Cardiac Myocyte-Specific Overexpression of FASTKD1 Prevents Ventricular Rupture After Myocardial Infarction.

Background The mitochondrial mRNA-binding protein FASTKD1 (Fas-activated serine/threonine [FAST] kinase domain-containing protein 1) protects myocytes from oxidative stress in vitro. However, the role of FASTKD1 in the myocardium in vivo is unknown. Therefore, we developed cardiac-specific FASTKD1 transgenic mice to test the effects of this protein on experimental myocardial infarction (MI). Methods and Results Transgenic mouse lines with cardiac myocyte-specific overexpression of FASTKD1 to varying degrees were generated. These mice displayed normal cardiac morphological features and function at the gross and microscopic levels. Isolated cardiac mitochondria from all transgenic mouse lines showed normal mitochondrial function, ATP levels, and permeability transition pore activity. Male nontransgenic and transgenic mice from the highest-expressing line were subjected to 8 weeks of permanent coronary ligation. Of nontransgenic mice, 40% underwent left ventricular free wall rupture within 7 days of MI compared with 0% of FASTKD1-overexpressing mice. At 3 days after MI, FASTKD1 overexpression did not alter infarct size. However, increased FASTKD1 resulted in decreased neutrophil and increased macrophage infiltration, elevated levels of the extracellular matrix component periostin, and enhanced antioxidant capacity compared with control mice. In contrast, markers of mitochondrial fusion/fission and apoptosis remained unaltered. Instead, transcriptomic analyses indicated activation of the integrated stress response in the FASTKD1 transgenic hearts. Conclusions Cardiac-specific overexpression of FASTKD1 results in viable mice displaying normal cardiac morphological features and function. However, these mice are resistant to MI-induced cardiac rupture and display altered inflammatory, extracellular matrix, and antioxidant responses following MI. Moreover, these protective effects were associated with enhanced activation of the integrated stress response.

The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death.

Opening of the mitochondrial permeability transition (MPT) pore leads to necrotic cell death. Excluding cyclophilin D (CypD), the makeup of the MPT pore remains conjecture. The purpose of these experiments was to identify novel MPT modulators by analyzing proteins that associate with CypD. We identified Fas-activated serine/threonine phosphoprotein kinase domain-containing protein 1 (FASTKD1) as a novel CypD interactor. Overexpression of FASTKD1 protected mouse embryonic fibroblasts (MEFs) against oxidative stress-induced reactive oxygen species (ROS) production and cell death, whereas depletion of FASTKD1 sensitized them. However, manipulation of FASTKD1 levels had no effect on MPT responsiveness, Ca2+-induced cell death, or antioxidant capacity. Moreover, elevated FASTKD1 levels still protected against oxidative stress in CypD-deficient MEFs. FASTKD1 overexpression decreased Complex-I-dependent respiration and ΔΨm in MEFs, effects that were abrogated in CypD-null cells. Additionally, overexpression of FASTKD1 in MEFs induced mitochondrial fragmentation independent of CypD, activation of Drp1, and inhibition of autophagy/mitophagy, whereas knockdown of FASTKD1 had the opposite effect. Manipulation of FASTKD1 expression also modified oxidative stress-induced caspase-3 cleavage yet did not alter apoptotic death. Finally, the effects of FASTKD1 overexpression on oxidative stress-induced cell death and mitochondrial morphology were recapitulated in cultured cardiac myocytes. Together, these data indicate that FASTKD1 supports mitochondrial homeostasis and plays a critical protective role against oxidant-induced death.

Saxagliptin and Tadalafil Differentially Alter Cyclic Guanosine Monophosphate (cGMP) Signaling and Left Ventricular Function in Aortic-Banded Mini-Swine.

BACKGROUND: Cyclic guanosine monophosphate-protein kinase G-phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl-peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function. METHODS AND RESULTS: We assessed LV hypertrophy and function at the organ and cellular level in aortic-banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end-systolic pressure-volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil-treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening-frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium-retention capacity and Complex II-dependent respiratory control, was present in both HF and tadalafil-treated animals. CONCLUSIONS: Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function.

