Anne A. Knowlton

Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia.

The effect of brief myocardial ischemia on the expression of heat shock protein (HSP 70) was examined in an in vivo rabbit model of myocardial ischemia using Northern blotting. Functional studies were carried out in the open-chested anesthetized rabbit. The large marginal branch of the left circumflex was occluded four times for 5 min. Using piezoelectric crystals implanted midwall in the ischemic zone, end-diastolic length, end-systolic length, and percent segmental shortening were assessed. Expression of HSP 70 was measured by Northern blotting. A single 5-min coronary occlusion doubled the expression of HSP 70 whereas four cycles of 5 min of ischemia/5 min of reperfusion resulted in a threefold increase in HSP 70 mRNA (P less than 0.001). Measurements with the piezoelectric crystals showed mild myocardial dysfunction concomitant with the increase in HSP 70. This increase in HSP 70 mRNA after repetitive brief ischemia was transient, occurring as early as 1 h and returning to baseline by 24 h after ischemia. Western blot analysis with a monoclonal antibody to HSP 70 was used to compare sham and postischemic myocardial HSP 70 levels. Changes in the amount of HSP 70 were evident as early as 2 h and were even more striking at 24 h.

Cytosolic Heat Shock Protein 60, Apoptosis, and Myocardial Injury

Background—Heat shock proteins (HSPs) are well known for their ability to “protect” the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect. Methods and Results—To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases. Conclusions—These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.

Complications of inappropriate use of spironolactone in heart failure: when an old medicine spirals out of new guidelines

OBJECTIVES This study was designed to investigate the appropriateness and complications of the use of spironolactone for heart failure (HF) in clinical practice. BACKGROUND Spironolactone was reported by one prospective randomized trial to decrease morbidity and mortality in patients with New York Heart Association (NYHA) class III and IV HF. With this report (Randomized Spironolactone Evaluation Study [RALES] trial), we noted a marked increase in widespread use of spironolactone in patients with HF. Long-term outcome data with respect to safety and utilization of this medication in HF are not available. METHODS To investigate the use of spironolactone for HF in a clinical setting, we analyzed the application of the RALES trial protocol to the care of 104 patients, whom we identified as being started on spironolactone for HF after prerelease of the RALES trial. RESULTS We found broader use, less intensive follow-up, and increased complications with spironolactone treatment compared with the RALES trial. Cardiologists provided more appropriate care than did primary care providers. CONCLUSIONS These data suggest that spironolactone is being used widely in HF without consideration of the NYHA class and ejection fraction, and without optimization of background treatment with angiotensin-converting enzyme inhibitors and beta-blockers. Clinical follow-up does not adhere to the RALES trial guidelines, resulting in higher complications. We conclude that long-term studies with further safety and efficacy data are needed.

Mitochondrial OPA1, apoptosis, and heart failure

AIMS Mitochondrial fusion and fission are essential processes for preservation of normal mitochondrial function. We hypothesized that fusion proteins would be decreased in heart failure (HF), as the mitochondria in HF have been reported to be small and dysfunctional. METHODS AND RESULTS Expression of optic atrophy 1 (OPA1), a mitochondrial fusion protein, was decreased in both human and rat HF, as observed by western blotting. OPA1 is important for maintaining normal cristae structure and function, for preserving the inner membrane structure and for protecting cells from apoptosis. Confocal and electron microscopy studies demonstrated that the mitochondria in the failing hearts were small and fragmented, consistent with decreased fusion. OPA1 mRNA levels did not differ between failing and normal hearts, suggesting post-transcriptional control. Simulated ischaemia in the cardiac myogenic cell line H9c2 cells reduced OPA protein levels. Reduction of OPA1 expression with shRNA resulted in increased apoptosis and fragmentation of the mitochondria. Overexpression of OPA1 increased mitochondrial tubularity, but did not protect against simulated ischaemia-induced apoptosis. Cytochrome c release from the mitochondria was increased both with reduction in OPA1 and with overexpression of OPA1. CONCLUSION This is the first report, to our knowledge, of changes in mitochondrial fusion/fission proteins in cardiovascular disease. These changes have implications for mitochondrial function and apoptosis, contributing to the cell loss which is part of the downward progression of the failing heart.

