Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide

Objective

Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.

Methods

Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca²⁺-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca²⁺ and TPP⁺ ion-selective electrodes.

Results

Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca²⁺ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca²⁺-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca²⁺/mg protein in SSM and IFM, respectively), suggestive of increased Ca²⁺ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca²⁺ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).

Conclusion

These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca²⁺-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.     

Subsarcolemmal mitochondrial flashes induced by hypochlorite stimulation in cardiac myocytes

Abstract

Mitochondrial superoxide flash (mitoflash) reflects quantal and bursting superoxide production and concurrent membrane depolarization triggered by transient mitochondrial permeability transition in many types of cells, at the level of single mitochondria. Here we investigate reactive oxygen species (ROS)-mediated modulation of mitoflash activity in cardiac myocytes and report a surprising finding that hypochlorite ions potently and preferentially triggered mitoflashes in the subsarcolemmal mitochondria (SSM), whereas hydrogen peroxide (H₂O₂) elicited mitoflash activity uniformly among SSM and interfibrillar mitochondria (IFM). The striking SSM mitoflash response to hypochlorite stimulation remained intact in cardiac myocytes from NOX2-deficient mice, excluding local NOX2-mediated ROS as the major player. Furthermore, it occurred concomitantly with SSM Ca²⁺ accumulation and local Ca²⁺ and CaMKII signaling played an important modulatory role by altering frequency and unitary properties of SSM mitoflashes. These findings underscore the functional heterogeneity of SSM and IFM and the oxidant-specific responsiveness of mitochondria to ROS, and may bear important ramifications in devising therapeutic strategies for the treatment of oxidative stress-related heart diseases.     

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.     

Proteomic alterations of distinct mitochondrial subpopulations in the type 1 diabetic heart: contribution of protein import dysfunction

Diabetic cardiomyopathy is associated with increased risk of heart failure in type 1 diabetic patients. Mitochondrial dysfunction is suggested as an underlying contributor to diabetic cardiomyopathy. Cardiac mitochondria are characterized by subcellular spatial locale, including mitochondria located beneath the sarcolemma, subsarcolemmal mitochondria (SSM), and mitochondria situated between the myofibrils, interfibrillar mitochondria (IFM). The goal of this study was to determine whether type 1 diabetic insult in the heart influences proteomic make-up of spatially distinct mitochondrial subpopulations and to evaluate the role of nuclear encoded mitochondrial protein import. Utilizing multiple proteomic approaches (iTRAQ and two-dimensional-differential in-gel electrophoresis), IFM proteomic make-up was impacted by type 1 diabetes mellitus to a greater extent than SSM, as evidenced by decreased abundance of fatty acid oxidation and electron transport chain proteins. Mitochondrial phosphate carrier and adenine nucleotide translocator, as well as inner membrane translocases, were decreased in the diabetic IFM (P < 0.05 for both). Mitofilin, a protein involved in cristae morphology, was diminished in the diabetic IFM (P < 0.05). Posttranslational modifications, including oxidations and deamidations, were most prevalent in the diabetic IFM. Mitochondrial heat shock protein 70 (mtHsp70) was significantly decreased in diabetic IFM (P < 0.05). Mitochondrial protein import was decreased in the diabetic IFM with no change in the diabetic SSM (P < 0.05). Taken together, these results indicate that mitochondrial proteomic alterations in the type 1 diabetic heart are more pronounced in the IFM. Further, proteomic alterations are associated with nuclear encoded mitochondrial protein import dysfunction and loss of an essential mitochondrial protein import constituent, mtHsp70, implicating this process in the pathogenesis of the diabetic heart.     

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (∼10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C₂₀-, C₂₂-, and C₂₄-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.     

Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria

Mitochondria contribute to myocyte injury during ischemia. After 30 and 45 min of ischemia in the isolated perfused rabbit heart, subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, sustain a decrease in oxidative phosphorylation through cytochrome oxidase. In contrast, oxidation through cytochrome oxidase in interfibrillar mitochondria (IFM), located between the myofibrils, remains unaffected. Cytochrome oxidase activity in the intact membrane requires an inner mitochondrial membrane lipid environment enriched in cardiolipin. During ischemia, the content of cardiolipin decreased only in SSM, whereas the content of other phospholipids was preserved. Ischemia did not alter the composition of the cardiolipin that remained in SSM. Cardiolipin content was preserved in IFM during ischemia. Thus cardiolipin is a relatively early target of ischemic mitochondrial damage, leading to loss of oxidative phosphorylation through cytochrome oxidase in SSM.     

