Tetracycline-regulated cardiac gene expression in vivo.

Tight regulation of foreign genes expressed in vivo would facilitate studies of many biologic processes and would be useful for gene transfer-based therapies. To test the ability of a tetracycline-regulated gene expression system to function in vivo, we directly injected chimeric tet repressor-VP16 transactivator expression plasmids and luciferase target genes into the hearts of adult rats. Cardiac luciferase activity increased over two orders of magnitude in response to small changes in input tetracycline-controlled transactivator DNA. Transactivation was repressed to background levels by subtherapeutic concentrations of tetracycline in a dose-dependent manner. Target gene expression could be rapidly and reversibly controlled by manipulating antibiotic administration. This system may be particularly useful for in vivo studies of gene function or gene therapies where the timing or extent of expression are critical variables.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

The roles of corticotropin-releasing factor-related peptides and their receptors in the cardiovascular system.

Corticotropin-releasing factor (CRF), CRF-related peptides and their receptors are present in the central nervous system and in peripheral tissues including the immune, reproductive and cardiovascular systems. CRF and urocortin (urocortin 1) bind to the CRF receptor type 1 (CRF(1) receptor) and the CRF receptor type 2 (CRF(2) receptor), whereas urocortin 2 (formerly known as stresscopin related peptide) and urocortin 3 (formerly known as stresscopin) bind with high affinity to the CRF(2) receptor. Recent studies show that urocortin 1, urocortin 2 and urocortin 3 are potent regulators of cardiovascular function. This review highlights the role of cardiovascular CRF and related peptides and its relevance in mediating the adaptive response of the cardiovascular system to stressful conditions.

Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitor improves left ventricular function and alters the pattern of left ventricular dilatation after myocardial infarction.

Background- Left ventricular (LV) remodeling after myocardial infarction (MI) is associated with fibrosis, dilatation, and dysfunction. We postulated that prevention of fibrosis after MI with a prolyl 4-hydroxylase inhibitor (P4HI) would preserve LV function and attenuate LV enlargement. Methods and Results- Adult female rats (200 to 250 g) had experimental MI and were then randomized to treatment with P4HI (MI-FG041, n=29) or vehicle (MI-control, n=29) 48 hours after MI for 4 weeks in 2 phases. Echocardiograms were performed weekly with a 15-MHz linear transducer, and at 4 weeks, collagen isoform determinations and in vivo hemodynamics were performed. At randomization, the infarct size and LV function and size were similar in MI-FG041 and MI-control but significantly different from shams (n=9). At week 4, the LV function in MI-FG041 was significantly better than in MI-controls (fractional shortening 21% versus 16%, P=0.01; fractional area change 30% versus 19%, P=0.002; ejection fraction 35% versus 23%, P=0.001). In the FG041 group, LV area in systole was less (P<0.05), the dP/dt(max) after isoproterenol was higher (P<0.05), and types I and III collagen in noninfarcted LV were less than in MI-control. The hydroxyproline/proline ratio was increased by 64% in MI-control and reduced to the sham value in MI-FG041 rats. In the scar tissue, it was reduced by 24% in MI-FG041. Conclusions- This study demonstrates that prevention of interstitial fibrosis with a P4H inhibitor alters the pattern of LV enlargement and produces partial recovery of LV function after MI.

Effects of systolic overload and swim training on cardiac mechanics and biochemistry in rats.

We have previously shown that swim conditioning corrects the depressed mechanical function and myosin adenosinetriphosphatase (ATPase) activities associated with renovascular hypertension (HTN) in the rat. The present study was designed to assess the effects of swim conditioning on another form of systolic overload, subdiaphragmatic suprarenal aortic stenosis. Cardiac mechanics in an isolated working heart apparatus and myosin enzymology were studied in four groups of rats: controls (C), animals with chronic systolic overload secondary to aortic constriction (St), swim-conditioning animals (Sw), and animals exposed to a combined load (St-Sw). Heart weight was increased by 23% in St, 27% in Sw, and 36% in St-Sw. In contrast to HTN, cardiac pump and muscle function were not depressed in St. Sw was associated with improved cardiac output, stroke work, and velocity of circumferential fiber shortening. St-Sw showed improved mechanical cardiac performance relative to both C and St. The percent of ventricular myosin of the V1 type and Ca2+-activated myosin ATPase activity relative to C was unchanged in Sw but was depressed in St and St-Sw. These data demonstrate that the salutory mechanical effects of Sw can be superimposed on the systolic overload of St. However, the dissociation between mechanics and myosin enzymology suggests that factors in excitation-contraction coupling other than myosin isoenzyme shifts are responsible for this finding.

