Patti L. Johnson

Isolation of skeletal muscle mitochondria from hamsters using an lonic medium containing ethylenediarninetetraacetic acid and nagarse

An improved procedure is reported for the isolation of skeletal muscle mitochondria from hamsters and compared with our previous method. This procedure utilizes 20 mg% Nagarse in an ionic medium containing 100 mM sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46 mM KCl, and 0.5% bovine serum albumin (BSA), at pH 7.4 (medium-B). Oxidative phosphorylation was studied by measuring ADP/O ratio and respiratory control ratio (RCR) using NAD(+)-linked pyruvate-malate (PM), as well as FAD-linked succinate (SUCC) as substrates. The mitochondria isolated in medium-B exhibited high RCR and high ADP phosphorylation capacity, and were superior to those prepared by our previous method. Electron micrographs of organelles isolated in medium-B revealed intact mitochondrial membrane and structural integrity, whereas those isolated with medium-A containing 50 mg% Nagarse depicted considerable damage including swelling, ruptured membrane, and loss of intramitochondrial matrix. Previously, we used a nonionic medium containing 210 mM mannitol, 70 mM sucrose, 0.1 mM EDTA, 10 mM Tris-HCl, 50 mg% Nagarse, and 0.5% BSA, at pH 7.4 (medium-A). Mitochondria isolated with medium-B yielded mean RCR values of 7.3 to 8.3 with PM, and values of 3.7 to 4.7 with SUCC as substrates, compared to 1.6 and 1.8 with PM, and 1.4 and 1.7 with SUCC for the organelles isolated using medium-A, respectively. Likewise, the ADP/O ratios were 2.6 to 2.7 with PM, and 1.6 to 1.7 with SUCC for medium-B preparations, compared to 1.5 and 1.8 with PM and 1.0 and 1.2 with SUCC for medium-A preparations, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupled calcium and zinc dyshomeostasis and oxidative stress in cardiac myocytes and mitochondria of rats with chronic aldosteronism.

A dyshomeostasis of extra- and intracellular Ca2+ and Zn2+ occurs in rats receiving chronic aldosterone/salt treatment (ALDOST). Herein, we hypothesized that the dyshomeostasis of intracellular Ca2+ and Zn2+ is intrinsically coupled that alters the redox state of cardiac myocytes and mitochondria, with Ca2+ serving as a pro-oxidant and Zn2+ as an antioxidant. Toward this end, we harvested hearts from rats receiving 4 weeks of ALDOST alone or cotreatment with either spironolactone (Spiro), an aldosterone receptor antagonist, or amlodipine (Amlod), an L-type Ca2+ channel blocker, and from age/sex-matched untreated controls. In each group, we monitored cardiomyocyte [Ca2+]i and [Zn2+]i and mitochondrial [Ca2+]m and [Zn2+]m; biomarkers of oxidative stress and antioxidant defenses; expression of Zn transporters, Zip1 and ZnT-1; metallothionein-1, a Zn2+-binding protein; and metal response element transcription factor-1, a [Zn2+]i sensor and regulator of antioxidant defenses. Compared with controls, at 4-week ALDOST, we found the following: (a) increased [Ca2+]i and [Zn2+]i, together with increased [Ca2+]m and [Zn2+]m, each of which could be prevented by Spiro and attenuated with Amlod; (b) increased levels of 3-nitrotyrosine and 4-hydroxy-2-nonenal in cardiomyocytes, together with increased H2O2 production, malondialdehyde, and oxidized glutathione in mitochondria that were coincident with increased activities of Cu/Zn superoxide dismutase and glutathione peroxidase; and (c) increased expression of metallothionein-1, Zip1 and ZnT-1, and metal response element transcription factor-1, attenuated by Spiro. Thus, an intrinsically coupled dyshomeostasis of intracellular Ca2+ and Zn2+ occurs in cardiac myocytes and mitochondria in rats receiving ALDOST, where it serves to alter their redox state through a respective induction of oxidative stress and generation of antioxidant defenses. The importance of therapeutic strategies that can uncouple these two divalent cations and modulate their ratio in favor of sustained antioxidant defenses is therefore suggested.

