Myocardial Protein Degradation Research Articles

Changes in AMPKα and Ubiquitin Ligases in Myocyte Reverse Remodeling after Surgical Ventricular Reconstruction in rats with ischemic cardiomyopathy.

The change in myocardial protein degradation systems after ventricular unloading has been unknown. We aimed to evaluate the anti-hypertrophic protein adenosine monophosphate-activated protein kinase (AMPK) and two major protein degradation systems (ubiquitin proteasome system and autophagy) in a model of surgical ventricular reconstruction (SVR) in rats with ischemic cardiomyopathy. Rats were randomized into the following groups: sham/sham (control group), myocardial infarction (MI)/sham (sham group) and MI/SVR (SVR group), with an interval of 4 weeks. Two (early, n = 5 for each) and 28 days (late, n = 5 for each) after SVR, ventricular size, and wall stress were assessed. Myocyte area, protein expression of AMPKα and autophagy markers, and gene expression of ubiquitin ligases (Atrogin-1 and Murf-1) were evaluated in the late phase. In the early phase, left ventricular dimensions and wall stress were smaller in the SVR group than in the sham group, whereas they were comparable in the late period. Myocyte area in the SVR group was reduced to the value in the control group, while it was larger in the sham group than in the control group. Total-AMPKα, p-AMPKα, and AMPKα phosphorylation rates were higher, and Atrogin-1 and Murf-1 were lower in the SVR group than in the sham group, while the autophagy markers were not different between the groups. p-AMPKα had strong negative correlations with myocyte area, Atrogin-1, and Murf-1. In myocyte reverse remodeling after SVR, AMPKα phosphorylation increased in association with reduced gene expression of ubiquitin ligases.

Temporally Distinct Regulation of Pathways Contributing to Cardiac Proteostasis During the Acute and Recovery Phases of Sepsis.

Cardiac dysfunction is a common manifestation of sepsis and is associated with early increases in inflammation and decreases in myocardial protein synthesis. However, little is known regarding the molecular mechanisms regulating protein homeostasis during the recovery phase after the removal of the septic nidus. Therefore, the purpose of this study was to investigate diverse signal transduction pathways that regulate myocardial protein synthesis and degradation. Adult male C57BL/6 mice were used to identify potential mechanisms mediating the acute (24 h) effect of cecal ligation and puncture as well as long-term changes that manifest during the chronic (10 days) recovery phase. Sepsis acutely decreased cardiac protein synthesis that was associated with reduced phosphorylation of S6K1/S6 but not 4E-BP1. Sepsis also decreased proteasome activity, although with no change in MuRF1 and atrogin-1 mRNA expression. Sepsis acutely increased apoptosis (increased caspase-3 and PARP cleavage), autophagosome formation (increased LC3B-II), and canonical inflammasome activity (increased NLRP3, TMS1, cleaved caspase-1). In contrast, during the recovery phase, independent of a difference in food consumption, global protein synthesis was increased, the early repression in proteasome activity was restored to basal levels, whereas stimulation of apoptosis, autophagosome formation, and the canonical inflammasome pathway had abated. However, during recovery there was a selective stimulation of the noncanonical inflammasome pathway as evidenced by activation of caspase-11 with cleavage of Gasdermin D. These data demonstrate a temporally distinct homeostatic shift in the cardiac proteostatic response to acute infection and recovery.

Alcoholic Cardiomyopathy: Disrupted Protein Balance and Impaired Cardiomyocyte Contractility.

Alcoholic cardiomyopathy (ACM) can develop after consumption of relatively large amounts of alcohol over time or from acute binge drinking. Of the many factors implicated in the etiology of ACM, chronic perturbation in protein balance has been strongly implicated. This review focused on recent contributions (since 2010) in the area of protein metabolism and cardiac function related to ACM. Data reviewed include that from invitro and preclinical invivo animal studies where alcohol or an oxidative metabolite was studied and outcome measures in either cardiomyocytes or whole heart pertaining to protein synthesis or degradation were reported. Additionally, studies on the contractile properties of cardiomyocytes were also included to link signal transduction with function. Methodological differences including the potential impact of sex, dosing, and duration/timing of alcohol administration are addressed. Acute and chronic alcohol consumption decreases cardiac protein synthesis and/or activation of proteins within the regulatory mammalian/mechanistic target of rapamycin complex pathway. Albeit limited, evidence suggests that myocardial protein degradation via the ubiquitin pathway is not altered, while autophagy may be enhanced in ACM. Alcohol impairs exvivo cardiomyocyte contractility in relation to its metabolism and expression of proteins within the growth factor pathway. Dysregulation of protein metabolism, including the rate of protein synthesis and autophagy, may contribute to contractile deficits and is a hallmark feature of ACM meriting additional sex-inclusive, methodologically consistent studies.

