Kelly A. O'Connell

Cardiomyocyte deletion of mitofusin-1 leads to mitochondrial fragmentation and improves tolerance to ROS-induced mitochondrial dysfunction and cell death

Molecular studies examining the impact of mitochondrial morphology on the mammalian heart have previously focused on dynamin related protein-1 (Drp-1) and mitofusin-2 (Mfn-2), while the role of the other mitofusin isoform, Mfn-1, has remained largely unexplored. In the present study, we report the generation and initial characterization of cardiomyocyte-specific Mfn-1 knockout (Mfn-1 KO) mice. Using electron microscopic analysis, we detect a greater prevalence of small, spherical mitochondria in Mfn-1 KO hearts, indicating that the absence of Mfn-1 causes a profound shift in the mitochondrial fusion/fission balance. Nevertheless, Mfn-1 KO mice exhibit normal left-ventricular function, and isolated Mfn-1 KO heart mitochondria display a normal respiratory repertoire. Mfn-1 KO myocytes are protected from mitochondrial depolarization and exhibit improved viability when challenged with reactive oxygen species (ROS) in the form of hydrogen peroxide (H(2)O(2)). Furthermore, in vitro studies detect a blunted response of KO mitochondria to undergo peroxide-induced mitochondrial permeability transition pore opening. These data suggest that Mfn-1 deletion confers protection against ROS-induced mitochondrial dysfunction. Collectively, we suggest that mitochondrial fragmentation in myocytes is not sufficient to induce heart dysfunction or trigger cardiomyocyte death. Additionally, our data suggest that endogenous levels of Mfn-1 can attenuate myocyte viability in the face of an imminent ROS overload, an effect that could be associated with the ability of Mfn-1 to remodel the outer mitochondrial membrane.

Dietary fat and heart failure: moving from lipotoxicity to lipoprotection.

There is growing evidence suggesting that dietary fat intake affects the development and progression of heart failure. Studies in rodents show that in the absence of obesity, replacing refined carbohydrate with fat can attenuate or prevent ventricular expansion and contractile dysfunction in response to hypertension, infarction, or genetic cardiomyopathy. Relatively low intake of n-3 polyunsaturated fatty acids from marine sources alters cardiac membrane phospholipid fatty acid composition, decreases the onset of new heart failure, and slows the progression of established heart failure. This effect is associated with decreased inflammation and improved resistance to mitochondrial permeability transition. High intake of saturated, monounsaturated, or n-6 polyunsaturated fatty acids has also shown beneficial effects in rodent studies. The underlying mechanisms are complex, and a more thorough understanding is needed of the effects on cardiac phospholipids, lipid metabolites, and metabolic flux in the normal and failing heart. In summary, manipulation of dietary fat intake shows promise in the prevention and treatment of heart failure. Clinical studies generally support high intake of n-3 polyunsaturated fatty acids from marine sources to prevent and treat heart failure. Additional clinical and animals studies are needed to determine the optimal diet in terms of saturated, monounsaturated, and n-6 polyunsaturated fatty acids intake for this vulnerable patient population.

Cardiomyocyte-specific perilipin 5 overexpression leads to myocardial steatosis and modest cardiac dysfunction.

Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment.

Improved Mitochondrial Function with Diet-Induced Increase in Either Docosahexaenoic Acid or Arachidonic Acid in Membrane Phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.

High intake of saturated fat, but not polyunsaturated fat, improves survival in heart failure despite persistent mitochondrial defects

AIMS The impact of a high-fat diet on the failing heart is unclear, and the differences between polyunsaturated fatty acids (PUFA) and saturated fat have not been assessed. Here, we compared a standard low-fat diet to high-fat diets enriched with either saturated fat (palmitate and stearate) or PUFA (linoleic and α-linolenic acids) in hamsters with genetic cardiomyopathy. METHODS AND RESULTS Male δ-sarcoglycan null Bio TO2 hamsters were fed a standard low-fat diet (12% energy from fat), or high-fat diets (45% fat) comprised of either saturated fat or PUFA. The median survival was increased by the high saturated fat diet (P< 0.01; 278 days with standard diet and 361 days with high saturated fat)), but not with high PUFA (260 days) (n = 30-35/group). Body mass was modestly elevated (∼10%) in both high fat groups. Subgroups evaluated after 24 weeks had similar left ventricular chamber size, function, and mass. Mitochondrial oxidative enzyme activity and the yield of interfibrillar mitochondria (IFM) were decreased to a similar extent in all TO2 groups compared with normal F1B hamsters. Ca(2+)-induced mitochondrial permeability transition pore opening was enhanced in IFM in all TO2 groups compared with F1B hamsters, but to a significantly greater extent in those fed the high PUFA diet compared with the standard or high saturated fat diet. CONCLUSION These results show that a high intake of saturated fat improves survival in heart failure compared with a high PUFA diet or low-fat diet, despite persistent mitochondrial defects.

