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We examined the role of the transforming growth factor (TGF)-β1signaling inhibitor Smad 7 in cardiac fibrosis. TGF-β1(10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-β type I receptor (TβRI), and TGF-β1proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TβRI expression were decreased in scar tissue, whereas TGF-β1expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.

We examined the effect of fibroblast growth factor (FGF)-2 on myocardial resistance to injury when administered after the onset of ischemia , in vivo and ex vivo, and the role of FGF-2 receptors and protein kinase C (PKC). FGF-2 was injected into the left ventricle of rats undergoing permanent surgical coronary occlusion leading to myocardial infarction (MI). After 24 h, FGF-2-treated hearts displayed significantly reduced injury, determined by histological staining and troponin T release, and improved developed pressure compared with untreated controls. An FGF-2 mutant with diminished affinity for the tyrosine kinase FGF-2 receptor 1 (FGFR1) was not cardioprotective. FGF-2-treated hearts retained improved function and decreased damage at 6 wk after MI. In the ex vivo heart, FGF-2 administration during reperfusion after 30-min ischemia improved functional recovery and increased relative levels of PKC subtypes α, ε, and ζ in the particulate fraction, in a chelerythrine-preventable mode; it also decreased loss of energy metabolites. We conclude that intramyocardial FGF-2 administration shortly after the onset of ischemia confers protection from acute and chronic cardiac dysfunction and damage; FGF-2 delivered during reperfusion protects from ischemia-reperfusion injury; and protection by FGF-2 requires intact binding to FGFR1 and is likely mediated by PKC.

Angiotensin II (angiotensin) and transforming growth factor-β1 (TGF-β1) play an important role in cardiac fibrosis and infarct scar remodeling after myocardial infarction (MI). We characterized 8 week post-MI rat hearts for altered expression of Smad proteins with and without losartan treatment. AT1 blockade was associated with attenuated activation of the latent form of TGF-β1 in remnant (viable) myocardium and infarct scar. Immunofluorescence (IF) studies revealed Smad 2 localization to myofibroblasts in target tissues with less intense staining in cardiac myocytes. Losartan administration (15mg/kg/day) for 8 weeks was associated with normalization of total cellular Smad 2 and Smad 4 overexpression in the infarct scar as well as Smad 2 overexpression in remnant heart tissue. On the other hand, phosphorylated Smad 2 (P-Smad 2) staining was reduced in cytosolic fractions from failing experimental heart tissues vs controls and these trends were normalized in the presence of losartan, suggesting augmented P-Smad 2 movement into nuclei in untreated hearts. Using cultured adult primary rat fibroblasts treated with 10-6M angiotensin, we noted rapid translocation (15 min) of P-Smad 2 into the cellular nuclei from the cytosol. Nuclear P-Smad 2 protein levels were increased in cultured fibroblasts following 15 min angiotensin treatment, and this response was blocked by losartan treatment. We conclude that angiotensin may influence total Smad 2 and 4 expression in post-MI heart failure, and that angiotensin treatment is associated with rapid P-Smad 2 nuclear translocation in isolated fibroblasts. This study suggests that crosstalk between angiotensin and Smad signaling are associated with fibrotic events in post-MI hearts.

We have previously shown that myofibroblasts of the healed 8 week infarct scar overexpress transduction proteins that may be linked to elevated deposition of extracellular matrix components in this tissue. Other work suggests that TGFβ1, may be involved in cardiac fibrosis and myocyte hypertrophy. The significance of the altered TGFβ signaling in heart failure in the chronic phase of post-myocardial infarction (MI), particularly in the ongoing remodeling of the infarct scar, remains unexplored. Patterns of cardiac TGFβ1 and Smad 2, 3, and 4 protein expression were investigated 8 weeks after MI and were compared to relative collagen deposition in border tissues (containing remnant myocytes) and the infarct scar (nonmyocytes dominated by myofibroblasts). Both TGFβ1 mRNA abundance (Northern analysis) and protein levels (ELISA) were significantly increased in the infarct scar versus control values, and this trend was positively correlated to increased collagen type I expression in this tissues. Cardiac Smad 2, 3, and 4 proteins were significantly increased in border and scar tissues versus control values. Immunofluorescent studies indicated that Smad proteins were proximally localized to cellular nuclei in the infarct scar. TβRI (53 kDa) protein expression was significantly reduced in the scar, while the 75kDa and 110kDa isoforms of TβRII were unchanged and significantly increased respectively, in the infarct scar. These results indicate that TGFβ/Smad signaling may be involved in the remodeling of the infarct scar after the completion of wound healing per se, via ongoing stimulation of matrix deposition.

