Gohar Azhar

Bcl-2 and Bax expression in adult rat hearts after coronary occlusion: age-associated differences

It has been reported that programmed cell death (apoptosis) occurs during myocardial infarction. The influence of age on programmed cell death or DNA fragmentation after coronary occlusion has not been extensively characterized. To test the hypothesis that there are age-related differences in susceptibility to DNA fragmentation during ischemia-infarction, we studied DNA fragmentation in young adult and old male F344 rat hearts after acute coronary artery occlusion. Hearts were studied at 1, 3, and 5 h and 1 and 7 days after coronary ligation. The percentage of apoptotic cells was determined by the in situ end-labeling technique, and internucleosomal fragmentation (DNA laddering) pattern was also analyzed. Our results show that 1) DNA fragmentation began earlier and peaked earlier in the old compared with young adult hearts during infarction; 2) there was heightened expression of both Bcl-2 and Bax in the old hearts at baseline; and 3) the Bcl-2-to-Bax ratio was higher in the older heart after coronary ligation. These results suggest that, compared with the young adult heart, the aged heart may be more susceptible to ischemia-induced DNA fragmentation.It has been reported that programmed cell death (apoptosis) occurs during myocardial infarction. The influence of age on programmed cell death or DNA fragmentation after coronary occlusion has not been extensively characterized. To test the hypothesis that there are age-related differences in susceptibility to DNA fragmentation during ischemia-infarction, we studied DNA fragmentation in young adult and old male F344 rat hearts after acute coronary artery occlusion. Hearts were studied at 1, 3, and 5 h and 1 and 7 days after coronary ligation. The percentage of apoptotic cells was determined by the in situ end-labeling technique, and internucleosomal fragmentation (DNA laddering) pattern was also analyzed. Our results show that 1) DNA fragmentation began earlier and peaked earlier in the old compared with young adult hearts during infarction; 2) there was heightened expression of both Bcl-2 and Bax in the old hearts at baseline; and 3) the Bcl-2-to-Bax ratio was higher in the older heart after coronary ligation. These results suggest that, compared with the young adult heart, the aged heart may be more susceptible to ischemia-induced DNA fragmentation.

The Expression of microRNA and microRNA Clusters in the Aging Heart

Background The microRNAs have been implicated in the process of cardiac development, cardiac hypertrophy, and heart failure. However, the impact of adult aging on cardiac expression of miRNA clusters, as well as both miRNA guide (miR) and passenger (miR*) strands has not been well established. Methods/Results We explored the expression profile of both miR and miR* in the hearts of young adult versus old mice. We found that 65 miRNAs were differentially expressed in the old versus young adult hearts; approximately half of them were clustered miRNAs that were distributed in 11 miRNA clusters. Each miRNA cluster contained from 2 to as many as 71 miRNA genes. The majority of the clusters displayed similar expression, with most cluster members within a cluster being either increased or decreased together, suggesting that most clusters are likely to be regulated by a common signaling mechanism and that the combined expression of multiple miRNA genes in a cluster could pose an impact on a broad range of targets during aging. We also found age-related changes in the expression of miR*s. The expression of both miR and miR* correlated with that of pri-miRNA transcript over the time course from development and maturation through adult aging. Age-related changes in the expression of Ago1 and Ago2 proteins in the heart were also observed. Transfection assay revealed that both Ago1 and Ago2 synergistically induced miR-21 and miR-21* when the mir-21 plasmid was co-transfected with either. Conclusion The data revealed age-related changes in the expression of pri-miRNA transcript, Argonaut proteins and both miR and miR* strands. The major changes occurred later in life, from middle to old age. It is likely that the expression of miR and miR* is regulated by both pri-miRNA transcription as well as Ago1 and Ago2 proteins during adult aging.

Identification of a Novel Serum Response Factor Cofactor in Cardiac Gene Regulation

The transcription factor serum response factor (SRF) plays an important role in the regulation of a variety of cardiac genes during development and during adult aging. A novel SRF cofactor, herein called p49/STRAP, for SRF-dependent transcription regulation-associated protein, was recently identified in our laboratory. This protein interacted mainly with the transcriptional activation domain of the SRF protein and was found to bind to SRF or to the complex of SRF and another cofactor, such as myocardin or Nkx2.5. The expression of p49/STRAP affected the promoter activity of SRF target genes in a non-uniform manner. For example, p49 activated MLC2v and cardiac actin promoters when it was co-transfected with SRF, but it repressed atrial natriuretic factor promoter activity, which was strongly induced by myocardin. The p49/STRAP mRNA was observed to be highly expressed in fetal, adult, and senescent human hearts, and also in hearts of young adult and old mice, suggesting that p49/STRAP may be an important SRF cofactor in the transcriptional regulation of mammalian cardiac muscle genes throughout the life span.

Nutrition and cardiac cachexia.

Purpose of reviewCongestive heart failure is a leading cause of morbidity and mortality, especially in older persons. In advanced stages of the disease, congestive heart failure can be associated with serious complications such as cardiac cachexia (defined here as weight loss of more than 6% in 6 months). This review will discuss recent insights into the pathophysiology, anthropometric predictors and potential management of cardiac cachexia. Recent findingsCardiac cachexia and the associated progressive weight loss are sometimes overlooked by care providers. A delay in diagnosis often results in further loss of vital tissues, progressive weakness, fall-related injuries and potentially long-term care institutionalization and/or death. Emerging data suggest that congestive heart failure is a dynamic disorder of many organ systems, including the myocardial, neurohormonal, immune, vascular, gastrointestinal, renal and musculoskeletal systems. It is becoming more widely appreciated that it is the deterioration of this interactive multisystem complex that results in the systemic inflammation and progressive wasting and atrophy of muscle and other organ tissues, which is the hallmark of cardiac cachexia. SummaryCardiac cachexia in congestive heart failure patients may be associated with a low level of physical activity. A high systemic inflammatory state is another marker of cardiac cachexia. Prudent anti-inflammatory nutrition, dietary supplements and exercise can serve to ameliorate and/or potentially prevent progressive wasting. A better understanding of factors contributing to the development of cardiac cachexia will enable us to design preventive strategies and provide improved care for individuals with this debilitating condition.