Mitochondrial iron accumulation with age and functional consequences

During the aging process, an accumulation of non-heme iron disrupts cellular homeostasis and contributes to the mitochondrial dysfunction typical of various neuromuscular degenerative diseases. Few studies have investigated the effects of iron accumulation on mitochondrial integrity and function in skeletal muscle and liver tissue. Thus, we isolated liver mitochondria (LM), as well as quadriceps-derived subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), from male Fischer 344 x Brown Norway rats at 8, 18, 29 and 37 months of age. Non-heme iron content in SSM, IFM and LM was significantly higher with age, reaching a maximum at 37 months of age. The mitochondrial permeability transition pore (mPTP) was more susceptible to the opening in aged mitochondria containing high levels of iron (i.e. SSM and LM) compared to IFM. Furthermore, mitochondrial RNA oxidation increased significantly with age in SSM and LM, but not in IFM. Levels of mitochondrial RNA oxidation in SSM and LM correlated positively with levels of mitochondrial iron, whereas a significant negative correlation was observed between the maximum Ca(2+) amounts needed to induce mPTP opening and iron contents in SSM, IFM and LM. Overall, our data suggest that age-dependent accumulation of mitochondrial iron may increase mitochondrial dysfunction and oxidative damage,thereby enhancing the susceptibility to apoptosis.     

Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria

Mitochondrial-derived oxidative injury contributes to cellular aging as well as to reperfusion-induced tissue damage. While the aging-heart suffers greater tissue damage following ischemia and reperfusion than the adult heart, the occurrence of aging-related alterations in mitochondrial oxidative metabolism in the elderly heart has remained uncertain. We determined if aging altered oxidative metabolism in either of the two populations of cardiac mitochondria, subsarcolemmal mitochondria (SSM) that reside beneath the plasma membrane or interfibrillar mitochondria (IFM) located between the myofibrils. SSM and IFM were isolated from 6-month adult and 24- and 28-month elderly Fischer 344 rat hearts. Aging-related alterations were limited to IFM, while SSM remained unaffected. Aging decreased the rate of oxidative phosphorylation in IFM, including when stimulated by electron donors specific for cytochrome oxidase. Cytochrome oxidase enzyme activity was decreased in IFM from aging hearts, while activity in SSM remained similar to adult controls. These findings allow future studies of aging-related decrements in oxidative function to focus upon IFM, while SSM provide an inherent control group of mitochondria that are free of aging-related alterations in oxidative function. The selective alteration of IFM during aging raises the possibility that the consequences of aging-induced mitochondrial dysfunction will be enhanced in specific subcellular regions of the senescent myocyte.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

Palmitic acid methyl ester induces cardiac hypertrophy through activating the GPR receptor-mediated changes of intracellular calcium concentrations and mitochondrial functions

Myocardial hypertrophy is associated with a significant increase in intracellular Ca²⁺, which can be induced by long-chain fatty acid. Palmitic acid methyl ester (PAME), a fatty acid ester released from adipose tissue, superior cervical ganglion, and retina, has been found to have anti-inflammation, antifibrosis, and peripheral vasodilation effects. However, the effects of PAME on cardiomyocytes are still unclear. The aim of this study was to determine whether PAME could disrupt the intracellular Ca²⁺ balance, leading to cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes were treated with various concentrations (10–100 μM) of PAME for 1–4 days. Cytosolic Ca²⁺ and mitochondrial Ca²⁺ concentrations were examined using Fura-2 AM and Rhod-2, respectively. After treatment with PAME for 4 days, mitochondrial Ca²⁺, an indicator of the state of mitochondrial permeability transition pore (MPTP), and cell death were monitored by flow cytometric analysis. ATP levels were detected using the ATP assay kit. Cardiomyocyte hypertrophy was analyzed by measuring the cardiac hypertrophy biomarker and cell area using quantitative real time-polymerase chain reaction, Western Blot analysis and immunofluorescence analysis. Our results show that PAME concentration- and time-dependently increased cytosolic and mitochondria Ca²⁺ through the mitochondrial calcium uniporter. Moreover, treatment with PAME for 4 days caused MPTP opening, thereby reducing ATP production and enhancing reactive oxygen species (ROS) generation, and finally led to cardiomyocyte hypertrophy. These effects caused by PAME treatment were attenuated by the G-protein coupled receptor 40 (GPR40) inhibitor. In conclusion, PAME impaired mitochondrial function, which in turn led to cardiomyocyte hypertrophy through increasing the mitochondrial Ca²⁺ levels mediated by activating the GPR40 signaling pathway.     

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Structural differences in two biochemically defined populations of cardiac mitochondria

To determine whether there are structural differences in two topologically separated, biochemically defined mitochondrial populations in rat heart myocytes, the interior of these organelles was examined by high-resolution scanning electron microscopy. On the basis of a count of 159 in situ subsarcolemmal mitochondria (SSM, i.e., those that directly abut the sarcolemma), these organelles possess mainly lamelliform cristae (77%), whereas the cristae in in situ interfibrillar mitochondria (IFM, i.e., those situated between the myofibrils, n = 300) are mainly tubular (55%) or a mixture of tubular and lamelliform (24%). Isolated SSM (n = 374), similar to their in situ counterparts, have predominantly lamelliform cristae (75%). The proportions of crista types in isolated IFM (n = 337) have been altered, with only 20% of these organelles retaining exclusively tubular cristae, whereas 58% are mixed; of the latter, lamelliform cristae predominate. This finding suggests that, in contrast to SSM, the cristae in IFM are structurally plastic, changing during isolation. These observations on >1,000 organelles provide the first quantitative morphological evidence for definitive differences between the two populations of cardiac mitochondria.     