Combined effects of hypertension and conditioning on coronary vascular reserve in rats.

To evaluate the combined effects of cardiac overload imposed by hypertension and chronic swim training on coronary vascularity, female rats were made hypertensive by unilateral renal artery stenoses and were exercised in an 8- to 10-wk swimming program. Maximal coronary flow was assessed in isolated retrograde buffer-perfused hearts under conditions of minimal coronary resistance (15 microM adenosine or anoxia). Sedentary normotensive animals, sedentary hypertensive animals, and normotensive animals exposed to a swimming program were also studied. Swimming was associated with an 18% increase in heart weight and with increases in both absolute (ml/min) and relative (ml X g-1 X min-1) maximal coronary flow. Hypertension was associated with a 32% increase in heart weight but with a decrease in absolute and relative coronary flow compared with controls. The combined stimuli resulted in a 63% myocardial hypertrophy and a 19% increase in absolute flow. Relative coronary flow (g tissue-1) was similar in hearts from hypertensive sedentary animals and hypertensive swimmers. These data indicate that the coronary vascular deficit that accompanies the cardiac hypertrophy of hypertension is not worsened by the superimposition of an exercise load that exaggerates the hypertrophy.

Angiotensin receptor 1 blockade does not prevent physiological cardiac hypertrophy in the adult rat.

The renin-angiotensin system has been implicated in the hypertrophic adaptation of the heart to exogenous pathological loads, such as hypertension and aortic stenosis; however, the role of this hormonal system in the cardiac adaptations to physiological loads, such as chronic exercise conditioning, has not been established. We therefore studied the effect of angiotensin receptor 1 (AT1) blockade on the chronic cardiac responses of rats subjected to an 8-wk swimming program. Compared with matched sedentary controls, untreated swimmers increased their left ventricular weights by 13%, and swimmers treated with the AT1 antagonist L-158809 increased their left ventricular weights by 11% (both P < 0.05 vs. sedentary controls). The incorporation of labeled amino acids into the heart at the time of death was unchanged in all groups, and therefore the increase in heart weight in both swim-conditioned groups appeared to reflect a decrease in the rate of protein degradation in the heart. Hearts from both swim-conditioned groups manifested an increase in the V1-predominant myosin isoform pattern but not an increase in atrial natriuretic factor mRNA expression or protein kinase C translocation. The fact that these patterns of adaptation are preserved in exercised conditioned animals treated with an AT1 antagonist suggests that the chronic hypertrophic response of the heart to physiological loads is not influenced by the renin-angiotensin system.

Early increases in coronary vascular reserve in exercised rats are independent of cardiac hypertrophy.

To evaluate the relationship between the physiological cardiac hypertrophy associated with physical training and the increases in vascular capacitance associated with this stimuli, male and female rats trained by a swimming program were studied. Both sexes were used so that the coronary vascular response to exercise could be studied in the presence (females) and absence (males) of cardiac hypertrophy. Coronary vascular reserve was assessed in isolated retrograde buffer-perfused hearts under conditions of minimal coronary resistance (15 microM adenosine or anoxia). Both groups demonstrated an increase in coronary vascular reserve after 8 wk of exercise swim training, male animals increasing flow (per g of myocardium) by 15% and females by 18%. When the time course of this response was compared in female animals with the time course of the development of myocardial hypertrophy, it was evident that the vascular changes occurred early, greater than 80% of the response was seen within the first 10 days of exercise, compared with an approximately 35% increase in cardiac mass. These data suggest that the vascular response to exercise swim training is independent of the hypertrophic response and further that the increase in coronary vascularity is an early event in the cardiac adaptation to a physiological load.

Physiologic, biochemical, and coronary adaptation to exercise conditioning.

Although more studies need to be done, particularly using sensitive nuclear techniques in humans, the data available demonstrate that conditioning has multiple salutory effects on the heart and suggest that exercise training may be an important adjuvant therapy for a variety of clinical disorders.

Gene transfer in the cardiovascular system: update 2000.