Biomarkers of Cardiotoxicity Among Multiple Myeloma Patients Subsequently Treated with Proteasome Inhibitor Therapy

BACKGROUNDCardiovascular (CV) events are a known complication to proteasome inhibitor therapy in myeloma. Underlying mechanisms are unknown. We recently completed an investigator initiated, single institution Phase II study of high dose carfilzomib (56mg/m2) in patients with relapsed/refractory MM (NCT01351623). Among 42 response evaluable patients, 11 patients (25%) developed treatment-emergent heart failure of any grade, and 5 patients (11%) developed severe heart failure requiring mechanical ventilation. We undertook a study to identify potential biomarkers that may point to underlying mechanisms of CV events among multiple myeloma patients treated with carfilzomib therapy. METHODSWe performed a nested case-control study with 7 patients who experienced a CV event on our high dose carfilzomib study and had pre-treatment (baseline) plasma stored and 19 case matched controls treated on the same study who did not have a CV event. We screened for 90 proteins known to be associated with CV disease using O-linked glycosylation. We used the Proseek Multiplex CVD I 96x96 platform which is based on the Proximity Extension Assay (PEA) technique. PEA is a 96-plex immunoassay that allows high throughput detection of protein biomarkers in liquid samples. For each biomarker, a matched pair of antibodies linked to unique oligonucleotides (proximity probes) binds to the respective protein target. Upon binding, the unique proximity probes can hybridize to each other and subsequently be detected and quantified by real-time PCR. Mean biomarker levels were compared using a t-test. False discovery rate (FDR) was used for multiple comparisons adjustment. RESULTSUsing samples collected prior to initiation of carfilzomib therapy, in an agnostic statistical model we identified the following four proteins to have altered levels in myeloma patients who developed CV events (p=0.002-0.004, unadjusted; p=0.089, after FDR correction): matrix metalloproteinase-1 (MMP-1, heparin-binding EGF-like growth factor (HB-EGF), TNF-related apoptosis-inducing ligand (TRAIL), and myoglobin (MB). Myeloma patients who developed CV events had 37% lower MMP-1, 15% lower MB, and 4% lower HB-EGF, while TRAIL was 7% higher in patients who developed CV events. Matrix metalloproteinases are a family of proteolytic enzymes responsible, among other functions, for myocardial extracellular protein degradation. Interestingly, several MMP species, including MMP-1, have been identified within the human myocardium and are thought to be dysregulated in congestive heart failure. HB-EGF is a mitogenic and chemotactic glycoprotein that is essential for maintaining normal cardiac function and is known to play an important role in myocardial remodeling. CONCLUSIONSWe found that there was a trend towards lower MMP-1, HB-EGF, and MB levels and higher TRAIL levels in patients with CV events while receiving proteasome therapy. MMP-1 appears to be the most promising potential biomarker based on our data. Our study supports further investigation of these proteins as potential biomarkers for patients at risk of CV events when treated with carfilzomib.Table 1CV eventNo CV eventN=7N=19CKD Proteins1Mean (SD)Mean(SD)Unadjusted P-valueAdjusted P-valueMMP_11.7 (0.5)2.7 (0.9)0.0020.089HB_EGF6.9 (0.2)7.2 (0.3)0.0040.089TRAIL8 (0.3)7.5 (0.5)0.0040.089MB5 (0.5)5.9 (0.8)0.0040.089HSP_272.2 (0.3)2.7 (0.8)0.0320.528PDGF_subunit_B4 (0.7)5 (1.5)0.0360.528CD40_L3.4 (0.6)4.2 (1.2)0.0420.533EGF3.7 (0.9)4.7 (1.4)0.0530.592CX3CL15.9 (0.2)5.6 (0.6)0.0920.895TRAIL_R24.2 (0.4)4.6 (0.6)0.1010.8951. Proteins are listed based on the p-value associated with the difference between patients who did and did not have CV events, with lowest p-value on the top. The top 10 biomarkers are shown. DisclosuresEkman:Olink Bioscience: Employment. Grundberg:Olink Bioscience: Employment. Hassoun:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda: Research Funding; Novartis: Consultancy. Lesokhin:Aduro: Consultancy; Efranat: Consultancy; Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Landau:Janssen: Consultancy; Prothena: Consultancy, Honoraria; Janssen: Consultancy; Spectrum Pharmaceuticals: Honoraria; Onyx: Honoraria, Research Funding; Takeda: Research Funding. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding. Landgren:Onyx: Honoraria; Celgene: Honoraria; BMJ Publishing: Consultancy; International Myeloma Foundation: Research Funding; Bristol-Myers Squibb: Honoraria; Onyx: Research Funding; Medscape: Consultancy; Medscape: Honoraria; BMJ Publishing: Honoraria; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Losartan treatment attenuates tumor-induced myocardial dysfunction

Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin–angiotensin–aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. Methods and results: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8weeks of age. Simultaneously, mice were administered Losartan (10mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. Conclusions: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation.

Myocardial ER chaperone activation and protein degradation occurs due to synergistic, not individual, cold and hypoxic stress

Environmental stress at high altitude affects the myocardium at the physiological and molecular level. Characterized by hypobaric hypoxia and low temperatures, the cumulative impact of these stressors on the protein folding homeostasis in the heart is yet unexplored. The present study evaluates the collective effect of cold and hypoxia on the myocardial protein oxidation and activation of the endoplasmic reticulum (ER) stress response. Adult rats were exposed to either a singular acute stress of cold (10 °C; C), hypobaric hypoxia (7620 m; H) or simultaneously to both cold and hypobaric hypoxia (CH) for 6 h. Hypoxic stress amplified the free radical generation in H and CH groups, leading to enhanced HIF-1α expression. Coupled to cold stress, reduced oxygen availability caused substantial protein oxidative modifications, as well as cardiac tissue injury and matrix remodeling, evident in the histological staining. Presence of oxidized proteins caused a significant upregulation in expression of ER chaperones GRP78 and PDI in the cold hypoxia exposed animals. Enhanced proteolytic activity signaled the removal of misfolded proteins. Linked intricately to cellular stress response, cell survival kinases were expressed higher in CH group; however apoptotic CHOP (C/EBP homologous protein) expression remained unaltered. Administration of ER stress inducer, tunicamycin along with cold hypoxic stress, caused a discernible increase in protein oxidation and GRP78 expression, along with a significant elevation in proteasome and apoptotic activity. Highlighting the significance of a synergistic, rather than individual, effect of low oxygen and temperature on the protein folding machinery, our study provides evidence for the activation of ER stress response in the myocardium under acute high altitude stress.

Caspase Inhibition Modulates Left Ventricular Remodeling Following Myocardial Infarction Through Cellular and Extracellular Mechanisms

Myocyte death occurs by necrosis and caspase-mediated apoptosis in myocardial infarction (MI). In vitro studies suggest caspase activation causes myocardial contractile protein degradation without inducing apoptosis. Thus, caspase activation may evoke left ventricular (LV) remodeling through independent processes post-MI. The effects of caspase activation on LV geometry post-MI remain unclear. This project applied pharmacologic caspase inhibition (CASPI) to a porcine model of MI. Pigs (34 kg) were instrumented to induce 60 minutes of coronary artery occlusion followed by reperfusion and a 7-day follow-up period. Upon reperfusion, the pigs were randomized to saline (n = 12) or CASPI (n = 10, IDN6734, 6 mg/kg i.v., then 6 mg/kg/h for 24 hours). Plasma troponin-I values were reduced with CASPI compared with saline at 24 hours post-MI (133 +/- 15 vs. 189 +/- 20 ng/mL, respectively, P < 0.05). LV end-diastolic area (echocardiography) and interregional length (sonomicrometry) increased from baseline in both groups but were attenuated with CASPI by 40% and 90%, respectively (P < 0.05). Myocyte length was reduced with CASPI compared with saline (128 +/- 3 vs. 141 +/- 4 microm, respectively, P < 0.05). Plasma-free pro-matrix metalloproteinase-2 values increased from baseline with CASPI (27% +/- 6%, P < 0.05) indicative of reduced conversion to active MMP-2. Separate in vitro studies demonstrated that activated caspase species cleaved pro-MMP-2 yielding active MMP-2 forms and that MMP activity was increased in the presence of activated caspase-3. CASPI attenuated regional and global LV remodeling post-MI and altered viable myocyte geometry. Caspases increased MMP activity in vitro, whereas CASPI modified conversion of MMP-2 to the active form in vivo. Taken together, the results of the present study suggest that the elaboration of caspases post-MI likely contribute to LV remodeling through both cellular and extracellular mechanisms.