See article by Bruel et al. [4] (pages 400–408) in this issue. The classic mythological tale of the crimes of Prometheus in gifting mortal men with, among other things, the divine fire, serves to remind us never to anger anyone as powerful as Zeus. For his efforts, Prometheus was shackled to a mountain to be tormented daily by Zeus' pet eagle who, according to legend, preferred liver. Each subsequent evening, Prometheus' tissues regenerated, and the cycle of mechanical destruction and regeneration would repeat ad nauseum until finally Zeus regained a modicum of calm and released him. What is possibly more fascinating (and certainly much less gruesome) – the notion of rebuilding or regenerating the myocardium following either catastrophic myocyte loss (as in myocardial infarction or MI) or in progressive loss of contractile cells in various progressive cardiomyopathies – has long intrigued both clinical and basic investigators in cardiology and is currently stirring a considerable degree of interest and effort. In this vein a number of exciting developments have come to pass within the past 2years that deserve comment. New myocytes in the heart: stem cells vs. stimulation of primordial cells. The putative “carrot” within the approach of cardiac regeneration includes the reversal of … *Tel.: +1 204 235 3419. idixon{at}sbrc.ca

Background —Large transmural myocardial infarction (MI) leads to maladaptive cardiac remodeling and places patients at increased risk of congestive heart failure. Angiotensin II, endothelin, and α 1 -adrenergic receptor agonists are implicated in the development of cardiac hypertrophy, interstitial fibrosis, and heart failure after MI. Because these agonists are coupled to and activate G qα protein in the heart, the aim of the present study was to investigate G qα expression and function in cardiac remodeling and heart failure after MI. Methods and Results —MI was produced in rats by ligation of the left coronary artery, and G qα protein concentration, localization, and mRNA abundance were noted in surviving left ventricle remote from the infarct and in border and scar tissues from 8-week post-MI hearts with moderate heart failure. Immunohistochemical staining localized elevated G qα expression in the scar and border tissues. Western analysis confirmed significant upregulation of G qα proteins in these regions versus controls. Furthermore, Northern analysis revealed that the ratios of G qα /GAPDH mRNA abundance in both scar and viable tissues from experimental hearts were significantly increased versus controls. Increased expression of phospholipase C (PLC)-β 1 and PLC-β 3 proteins was apparent in the scar and viable tissues after MI versus controls and is associated with increased PLC-β 1 activity in experimental hearts. Furthermore, inositol 1,4,5-tris-phosphate is significantly increased in the border and scar tissues compared with control values. Conclusions —Upregulation of the G qα /PLC-β pathway was observed in the viable, border, and scar tissues in post-MI hearts. G qα and PLC-β may play important roles in scar remodeling as well as cardiac hypertrophy and fibrosis of the surviving tissue in post-MI rat heart. It is suggested that the G qα /PLC-β pathway may provide a possible novel target for altering postinfarct remodeling.

An explanation of the molecular mechanisms that trigger the development of pathological cardiac fibrosis, myocyte hypertrophy, and heart failure associated with common ailments such as chronic postinfarction has been sought at the benchtop and clinic for the past forty years and is a current topic of intensive investigative activity. Among the information garnered from these studies is that (1) growth factors including angiotensin (Ang) II are involved in modulation of gene products specifically expressed by cardiac fibroblasts in vitro and (2) their enhanced presence has been associated with myocardial stress, inappropriate cardiac growth, and fibrosis in vivo. Although increased deposition of collagen proteins has been described after myocardial infarction (MI), little is known of (1) time-dependent transcriptional alteration of specific cardiac collagen subtypes or (2) the degradative mechanisms for cardiac collagens in right and left ventricular myocardium remote to large left ventricular infarction. We have investigated collagen mRNA abundance and the deposition of specific collagen subtypes in noninfarcted left and right rat heart muscle at different times after MI. We have also assessed the activity of different myocardial matrix metalloproteinases (MMPs) using zymography to gain some information about degradative pathways for collagen. Furthermore, we assessed passive compliance properties of the right ventricle in experimental hearts. Finally, the efficacy of an angiotensin-converting enzyme (ACE) inhibitor (ramipril) and that of an Ang II receptor type I antagonist (losartan) was compared in experimental animals. We observed that the mRNA abundance of types I and III collagen were increased 3 days after myocardial infarction in both viable left and uninfarcted right ventricular tissues and were maintained at relatively high levels throughout the duration of our studies. Stiffness of the right ventricular myocardium was significantly increased in the 56 days experimental group when compared with that of control values; this was positively correlated with increased immunoreactive collagens in surviving right (and left) cardiac tissue of 14, 28, and 56 day experimental groups. The elevation of fibrillar collagen mRNA abundance in noninfarcted muscle from ventricular chambers was not normalized by treatments with either ramipril or losartan. MMP activity was increased in viable left ventricle at 14, 28, and 56 days and at 14 days in the right ventricle in experimental animals when compared with controls. Finally, we observed that AT1 receptor blockade in infarcted hearts was associated with normalization of elevated cardiac 4-prolyl hydroxylase protein expression compared with nontreated post-MI rats. Thus, increased expression of collagen types I and III occurs in noninfarcted heart after MI, followed thereafter by deposition of collagen proteins. Increased MMP activity is present in chronic experimental hearts; MMP activation may be important in remodeling of the noninfarcted cardiac stroma. Because losartan treatment was not associated with any normalization of elevated collagen mRNA abundance, a clear causal relationship between the upregulation of fibrillar collagen mRNA abundance and the AT1 receptor was not apparent. On the other hand, the reduction of cardiac fibrosis mediated by ACE inhibition and losartan treatment may reside at the posttranslational level in cardiac collagen metabolism.