It has been slightly more than 10 years since the first proof-of-concept studies were performed, which demonstrated the feasibility of gene transfer into the heart and vasculature of experimental animals. Since that time there has been a dramatic increase in the nature and sophistication of gene transfer techniques and also in the number of cardiovascular diseases that are potential targets for gene-based therapies. In this article, the authors review the current strategies for gene delivery, including viral and nonviral approaches. The authors also highlight several biologic processes within the cardiovascular system, including restenosis, experimental angiogenesis, heart failure, and atherosclerosis-conditions for which gene therapy shows promise. It is hoped that this will provide an update of this therapeutic strategy for the year 2000.

Protein kinase C(epsilon) modulates apoptosis induced by beta -adrenergic stimulation in adult rat ventricular myocytes via extracellular signal-regulated kinase (ERK) activity.

Beta-adrenergic stimulation of ventricular myocytes has been shown to induce apoptosis; however, the cellular mechanisms involved in this pathway have not been completely characterized. This study examines the role of protein kinase C (PKC) in the signaling cascade that mediates beta-adrenergic stimulation-induced apoptosis. Stimulation of beta-adrenergic receptors using isoproterenol (ISO, 1-10 microm, 24 h) induced apoptosis in cultured adult rat ventricular myocytes (ARVM) in a dose-dependent manner. Treatment with ISO significantly resulted in the membrane translocation of PKC(epsilon), but not of PKC alpha or delta in ARVM. The activation of PKC(epsilon) by ISO was confirmed using an immune complex kinase assay. To address whether PKC(epsilon) is involved in the mechanism of ISO-induced apoptosis, we used the PKC(epsilon)-specific translocation inhibitor peptide, epsilonV1-2. Peptide epsilonV1-2 significantly blocked the translocation of PKC(epsilon), as well as the enzymatic action of PKC(epsilon), resulting from ISO stimulation. The inhibition of PKC(epsilon) attenuated ISO-induced apoptosis as measured by terminal deoxynucleotidyltransferase nick-end labeling (TUNEL) assay (18.2+/-3.8%v 49.0+/-2.4%P<0.05), while a PKC delta-specific peptide translocation inhibitor (delta V1-1) failed to do so (39.8+/-7.8%). In the presence of ISO, PKC(epsilon) inhibition by epsilonV1-2 was found to significantly enhance activity of ERK, but not that of Akt/PKB. Inhibition of ERK activation by PD 98059 (10-50 microm) attenuated the epsilonV1-2 peptide-mediated anti-apoptotic effect, thus suggesting that ERK activation is involved in this anti-apoptotic effect. Therefore, our results suggest that activation of PKC(epsilon) downstream of beta-adrenergic stimulation promotes apoptosis largely via inhibition of an ERK activation-dependent anti-apoptotic effect.

Ventricular myosin light chain 1 is developmentally regulated and does not change in hypertension.

Cardiac myosin heavy chain (MHC) isoform distribution has been shown to undergo changes during development, in response to hormonal stimuli, and during pathologic states like hypertension. We initiated a study of myosin light chain 1 (MLC1) expression in cardiac tissue to determine whether MLC1 undergoes changes similar to those seen for MHC. We isolated a full length cDNA for the predominant MLC1 sequence in rat hearts. This gene is expressed in ventricular tissue at much higher levels than in atrial tissue. Based on its expression pattern and sequence homology, this cDNA encodes the rat ventricular MLC1 and has been named RVMLC1. RVMLC1 is expressed at very low levels in cardiac tissue during early development and is expressed abundantly after birth and in adult hearts. The expression of RVMLC1 was found not to change in the hearts of rats with renovascular hypertension.

PKC-lambda is the atypical protein kinase C isoform expressed by immature ventricle.

We recently identified a developmental decline in protein kinase C (PKC) isoform expression, at the level of the protein, in rat ventricular myocardium. To investigate mechanisms regulating PKC isoform expression in cardiac tissue, this study uses Northern blot analysis to compare the abundance of PKC isoform mRNAs in neonatal and adult rat ventricular myocardium. PKC-epsilon protein and mRNA were detected in both neonatal and adult rat ventricular myocardial preparations. In contrast, coordinate postnatal declines in the abundance of PKC-alpha and PKC-delta proteins and transcripts were identified. An antiserum raised against the COOH-terminal sequence of PKC-zeta detected abundant immunoreactivity in neonatal, but not adult, ventricular myocytes. However, PKC-zeta transcripts were not detectable in the heart either by Northern blot analysis or a reverse transcriptase-polymerase chain reaction approach, indicating that neither the myocytes nor the contaminating cellular elements in the heart express PKC-zeta. Rather, PKC-lambda, another atypical PKC isoform that is structurally highly homologous to PKC-zeta, was detected at the protein and mRNA level in neonatal, but not adult, ventricular myocardium. Taken together, these results establish that developmental declines in calcium-sensitive, novel, and atypical PKC isoforms are paralleled by changes in the levels of the mRNAs encoding these proteins, suggesting transcriptional regulation of PKC during normal cardiac development. The results of this study further identify PKC-lambda as the atypical PKC isoform expressed by the immature ventricle.

