Ashwani Malhotra

Pacing-Induced Heart Failure in Dogs Enhances the Expression of p53 and p53-Dependent Genes in Ventricular Myocytes

BACKGROUNDnRapid ventricular pacing in dogs is characterized by a dilated myopathy in which myocyte cell death by apoptosis may play a significant role in the impairment of cardiac pump function. However, the molecular mechanisms implicated in the modulation of programmed cell death under this setting remain to be identified. Moreover, questions have been raised on the specificity and sensitivity of the histochemical detection of DNA strand breaks in nuclei by the terminal deoxynucleotidyl transferase (TdT) reaction.nnnMETHODS AND RESULTSnChanges in the expression of Bcl-2 and Bax and their transcriptional regulator, p53, were determined by Western blot analysis in myocytes isolated from dogs affected by pacing-induced heart failure. A mobility shift assay for p53 binding activity was also performed. In addition, apoptosis was measured by confocal microscopy, which allowed the simultaneous detection of chromatin alterations and DNA damage. p53 DNA binding activity to the bax promoter was increased in nuclear extracts from myocytes obtained from failing hearts, and this response was associated with enhanced expression of Bax protein, 52%, and attenuation of Bcl-2, -92%. Immunolabeling of p53 in myocyte nuclei, measured by confocal microscopy, was 100% higher in cells from paced hearts. The combination of the TdT assay and confocal microscopy demonstrated that 20 myocyte nuclei per 10(6) were undergoing apoptosis in control myocardium and 4000 per l0(6) after pacing. Moreover, DNA laddering was shown in myocytes by agarose gel electrophoresis of DNA fragments.nnnCONCLUSIONSnThe activation of p53 and p53-dependent genes may be critical in the modulation of myocyte apoptosis in pacing-induced heart failure.

Effects of Constitutive Overexpression of Insulin-Like Growth Factor-1 on the Mechanical Characteristics and Molecular Properties of Ventricular Myocytes

Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca2+ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1 in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.

Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis.

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

Reversibility of diabetic cardiomyopathy with insulin in rats

Diabetes appears to cause a cardiomyopathy independent of atherosclerotic coronary artery disease and hypertension. Left ventricular papillary muscle function studies in rats made severely diabetic with streptozotocin have shown a slowing of relaxation and a depression of shortening velocity. However, the effects of insulin therapy on the myocardial mechanics of diabetic rats have not been studied. Therefore, rats diabetic for 6–10 weeks were treated with PZI insulin for 2, ft, 10, or 28 days and the mechanical performance of their left ventricular papillary muscles was compared to that of untreated diabetics and age-matched controls; cardiac contractile protein enzymatic activity was also measured. Neither 2 nor 6 days of therapy had any effects on the depressed cardiac muscle performance of diabetic animals, although plasma glucose concentration was restored to normal. By 10 days of therapy, recovery of mechanical performance was nearly complete, and by 28 days of therapy, complete reversal of the altered myocardial mechanics was observed. Crystalline insulin added to the bath (9 mU/ml) had no effect on myocardial mechanics in either diabetics or controls. A gradual recovery of actomyosin and myosin ATPase activity In the hearts of insulin-treated diabetic aoinials was also found, complementing the mechanical studies. In addition to demonstrating a gradual but complete reversibility of the abnormalities in papillary muscle function in diabetic rats (although control of hyperglycemia was less than ideal), this study confirms that this model of a cardiomyopathy is not a result of streptozotocin-induced cardiac toxicity. Additional data are provided indicating that depressed thyroid hormone levels in diabetic rats are not responsible for the mechanical changes observed.