Long-Term caspase inhibition ameliorates apoptosis, reduces myocardial troponin-I cleavage, protects left ventricular function, and attenuates remodeling in rats with myocardial infarction

ObjectivesThis study was designed to evaluate whether in vivo caspase inhibition can prevent myocardial contractile protein degradation, improve myocardial function, and attenuate ventricular remodeling. BackgroundApoptosis is thought to play an important role in the development and progression of heart failure (HF) after a myocardial infarction (MI). However, it is not known whether inhibiting apoptosis can attenuate left ventricular (LV) remodeling and minimize systolic dysfunction. MethodsA 28-day infusion of caspase inhibitor (n = 12) or vehicle (n = 9) was administered to rats immediately after an anterior MI. In addition, five sham-operated rats given the caspase inhibitor were compared with 17 untreated sham-operated animals to study effects in non-MI rats. Left ventricular function, remodeling parameters, and hemodynamics were studied four weeks later. Myocardial caspase 3 activation and troponin-I contractile protein cleavage were studied in the non-infarct, remote LV myocardium using Western blots. Apoptosis was assessed using immunohistochemistry for activated caspase-positive cells as well as the TUNEL method. Collagen volume was estimated using morphometry. ResultsCaspase inhibition reduced myocardial caspase 3 activation. This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus, and fewer apoptotic cardiomyocytes. Furthermore, caspase inhibition reduced LV-weight-to-body-weight ratio, decreased myocardial interstitial collagen deposition, attenuated LV remodeling, and better preserved LV systolic function after MI. ConclusionsCaspase inhibition, started soon after MI and continued for four weeks, preserves myocardial contractile proteins, reduces systolic dysfunction, and attenuates ventricular remodeling. These findings may have important therapeutic implications in post-MI HF.

Redox pacing of proteome turnover: influences of glutathione and ketonemia

In early starvation tissue protein degradation increases, however in later starvation proteolysis declines so as to pace gradual atrophy during synthetic failure. Secondary decline of proteolytic pathways under progressive nutritional desperation is unexplained. After several days of starvation tissue GSH is partly depleted and GSSG/GSH is increased, followed by onset of ketonemia from fat breakdown. Ketone bodies inexplicably delay net muscle protein loss. Recent studies identify a proteome subset of more than 200 proteins with reactive sulfhydryl sites as candidates for coordinate redox control of diverse cell functions. Ketones cause protein sulfhydryl oxidation and protein S-glutathionylation. Here, redox-responsive proteolytic pathways were bio-assayed by release of [ 3H]leucine from rat myocardium under non-recirculating perfusion. More than 75% of myocardial protein degradation was inhibited and defined by infusion of diamide (100 μM) under constant physiologic concentrations of complete amino acids. Diamide-inhibitable proteolysis includes all lysosomal and some extra-lysosomal proteolysis. Following diamide washout, the reversal of proteolytic inhibitory action was greatly enhanced by artificial repletion of GSH by supra-physiologic extra-cellular GSH (1 mM) exposure. Therefore, GSH maintains much of constitutive protein degradation in a primary tissue bioassay. Physiologic acetoacetate infusion (5 mM) inhibited redox-responsive protein degradation. Uniformly [ 3H]leucine labeled 3T3 cells exhibited similar redox-dependent and redox-independent subcomponents of protein degradation. Independent of ketones, steady state cathepsin B reaction rate ex vivo was graded in proportion to the GSH concentration without GSSG, and inversely proportional to the GSSG/GSH redox ratio with inhibitory threshold at 0.5% oxidized. Linkage of some cysteine protease reaction rates to the interplay between GSH-GSSG/GSH status and ketonemia is suggested among transcendent mechanisms coordinating and pacing proteome turnover under prolonged starvation. The possibility of pre-emptive, redox coordination of distinct proteolytic pathways is speculatively discussed.