Endoplasmic reticulum stress-mediated mitochondrial dysfunction in aged hearts

Aging impairs the mitochondrial electron transport chain (ETC), especially in interfibrillar mitochondria (IFM). Mitochondria are in close contact with the endoplasmic reticulum (ER). Induction of ER stress leads to ETC injury in adult heart mitochondria. We asked if ER stress contributes to the mitochondrial dysfunction during aging. Subsarcolemmal mitochondria (SSM) and IFM were isolated from 3, 18, and 24 mo. C57Bl/6 mouse hearts. ER stress progressively increased with age, especially in 24 mo. mice that manifest mitochondrial dysfunction. OXPHOS was decreased in 24 mo. IFM oxidizing complex I and complex IV substrates. Proteomic analysis showed that the content of multiple complex I subunits was decreased in IFM from 24 mo. hearts, but remained unchanged in in 18 mo. IFM without a decrease in OXPHOS. Feeding 24 mo. old mice with 4-phenylbutyrate (4-PBA) for two weeks attenuated the ER stress and improved mitochondrial function. These results indicate that ER stress contributes to the mitochondrial dysfunction in aged hearts. Attenuation of ER stress is a potential approach to improve mitochondrial function in aged hearts.     

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.     

Mitochondrial Ca2+ uptake in skeletal muscle health and disease

Muscle uses Ca²⁺ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca²⁺ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca²⁺ from their surroundings, a process called mitochondrial Ca²⁺ uptake. Under physiological conditions, Ca²⁺ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca²⁺ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca²⁺ uptake could shape spatio-temporal patterns of intracellular Ca²⁺ signaling. Malfunction of mitochondrial Ca²⁺ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca²⁺ levels. Besides the sudden elevation of Ca²⁺ level induced by action potentials, Ca²⁺ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as minutes during tetanic contraction, which raises the question whether mitochondrial Ca²⁺ uptake is fast and big enough to shape intracellular Ca²⁺ signaling during excitation-contraction coupling and creates technical challenges for quantification of the dynamic changes of Ca²⁺ inside mitochondria. This review focuses on characterization of mitochondrial Ca²⁺ uptake in skeletal muscle and its role in muscle physiology and diseases.     

Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III

The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. Aging decreases oxidative function in interfibrillar mitochondria (IFM) that reside among the myofibers, while subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. Aging decreases complex III activity selectively in IFM via alteration of the cytochrome c binding site. With 25 min of global ischemia, complex III activity decreases in SSM and further decreases in IFM in the aging heart. Ischemia leads to a marked decrease in the electron paramagnetic resonance signal of the iron-sulfur protein (ISP) in both SSM and IFM, despite a preserved content of ISP peptide. Thus, ischemia results in a functional decrease in the iron-sulfur center in ISP without subunit peptide loss. In the aging heart, at the onset of reperfusion, IFM contain two tandem defects in the path of electron flow through complex III, providing a likely mechanism for enhanced oxidant production and reperfusion damage.     

Interactions of Adriamycin aglycones with mitochondria may mediate Adriamycin cardiotoxicity

Adriamycin and related anthracyclines are potent oncolytic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone metabolites of Adriamycin (5–20 μM) induce a Ca²⁺-dependent increase in the permeability of the inner mitochondrial membrane of both heart and liver mitochondria to small (< 1500 Da) solutes; this phenomenon is accompanied by release of mitochondrial Ca²⁺, mitochondrial swelling, collapse of the membrane potential, oxidation of mitochondrial pyridine nucleotides [NAD(P)H], uncoupling, and a transition from the condensed to the orthodox conformation and is inhibited by ATP, dithiothreitol, the immunosuppressant cyclosporin A, and the ubiquitous polyamine spermine. Aglycones also modify mitochondrial sulfhydryl groups and induce a Ca²⁺ independent oxidation of mitochondrial NAD(P)H which appears to reflect electron transport from NADH to oxygen, mediated by the aglycones and resulting in the production of Superoxide (O₂⁻). Selenium deficiency and butylated hydroxytoluene inhibit aglycone-induced Ca²⁺ release from liver, but not heart, mitochondria, suggesting that the interactions of the aglycones with mitochondria diner in these two tissues. It can be proposed that the effects of Adriamycin aglycones on heart mitochondria are responsible for the cardiotoxicity of the parent drug.