Objective: Discrete morphologic, enzymatic and functional changes in skeletal muscle mitochondria have been demonstrated in patients with peripheral arterial disease (PAD). We examined mitochondrial respiration in the gastrocnemius muscle of nine patients (10 legs) with advanced PAD and in nine control patients (nine legs) without evidence of PAD. Methods: Mitochondrial respiratory rates were determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Muscle samples were obtained from the anteromedial aspect of the gastrocnemius muscle, at a level 10 cm distal to the tibial tuberosity. Mitochondria respiratory rate, calculated as nanoatoms of oxygen consumed per minute per milligram of noncollagen protein, were measured at baseline (V 0 ), after addition of substrates (malate and glutamate; (V SUB ), after addition of adenosine diphosphate (ADP) (V ADP ), and finally, after adenine nucleotide translocase inhibition with atractyloside (V AT ). The acceptor control ratio, a sensitive indicator of overall mitochondrial function, was calculated as the ratio of the respiratory rate after the addition of ADP to the respiratory rate after adenine nucleotide translocase inhibition with atractyloside (V ADP / V AT ). Results: Respiratory rate in muscle mitochondria from patients with PAD were not significantly different from control values at baseline (0.31 ؎ 0.06 vs 0.55 ؎ 0.12; P ‫؍‬ .09), but V sub was significantly lower in patients with PAD compared with control subjects (0.43 ؎ 0.07 vs 0.89 ؎ 0.20; P < .05), as was V ADP (0.69 ؎ 0.13 vs 1.24 ؎ 0.20; P < .05). Respiratory rates after atractyloside inhibition in patients with PAD were no different from those in control patients (0.47 ؎ 0.07 vs 0.45 ؎ P ‫؍‬ .08). Compared with control values, mitochondria from patients with PAD had a significantly lower acceptor control ratio (1.41 ؎ 0.10 vs 2.90 ؎ 0.20; P < .001). Conclusion: Mitochondrial respiratory activity is abnormal in lower extremity skeletal muscle in patients with PAD. When considered in concert with the ultrastructural and enzymatic abnormalities previously documented in mitochondria of chronically ischemic muscle, these data support the concept of defective mitochondrial function as a pathophysiologic component of PAD.

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, nϭ7) or no therapy at all (control, nϭ7). 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 (62Ϯ4 versus 68Ϯ4 mL, PϽ0.001, and 38Ϯ3 versus 47Ϯ3 mL, PϽ0.001, respectively), and EF decreased significantly (38Ϯ1% versus 31Ϯ2%, PϽ0.001). 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.

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Abstract-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 Ca 2ϩ -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 ␣/␤ mitogen-activated protein kinase), increased myocyte hypertrophy, reduced SERCA2a activity with unchanged affinity for calcium, reduced proportion of mRNA gene expression for ␣-MHC, and increased proportion of ␤-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 ␣-and ␤-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. Key Words: heart failure Ⅲ myocyte hypertrophy Ⅲ sarcoplasmic reticulum Ⅲ myosin heavy chain H eart failure (HF) is a progressive disorder mediated through multiple signaling pathways. Once initiated, HF is characterized by increased neurohumoral activation and ventricular dilation. Although such compensatory changes are initially beneficial, over the long-term they cause adverse structural and functional changes collectively referred to as ventricular remodeling. Ventricular dilation also causes increased mechanical stress and myocardial stretch. Upregulation of stretch response proteins, such as p21ras, 1 c-fos, 2,3 and p38 ␣/␤ mitogen-activated protein kinase (MAPK), 4 have been shown to induce cardiomyocyte hypertrophy.The Acorn Cardiac Support Device (CSD) has been shown to halt progressive left ventricular (LV) dilation and improve ejection fraction. [5][6][7] However, the mechanism(s) underlying the improved cardiac function has not been elucidated. In the present study, we tested the hypothesis that improvement in LV systolic function in dogs with HF after long-term therapy with the CSD results, in part, from downregulation of stretch response proteins, attenuation of cardiomyocyte hypertrophy, 1-4 and improvement of sarcoplasmic reticulum (SR) calcium cycling. To further understand the mechanisms for the improvement in LV systolic function, we also explored the influence of ...

Objective-We used isolated cardiomyocytes to investigate a possible role of mitochondrial permeability transition pore in mitochondrial abnormalities associated with heart failure.Methods-Cardiomyocytes were isolated from LV myocardium of normal control dogs and dogs with heart failure produced by intracoronary microembolizations. Mitochondrial permeability transition was measured in isolated cardiomyocytes with intact sarcolemma with and without 0.2 μM Cyclosporin A using calcein AM and the fluorometer. State-3 mitochondrial respiration was also measured with the Clark electrode. Mitochondrial membrane potential was measured with JC-1 probe using the fluorometer. Propidium iodide was used to ensure sarcolemma integrity.Results-200 minutes after loading with calcein AM, mitochondria of failing cardiomyocytes showed only 50% of maximal level of calcein fluorescence while it remained unchanged in normal cells. The mitochondrial membrane potential in failing cardiomyocytes was significantly decreased by 38% compared to normal cardiomyocytes. Cyclosporine A significantly slowed the exit of calcein from mitochondria of failing cardiomyocytes and increased mitochondrial membrane potential by 29%. State-3 respiration was not affected with Cyclosporine A in normal cardiomyocytes while it was significantly increased in failing cardiomyocytes by 20%.Conclusions-Exit of calcein (m.w. 1.0 kDa) from mitochondria of viable failing cardiomyocytes with intact sarcolemma suggests an existence of a reversible transitory permeability transition opening in high conductance mode. Attenuation of calcein exit, ΔΨ m and improvement of state-3 respiration achieved with CsA (0.2 μM) show that permeability transition opening could be a cause of mitochondrial dysfunction described in the failing heart.