Difference between revisions of "Hoppel 2018 MiPschool Tromso D1"

Revision as of 10:22, 5 October 2018

Two populations of muscle mitochondria: heart and skeletal muscle.

Link: MitoEAGLE

Hoppel CL (2018)

Event: MiPschool Tromso-Bergen 2018

Based on morphology of cardiac muscle cells, mitochondria are located under the sarcolemma, between the myofibrils, and in the central perinuclear region. Using concepts of cellular enzymology, protein yields, transmission electron microscopy (TEM) by Drs. Bernard Tandler and Hisashi Fujioka, high-resolution scanning electron microscopy (HRSEM) with Dr. Alessandro Riva, and functional studies with Dr. June Palmer, methods were devised to remove the sarcolemma from heart tissue producing a skinned myofibril releasing of a specific population of mitochondria, dubbed subsarcolemmal mitochondria (SSM). The resultant myofibril pellet was then subjected to protease treatment to disrupt the fibrils, leading to liberation of mitochondria from the interfibrillar space (IFM). The overall recovery of mitochondria was 70 -80% with only 5% released into the supernatants and the remaining ~20% entrapped in the tissue debris consisting largely of digested myofibrils. The content of mitochondrial enzymes and activity of oxidative phosphorylation is @150% in IFM compared to the SSM. Of the multiple examples of pathophysiological changes found only in one population, we will focus on the cardiomyopathy in the Syrian Hamster (only IFM affected), diabetes mellitus in the rat (only SSM affected), and aging in the rat heart (only the IFM involved). Because of the location of the IFM and t-tubule system in proximity of the intercalated discs, the involvement and interaction of these with Ca2+ signaling in cardiac excitation-contraction coupling (E-C) has been explored.

In skeletal muscle there is controversy as to whether the mitochondria exist as a reticulum or as distinct entities. There are more studies published using the two populations of skeletal muscle mitochondria than from heart. We were unable to prepare purified SSM from skeletal muscle as judged by transmission electron microscopy, because of the presence of vesicles of undetermined origin. Attempts to remove these contaminants were unsuccessful (unpublished with Dr. Linda Brady). When we switched from using the Polytron homogenizer to disrupt the sarcolemma in muscle tissue to using proteolytic enzymes (collagenase for heart and dispase for skeletal muscle) to expose the sarcolemma so that a gentle physical force could be applied (Potter-Elvehjem homogenizer) to disrupt that plasma membrane, we observed a remarkable diminution in the presence of vesicles in the SSM. Nicola Lai modified the skeletal muscle procedure to maximize SSM and IFM recovery (overall ~80%) while preserving mitochondrial integrity, function, and structure. We will discuss the skeletal muscle mitochondrial observations in a model of pacing-induced heart failure in a dog model. The decrease in respiratory rates of skeletal muscle SSM are neither relieved upon collapsing the mitochondrial potential with an uncoupler nor increased in the presence of maximal ADP concentrations, showing a defect in the ETC. In contrast, respiratory rates of skeletal muscle IFM from HF were relieved with the uncoupler and partially improved in the presence of maximal ADP concentrations. These IFM alterations in the phosphorylation apparatus were detected with a decreased amount of ANT isoform 2 and increased amount of isoform 1. The IFM dysfunction may be explained by this shift in ANT isoforms.

In conclusion, the study of the two populations of muscle mitochondria is necessary to truly understand the presence and type of dysfunction.

• Bioblast editor: Beno M, Plangger M • O2k-Network Lab: US OH Cleveland Hoppel CL

Labels:

Stress:Ischemia-reperfusion Organism: Rat

Affiliations

Dept Pharmacology Medicine, Case Western Reserve Univ School Medicine, Cleveland, OH, USA