Uncoupling the coupled calcium and zinc dyshomeostasis in cardiac myocytes and mitochondria seen in aldosteronism.

Intracellular [Ca2+]i overloading in cardiomyocytes is a fundamental pathogenic event associated with chronic aldosterone/salt treatment (ALDOST) and accounts for an induction of oxidative stress that leads to necrotic cell death and consequent myocardial scarring. This prooxidant response to Ca2+ overloading in cardiac myocytes and mitochondria is intrinsically coupled to simultaneous increased Zn2+ entry serving as an antioxidant. Herein, we investigated whether Ca2+ and Zn2+ dyshomeostasis and prooxidant to antioxidant dysequilibrium seen at 4 weeks, the pathologic stage of ALDOST, could be uncoupled in favor of antioxidants, using cotreatment with a ZnSO4 supplement; pyrrolidine dithiocarbamate (PDTC), a Zn2+ ionophore; or ZnSO4 in combination with amlodipine (Amlod), a Ca2+ channel blocker. We monitored and compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i together with biomarkers of oxidative stress in cardiac myocytes and mitochondria. At week 4 of ALDOST and compared with controls, we found (1) an elevation in [Ca2+]i coupled with [Zn2+]i and (2) increased mitochondrial H2O2 production and increased mitochondrial and cardiac 8-isoprostane levels. Cotreatment with the ZnSO4 supplement alone, PDTC, or ZnSO4+Amlod augmented the rise in cardiomyocyte [Zn2+]i beyond that seen with ALDOST alone, whereas attenuating the rise in [Ca2+]i, which together served to reduce oxidative stress. Thus, a coupled dyshomeostasis of intracellular Ca2+ and Zn2+ was demonstrated in cardiac myocytes and mitochondria during 4-week ALDOST, where prooxidants overwhelm antioxidant defenses. This intrinsically coupled Ca2+ and Zn2+ dyshomeostasis could be uncoupled in favor of antioxidant defenses by selectively increasing free [Zn2+]i and/or reducing [Ca2+]i using cotreatment with ZnSO4 or PDTC alone or ZnSO4+Amlod in combination.

Vitamin D Analogs 17,20S(OH)2pD and 17,20R(OH)2pD Are Noncalcemic and Exhibit Antifibrotic Activity

