Shirish Damle

Regional Distribution of Mitochondrial Dysfunction and Apoptotic Remodeling in Pacing-Induced Heart Failure

BACKGROUND Specific myocardial mitochondrial enzymatic dysfunction and apoptotic remodeling occur in pacing-induced heart failure. We sought to define their regional distribution and molecular basis in the failing heart. METHODS AND RESULTS Enzyme dysfunction was assessed in mitochondrial subpopulations and immunoblot analysis was performed using homogenate proteins from the left atria (LA) and left ventricle (LV) of paced and control mongrel dogs. A greater range of enzymatic defects (complex I, III, and V) was found in mitochondria subpopulations from the LV as compared with the LA (where only complex V was defective). Analysis of paced LV proteins demonstrated a downregulated expression of both mitochondrial genes (eg, cytochrome b) and nuclear genes (eg, ATP synthase beta subunit, mitochondrial creatine kinase). Protease-activated products of both mitochondrial (eg, apoptosis inducing factor) and cytosolic (eg, caspase-3) apoptogenic proteins were increased in both the LA and LV. Nuclear-localized apoptotic markers (eg, p53, p21) were also significantly increased in the LV of paced dogs. CONCLUSION Abnormal activity of several mitochondrial enzymes and increased apoptogenic pathway appear to be mediated, at least in part, by an orchestrated shift in expression (both nuclear and mitochondrial DNA) of respiratory chain subunits (eg, cyt b, ATP-beta), mitochondrial bioenergetic enzymes (eg, mitochondrial creatine kinase), global transcription factor (eg, PGC-1), and apoptotic proteins (eg, p53, p21) with distinct differences in their regional distribution and in the subpopulations of mitochondria affected.

Non-invasive evaluation of buccal respiratory chain enzyme dysfunction in mitochondrial disease: comparison with studies in muscle biopsy.

Making a diagnosis of mitochondrial disease (MD) is extremely challenging and often employs the analysis of respiratory complex (RC) activities in biopsied skeletal muscle. Given both the invasive nature and expense of biopsied-muscle based testing for mitochondrial defects, buccal swab enzyme analysis has been explored as an alternative approach to the more invasive muscle biopsy. Case studies have recently suggested that buccal swabs from patients can be used to accurately assess mitochondrial enzyme activities including RC I and RC IV using a dipstick methodology combined with spectrophotometric analysis. In this study, forty patients with suspected MD who have previously been found to have significant defects in either RC I or RC IV in skeletal muscle were assessed by buccal swab analysis and compared to enzyme values obtained with unaffected controls (n=106) in the same age range. Buccal citrate synthase was used as an indicator of overall mitochondrial content, correlating well with overall buccal mitochondrial frataxin levels and was found to be elevated above control levels in 28% of the patients in this cohort. Of 26 cases with significant muscle RC I deficiency, 20 displayed significantly reduced levels of buccal RC I activity. All 7 of the patients with muscle RC IV deficiency showed significant buccal RC IV defect and 6 of the 7 patients with combined defects in muscle RC I and IV activity levels also exhibited analogous deficiencies in both buccal RC I and RC IV activities. In conclusion, the relatively high correlation (over 82%) of buccal and muscle RC deficiencies further supports the validity of this non-invasive approach as a potentially useful tool in the diagnosis of MD.

Mitochondrial enzyme dysfunction in autism spectrum disorders; a novel biomarker revealed from buccal swab analysis

AIM Mitochondrial function studies in autism spectrum disorders (ASD) have detected skeletal muscle mitochondrial enzyme deficiencies in respiratory complex (RC) activities. As a muscle biopsy is expensive and invasive, we assessed RC-I and RC-IV activities in buccal swabs. METHODS 92 children with ASD and 68 controls were studied with immunocapture for RC-I and microspectrophotometry for RC-IV. RESULTS Significant RC activity deficiencies were found in 39 (42%) ASD patients (p < 0.01) and more prevalent in more severe cases. Aberrant RC overactivity was seen in 9 children. RC-I/RC-IV activity ratio was significantly increased in 64% of the entire ASD cohort including 76% of those more severely affected (p < 0.05). CONCLUSION Buccal swab analysis revealed extensive RC abnormalities in ASD providing a noninvasive biomarker to assess mitochondrial function in ASD patients.

