeling. Eplerenone is a novel selective aldosterone blocker. The present study examined the effects of long-term monotherapy with eplerenone on the progression of LV dysfunction and remodeling in dogs with chronic HF. METHODS AND RESULTS: HF was produced in 14 dogs by intracoronary microembolizations that were discontinued when LV ejection fraction (EF) was between 30% and 40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with eplerenone (10 mg/kg twice daily, n=7) or no therapy at all (control, n=7). Hemodynamic measurements were made just before randomization and were repeated at the end of 3 months of therapy. In control dogs, LV end-diastolic and end-systolic volume increased significantly (62+/-4 versus 68+/-4 mL, P<0.001, and 38+/-3 versus 47+/-3 mL, P<0.001, respectively), and EF decreased significantly (38+/-1% versus 31+/-2%, P<0.001). In contrast, end-diastolic volume, end-systolic volume, and EF remained unchanged during the 3 months of treatment in eplerenone-treated dogs. LV end-diastolic wall stress increased significantly in control dogs but decreased significantly in eplerenone-treated dogs. Compared with control, eplerenone was associated with a 28% reduction in cardiomyocyte cross-sectional area, a 37% reduction of volume fraction of reactive interstitial fibrosis, and a 34% reduction of volume fraction of replacement fibrosis. CONCLUSIONS: Our results indicate that long-term therapy with eplerenone prevents progressive LV dysfunction and attenuates LV remodeling in dogs with chronic HF.

Effects of chronic neutral endopeptidase inhibition on the progression of left ventricular dysfunction and remodeling in dogs with moderate heart failure.

BACKGROUND: The diuretic actions of endogenously produced atrial natriuretic factor (ANF) may be beneficial in the treatment of chronic heart failure (CHF). Neutral endopeptidase (NEP) is the primary enzyme responsible for the degradation of ANF. The present study investigates the effects of long-term NEP inhibition on the progression of left ventricular (LV) dysfunction and remodeling in dogs with moderate heart failure. METHODS: LV dysfunction was produced in 12 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LV ejection fraction (EF) was between 30-40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with the NEP inhibitor ecadotril (100 mg, once daily, n = 6) or to no therapy at all (control, n = 6). RESULTS: During the 3 months of follow-up, LV EF in control dogs decreased from 37 +/- 1% to 28 +/- 1% (P < 0.01) and LV end-diastolic volume (EDV) and end-systolic volume (ESV) increased (EDV: 72 +/- 3 vs. 84 +/- 5 ml, P < 0.01); ESV: 45 +/- 1 vs. 60 +/- 4 ml, P < 0.01). In dogs treated with ecadotril, LV EF (34 +/- 1% vs. 37 +/- 2%), EDV (79+/- 5 vs. 78+/- 6 ml) and ESV (52 +/- 3 vs. 49 +/- 4) remained essentially unchanged after 3 months of therapy. Histomorphometric measurements at the termination of the study showed that ecadotril was associated with significantly reduced cardiomyocyte hypertrophy compared to control. CONCLUSION: Early, long-term NEP inhibition with ecadotril prevents the progression of LV dysfunction and attenuates progressive LV remodeling in dogs with moderate heart failure.

Hypoxia-induced cleavage of caspase-3 and DFF45/ICAD in human failed cardiomyocytes.

It has been proposed that the hemodynamic deterioration associated with heart failure (HF) may be due in part to ongoing loss of viable cardiac myocytes through apoptosis. Hypoxia has been shown to promote apoptosis in normal cardiomyocytes. Adaptation and maladaptations inherent to heart failure can modify the susceptibility of cells to different stress factors. We hypothesized that HF modifies the threshold of cardiomyocytes to hypoxia-induced apoptosis. Cardiomyocytes were isolated from 18 human hearts explanted at the time of cardiac transplantation due to either ischemic cardiomyopathy (ICM) (n = 9) or idiopathic dilated cardiomyopathy (IDC) (n = 9). Tissue from five normal donor hearts (NL) for whom no suitable recipient was available was used as control. Cardiomyocytes were incubated for 3 h under normoxic (95% air-5% CO(2)) or hypoxic (95% N(2)-5% CO(2)) conditions. Expression of caspase-3 and DNA fragmentation factor-45 (DFF45)/inhibitor of caspase-3-activated DNase (ICAD) was detected by Western blot analysis. Three hours of hypoxia did not affect the expression of these proteins in NL cardiomyocytes. In contrast, hypoxia led to cleavage of caspase-3 and DFF45/ICAD both in ICM and IDC. In conclusion, failing cardiomyocytes exhibit increased susceptibility to hypoxia-induced apoptosis.

Effects of long-term monotherapy with metoprolol CR/XL on the progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure.

We examined the effects of long-term monotherapy with the beta-blocker, metoprolol controlled release/extended release (CR/XL), on the progression of LV dysfunction as well as on global and cellular remodeling in dogs with heart failure (HF). Chronic HF was produced by intracoronary microembolizations that were discontinued when LV ejection fraction (EF) was between 30% and 40%. Dogs were randomized to 3 months oral monotherapy with metoprolol CR/XL (100 mg once daily, n = 7) or no therapy at all (control, n = 7). In control dogs, EF decreased from 38 +/- 1% to 31 +/- 2% (p = 0.002), and LV end-systolic volume (ESV) and LV end-diastolic volume (EDV) increased (37 +/- 2 vs 45 +/- 2 ml, p = 0.001; 59 +/- 3 vs 65 +/- 3 ml, p = 0.001; respectively) during the 3 month follow-up period. In dogs treated with metoprolol CR/XL, EF increased after 3 months from 36 +/- 1% to 43 +/- 1% (p = 0.001), and ESV decreased (42 +/- 2 vs 38 +/- 2 ml, p = 0.003), whereas EDV remained unchanged. Compared to controls, treatment with metoprolol CR/XL showed 46% reduction in replacement fibrosis, 54% reduction in interstitial fibrosis and 20% reduction in myocyte cross-sectional area, a measure of myocyte hypertrophy. These findings indicate that metoprolol CR/XL improves LV function and attenuates progressive global and cellular LV remodeling in dogs with HF. The benefits are fully attributable to beta-blockade alone as no other adjunctive therapy was used.