LETTER TO THE EDITOR The active form of vitamin D, 1,25(OH)2D3, has a number of desirable properties in addition to its classical effect on bone and calcium metabolism. These include immunomodulatory, anti-inflammatory and anti-fibrotic properties, however, its use is limited because of the toxic (calcemic) effect at therapeutically relevant doses (Bikle, 2010; Szodoray et al., 2008). Our goal of this and related studies has been to identify natural noncalcemic vitamin D analogues for use as therapeutic agents to treat diseases like scleroderma and/or autoimmune diseases. We embarked upon a strategy to produce such agents with a short or long side chain. Specifically, we defined a novel steroidogenic pathway initiated by cleavage of the side chain of 7-dehydrocholestrol by P450scc to produce 7-dehydropregnenolone (7DHP), which could be further converted to hydroxy-5,7-dienal intermediates by classical steroidogenic enzymes (Slominski et al., 2004; Slominski et al., 2009). An example of the latter is 17,20(OH)27DHP (Shackleton et al., 2002), for which we established a chemical route of synthesis with UVB induced transformation to vitamin D–like 5Z,7E-3β,17,20-trihydroxy-9,10-secopregna-5,7,10 triene (17,20(OH)2pD) and lumisterol-like structures (Zmijewski et al., 2009). Since 17,20(OH)27DHP is produced in the body, at least under pathologic conditions (Smith Lemli Optiz Syndrome) (Shackleton et al., 2002), and it can be transformed in the skin to corresponding secosteroids when exposed to the UVB (Slominski et al., 2004; Zmijewski et al., 2009), these vitamin D-like hydroxyproducts are good candidates to serve as natural products, which by definition would have low toxicity. Supporting this, shortening of the side chain of vitamin D eliminates its calcemic activity (Holick et al., 1975). Thus, these novel vitamin D analogues could serve as candidates for treatment of autoimmune or fibrosing diseases since vitamin D3 displays well documented effects on these conditions in animal models (Moro et al., 2008). 17,20(OH)2D and lumisterol derivatives were synthesized as described previously (Zmijewski et al., 2009). For details see Figure S1. Female C57BL/6 mice 6 wks old were purchased from Jackson Labs and maintained on a regular laboratory chow diet. Mice were divided into groups of three to receive one of the following treatments by daily intraperitoneal injection for 14 days: Sterile sesame oil (50 µl) (Sigma Chemical Co., St. Louis, MO) or 3 µg/kg 17,20R(OH)2D3 or 17,20S(OH)2D3 dissolved in 50 µl sterile sesame oil. Mice tolerated the injections well without signs of clinical distress. On day 15 mice were euthanized, and sera were obtained for measurement of total Ca++ by atomic absorption spectrophotometry (Bhattacharya, 1977), inorganic phosphate (iP) by Malachite Green Assay Kit (Cayman Chemical) and fibroblast growth factor (FGF) 23 by ELISA (Immuntopics, San Clemente, CA). Bone mineral density (BMD) and bone mineral content (BMC) were determined for whole body (excluding head) and excised soft tissue-cleaned femurs by dual-energy X - ray absorptiometry (DEXA) using a GE Lunar PIXImus (GE Healthcare); quality control and calibration were carried out within 24 hours of each scanning period. An Instron Universal Test System (Instron Corp) was used for three-point bending (gap size, 7 mm) to determine the flexure stress of mouse femoral diaphyses. The load was applied at a constant displacement of 5 mm/min to failure. The femurs broke in the mid-diaphyseal region, and data were recorded as flexure stress in megapascals (MPa). Hearts, livers, spleens and kidneys were processed for histology. Human dermal fibroblasts were grown in 24 well Costar tissue culture plates for experiments to assess effect of vitamin D analogues on total collagen protein (Rahow et al., 1987), hyaluronan (Postlethwaite et al, 1989), type I collagen by ELISA (Chondrex) or Col1A1 mRNA by quantitative real time qPCR. We previously showed that 20(OH)D3 at a dose as high as 3.0 µg/kg in rats, has no calcemic activity (calcium = 10.4 ± 1.5 mg/dL(mean +SD) vs 9.3 ± 1.3 mg/dL for control) whereas 1,25(OH)2D3 at the same dose had the expected strong calcemic effect raising calcium to 16.0 ± 1.2 mg/dL (Slominski et al., 2010). In this study, we have found that 17,20S(OH)2pD and 17,20R(OH)2 pD at doses of 3 µg/kg had no significant effects on sera total Ca++ levels (Table 1). Examination of histologic sections of the hearts, kidneys, liver and spleen revealed lack of calcifications or identifiable toxicity (not shown). Thus, 17,20R(OH)2pD and 17,20R(OH)2pD show the same noncalcemic properties as the structurally similar 20(OH)pD (Holick et al., 1975) and 20(OH)D3 (Slominski et al., 2010). Table 1 Serum Calcium Levels, Serum, FGF-23 Levels and Bone Measurements in Mice Serum levels of the phosphatonin factor, FGF23 were not different in mice treated with sesame oil or 17,20S(OH)2pD (Table 1). However, 17,20R(OH)2pD did induce a significant elevation in serum levels of FGF23 (Table 1). When rats are administered 1,25(OH)2D3 serum levels of FGF23 increase dramatically [>5-fold increase by 0.3 µg/kg 1,25(OH)2D3], and iP in sera is markedly reduced (Saito et al., 2005). In vitro, addition of 1,25(OH)2D3 to cultures of osteoblasts increases FGF23 mRNA levels (Liu et al., 2006). In this study, levels of iP in sera were not significantly different between mice treated with sesame oil vs 17,20R(OH)2pD or 17,20S(OH)2pD (Table 1). The data suggest that 17,20R(OH)2pD has retained a weak ability to modestly increase FGF23 levels in serum but not sufficiently high to significantly reduce serum iP, while 17,20S(OH)2pD does not possess this FGF23 regulatory property of 1,25(OH)2D3. There were no differences in BMD, BMC or flexure stress between 17,20S(OH)2D3, 17,20R(OH)2D3 and sesame oil treated mice (Table 1). Table 2 shows that R and S forms of 17,20(OH)2pD and 17,20(OH)2pL, like 1,25(OH)2D3, significantly inhibit TGF-β1-induced total collagen protein and hyaluronan production by human dermal fibroblasts. We found no significant differences in fibroblast numbers per well and no significant differences in trypan blue exclusion. In agreement, 10−7 M 17,20S(OH)2pD or 17,20R(OH)2pD inhibited the TGF-β1-stimulated expression of Col1A1 mRNA (Figure S2), and secreted type I collagen protein (Figure S2, insert). Table 2 Inhibition in Human Dermal Fibroblasts Stimulated by TGF-β1* of Total Collagen and Hyaluronan Production by Novel Secosteroids In conclusion, novel dihydroxysecosteroidal derivatives with a short side chain are noncalcemic and exhibit antifibrotic activity; therefore they are excellent candidates for further testing in in vivo models of fibrosis and scleroderma.