Marine n3 polyunsaturated fatty acids enhance resistance to mitochondrial permeability transition in heart failure but do not improve survival

Mitochondrial dysfunction in heart failure includes greater susceptibility to mitochondrial permeability transition (MPT), which may worsen cardiac function and decrease survival. Treatment with a mixture of the n3 polyunsaturated fatty acids (n3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is beneficial in heart failure patients and increases resistance to MPT in animal models. We assessed whether DHA and EPA have similar effects when given individually, and whether they prolong survival in heart failure. Male δ-sarcoglycan null cardiomyopathic hamsters were untreated or given either DHA, EPA, or a 1:1 mixture of DHA + EPA at 2.1% of energy intake. Treatment did not prolong survival: mean survival was 298 ± 15 days in untreated hamsters and 335 ± 17, 328 ± 14, and 311 ± 15 days with DHA, EPA, and DHA + EPA, respectively (n = 27-32/group). A subgroup of cardiomyopathic hamsters treated for 26 wk had impaired left ventricular function and increased cardiomyocyte apoptosis compared with normal hamsters, which was unaffected by n3 PUFA treatment. Evaluation of oxidative phosphorylation in isolated subsarcolemmal and interfibrillar mitochondria with substrates for complex I or II showed no effect of n3 PUFA treatment. On the other hand, interfibrillar mitochondria from cardiomyopathic hamsters were significantly more sensitive to Ca(2+)-induced MPT, which was completely normalized by treatment with DHA and partially corrected by EPA. In conclusion, treatment with DHA or EPA normalizes Ca(2+)-induced MPT in cardiomyopathic hamsters but does not prolong survival or improve cardiac function. This suggest that greater susceptibility to MPT is not a contributor to cardiac pathology and poor survival in heart failure.

Enhanced resistance to permeability transition in interfibrillar cardiac mitochondria in dogs: effects of aging and long-term aldosterone infusion

Functional differences between subsarcolemmal and interfibrillar cardiac mitochondria (SSM and IFM) have been observed with aging and pathological conditions in rodents. Results are contradictory, and there is little information from large animal models. We assessed the respiratory function and resistance to mitochondrial permeability transition (MPT) in SSM and IFM from healthy young (1 yr) and old (8 yr) female beagles and in old beagles with hypertension and left ventricular (LV) wall thickening induced by 16 wk of aldosterone infusion. MPT was assessed in SSM and IFM by Ca(2+) retention and swelling. Healthy young and old beagles had similar mitochondrial structure, respiratory function, and Ca(2+)-induced MPT within SSM and IFM subpopulations. On the other hand, oxidative capacity and resistance to Ca(2+)-induced MPT were significantly greater in IFM compared with SSM in all groups. Old beagles treated with aldosterone had greater LV wall thickness and worse diastolic filling but normal LV chamber volume and systolic function. Treatment with aldosterone did not alter mitochondrial respiratory function but accelerated Ca(2+)-induced MPT in SSM, but not IFM, compared with healthy old and young beagles. In conclusion, in a large animal model, oxidative capacity and resistance to MPT were greater in IFM than in SSM. Furthermore, aldosterone infusion increased susceptibility to MPT in SSM, but not IFM. Together this suggests that SSM are less resilient to acute stress than IFM in the healthy heart and are more susceptible to the development of pathology with chronic stress.

Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzymopathy that affects cellular redox status and may lower flux into nonoxidative pathways of glucose metabolism. Oxidative stress may worsen systemic glucose tolerance and cardiometabolic syndrome. We hypothesized that G6PD deficiency exacerbates diet-induced systemic metabolic dysfunction by increasing oxidative stress but in myocardium prevents diet-induced oxidative stress and pathology. WT and G6PD-deficient (G6PDX) mice received a standard high-starch diet, a high-fat/high-sucrose diet to induce obesity (DIO), or a high-fructose diet. After 31 wk, DIO increased adipose and body mass compared with the high-starch diet but to a greater extent in G6PDX than WT mice (24 and 20% lower, respectively). Serum free fatty acids were increased by 77% and triglycerides by 90% in G6PDX mice, but not in WT mice, by DIO and high-fructose intake. G6PD deficiency did not affect glucose tolerance or the increased insulin levels seen in WT mice. There was no diet-induced hypertension or cardiac dysfunction in either mouse strain. However, G6PD deficiency increased aconitase activity by 42% and blunted markers of nonoxidative glucose pathway activation in myocardium, including the hexosamine biosynthetic pathway activation and advanced glycation end product formation. These results reveal a complex interplay between diet-induced metabolic effects and G6PD deficiency, where G6PD deficiency decreases weight gain and hyperinsulinemia with DIO, but elevates serum free fatty acids, without affecting glucose tolerance. On the other hand, it modestly suppressed indexes of glucose flux into nonoxidative pathways in myocardium, suggesting potential protective effects.

Dietary saturated fat and docosahexaenoic acid differentially effect cardiac mitochondrial phospholipid fatty acyl composition and Ca2+ uptake, without altering permeability transition or left ventricular function

High saturated fat diets improve cardiac function and survival in rodent models of heart failure, which may be mediated by changes in mitochondrial function. Dietary supplementation with the n3‐polyunsaturated fatty acid docosahexaenoic acid (DHA, 22:6n3) is also beneficial in heart failure and can affect mitochondrial function. Saturated fatty acids and DHA likely have opposing effects on mitochondrial phospholipid fatty acyl side chain composition and mitochondrial membrane function, though a direct comparison has not been previously reported. We fed healthy adult rats a standard low‐fat diet (11% of energy intake from fat), a low‐fat diet supplemented with DHA (2.3% of energy intake) or a high‐fat diet comprised of long chain saturated fatty acids (45% fat) for 6 weeks. There were no differences among the three diets in cardiac mass or function, mitochondrial respiration, or Ca2+‐induced mitochondrial permeability transition. On the other hand, there were dramatic differences in mitochondrial phospholipid fatty acyl side chains. Dietary supplementation with DHA increased DHA from 7% to ~25% of total phospholipid fatty acids in mitochondrial membranes, and caused a proportional depletion of arachidonic acid (20:4n6). The saturated fat diet increased saturated fat and DHA in mitochondria and decreased linoleate (18:2n6), which corresponded to a decrease in Ca2+ uptake by isolated mitochondria compared to the other diet groups. In conclusion, despite dramatic changes in mitochondrial phospholipid fatty acyl side chain composition by both the DHA and high saturated fat diets, there were no effects on mitochondrial respiration, permeability transition, or cardiac function.