Necroptosis: is there a role for mitochondria?

Once thought to be a random process of cell death, necrosis can proceed via a defined molecular mechanism and is integral to physiological and pathological states. In particular a form of necrosis called necroptosis has been the subject of intense investigation. Necroptosis is initiated by tumor necrosis factor-α (TNFα), which leads to the activation of the kinase receptor-interacting protein 1 (RIP1). RIP1 then binds with and activates RIP3 to form the necrosome. RIP3 in turn interacts with and activates the pseudokinase mixed lineage kinase domain-like (MLKL). This complex has then been proposed to induce necrotic death via the induction of mitochondrial dysfunction, with a variety of mechanisms being put forth including: production of mitochondrial reactive oxygen species (ROS), activation of the mitochondrial phosphatase PGAM5, or induction of mitochondrial permeability transition (MPT). However, recent evidence suggests that none of these are involved in necroptosis, and that mitochondria may in fact be dispensable for this process. Therefore, the purpose of this perspective is to discuss the current understanding of necroptosis, and more specifically, what role if any do mitochondria play in this mechanism of cell death.

A new twist on an old idea part 2: cyclosporine preserves normal mitochondrial but not cardiomyocyte function in mini-swine with compensated heart failure.

We recently developed a clinically relevant mini-swine model of heart failure with preserved ejection fraction (HFpEF), in which diastolic dysfunction was associated with increased mitochondrial permeability transition (MPT). Early diastolic function is ATP and Ca(2+)-dependent, thus, we hypothesized chronic low doses of cyclosporine (CsA) would preserve mitochondrial function via inhibition of MPT and subsequently maintain normal cardiomyocyte Ca(2+) handling and contractile characteristics. Left ventricular cardiomyocytes were isolated from aortic-banded Yucatan mini-swine divided into three groups; control nonbanded (CON), HFpEF nontreated (HF), and HFpEF treated with CsA (HF-CsA). CsA mitigated the deterioration of mitochondrial function observed in HF animals, including functional uncoupling of Complex I-dependent mitochondrial respiration and increased susceptibility to MPT. Attenuation of mitochondrial dysfunction in the HF-CsA group was not associated with commensurate improvement in cardiomyocyte Ca(2+) handling or contractility. Ca(2+) transient amplitude was reduced and transient time to peak and recovery (tau) prolonged in HF and HF-CsA groups compared to CON. Alterations in Ca(2+) transient parameters observed in the HF and HF-CsA groups were associated with decreased cardiomyocyte shortening and shortening rate. Cellular function was consistent with impaired in vivo systolic and diastolic whole heart function. A significant systemic hypertensive response to CsA was observed in HF-CsA animals, and may have played a role in the accelerated the development of heart failure at both the whole heart and cellular levels. Given the significant detriment to cardiac function observed in response to CsA, our findings suggest chronic CsA treatment is not a viable therapeutic option for HFpEF.

Proteomic mapping of proteins released during necrosis and apoptosis from cultured neonatal cardiac myocytes.