Tumor Necrosis Factor-α Confers Resistance to Hypoxic Injury in the Adult Mammalian Cardiac Myocyte

BACKGROUND Previous studies in isolated cardiac myocytes have shown that tumor necrosis factor (TNF)-alpha provokes increased expression of 27- and 70-kD stress proteins as well as manganese superoxide dismutase, suggesting that TNF-alpha might play a role in mediating stress responses in the heart. METHODS AND RESULTS To determine whether TNF-alpha stimulation would protect isolated cardiac myocytes against environmental stress, myocyte cultures were pretreated with TNF-alpha for 12 hours and then subjected to continuous hypoxic injury (O2 content, 3 to 5 ppm) for 12 hours, followed by reoxygenation. Cell injury was assessed in terms of lactic dehydrogenase (LDH) release, 45Ca2+ uptake, and MTT metabolism. Pretreatment with TNF-alpha concentrations > or = 50 U/mL significantly attenuated LDH release by hypoxic cells compared with diluent-treated hypoxic cells. Similar findings were observed with respect to 45Ca2+ uptake and MTT metabolism in TNF-alpha-pretreated cells that were subjected to prolonged hypoxia. To determine the mechanism for the TNF-alpha-induced protective effect, the cells were pretreated with heat shock protein (HSP) 72 antisense oligonucleotides. These studies showed that the protective effect of TNF-alpha was not inhibited by antisense oligonucleotides, despite use of a concentration of antisense that was sufficient to attenuate the TNF-alpha-induced increase in HSP 72 expression. Subsequent studies using mutated TNF ligands showed that activation of both types 1 and 2 TNF receptors was sufficient to confer a protective response in isolated cardiac myocytes through an as yet unknown pathway(s). CONCLUSIONS Taken together, the above observations demonstrate that TNF-alpha pretreatment confers resistance to hypoxic stress in the adult cardiac myocyte through a novel mechanism that appears to be different from but not necessarily exclusive of the protective response conferred by HSP 72 expression.

Cytosolic Heat Shock Protein 60, Hypoxia, and Apoptosis

Background—Heat shock protein (HSP)60 is an abundant protein found primarily in the mitochondria, though 15% to 20% is found in the cytosol. Previously we observed that HSP60 complexes with bax in the cytosol. Reduction in HSP60 precipitates translocation of bax to the mitochondria and apoptosis. We hypothesized that HSP60 would decrease with hypoxia/reoxygenation and that this would precipitate bax translocation to the mitochondria and release of cytochrome c. Methods and Results—Adult rat cardiac myocytes were studied at end-hypoxia and at 10 and 24 hours of reoxygenation. HSP60 levels were unchanged at end-hypoxia and decreased 33% and 40% at 10 and 24 hours of reoxygenation, whereas HSP72 increased 80% and 110%. Bax and bcl-2 decreased during reoxygenation. However, cytochrome c release occurred at end-hypoxia, before reoxygenation. Cell fractionation was done to analyze this further. In normal myocytes, bax and HSP60 were present in the cytosol, and bax coimmunoprecipitated with cytosolic HSP60. At end-hypoxia, mitochondrial HSP60 was unchanged, but cytosolic HSP60 had disappeared and was now in the plasma membrane fraction. Concurrently, bax was no longer in the cytosol but now in the mitochondria. Thus, although total HSP60 remained the same, it no longer complexed with bax, and bax was free to translocate to the mitochondria and precipitate apoptosis. Reduction in ATP had a similar effect. Conclusions—These studies show that hypoxia results in disassociation of the HSP60-bax complex with translocation of cytosolic HSP60 to the plasma membrane and bax to the mitochondria. This is sufficient to trigger apoptosis.