Regulation of membrane-mediated chronic muscle degeneration in dystrophic hamsters by calcium-channel blockers: diltiazem, nifedipine and verapamil

Membrane-mediated excessive intracellular calcium accumulation (EICA) is a fundamental pathogenetic event associated with chronic muscle degeneration in patients with Duchenne muscular dystrophy (DMD), and in animals with hereditary muscular dystrophy (HMD). Because of potential Ca(2+)-channel blocking properties, we investigated the relative efficacies of chronic diltiazem (DTZM) (50 mg/kg/d), nifedipine (NFDN) (6 mg/kg/d), and verapamil (VPML) (25 mg/kg/d) therapies in reducing EICA and improving dystrophic pathobiology beginning in 30-day-old male BIO-14.6 strain dystrophic hamsters (DH). Each agent, and sterile distilled water as vehicle control, was given in a single daily oral dose for 180 days to four groups each of DH and BIO-F1B strain normal hamsters (NH). Plasma [Ca] and [Mg]; plasma aldolase (ALD), creatine kinase (CK), and lactate dehydrogenase (LDH) activities; relative cardiac hypertrophy and relative soleus hypertrophy; tissue [Ca] and [Mg] of the heart and rectus femoris muscle, histology of rectus femoris, and overall mortality rate were quantitated. Muscle Mg was not modified in DH, or by any of these agents. NFDN produced significant edema in the soleus and myocardium. During the 6-month therapeutic trial, 45% DH and 18% NH died on VPML, 27% DH and 9% NH on NFDN, and 20% DH controls on distilled water, but none on DTZM; suggesting that DTZM treated DH lived longer than DH controls. Relative efficacy in regulating EICA in both the cardiac and skeletal muscles; plasma ALD, CK, and LDH; and improving associated dystrophic pathobiology was found to be DTZM >>> NFDN > VPML. DTZM appears to be the most effective and safest agent in mitigating EICA in cardiac and skeletal muscles, efflux of intracellular enzymes, histopathology of dystrophic muscle with sporadic necrosis, and chronic muscle degeneration in DH with HMD. DTZM therapy also halted the high morbidity and mortality associated with the dystrophic pathobiology inherent in DH.