Extracellular degradative pathways in myocardial remodeling and progression to heart failure

A milestone in the progression of congestive heart failure (CHF) is myocardial remodeling. Left ventricular (LV) remodeling during the progression of CHF is accompanied by changes in the structure of the myocardial extracellular matrix. The myocardial extracellular matrix is composed of structural proteins and biologically active molecules that contribute to the maintenance of LV myocardial architecture and function. A family of proteolytic enzymes responsible for myocardial extracellular protein degradation is the matrix metalloproteinases (MMPs). A number of MMP species have been identified within the human myocardium and are increased with the development of CHF. Certain MMPs that are expressed at very low levels in normal myocardium such as collagenase-3 (MMP-13) and the membrane-type MMPs (MT-MMPs) are substantially upregulated in CHF and likely contribute to the pathologic remodeling that occurs within the myocardium. However, not all MMP species are uniformly increased in patients with end-stage CHF, suggesting that a specific portfolio or cassette of MMPs are expressed in the failing myocardium. The necessary next steps for improving our understanding of this myocardial MMP system include (1) the identification of the MMP species that are upregulated in CHF, (2) elucidation of the upstream signaling pathways that contribute to MMP induction, (3) the development of methods to detect early myocardial MMP activation in patients with developing CHF, and (4) the examination of the effects of pharmacologic modulators of the MMP system in animal models of CHF. The results from these future studies may yield some novel therapeutic strategies by which to control myocardial extracellular remodeling and thereby slow the progression of the CHF process.

Matrix metalloproteinases: regulation and dysregulation in the failing heart.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible for myocardial extracellular protein degradation. Several MMP species identified within the human myocardium may be dysregulated in congestive heart failure (CHF). For example, MMPs that are expressed at very low levels in normal myocardium, such as collagenase-3 (MMP-13) and the membrane-type-1 MMPs, are substantially upregulated in CHF. However, MMP species are not uniformly increased in patients with end-stage CHF, suggesting that a specific portfolio of MMPs are expressed in the failing myocardium. With the use of animal models of CHF, a mechanistic relationship has been demonstrated with respect to myocardial MMP expression and the left ventricular (LV) remodeling process. The tissue inhibitors of the MMPs (TIMPs) are locally synthesized proteins that bind to active MMPs and thereby regulate net proteolytic activity. However, there does not appear to be a concomitant increase in myocardial TIMPs during the LV remodeling process and progression to CHF. This disparity between MMP and TIMP levels favors a persistent MMP activation state within the myocardium and likely contributes to the LV remodeling process in the setting of developing CHF. The elucidation of upstream signaling mechanisms that contribute to the selective induction of MMP species within the myocardium as well as strategies to normalize the balance between MMPs and TIMPs may yield some therapeutic strategies by which to control myocardial extracellular remodeling and thereby slow the progression of the CHF process.

Pathogenesis of early cardiac myocyte damage after severe burns.

The importance of early cardiac myocyte damage during postburn trauma has been emphasized in recent years. However, its pathogenesis, prevention, and treatment have not been fully clarified. The aim of this study is to define its pathogenesis. Rats with 30% third-degree burns were used. Cardiac biochemical markers reflecting cardiac myocyte damage including troponin T, cardiac myosin light chain 1, creatinine kinase and its cardiac-specific isoenzyme compound, as well as inflammatory mediators such as tumor necrosis factor, endothelin/nitric oxide ratio, malondialdehyde, and superoxide dismutase, were determined. Cardiac biochemical markers reflecting cardiac myocyte damage, including troponin T, cardiac myosin light chain 1, cardiac-specific isoenzyme compound, were all significantly elevated between 3 hours and 24 hours after burn. Changes in tumor necrosis factor, endothelin/nitric oxide ratio, and malondialdehyde were similar to those of cardiac biochemical markers. In contrast, levels of superoxide dismutase declined markedly after burn. The findings of this study showed that considerable amounts of myocardial constructive protein degradation and release due to destruction of cardiac myocytes occurred early after severe burns. The inflammatory mediators released after burn injury may be involved in the pathogenesis of myocardial destruction.