Function and bioenergetics in isolated perfused trained rat hearts.

To evaluate the resistance of physiologically hypertrophied hearts to hypoxic insult, we quantified the development of functional deficits during hypoxia and reoxygenation in hypertrophied hearts from swim-trained female rats and we correlated this with assessment of high-energy phosphate (HEP) metabolites from simultaneous 31P nuclear magnetic resonance (NMR) measurements. Furthermore, in vivo enzymatic studies were carried out with saturation transfer NMR under well-oxygenated perfusion conditions for both beating and KCl-arrested hearts. Finally, in vitro enzymatic assays were performed. During hypoxia, the trained hearts exhibited improved systolic and diastolic function compared with hearts from sedentary animals. After 16 min of hypoxia, left ventricular (LV) developed pressure fell to 9% of baseline in control hearts but to only 21% of baseline in trained hearts (P < 0.01). LV diastolic function was also improved by training, increasing during hypoxia from a baseline of 10 to 71.0 +/- 3.3 mmHg in control hearts and to 55.3 +/- 4.8 mmHg in trained hearts (P < 0.05). Trained hearts also showed more rapid and complete recovery of function during reoxygenation and greater coronary flow per gram of heart throughout the entire protocol. Functional differences were not accompanied by differences in HEP at baseline; moreover, ATP and phosphocreatine (PCr) loss during hypoxia was similar between control and trained hearts, as was the recovery of PCr during reoxygenation. Saturation transfer experiments showed an increase in the forward creatine kinase (CrK) rate constant in trained hearts of 18% while beating, whereas in vitro enzymatic analysis revealed a 16% increase in the ratio of mitochondrial CrK to citrate synthase activity in LV tissue. Thus the relative preservation of function in hearts from trained rats could not be accounted for by overall HEP levels but may reflect adaptations in the CrK system.

Behavior of genes directly injected into the rat heart in vivo.

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of promoter elements of the rabbit cardiac sarcoplasmic reticulum Ca(2+)-ATPase gene required for expression in cardiac muscle cells.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in the contractile performance of cardiac and slow-twitch skeletal muscle by restoring cytosolic calcium to low resting levels during the contractile cycle. We have previously shown that SERCA2 expression in the heart is altered by a number of pathophysiological stimuli. In an effort to define molecular mechanisms regulating expression of the SERCA2 gene in cardiac muscle cells, deletions of a 1460-bp promoter fragment were generated and inserted into a luciferase reporter plasmid. Promoter constructs were transiently transfected into embryonic cardiocytes and skeletal muscle cell lines Sol 8 and C2C12 in vitro and injected into adult myocardium in vivo. Results demonstrate that sequences from the transcription start site to -284 are both necessary and sufficient for high-level transcription of the reporter gene in differentiating muscle cells and in fetal cardiocytes in culture. We further demonstrate that this promoter fragment is highly active in vivo when injected into rat hearts, suggesting that the same regulatory elements are functional in vivo as well as in vitro. The region of the gene from -284 to -658 exerts a modest positive effect in cardiocytes and Sol 8 myotubes but exerts a negative effect in C2C12 fast skeletal muscle cells. This initial analysis of transcriptional regulation of the SERCA2 gene will serve as a foundation for the study of alterations of expression of the gene in pathological conditions.

Ventricular pacing attenuates but does not reverse cardiac atrophy and an isomyosin shift in the rat heart.