Molecular Coupling of a Ca2+-Activated K+ Channel to L-Type Ca2+ Channels via α-Actinin2

Cytoskeletal proteins are known to sculpt the structural architecture of cells. However, their role as bridges linking the functional crosstalk of different ion channels is unknown. Here, we demonstrate that a small conductance Ca2+-activated K+ channels (SK2 channel), present in a variety of cells, where they integrate changes in intracellular Ca2+ concentration [Ca2+i] with changes in K+ conductance and membrane potential, associate with L-type Ca2+ channels; Cav1.3 and Cav1.2 through a physical bridge, α-actinin2 in cardiac myocytes. SK2 channels do not physically interact with L-type Ca2+ channels, instead, the 2 channels colocalize via their interaction with α-actinin2 cytoskeletal protein. The association of SK2 channel with α-actinin2 localizes the channel to the entry of external Ca2+ source, which regulate the channel function. Furthermore, we demonstrated that the functions of SK2 channels in atrial myocytes are critically dependent on the normal expression of Cav1.3 Ca2+ channels. Null deletion of Cav1.3 channel results in abnormal function of SK2 channel and prolongation of repolarization and atrial arrhythmias. Our study provides insight into the molecular mechanisms of the coupling of SK2 channel with voltage-gated Ca2+ channel, and represents the first report linking the coupling of 2 different types of ion channels via cytoskeletal proteins.

Estrogen and the cardiovascular system

Estrogen is a potent steroid with pleiotropic effects, which have yet to be fully elucidated. Estrogen has both nuclear and non-nuclear effects. The rapid response to estrogen, which involves a membrane associated estrogen receptor(ER) and is protective, involves signaling through PI3K, Akt, and ERK 1/2. The nuclear response is much slower, as the ER-estrogen complex moves to the nucleus, where it functions as a transcription factor, both activating and repressing gene expression. Several different ERs regulate the specificity of response to estrogen, and appear to have specific effects in cardiac remodeling and the response to injury. However, much remains to be understood about the selectivity of these receptors and their specific effects on gene expression. Basic studies have demonstrated that estrogen treatment prevents apoptosis and necrosis of cardiac and endothelial cells. Estrogen also attenuates pathologic cardiac hypertrophy. Estrogen may have great benefit in aging as an anti-inflammatory agent. However, clinical investigations of estrogen have had mixed results, and not shown the clear-cut benefit of more basic investigations. This can be explained in part by differences in study design: in basic studies estrogen treatment was used immediately or shortly after ovariectomy, while in some key clinical trials, estrogen was given years after menopause. Further basic research into the underlying molecular mechanisms of estrogens actions is essential to provide a better comprehension of the many properties of this powerful hormone.

Rapid expression of fibronectin in the rabbit heart after myocardial infarction with and without reperfusion.

The expression of fibronectin in the repair process after myocardial infarction was studied using two protocols of coronary occlusion in the rabbit: a permanent occlusion or 3 h of occlusion followed by reperfusion (too late for salvage). We found a rapid and progressive increase in cardiac fibronectin expression in the infarcted region of the ventricle. Steady-state mRNA levels for fibronectin increased 13- and 16-fold, respectively, in the permanent and reperfused infarcts 1 d postinfarction. Immunological detection of the protein with a polyclonal antibody against plasma fibronectin showed significant increases of the protein fibronectin in the infarcted myocardium by day 3 in the reperfused group and by day 5 in the permanent coronary occlusion group. Ribonuclease protection assays established the induction of EIIIB containing fibronectin mRNA in both models by day 1 and use of a monoclonal antibody showed an increase in the EIIIA isoform 2 d postinfarction. Increases in steady-state mRNA levels for several collagen types were found in both groups, but these changes occurred after those noted for fibronectin. Thus fibronectin mRNA and protein expression increased rapidly postinfarction suggesting a functional role in the repair process.

Triglyceride-Rich Lipoproteins Prime Aortic Endothelium for an Enhanced Inflammatory Response to Tumor Necrosis Factor-α

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-α. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor–associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-&kgr;B, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.