Cardiac injury induces myocyte apoptosis and necrosis, resulting in the secretion and/or release of intracellular proteins. Currently, myocardial injury can be detected by analysis of a limited number of biomarkers in blood or coronary artery perfusate. However, the complete proteomic signature of protein release from necrotic cardiac myocytes is unknown. Therefore, we undertook a proteomic-based study of proteins released from cultured neonatal rat cardiac myocytes in response to H2O2 (necrosis) or staurosporine (apoptosis) to identify novel specific markers of cardiac myocyte cell death. Necrosis and apoptosis resulted in the identification of 147 and 79 proteins, respectively. Necrosis resulted in a relative increase in the amount of many proteins including the classical necrotic markers lactate dehydrogenase (LDH), high-mobility group B1 (HMGB1), myoglobin, enolase, and 14-3-3 proteins. Additionally, we identified several novel markers of necrosis including HSP90, α-actinin, and Trim72, many of which were elevated over control levels earlier than classical markers of necrotic injury. In contrast, the majority of identified proteins remained at low levels during apoptotic cell death, resulting in no candidate markers for apoptosis being identified. Blotting for a selection of these proteins confirmed their release during necrosis but not apoptosis. We were able to confirm the presence of classical necrotic markers in the extracellular milieu of necrotic myocytes. We also were able to identify novel markers of necrotic cell death with relatively early release profiles compared with classical protein markers of necrosis. These results have implications for the discovery of novel biomarkers of necrotic myocyte injury, especially in the context of ischemia-reperfusion injury.

Heart failure with preserved ejection fraction: chronic low-intensity interval exercise training preserves myocardial O2 balance and diastolic function.

We have previously reported chronic low-intensity interval exercise training attenuates fibrosis, impaired cardiac mitochondrial function, and coronary vascular dysfunction in miniature swine with left ventricular (LV) hypertrophy (Emter CA, Baines CP. Am J Physiol Heart Circ Physiol 299: H1348-H1356, 2010; Emter CA, et al. Am J Physiol Heart Circ Physiol 301: H1687-H1694, 2011). The purpose of this study was to test two hypotheses: 1) chronic low-intensity interval training preserves normal myocardial oxygen supply/demand balance; and 2) training-dependent attenuation of LV fibrotic remodeling improves diastolic function in aortic-banded sedentary, exercise-trained (HF-TR), and control sedentary male Yucatan miniature swine displaying symptoms of heart failure with preserved ejection fraction. Pressure-volume loops, coronary blood flow, and two-dimensional speckle tracking ultrasound were utilized in vivo under conditions of increasing peripheral mean arterial pressure and ß-adrenergic stimulation 6 mo postsurgery to evaluate cardiac function. Normal diastolic function in HF-TR animals was characterized by prevention of increased time constant of isovolumic relaxation, normal LV untwisting rate, and enhanced apical circumferential and radial strain rate. Reduced fibrosis, normal matrix metalloproteinase-2 and tissue inhibitors of metalloproteinase-4 mRNA expression, and increased collagen III isoform mRNA levels (P < 0.05) accompanied improved diastolic function following chronic training. Exercise-dependent improvements in coronary blood flow for a given myocardial oxygen consumption (P < 0.05) and cardiac efficiency (stroke work to myocardial oxygen consumption, P < 0.05) were associated with preserved contractile reserve. LV hypertrophy in HF-TR animals was associated with increased activation of Akt and preservation of activated JNK/SAPK. In conclusion, chronic low-intensity interval exercise training attenuates diastolic impairment by promoting compliant extracellular matrix fibrotic components and preserving extracellular matrix regulatory mechanisms, preserves myocardial oxygen balance, and promotes a physiological molecular hypertrophic signaling phenotype in a large animal model resembling heart failure with preserved ejection fraction.

Replicative aging induces endothelial to mesenchymal transition in human aortic endothelial cells: potential role of inflammation.

Thickening of the intimal layer of arteries characterized by expression of smooth muscle α-actin (SMαA), collagen deposition, and inflammation is an important pathophysiological change with aging assumed to be mediated by smooth muscle cells migrating from the medial layer. We tested the novel hypothesis that these characteristics could also reflect an endothelial-mesenchymal (smooth muscle-like) transition (EnMT). Late ('old') compared with early ('young') passage (45.0 ± 1.2 vs. 27.1 ± 0.5 population doublings) human aortic endothelial cells demonstrated greater smooth muscle (spindle) morphological changes, expression of SMαA and collagen I, nuclear factor-κB activation, and transforming growth factor-ß (TGF-ß) (all p < 0.05). Based on increases in SMαA, stimulation with the proinflammatory cytokine tumor necrosis factor-α, but not with TGF-ß, induced EnMT in early passage cells similar to that observed in late passage cells. Here, we present the first evidence for EnMT induced in a model of endothelial cell aging and provide support for proinflammatory signaling in mediating this phenotypic change.