Myocyte Cell Loss in Ischemic Cardiomyopathy: Role of Apoptosis

Ischemic cardiomyopathy is an anatomic condition initiated by primary defects in the coronary circulation that result in myocyte loss, scarring, and ventricular failure. A sudden occlusion of a major epicardial coronary artery leads to loss of function in the supplied myocardium, impairing ventricular hemodynamics in proportion to the tissue involved in the ischemic event [1–3]. Infarct size affects the heart in three ways: 1) the number of cells destined to die [4]; 2) the nature and timing of myocyte cell death [5]; and 3) the extent of scarring with thinning of the wall [6–10]. These mechanisms of restructuring of the heart are critical in the acute myocardial response to infarction [7,9] but contribute less to ventricular remodeling after healing is completed [1–3,11,12]. The tissue and cellular adaptations occurring in the surviving myocardium are the major determinants of the changes in cardiac size and shape that characterize the long-term evolution of the postinfarcted heart [1–3]. However, infarct dimension conditions the adaptations of the viable portions of the wall [3,4,10]. Thus, infarct size dictates prognosis [13] and in_uences the outcome of the cardiac myopathy. Before discussing the multiple processes implicated in the structural modi~cations of the remaining viable region of the heart, the consequences of coronary artery occlusion on the behavior of the ischemic myocardium and the etiology of myocyte cell death will be analyzed. Within one minute after coronary artery occlusion, the ischemic myocardium fails to contract, losing its ability to oppose the mechanical stimuli generated throughout the cardiac cycle. The central ischemic zone bulges during systole, being exposed to the physical forces resulting from systolic and diastolic wall stress [2,5]. This area of the ventricle is subject to both pathologic levels of myocardial loading and tissue ischemia, which independently or in concert may promote irreversible cellular damage and myocyte death [14–16]. Since myocyte necrosis and apoptosis may contribute separately to infarct size, these two forms of myocyte cell death were measured quantitatively at different time points following coronary artery ligation in the rat model [5]. A signi~cant issue concerned the identi~cation of the timing and nature of myocyte death. This information is critical for both the characterization of the period available for intervention between the coronary event and cell death and the possible interference with the molecular mechanisms implicated in the transmission of a death signal to myocytes. To address these clinically relevant problems, a methodology had to be employed that allowed the distinction to be made between the two forms of myocyte death; namely, apoptosis and necrosis. Cell death by apoptosis is activated by an endogenous endonuclease that results in endonucleolysis [17]. DNA degradation, triggered by this mechanism, is speci~c to the spacer regions, leaving intact the DNA associated with the nucleosomes [18]. The detection in the cells of DNA fragments of a size equivalent to that of the mononucleosome combined with its multiplicity, i.e., the nucleosomal ladder, is frequently considered to be the trademark of apoptosis [18]. The presence of pieces of DNA of 200 bp and multiples of 200 bp is characteristic of apoptosis [17]. This pattern of DNA cleavage can be detected biochemically by gel electrophoresis and morphologically by the terminal deoxynucleotidyl transferase (TdT) assay [19]. These techniques identify early stages of apoptosis, documenting irreversible double-strand breaks in the DNA. However, their sensitivity is unknown [20]. This nuclear modi~cation is accompanied by a preservation of the cytoplasmic structures of the cell [21], indicating that DNA damage is the primary event in the initiation of myocyte cell death [17]. Importantly, the histochemical detection of apoptosis by the TdT reaction can be complemented with the identi~cation of alterations in the chromatin pattern in the same nucleus by confocal microscopy. Such an approach, illustrated in Figure 1, permits the simultaneous recognition of two fundamental aspects of programmed cell death [22]: chromatin fragmentation and internucleosomal DNA cleavage. Additionally, cleavage of the DNA characterized by the generation of 39 overhangs can be labeled through the ligation of double-stranded DNA fragments with a single 39 base extension [23]. This probe can be obtained by Taq DNA polymerase in the polymerase chain reaction. Of relevance, this type of internucleosomal cleavage is the

Effect of Diabetes on Protein Synthesis in the Myocardium

Protein turnover in muscle represents the net result of synthesis of new protein from a precursor pool of amino acids and the degradation of contractile, regulatory and other proteins into their constituent amino acids. In muscle, an array of hormonal, mechanical, and metabolic factors influence protein synthesis and degradation. Insulin, for example, is a potent stimulus of protein turnover increasing synthesis and attenuating degradation [1,2]. Fractional synthesis (Ks) and degradation (Kd) rate represent the fraction of the available precursor that is synthesized into protein or the protein product which is degraded into amino acids, respectively. Fractional synthesis rate is often expressed as a percent of the amino acid precursor pool utilized over time.

Rat Heterotopic Cardiac Isograft Model: What Atrophy Teaches Us about Hypertrophy

Cardiac hypertrophy is a fundamental adaptive response of the adult mammalian heart to superimposed mechanical and neurohumoral loads [1–4]. The precise molecular genetic, biochemical, and physiologic characteristics of adaptive cardiac hypertrophy are, in large measure, a reflection of the nature of the inciting stimulus. Nowhere is this more clear than when contrasting the cardiac response to chronic exercise conditioning with that secondary to chronic pressure overload [5,6]. In rats, these stimuli elicit coordinate changes in expression of a number of cardiac restricted genes and proteins. For example, α myosin heavy chain, sarcoplasmic reticular calcium ATPase, and cardiac troponin I mRNA and/or protein increase with chronic physical conditioning, whereas with pressure overload, coordinated decreases in all of these components (and increases in β myosin heavy chain and in ANF gene and protein expression) are seen.