Although increased deposition of collagen proteins has been described after myocardial infarction (MI), little is known of time-dependent transcriptional alteration of specific cardiac collagen sub-types as well as the degradative mechanisms for cardiac collagens in right and left ventricular myocardium remote to large left ventricular infarction. We sought to study collagen mRNA abundance and the deposition of specific collagen subtypes in noninfarcted left and right rat heart muscle at different times after MI. We also assessed the activity of different myocardial matrix metalloproteinases (MMP) using zymography to gain some information about degradative pathways for collagen. Furthermore, we assessed passive compliance properties of the right ventricle in experimental hearts. Finally we investigated the role of the renin angiotensin system in the collagen gene expression by administration of an angiotensin converting enzyme (ACE) inhibitor (ramipril) and an angiotensin II receptor type I antagonist (losartan) in experimental animals. We observed that the mRNA abundance of types I and III collagen were increased 3 days after myocardial infarction in both viable left and uninfarcted right ventricular tissues, that they peaked at 7-14 days, and were maintained at relatively high levels in the 28 and 56 days experimental groups. Stiffness of the right ventricular myocardium was significantly increased in the 56 days experimental group when compared to that of control values. These findings correlated with increased immunohistochemical staining patterns of different collagen species in the surviving right (and left) cardiac interstitium of 14, 28, and 56 day experimental cardiac groups. The elevation of fibrillar collagen mRNA abundance in noninfarcted muscle from ventricular chambers was not significantly altered after treatment of experimental animals with ramipril and losartan for up to 14 days. MMP activity was increased in viable left ventricle at 14, 28 and 56 days and at 14 days in the right ventricle in experimental animals when compared to controls. These results indicated that (1) activation of transcription of collagen types I and III gene occurs in acute and chronic MI, and that fibrillar collagen proteins are deposited in the noninfarcted cardiac interstitium after a lag period relative to increased corresponding mRNA abundance; (2) an increase in MMP activity in chronic experimental hearts indicates that increased collagen deposition may be due to an increment in collagen synthesis rather by reduced degradation of collagen, and that MMP activation may be important in remodeling of the noninfarcted cardiac stroma; (3) an increase of right ventricular stiffness was associated with increased deposition of collagen; (4) as losartan treatment is not associated with any normalization of elevated collagen mRNA abundance, the upregulation of collagen gene expression in this model is not mediated by AT1 receptor; and (5) the reduction of cardiac fibrosis mediated by ACE inhibition and losartan treatment may reside at the post-translational level in cardiac collagen metabolism.

The efficacy of angiotensin converting enzyme (ACE) inhibitors is well known to prevent the formation of angiotensin II (Ang II) by these agents. The objective of the present study was to evaluate the hemodynamic, biochemical, and morphological responses to Ang II receptor blockade with E-4177, 3-[(2′-carboxybiphenyl-4-yl) methyl]-2-cyclopropyl-7-methyl 3H-imidazol[4,5-b] pyridine, in rats with a healing myocardial infarction that had been induced by the surgical occlusion of the left main coronary artery. The left ventricular weight increased 8 and 12 weeks after infarction in comparison to that in sham-operated rats. Among the rats with experimental infarction, treatment with E-4177 significantly decreased the left ventricular weight. Although the infarct size was not affected by E-4177, its administration ameliorated the elevated end-diastolic pressure and reduced the systolic pressure. The effects of this agent on the levels of Ang II type 1 (AT1) receptor mRNA and ACE mRNA were evaluated in the non-infarcted myocardium by reverse transcriptase polymerase chain reaction and binding assays. Treatment with E-4177 reduced both the elevated AT1 mRNA and the number of Ang II receptors, but not the ACE mRNA or ACE activity. While the receptor affinity remained unchanged with this agent, the collagen concentration was decreased. On the other hand, the depressed Na + /Ca 2+ exchange activity was restored in the non-infarcted myocardium at 8 and 12 weeks after injury to the level seen in the sham-operated rats. These findings suggest that the AT1 receptor antagonist, E-4177, has a beneficial effect on the hemodynamics in spite of the lack of any improvement in the infarct size. These observations may be partly attributed to the prevention of angiotensin II formation during the period of post-infarction healing.