Hyperinsulinemia inhibits myocardial protein degradation in patients with cardiovascular disease and insulin resistance.

Insulin resistance, hyperinsulinemia, and myocardial hypertrophy frequently coexist in patients. Whether hyperinsulinemia directly affects myocardial protein metabolism in humans has not been examined, however. To test the hypothesis that hyperinsulinemia is anabolic for human heart protein, we examined the effects of insulin infusion on myocardial protein synthesis, degradation, and net balance in patients with ischemic heart disease. Eleven men (aged 57 +/- 3 years) with coronary artery disease who had fasted for 12 to 16 hours received a constant infusion of insulin (50 mU.m-2.min-1) while plasma concentrations of glucose and amino acids were kept constant. Rates of myocardial protein synthesis, degradation, and net balance were estimated from steady state extraction and isotopic dilution of L-[ring-2,6-3H]phenylalanine across the heart basally and 90 minutes into infusion. Subjects had elevated fasting plasma insulin concentrations (173 +/- 21 pmol/L) and used little exogenous glucose during insulin infusion, suggesting resistance to the effects of insulin on whole-body carbohydrate metabolism. Basally, myocardial protein degradation, as estimated by phenylalanine release (133 +/- 28 nmol/min), exceeded protein synthesis, estimated by phenylalanine uptake (31 +/- 15 nmol/min), resulting in net negative phenylalanine balance (-102 +/- 17 nmol/min). Insulin infusion reduced myocardial protein degradation by 80% but did not affect protein synthesis, returning net phenylalanine balance to neutral. Acute hyperinsulinemia markedly suppresses myocardial protein degradation in patients with cardiovascular disease who are resistant to its effects on whole-body glucose metabolism. This antiproteolytic action represents a potential mechanism by which hyperinsulinemia could contribute to the development of myocardial hypertrophy in patients with cardiovascular disease.

Effects of insulin, biguanide antihyperglycaemic agents and β-adrenergic agonists on pathways of myocardial proteolysis

Pathways of bulk protein degradation controlled by insulin and isoprenaline (isoproterenol) were distinguished in Langendorff-perfused rat hearts. Proteins were biosynthetically labelled in vitro with [3H]leucine, followed by addition of 2 mM non-radioactive leucine to competitively prevent reincorporation. Rapidly degraded proteins were eliminated during a 3 h preliminary perfusion period without insulin. One third of bulk myocardial protein degradation was inhibited by isoprenaline as described previously. An insulin concentration of 5 nM maximally inhibited proteolysis, beginning within 2 min. Inhibition reached 32% within 1.25 h and 35% after 1.5 h. The minimum effective insulin concentration was approx. 10-50 pM, which caused 10-20% inhibition. Following 3 h of perfusion without insulin, the lysosomal inhibitor, chloroquine (30 microM), inhibited 38% of bulk degradation. The 35% proteolytic inhibition caused by insulin was followed by very little further inhibition on subsequent concurrent infusion of chloroquine, i.e. the inhibitory effects of insulin and chloroquine were not additive. In contrast, prior inhibition of lysosomal proteolysis by insulin or chloroquine did not prevent the subsequent additive inhibition caused by isoprenaline. Insulin and beta-agonists additively inhibited approx. two-thirds of bulk degradation. The biguanide antihyperglycaemic agent phenformin (2 microM) inhibited 35% of bulk degradation, beginning at 2 min and reaching a near maximum at approx. 1.25-1.5 h. Following inhibition of proteolysis with phenformin (20 microM), subsequent infusion of chloroquine (30 microM) produced only a slight additional inhibition. Following inhibition of 35% of degradation by 1.5 h of perfusion with insulin (5 nM), subsequent exposure to phenformin (2 microM) produced only a slight additional inhibition which did not exceed 38% of basal proteolysis. Thus insulin and phenformin both inhibit lysosomal proteolysis; however, the adrenergic-responsive pathway is distinct.

Regional differences in the in vivo synthesis and degradation of myosin subunits in rabbit ventricular myocardium.