The heterotopically transplanted rat heart (TH) undergoes rapid muscle atrophy and a concurrent shift from alpha- to beta-myosin heavy chain (MHC) by 1 wk after surgery. In the current experiments, TH were continuously paced (420 beats/min) for 1 wk beginning 24 h after surgery or for 1 wk beginning 14 days after surgery to determine the role of increased heart rate in preventing or reversing cardiac atrophy. Left ventricular (LV) wet weight (283 vs. 256 mg paced vs. nonpaced) and protein content (32 vs. 23 mg paced vs. nonpaced, P < 0.05) were significantly elevated in TH paced 1 wk after surgery but were unchanged (211 vs. 198 mg and 24 vs. 23 mg LV wet wt and protein content, respectively) in TH paced 2 wk after surgery. Total cardiac protein synthesis in the TH paced immediately after surgery was increased compared with the corresponding nonpaced hearts (5.6 vs. 4.0 mg.mg LV wet wt-1.day-1, P < 0.05), while in the TH, where pacing was initiated 2 wk after surgery, it was unchanged (3.6 vs. 3.7 mg.mg LV wet wt-1.day-1). Fractional synthesis rate was elevated in TH and was not altered by pacing. Pacing the TH also attenuated the shift in alpha-MHC in the first 7 days after surgery but did not reverse the shift 2 wk later. The increase in protein synthesis combined with an unchanged fractional synthesis rate suggests that pacing attenuates cardiac mass by decreasing protein degradation and that once the atrophic process is established, neither synthesis rate nor isomyosin shift can be altered by continuous pacing.

Functional analysis of the connexin43 gene promoter in vivo and in vitro.

Connexin43 is the principal gap junction protein found in the mammalian heart. The regulation of connexin43 gene expression may be an important determinant of normal and altered cardiac development and electrophysiology. We constructed a series of chimeric luciferase reporter genes containing nested deletions from the human connexin43 gene (-2400 to -50 basepairs, relative to transcription initiation). The transcriptional activity of the chimeric genes was assayed in several cell types and systems, including rat cardiocytes in vitro and adult rat heart in vivo. In both systems, high levels of luciferase activity required at least 175 base-pairs of 5'-flanking sequence. Constructs including 2400 base-pairs of upstream sequence increased activity two-fold in vivo, but not in vitro. Chimeric genes were also assayed in cultured cardiac non-myocytes. This population of cells accumulates significant levels of connexin43 mRNA when propagated in vitro and promoter constructs were correspondingly active. Elements within the proximal promoter may also confer tissue-specificity. Transfectional analysis of the SKHep1 cell line, which does not express connexin43, demonstrated reduced reporter gene activity, especially for the longest constructs. These studies begin to define the cis-acting elements of the connexin43 gene which regulate its strength and specificity of transcription.

Myosin isoenzyme distribution in overloaded human atrial tissue.

Using nondenaturing polyacrylamide gel electrophoresis, we have identified two distinct myosin isoenzymes in human atrial tissue that correspond to the V1 and V3 isomyosins found in rat ventricular tissue. Normal left and right atrial appendages have approximately 50% V3. When the left atrium was exposed to hemodynamic overload secondary to mitral stenosis, the percent V3 increased to 77 +/- 10% (n = 10); exposure to hemodynamic overload secondary to mitral regurgitation caused an increase to 70 +/- 14% (n = 6). Changes in the isoenzyme pattern were seen in the right atria of patients with mitral stenosis and markedly elevated pulmonary arterial pressures compared with control subjects and patients with mitral stenosis without severe pulmonary hypertension. Several clinical variables were examined to determine which factors might influence isoenzyme expression. Age, sex, the presence of atrial fibrillation, and pulmonary capillary wedge pressure did not predict the isoenzyme pattern. However, patients with mitral valvular disease and only slightly enlarged left atria tended to have a higher percent V3 than those with massively enlarged atria. These data confirm that human atrial tissue, like rat ventricular tissue, can alter its isomyosin composition in response to a hemodynamic load. The data further suggest that the isoenzyme shift is an early adaptation to the imposed load.

Increased heart rate prevents the isomyosin shift after cardiac transplantation in the rat.

The heterotopically transplanted rat heart undergoes significant atrophy and a shift from V1 to V3 isomyosin. The purpose of this study was to pace the cardiac isograft and determine whether an increase in heart rate would attenuate the changes in cardiac mass and isoenzyme distribution. Nonpaced transplanted hearts were compared with hearts in which pacing was initiated at 7 Hz, 24 hours after transplantation, and continued for 7 days. There was a 29% decrease in myosin ATPase activity and a 22% decrease in alpha-myosin in the nonpaced isograft; both decreases were completely prevented by pacing. The decrease in cardiac mass was also significantly attenuated. Pacing did not alter intrinsic heart rate, systolic pressure, dP/dt, or norepinephrine concentration in the isograft. These results suggest that the adaptation in both cardiac mass and isoenzymes may be related to the rate or the rate-pressure product in the transplanted paced heart independent of left ventricular pressure, tissue catecholamines, or neural activity.