Mitochondrial Dysfunction may explain symptom variation in Phelan-McDermid Syndrome

Phelan-McDermid Syndrome (PMS), which is defined by a deletion within 22q13, demonstrates significant phenotypic variation. Given that six mitochondrial genes are located within 22q13, including complex I and IV genes, we hypothesize that mitochondrial complex activity abnormalities may explain phenotypic variation in PMS symptoms. Complex I, II, II + III and IV activity was measured in 51 PMS participants. Caretakers completed questionnaires and provided genetic information through the PMS foundation registry. Complex activity was abnormal in 59% of PMS participants. Abnormalities were found in complex I and IV but not complex II + III and II activity, consistent with disruption of genes within the 22q13 region. However, complex activity abnormalities were not related to specific gene deletions suggesting a “neighboring effect” of regional deletions on adjacent gene expression. A specific combination of symptoms (autism spectrum disorder, developmental regression, failure-to-thrive, exercise intolerance/fatigue) was associated with complex activity abnormalities. 64% of 106 individuals in the PMS foundation registry who did not have complex activity measured also endorsed this pattern of symptoms. These data suggest that mitochondrial abnormalities, specifically abnormalities in complex I and IV activity, may explain some phenotypic variation in PMS individuals. These results point to novel pathophysiology mechanisms and treatment targets for PMS patients.

Hypoxia‐inducible factor‐1 improves inotropic responses of cardiac myocytes in ageing heart without affecting mitochondrial activity

Ageing reduces the ability of cardiac myocytes to respond to inotropic agents. We hypothesized that hypoxia‐inducible factor‐1 (HIF‐1) would improve the functional and Ca2+ transient responses of ageing myocytes to the inotropic agents and this would act, in part, through altered mitochondrial activity. Young (3–4 months) and older Fischer 344 rats (18–20 months) were used. Hypoxia‐inducible factor‐1α was upregulated with ciclopirox olamine (CPX, 50 mg kg−1 on 2 days). Hypoxia‐inducible factor‐1 upregulation was detected by Western blot. Cardiomyocyte contraction and Ca2+ transients were measured at baseline and after forskolin and ouabain. We also measured mitochondrial complex activities and production of reactive oxygen species (ROS). In the young group, forskolin (31%) and ouabain (31%) significantly increased percentage shortening. Similar changes were observed in the young + CPX group. Calcium transients also responded in a similar manner. However, in the older group, forskolin (12%) and ouabain (6%) did not significantly increase myocyte contractility or Ca2+ transients. In the older + CPX group, the effects of forskolin (34%) and ouabain (29%) were restored. In the young + CPX group, there was increased ROS production and mitochondrial complex I and III activity compared with the young group. These differences were not observed in older groups. These data demonstrate an impaired functional and Ca2+ effect of positive inotropic agents in older myocytes. Upregulation of HIF‐1 restored this blunted response, but this was not related to changed mitochondrial activity induced by HIF‐1. Thus, we found that HIF‐1 improved inotropy in older myocytes without requiring mitochondrial activity changes.

The Effect of Mitochondrial Supplements on Mitochondrial Activity in Children with Autism Spectrum Disorder

Treatment for mitochondrial dysfunction is typically guided by expert opinion with a paucity of empirical evidence of the effect of treatment on mitochondrial activity. We examined citrate synthase and Complex I and IV activities using a validated buccal swab method in 127 children with autism spectrum disorder with and without mitochondrial disease, a portion of which were on common mitochondrial supplements. Mixed-model linear regression determined whether specific supplements altered the absolute mitochondrial activity as well as the relationship between the activities of mitochondrial components. Complex I activity was increased by fatty acid and folate supplementation, but folate only effected those with mitochondrial disease. Citrate synthase activity was increased by antioxidant supplementation but only for the mitochondrial disease subgroup. The relationship between Complex I and IV was modulated by folate while the relationship between Complex I and Citrate Synthase was modulated by both folate and B12. This study provides empirical support for common mitochondrial treatments and demonstrates that the relationship between activities of mitochondrial components might be a marker to follow in addition to absolute activities. Measurements of mitochondrial activity that can be practically repeated over time may be very useful to monitor the biochemical effects of treatments.

Bioenergetic variation is related to autism symptomatology

Autism spectrum disorder (ASD) has been associated with mitochondrial dysfunction but few studies have examined the relationship between mitochondrial function and ASD symptoms. We measured Complex I and IV and citrate synthase activities in 76 children with ASD who were not receiving vitamin supplementation or medication. We also measured language using the Preschool Language Scales or Clinical Evaluation of Language Fundamentals, adaptive behavior using the Vineland Adaptive Behavioral Scale, social function using the Social Responsiveness Scale and behavior using Aberrant Behavior Checklist, Childhood Behavior Checklist and the Ohio Autism Clinical Impression Scale. Children with ASD demonstrated significantly greater variation in mitochondrial activity compared to controls with more than expected ASD children having enzyme activity outside of the normal range for Citrate Synthase (24%), Complex I (39%) and Complex IV (11%). Poorer adaptive skills were associated with Complex IV activity lower or higher than average and lower Complex I activity. Poorer social function and behavior was associated with relatively higher Citrate Synthase activity. Similar to previous studies we find both mitochondrial underactivity and overactivity in ASD. This study confirms an expanded variation in mitochondrial activity in ASD and demonstrates, for the first time, that such variations are related to ASD symptoms.