To test for regional differences in the rates of synthesis and degradation of contractile proteins during normal physiological growth of the heart in vivo, fractional rates of protein accumulation and synthesis were assessed for total protein, myosin heavy chain, myosin light chain, phosphorylatable myosin light chain, and actin in the right and left ventricular free walls of growing, New Zealand White rabbits. Fractional rates of protein accumulation were determined from regional protein content growth curves of total and individual myofibrillar proteins measured in 82 animals ranging from 1 day to 9 weeks of age. In vivo right and left ventricular fractional rates of protein synthesis were determined by the [3H]leucine constant infusion method in 9-week-old rabbits. At this age, right and left ventricular fractional accumulation rates for total protein, myosin subunits, and actin were found to be identical, thus allowing for the comparison of the effect of regional hemodynamic load on protein degradation independent of its effect on growth. Total protein and individual myofibrillar protein fractional degradative rates were then derived as the difference between fractional rates of synthesis and accumulation. The results indicate increased fractional synthesis and degradation of myosin heavy chain and light chains, but not actin, in the left compared to the right ventricular free wall. Accelerated left ventricular replacement of myosin subunits may be related to regional differences in hemodynamic load. These results help explain the apparent increase in the in vivo rate of myocardial protein degradation observed in several experimental models of ventricular hypertrophy.

Toxic cardiomyopathy: The effect of antipsychotic-antidepressant drugs and calcium on myocardial protein degradation and structural integrity

In the nonrecirculating isolated perfused rat heart it has recently been described that basal myocardial protein degradation is suppressed by 30% within 5 min of maximal β-adrenergic receptor occupancy under 5 × 10 −7 m isoproterenol ( Lockwood, 1985, Biochem. J. 231, 299–308). This adrenergic-controlled proteolytic process presumably contributes to the well-known normal coordination of myocardial protein mass with functional demand. It is presently reported that elevated intracellular calcium is among the messengers that somehow suppress protein degradation. Acute elevation of extracellular calcium to a maximal concentration of 9.0 m m mimicked the simultaneous effects of isoproterenol on increasing inotropy and decreasing protein degradation, although this concentration was eventually lethal. Conversely, infusion of trifluoperazine (TFP), a calmodulin-blocking antipsychotic drug, caused stimulation of protein degradation above basal levels within 5 min. The stimulation of degradation by 30–60% was transient at 5 × 10 −7 m and returned to the control level in 5–10 min. However, TFP produced massive irreversible release of amino acid peptides and proteins at 10 −5 m within 30 min, followed by grossly observable cell structural disruption and cell separation. The degradative stimulation caused by TFP was potentiated by lowering the normal 2.5-m m extracellular Ca 2+ concentration to 1.25 m m. Trifluoperazine at 10 −5 m caused longitudinal separation of myofibrils by disrupting lateral attachments between adjacent Z lines, leading to a loss of lateral myofibrillar registry followed by myofibrillar degeneration. Spot desmosomes were disrupted, leading to lateral cell separation; however, the fascia adherens region of the intercalated disks remained intact and cells maintained end-to-end attachment. Perfusion under the low extracellular Ca 2+ concentration of 0.1 m m for 0.5 hr caused separation of the fascia adherens region and spot desmosomes of the intercalated disks as well as disruption of cytoplasmic myofibrils and other changes. Although the structural disorganization caused by perfusion with low (0.1 m m) Ca 2+ were similar to those caused by TFP, cells also lost end to end attachment under low Ca 2+. Amitriptyline (10 −5 m), thioridazine (10 −5 m), and calmidazolium (10 −6 m) stimulated protein degradation and caused structural damage. It is speculated that the above Ca 2+-related phenomena describe the mechanism of the well-known toxic cardiomyopathy resulting from overdoses of some of the antipsychotic-antidepressant drugs. The antipsychotic agent, haloperidol, which has been reported to be noncardiotoxic and weakly inhibitory toward calmodulin, did not stimulate protein degradation, nor did it cause structural disruption even at a grossly supratherapeutic 8 × 10 −6 m concentration that completely suppressed contractile activity.

Simultaneous response of myocardial contractility and a major proteolytic process to beta-adrenergic-receptor occupancy in the Langendorff isolated perfused rat heart.