Trends in Percutaneous Endoscopic Gastrostomy Placement in the Elderly From 1993 to 2003

Objectives: To investigate national trends in percutaneous endoscopic gastrostomy (PEG) tube placement for hospitalized elderly patients from 1993to 2003. Methods: Retrospective analysis of patients ≥65 years of age with PEG tube placement from 1993 to 2003 from the Nationwide Inpatient Sample (NIS) database was utilized to calculate PEG placement rates per 1000 people. Results: Placement of PEG tube increased by 38% in elderly patients during the study period, from 2.71 procedures during hospitalization per 1000 people to 3.75 procedures during hospitalization per 1,000 people. Placement of PEG tube in patients with Alzheimer’s dementia doubled (5%-10%) over the study period. Conclusion: Over a 10-year period, PEG tube use in hospitalized elderly patients increased significantly. More importantly, approximately 1 in 10 PEG tube placements occurred in patients with dementia.

Regulation of cardiac microRNAs by serum response factor

Serum response factor (SRF) regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg) led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg) resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis.

The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults

We have determined whole body protein kinetics, i.e., protein synthesis (PS), breakdown (PB), and net balance (NB) in human subjects in the fasted state and following ingestion of ~40 g [moderate protein (MP)], which has been reported to maximize the protein synthetic response or ~70 g [higher protein (HP)] protein, more representative of the amount of protein in the dinner of an average American diet. Twenty-three healthy young adults who had performed prior resistance exercise (X-MP or X-HP) or time-matched resting (R-MP or R-HP) were studied during a primed continuous infusion of l-[(2)H5]phenylalanine and l-[(2)H2]tyrosine. Subjects were randomly assigned into an exercise (X, n = 12) or resting (R, n = 11) group, and each group was studied at the two levels of dietary protein intake in random order. PS, PB, and NB were expressed as increases above the basal, fasting values (mg·kg lean body mass(-1)·min(-1)). Exercise did not significantly affect protein kinetics and blood chemistry. Feeding resulted in positive NB at both levels of protein intake: NB was greater in response to the meal containing HP vs. MP (P < 0.00001). The greater NB with HP was achieved primarily through a greater reduction in PB and to a lesser extent stimulation of protein synthesis (for all, P < 0.0001). HP resulted in greater plasma essential amino acid responses (P < 0.01) vs. MP, with no differences in insulin and glucose responses. In conclusion, whole body net protein balance improves with greater protein intake above that previously suggested to maximally stimulating muscle protein synthesis because of a simultaneous reduction in protein breakdown.

Exposure to High or Low Glucose Levels Accelerates the Appearance of Markers of Endothelial Cell Senescence and Induces Dysregulation of Nitric Oxide Synthase

To test the hypothesis that aging impairs endothelial cell response to glucose stress, we utilized a human umbilical vein endothelial cell in vitro model in which clinically relevant concentrations of normal (5.5 mM), high (25 mM), and low (1.5mM) glucose were tested. With advancing population doubling, exposure to normal glucose gradually decreased endothelial nitric oxide synthase expression and activity, resulting in slow, progressive development of markers of cell senescence (by population doubling level [PDL] 44). High or low glucose treatment accelerated the appearance of markers of senescence (by ~PDL 35) along with declines in endothelial nitric oxide synthase expression and activity. Human umbilical vein endothelial cells exposed to alternating low and high glucose gave even more rapid acceleration in the appearance of markers of senescence (by ~PDL 18) and reduction in endothelial nitric oxide synthase levels. Thus, exposure to low and high glucose induces earlier appearance of markers of endothelial cell senescence and dysregulation of the nitric oxide synthase gene and protein expression and function. These findings will help to elucidate endothelial dysfunction associated with glucose intolerance and improve future therapy for diabetic seniors.

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

BackgroundThe p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart.ResultsIn the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor.ConclusionThese data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

Maintaining serum response factor activity in the older heart equal to that of the young adult is associated with better cardiac response to isoproterenol stress.

AbstractTo understand the effect of transcription regulation in modulating cardiac aging, we sought to study the role of serum response factor (SRF), a key transcription factor in the heart that is normally increased with senescence and also in congestive heart failure. A Tet-Off gene expression system was used for cardiac-specific over-expression of a mutant SRF protein. In these binary transgenic mice, there is no age-related increase in SRF protein expression; in fact, there appeared to be a mild reduction of SRF protein (Mild-R SRF Tg). The older, middle-aged (15 mo) Mild-R SRF Tg mice appeared healthier and were better able to maintain their left ventricular systolic pressure (LVSP) in response to moderate â-adrenergic stimulation compared with age-matched Non-Tg mice, which demonstrated a negative ionotropic response. The Mild-R SRF Tg hearts had lower mRNA expression of BNP (p < 0.05), and the sodium calcium exchanger (p < 0.05), compared to Non-Tg. Mild-R SRF Tg had higher mRNA levels of SERCA2 (p < 0.05) and ryanodine receptor 2 (p < 0.05) compared to Non-Tg hearts. These findings suggest that preventing the age-associated increase in SRF is associated with better preserved intracellular calcium handling and functional response to stress; it might be advantageous for the older adult heart. This mouse model could be helpful in elucidating the molecular mechanisms underlying certain age-related changes in cardiac reserve capacity and response to stress.