Mitochondria-targeted cardioprotection in aldosteronism

Chronic aldosterone/salt treatment (ALDOST) is accompanied by an adverse structural remodeling of myocardium that includes multiple foci of microscopic scarring representing morphologic footprints of cardiomyocyte necrosis. Our previous studies suggested that signal-transducer-effector pathway leading to necrotic cell death during ALDOST includes intramitochondrial Ca2+ overloading, together with an induction of oxidative stress and opening of the mitochondrial permeability transition pore (mPTP). To further validate this concept, we hypothesized that mitochondria-targeted interventions will prove to be cardioprotective. Accordingly, 8-week-old male Sprague-Dawley rats receiving 4 weeks ALDOST were cotreated with either quercetin, a flavonoid with mitochondrial antioxidant properties, or cyclosporine A (CsA), an mPTP inhibitor, and compared with ALDOST alone or untreated, age/sex-matched controls. We monitored mitochondrial free Ca2+ and biomarkers of oxidative stress, including 8-isoprostane and H2O2 production; mPTP opening; total Ca2+ in cardiac tissue; and collagen volume fraction to quantify replacement fibrosis, a biomarker of cardiomyocyte necrosis, and employed terminal deoxynucleotidyl transferase dUTP nick end labeling assay to address apoptosis in coronal sections of ventricular myocardium. Compared with controls, at 4 weeks ALDOST we found a marked increase in mitochondrial H2O2 production and 8-isoprostane levels, an increased propensity for mPTP opening, and greater concentrations of mitochondrial free [Ca2+]m and total tissue Ca2+, coupled with a 5-fold rise in collagen volume fraction without any terminal deoxynucleotidyl transferase dUTP nick end labeling-based evidence of cardiomyocyte apoptosis. Each of these pathophysiologic responses to ALDOST was prevented by quercetin or cyclosporine A cotreatment. Thus, mitochondria play a central role in initiating the cellular-subcellular mechanisms that lead to necrotic cell death and myocardial scarring. This destructive cycle can be interrupted and myocardium salvaged with its structure preserved by mitochondria-targeted cardioprotective strategies.

Calcium and zinc dyshomeostasis during isoproterenol-induced acute stressor state.

Acute hyperadrenergic stressor states are accompanied by cation dyshomeostasis, together with the release of cardiac troponins predictive of necrosis. The signal-transducer-effector pathway accounting for this pathophysiological scenario remains unclear. We hypothesized that a dyshomeostasis of extra- and intracellular Ca2+ and Zn2+ occurs in rats in response to isoproterenol (Isop) including excessive intracellular Ca2+ accumulation (EICA) and mitochondrial [Ca2+]m-induced oxidative stress. Contemporaneously, the selective translocation of Ca2+ and Zn2+ to tissues contributes to their fallen plasma levels. Rats received a single subcutaneous injection of Isop (1 mg/kg body wt). Other groups of rats received pretreatment for 10 days with either carvedilol (C), a β-adrenergic receptor antagonist with mitochondrial Ca2+ uniporter-inhibiting properties, or quercetin (Q), a flavonoid with mitochondrial-targeted antioxidant properties, before Isop. We monitored temporal responses in the following: [Ca2+] and [Zn2+] in plasma, left ventricular (LV) apex, equator and base, skeletal muscle, liver, spleen, and peripheral blood mononuclear cells (PBMC), indices of oxidative stress and antioxidant defenses, mitochondrial permeability transition pore (mPTP) opening, and myocardial fibrosis. We found ionized hypocalcemia and hypozincemia attributable to their tissue translocation and also a heterogeneous distribution of these cations among tissues with a preferential Ca2+ accumulation in the LV apex, muscle, and PBMC, whereas Zn2+ declined except in liver, where it increased corresponding with upregulation of metallothionein, a Zn2+-binding protein. EICA was associated with a simultaneous increase in tissue 8-isoprostane and increased [Ca2+]m accompanied by a rise in H2O2 generation, mPTP opening, and scarring, each of which were prevented by either C or Q. Thus excessive [Ca2+]m, coupled with the induction of oxidative stress and increased mPTP opening, suggests that this signal-transducer-effector pathway is responsible for Isop-induced cardiomyocyte necrosis at the LV apex.

Reversal of impaired oxidative phosphorylation and calcium overloading in the in vitro cardiac mitochondria of CHF-146 dystrophic hamsters with hereditary muscular dystrophy

