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MicroRNAs are posttranscriptional gene regulators that are differentially expressed during various diseases and have been implicated in the underlying pathogenesis. We report here that miR-199a is acutely downregulated in cardiac myocytes on a decline in oxygen tension. This reduction is required for the rapid upregulation of its target, hypoxia-inducible factor (Hif)-1α. Replenishing miR-199a during hypoxia inhibits Hif-1α expression and its stabilization of p53 and, thus, reduces apoptosis. On the other hand, knockdown of miR-199a during normoxia results in the upregulation of Hif-1α and Sirtuin (Sirt)1 and reproduces hypoxia preconditioning. Sirt1 is also a direct target of miR-199a and is responsible for downregulating prolyl hydroxylase 2, required for stabilization of Hif-1α. Thus, we conclude that miR-199a is a master regulator of a hypoxia-triggered pathway and can be exploited for preconditioning cells against hypoxic damage. In addition, the data demonstrate a functional link between 2 key molecules that regulate hypoxia preconditioning and longevity.

Abstract —Inotropic responsiveness to β-adrenergic stimulation is generally found to be impaired in left ventricular (LV) hypertrophy and failure. To investigate the mechanisms by which angiotensin-converting enzyme inhibitor therapy may modulate inotropic responsiveness with long-term pressure overload, we studied the effects of captopril treatment on cardiac gene expression, LV muscle mechanical contraction, and intracellular calcium (Ca 2+ ) transients from spontaneously hypertensive rats (SHR). LV papillary muscles from untreated SHR, age-matched normotensive Wistar-Kyoto rats (WKY), and SHR treated with captopril (CAP Rx started at 12, 18, and 21 months of age) were studied. All animals were studied at 24 months of age or when heart failure developed. In untreated SHR, α-myosin heavy chain (MHC) gene expression and protein were decreased, the Ca 2+ transient (with the bioluminescent indicator aequorin) was prolonged, and abundance of Na + /Ca 2+ exchanger mRNA levels increased in comparison to WKY. Active stress development at L max and the maximum rate of stress development were depressed and contractile duration prolonged in SHR relative to WKY. Isoproterenol administration further decreased active stress in untreated SHR despite an increase in intracellular Ca 2+ levels. In CAP Rx SHR, α- MHC gene expression and protein levels were increased, the Ca 2+ transient was not prolonged, Na + /Ca 2+ exchanger expression was downregulated, and papillary muscle function demonstrated increased active stress and maximum rate of stress development in response to isoproterenol. The increased abundance of α- MHC mRNA in conjunction with an increase in V 1 myosin isozyme suggests that captopril affects transcriptional regulation of cardiac gene expression. Restored LV inotropic responsiveness to β-adrenergic stimulation in CAP Rx SHR appears to be coupled to normalization of Na + /Ca 2+ exchanger mRNA expression, upregulation of V 1 myosin isozyme levels, and increased speed of contraction.

Abstract —Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)–1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB. Igf +/– mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB. Igf +/– mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB. Igf +/– mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. α-Myosin and β-myosin isoform mRNAs were affected by CAN, but α-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in α- and β-myosin isoform expression.

Abstract —Constitutive overexpression of insulin-like growth factor-1 (IGF-1) in myocytes protects them from apoptosis and interferes with myocyte hypertrophy in the normal and pathological heart. Conversely, angiotensin II (Ang II) triggers cell death and promotes myocyte hypertrophy. Moreover, activation of p53 upregulates the cellular renin-angiotensin system (RAS). Therefore, IGF-1 overexpression in FVB. Igf +/− mice may downregulate the local RAS through the attenuation of p53 and p53-inducible genes. On this basis, p53 DNA binding activity to angiotensinogen (Aogen), bax, and the AT 1 receptor was determined in left ventricular myocytes from FVB. Igf −/− and FVB. Igf +/− mice. The quantity of Bax, Bcl-2, Aogen, and AT 1 receptor in these cells was evaluated. The presence of Mdm2-p53 complexes was also established. Finally, Ang II levels in myocytes were measured. Upregulation of IGF-1 in myocytes was associated with a protein-to-protein interaction between Mdm2 and p53, which attenuated p53 transcriptional activity for bax, Aogen, and AT 1 receptor. Similarly, the amount of Bax, Aogen, and AT 1 receptor proteins in these cells decreased. In contrast, the expression of Bcl-2 remained constant. The downregulation of Aogen in myocytes from FVB. Igf +/− mice was characterized by a reduction in Ang II. In conclusion, IGF-1 negatively influences the myocyte RAS through the upregulation of Mdm2 and its binding to p53. This may represent the molecular mechanism responsible for the effects of IGF-1 on cell viability and myocyte hypertrophy in the nonpathological and pathological heart in vivo.

Abstract A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)–mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKCε isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate ( P P 1 ) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor–mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

The effects of renovascular hypertension on the biochemical, contractile, and electrical performance of myocardial tissue from rats of various ages has been examined. Male Fischer rats, 2, 7, 12, and 17 months old, were made hypertensive by constriction of the left renal artery. Ten weeks after the onset of hypertension, left ventricular papillary muscles were isolated from those four groups when 5, 10, 15, and 20 months old, respectively. Mechanical performance and transmembrane electrical events were recorded simultaneously. Contractile protein enzyme activity was determined in the same hearts from which papillary muscles were used for acquisition of mechanical and electrical information. There was a slight increase in blood pressure in control groups as a function of age while blood pressure maintained a range of approximately 179-188 mm Hg for all hypertensive groups. Heart weight of control animals increased significantly from 5 months to 20 months of age from 539 +/- 26 to 1088 +/- 56 mg, representing an increase of 101%. In hypertensive animals, heart weight increased 50% in 5-month-, 15% in 10-month-, 50% in 15-month-, and 11.7% in 20-month-old animals. Although control groups revealed alterations in mechanical, electrical, and biochemical parameters that increased as a function of age, the magnitude of the biochemical, contractile, and electrical response to hypertension varied monotonically with the extent of myocardial hypertrophy, rather than age per se. Adaptation to the stress of hypertension was observed in each age group, and was revealed as prolongation of mechanical and electrical timing parameters, depression of the load-velocity relation, and contractile protein enzyme activity. Thus, the stress of hypertension, which was tolerated by the 10- and 20-month-old animals with lesser relative hypertrophy and lesser changes in measured parameters, may represent a differential adaptation to the stress of hypertension.