The Langendorff isolated rat heart was adapted to the study of minute-to-minute percentage changes in bulk protein degradation by using non-recirculating perfusion. Hearts were perfused at 8 ml/min at 35 degrees C with Krebs-Henseleit buffer containing 11 mM-glucose, and only hearts with regular ventricular rhythm were employed. Proteins were labelled by infusion of [3H]leucine for 0.5 h in vitro. A complete amino acid mixture was then added at 3 times normal rat extracellular concentrations. After labelling, the re-incorporation of [3H]leucine was competitively inhibited by addition of either 4 mM-leucine or 20 microM-cycloheximide. The residual unincorporated radioactivity and the preferentially labelled rapid-turnover proteins were eliminated during a 3 h preliminary perfusion period. The basal rate of release of [3H]leucine and percentage changes were then determined at 1 min intervals, by using each heart as its own control. Leucine metabolism was inconsequential to results. Exchange of intracellular leucine pools with extracellular leucine and subsequent release in effluent perfusate was 95% complete within approx. 2 min. The basal rate of protein degradation was unchanged by electrical stimulation of the heart rate to 360 beats/min or cessation of contractile activity by membrane depolarization under 25 mM-KCl. Infusion of the beta-agonist isoprenaline at 5-500 nM caused a graded inhibition of myocardial protein degradation within 5-6 min, with a maximum inhibition of 30%. This inhibition was sustained for at least 1 h of drug administration and was reversed within 4-6 min of cessation of isoprenaline or simultaneous infusion of 1 microM of the beta-receptor antagonist propranolol. Minute-to-minute adrenergic proteolytic control was a simultaneous co-variable with beta-receptor-mediated inotropic changes in right-intraventricular systolic pressure. Stoppage of the heart in asystole by the Ca2+-channel blocker nifedipine (0.7 microM) delayed the onset, but did not cause sustained reversal, of adrenergic-inhibited degradation, indicating the absence of a direct obligatory mechanistic linkage between the events of the contraction-relaxation cycle and protein degradation in this preparation.

Sequential metabolic alterations in the myocardium during influenza and tularemia in mice.

Mice with generalized influenza or tularemia of similar lethality were studied in an effort to compare biochemical responses of the myocardium during infections of viral and bacterial etiology. A progressive loss of body weight characterized the course of both infections. Accompanying this, the myocardial content of protein and the activities of lactate dehydrogenase, citrate synthase, and cytochrome c oxidase all decreased. However, myocardial protein degradation appeared earlier and was more pronounced in influenza, and the protein changes were accompanied by a rapid decline of myocardial RNA. Activation of acid hydrolases, such as cathepsin D and beta-glucuronidase, occurred in tularemia but not in influenza, whereas leakage of beta-glucuronidase into the plasma occurred in both infections. Conversely, there was a considerably greater activation of myocardial catalase in influenza. These findings suggested that different control mechanisms or metabolic pathways were operative in the degradation of myocardial constituents in influenza as compared with tularemia. The absence of histological signs of myocarditis in either infection appeared to exclude any direct local effects of an inflammatory process on myocardial cells. Since the infections were of comparable lethality (based upon the inoculated dose of organisms), the observed differences in pattern and extent of metabolic responses of the myocardium to these infections may be attributed to different pathophysiological mechanisms evoked by the different microorganisms.

Rat myocardial protein degradation.

1. Myocardial protein degradation rates were determined by following tyrosine release from rat isolated left hemi-atria in vitro. 2. After two 20 min preincubations the rate of tyrosine release from hemi-atria was constant for 4 h. 3. Skeletal muscle protein degradation was determined by following tyrosine release from rat isolated hemi-diaphragm (Fulks, Li & Goldberg, 1975). 4. Insulin (10(-7) M) inhibited tyrosine release from hemi-atria and hemi-diaphragm to a similar extent. A 48 h fast increased tyrosine release rate from hemi-diaphragm and decreased tyrosine release rate from hemi-atria. Hemi-diaphragm tyrosine release was inhibited by 15 mmol/l D-glucose but a variety of concentrations of D-glucose (0, 5, 15 mmol/l) had no effect on tyrosine release from hemi-atria. Five times the normal plasma levels of the branched-chain amino acids leucine, isoleucine and valine had no effect on tyrosine release from either hemi-atria or hemi-diaphragm.

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH

PROCEEDINGS OF THE AUSTRALIAN SOCIETY FOR MEDICAL RESEARCH