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 |event=MiPschool Tromso-Bergen 2018
 |abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
-Multiple studies have documented that myocardial ischemia results in progressive damage to the mitochondrial electron transport chain.  Oxidative phosphorylation has been used to track the sequence and localize the area of damage.  In early ischemia, glutamate oxidation is decreased and appears due to a defect in complex I, as well as in complex V and the adenine nucleotide translocase.  These defects are resolved with reperfusion.  Subsequently, a decrease in succinate oxidation occurs with prolonged ischemia suggesting a defect distal to complex I, which persists with reperfusion.  The subsarcolemmal mitochondria (SSM) located under the sarcolemma are affected earlier in ischemia that those mitochondria between the myofibrils (interfibrillar, IFM).  In rabbit heart with prolonged ischemia, we have identified a defect in cytochrome c oxidase, a loss of the phospholipid, cardiolipin, as well as of cytochrome c from the SSM only.  Reperfusion did not further damage the mitochondria as these defects persist, but do not progress.  Blockade of the electron transport chain with the irreversible inhibitor, rotenone, modulated the ischemic damage with preservation of both cardiolipin and cytochrome c content of the SSM while improving the oxidation through complex IV.
+Based on morphology of cardiac muscle cells, mitochondria are located under the sarcolemma, between the myofibrils, and in the central perinuclear region.  Using concepts of cellular enzymology, protein yields, transmission electron microscopy (TEM) by Drs. Bernard Tandler and Hisashi Fujioka, high-resolution scanning electron microscopy (HRSEM) with Dr. Alessandro Riva, and functional studies with Dr. June Palmer, methods were devised to remove the sarcolemma from heart tissue producing a skinned myofibril releasing of a specific population of mitochondria, dubbed subsarcolemmal mitochondria (SSM).  The resultant myofibril pellet was then subjected to protease treatment to disrupt the fibrils, leading to liberation of mitochondria from the interfibrillar space (IFM).  The overall recovery of mitochondria was 70 -80% with only 5% released into the supernatants and the remaining ~20% entrapped in the tissue debris consisting largely of digested myofibrils.  The content of mitochondrial enzymes and activity of oxidative phosphorylation is @150% in IFM compared to the SSM. Of the multiple examples of pathophysiological changes found only in one population, we will focus on the cardiomyopathy in the Syrian Hamster (only IFM affected), diabetes mellitus in the rat (only SSM affected), and aging in the rat heart (only the IFM involved).  Because of the location of the IFM and t-tubule system in proximity of the intercalated discs, the involvement and interaction of these with Ca2+ signaling in cardiac excitation-contraction coupling (E-C) has been explored.
-In the isolated buffer-perfused rat heart, global ischemia decreases oxidative phosphorylation and damages the distal electron transport chain, with decreased complex III activity, cytochrome c content, and respiration through complex IV in both SSM and IFM.  The defect in complex III has been identified as a functional loss of the iron-sulfur center of the Rieske iron-sulfur protein (ISP) without loss of the subunit peptide.  In the aged rat heart, oxidative function is decreased in the IFM only with the defect localized to complex III at Qo site.  At the onset of reperfusion in the aged heart, IFM contain two tandem defect in Complex III, which provides a mechanism for the enhance oxidant production and reperfusion damage.  Resolution of the aging complex II defect in the aged heart resolves the combined defect and the treated-aged heart now behaves like a young heart.
+In skeletal muscle there is controversy as to whether the mitochondria exist as a reticulum or as distinct entities.   There are more studies published using the two populations of skeletal muscle mitochondria than from heart.  We were unable to prepare purified SSM from skeletal muscle as judged by transmission electron microscopy, because of the presence of vesicles of undetermined origin.  Attempts to remove these contaminants were unsuccessful (unpublished with Dr. Linda Brady).  When we switched from using the Polytron homogenizer to disrupt the sarcolemma in muscle tissue to using proteolytic enzymes (collagenase for heart and dispase for skeletal muscle) to expose the sarcolemma so that a gentle physical force could be applied (Potter-Elvehjem homogenizer) to disrupt that plasma membrane, we observed a remarkable diminution in the presence of vesicles in the SSM.  Nicola Lai modified the skeletal muscle procedure to maximize SSM and IFM recovery (overall ~80%) while preserving mitochondrial integrity, function, and structure.  We will discuss the skeletal muscle mitochondrial observations in a model of pacing-induced heart failure in a dog model.  The decrease in respiratory rates of skeletal muscle SSM are neither relieved upon collapsing the mitochondrial potential with an uncoupler nor increased in the presence of maximal ADP concentrations, showing a defect in the ETC. In contrast, respiratory rates of skeletal muscle IFM from HF were relieved with the uncoupler and partially improved in the presence of maximal ADP concentrations. These IFM alterations in the phosphorylation apparatus were detected with a decreased amount of ANT isoform 2 and increased amount of isoform 1. The IFM dysfunction may be explained by this shift in ANT isoforms.
+In conclusion, the study of the two populations of muscle mitochondria is necessary to truly understand the presence and type of dysfunction.
 |editor=[[Beno M]], [[Plangger M]],
 |mipnetlab=US OH Cleveland Hoppel CL
 }}
 {{Labeling
-|area=Respiration
 |injuries=Ischemia-reperfusion
-|organism=Rat, Rabbit
+|organism=Rat
-|tissues=Heart
-|instruments=Oxygraph-2k
 }}
 == Affiliations ==
 ::::Dept Pharmacology Medicine, Case Western Reserve Univ School Medicine, Cleveland, OH, USA