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging.

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100-300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Arterial stiffening with ageing is associated with transforming growth factor-ß1-related changes in adventitial collagen: reversal by aerobic exercise.

We tested the hypothesis that carotid artery stiffening with ageing is associated with transforming growth factor-ß1 (TGF-ß1)-related increases in adventitial collagen and reductions in medial elastin, which would be reversed by voluntary aerobic exercise. Ex vivo carotid artery incremental stiffness was greater in old (29­32 months, n = 11) vs. young (4­7 months, n = 8) cage control B6D2F1 mice (8.84 ± 1.80 vs. 4.54 ± 1.18 AU, P < 0.05), and was associated with selective increases in collagen I and III and TGF-ß1 protein expression in the adventitia (P < 0.05), related to an increase in smooth muscle α-actin (SMαA) (myofibroblast phenotype) (P < 0.05). In cultured adventitial fibroblasts, TGF-ß1 induced increases in superoxide and collagen I protein (P < 0.05), which were inhibited by Tempol, a superoxide dismutase. Medial elastin was reduced with ageing, accompanied by decreases in the pro-synthetic elastin enzyme, lysyl oxidase, and increases in the elastin-degrading enzyme, matrix metalloproteinase 2. Fibronectin was unchanged with ageing, but there was a small increase in calcification (P < 0.05). Increased incremental stiffness in old mice was completely reversed (3.98 ± 0.34 AU, n = 5) by 10­14 weeks of modest voluntary wheel running (1.13 ± 0.29 km day−1), whereas greater voluntary wheel running (10.62 ± 0.49 km day−1) had no effect on young mice. The amelioration of carotid artery stiffness by wheel running in old mice was associated with reductions in collagen I and III and TGF-ß1, partial reversal of the myofibroblast phenotype (reduced SMαA) and reduced calcification (all P < 0.05 vs. old controls), whereas elastin and its modulating enzymes were unaffected. Adventitial TGF-ß1-related oxidative stress may play a key role in collagen deposition and large elastic artery stiffening with ageing and the efficacious effects of voluntary aerobic exercise.

Low-intensity exercise training delays heart failure and improves survival in female hypertensive heart failure rats.

Exercise training improves functional capacity and quality of life in patients with heart failure. However, the long-term effects of exercise on mortality associated with hypertensive heart disease have not been well defined. In the present study, we investigated the effect of low-intensity exercise training on disease progression and survival in female spontaneously hypertensive heart failure rats. Animals with severe hypertension (16 months old) were treadmill trained (14.5 m/min, 45 min/d, 3 d/wk) until they developed terminal heart failure or were euthanized because of age-related complications. Exercise delayed mortality resulting from heart failure (P<0.001) and all causes (P<0.05) and transiently attenuated the systolic hypertension and contractile dysfunction observed in the sedentary animals but had no effect on cardiac morphology or contractile function in end-stage heart failure. Training had no effect on terminal myocardial protein expression of antioxidant enzymes, calcium handling proteins, or myosin heavy chain isoforms but was associated with higher cytochrome oxidase activity in cardiac mitochondria (P<0.05) and a greater mitochondrial content of cardiolipin, a phospholipid that is essential for optimal mitochondrial energy metabolism. In conclusion, low-intensity exercise training significantly delays the onset of heart failure and improves survival in female hypertensive heart failure rats without eliciting sustained improvements in blood pressure, cardiac function, or expression of several myocardial proteins associated with the cardiovascular benefits of exercise. The effects of exercise on cytochrome oxidase and cardiolipin provide novel evidence that training may improve prognosis in hypertensive heart disease by preserving mitochondrial energy metabolism.