Membrane-mediated excessive intracellular calcium accumulation (EICA), and diminished cellular energy charge are invariably present in the myocardium of CHF-146 strain dystrophic hamsters (DH) with hereditary muscular dystrophy (HMD) and hypertrophic cardiomyopathy (HC). Therefore, we investigated respiratory dysfunctions and Ca2+ overloading in the isolated cardiac mitochondria from young and old DH, and whether these abnormalities can be reversed by controlling EICA in the in vitro mitochondria upon chelating excessive Ca2+ from the isolation medium with EDTA. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as the disease controls. As an index of membrane-mediated EICA and chronic cellular degeneration, Ca and Mg concentrations were quantitated in the ventricular myocardium and in the cardiac mitochondria harvested in two different isolation media. Mitochondria from young and old DH, isolated in the absence of 10 mM EDTA (B0 medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control and ADP/O ratios, than those seen in NH. However, incorporation of 10 mM EDTA in the isolation medium (B medium) restored the mitochondrial functions and reduced massive Ca(2+)-overloading in the dystrophic organelles. Ca concentration in the in vitro mitochondria from DH was significantly higher than in NH, irrespective of the composition of the isolation medium and age of the hamsters. Furthermore, the dystrophic organelles isolated in B medium had a much lower Ca concentration, and markedly improved oxidative phosphorylation as seen in the cardiac mitochondria from NH, compared to those prepared using B0 medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of impaired oxidative phosphorylation and calcium overloading in the skeletal muscle mitochondria of CHF-146 dystrophic hamsters.

Membrane-mediated excessive intracellular calcium accumulation (EICA) and diminished cellular energy production are the hallmarks of dystrophic pathobiology in Duchenne and Becker muscular dystrophies. We reported reversal of respiratory damage and Ca(2+)-overloading in the in vitro cardiac mitochondria from CHF-146 dystrophic hamsters (DH) with hereditary muscular dystrophy (Bhattacharya et al., 1993). Here we studied respiratory dysfunctions in the skeletal muscle mitochondria from young and old DH, and whether these abnormalities can be reversed by reducing [Ca2+] in the isolation medium, thereby lowering intramitochondrial Ca(2+)-overloading. Age- and sex-matched CHF-148 albino normal hamsters (NH) served as controls. As an index of EICA and cellular degeneration, Ca and Mg levels were assayed in the skeletal muscle and mitochondria. Mitochondria from young and old DH, isolated without EDTA (BE medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control ratio and ADP/O ratios, compared to NH. Incorporation of 10 mM EDTA (Bo medium) in the isolation medium restored mitochondrial functions of the dystrophic organelles to a near-normal level, and reduced Ca(2+)-overloading. The mitochondrial Ca level in DH was significantly higher than in NH, irrespective of the medium. However, compared to Bo medium, the dystrophic organelles isolated in BE medium had lower Ca levels and markedly improved oxidative phosphorylation as seen in NH. Muscle Ca contents in the young and old DH were elevated relative to NH, showing a positive correlation with the increased mitochondrial Ca(2+)-sequestration. Dystrophic muscle also revealed Ca deposition with an abundance of Ca(2+)-positive and necrotic myofibers by light microscopy, and intramitochondrial Ca(2+)-overloading by electron microscopy, respectively. However, Mg levels in the muscle and mitochondria did not alter with age or dystrophy. These data parallel our observations in the heart, and suggest that functional impairments and Ca(2+)-overloading also occur in the skeletal muscle mitochondria of DH, and are indeed reversible if EICA is regulated by slow Ca(2+)-channel blocker therapy (Johnson and Bhattacharya, 1993).

Pathogenetic Roles of Intracellular Calcium and Magnesium in Membrane-Mediated Progressive Muscle Degeneration in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) in humans is a progressively crippling X-linked recessive neuromuscular disease with no effective treatment (Rowland, 1980; Moser, 1984). It is characterized by profound biochemical (Kar and Pearson, 1976; Bertoriniet al., 1982; Bhattacharya and Crawford, 1985), electrocardiographic (Sanyal and Johnson, 1982), histopathological (Bodensteiner and Engel, 1978; Emery and Burt, 1980; Bertoriniet al., 1982, 1984), and ultrastructural (Mokri and Engel, 1975; Oberc and Engel, 1977) abnormalities of skeletal and cardiac muscle, and a 70–80% reduced life expectancy. Although a “vascular hypothesis” implicating abnormal microvasculature has been presented in the past to explain many aspects of the dystrophic pathophysiology, the most tenable mechanism for the classical muscle degeneration in DMD is now widely attributed to a generalized functional and structural defect(s) in the plasma membrane integrity of myofibers (Mokri and Engel, 1975; Schotlandet al., 1977) and erythroeytes (Araki and Mawatari, 1971).