A significant body of evidence from observations in clinical and experimental models has accumulated to demonstrate that a progressive loss of intrinsic contractility occurs in surviving myocardium after large transmural myocardial infarction, and the degree of cardiac pump dysfunction in viable tissue has been correlated to the size of the initial infarct [1–4]; however, no common biochemical defect has been identified to explain the loss of cardiac function [5]. Previous work indicates that structural and biochemical remodeling of surviving cardiac tissue is characterized by concentric and eccentric hypertrophy of myocytes and altered biochemical characteristics of myosin protein [4,6–9]. Furthermore, defective sarcolemmal and sarcoplasmic reticular membrane functions have been described both in the clinical setting and in experimental models of heart failure, including the rat model of congestive heart failure following myocardial infarction [5]. Indeed, many previous investigative efforts that have addressed the pathology of myocardial infarction have been directed at mechanisms to explain the loss of contractility in surviving myocardium of infarcted hearts. The extracellular matrix has been suggested to play a role in the pathogenesis of heart failure, and recent work from this laboratory has revealed that the collagen, a major component of the matrix, is altered in remaining viable myocardium [10].

Recent studies suggest that angiotensin II (angiotensin) may be involved in the regulation of metabolism of the cardiac extracellular matrix (ECM). Two major components of ECM are collagen types I and III which play an important role in maintaining the structure and function of the heart. Although the cellular metabolism of collagen is very complex (especially at the posttranslational level), we chose to address events that occur relatively early in the synthesis of cardiac collagen molecules. To gain an understanding of the role of angiotensin in the regulation of cardiac collagen gene expression, we studied the effect of three different doses of angiotensin (12, 24, and 48 µg/kg/h) on adult heart and cultured neonatal cardiac fibroblasts. The steady-state mRNA abundance of collagen types I and III was monitored using Northern blot analysis in both left and right ventricular samples at day 3 of angiotensin infusion and in cultured cardiac fibroblasts stimulated with angiotensin. In all mRNA abundance studies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal was used to normalize the data for possible differences in loading and/or transfer of total RNA. Both collagen types I/GAPDH and III/GAPDH mRNA signal ratios were increased significantly in left ventricle in all dose regimens used for angiotensin infusion. Only the collagen type I/GAPDH mRNA signal ratio was increased in right ventricle with angiotensin infusion. Angiotensin (10−7–10−5 M) had no effect on the steady-state mRNA abundance of collagen genes in cultured neonatal cardiac fibroblasts after 24 h treatment in serum-free conditions. Our results confirm that infusion of angiotensin may upregulate steady-state collagen gene mRNA abundance in the heart. Angiotensin had no observable effect on collagen mRNA abundance in neonatal fibroblast culture. An explanation for the current results may be that angiotensin causes the release of undefined factors from cardiac myocytes, and that these secondary factors may be involved in either the activation of collagen gene transcription or in alteration of stability of collagen mRNA transcripts via a paracrine mechanism. Although our results indicate hemodynamic loading may potentiate the action of angiotensin, this scenario is unlikely as collagen type I gene expression was increased in the normotensive right ventricle.

There is increasing evidence that the renin-angiotensin system (RAS) may play a important role in the development of left ventricular remodeling and hypertrophy after myocardial infarction (MI). Previous studies have demonstrated that chronic treatment with angiotensin converting enzyme (ACE) inhibitors has beneficial effects for survival survival after MI. To elucidate the role of the RAS in the reactive hypertrophy after MI, we evaluated the effect of an angiotensin II (Ang II) receptor antagonist, E-4177 (AT-1 antagonist), on rat myocardium with infarct (IF) and noninfarcted (NI) regions, and also determined the relationship between the role of RAS and the trans-sarcolernmal Ca2+ transport activity because of the disorder in Na+-Ca2+ exchange] described previously. Myocardial infarctions were induced by surgical occlusion of the left main coronary artery in rat, and oral drug therapy was initiated beginning on the next day. Rats were killed 4, 8, and 12 weeks later, and left ventricular (LV) weight, LV to body weight (BW) ratio, and inter-ventricular septal (IVS) weight to BW were measured. The left ventricule was separated into three sections with IF (scar) or NI myocardium (LV free wall and IVS). At 4 weeks after MI, the ratio of IVS to BW was significantly higher in the MI group and indicated that reactive hypertrophy was observed in NI myocardium. Collagen concentration and ACE activity were significantly increased in NI myocardium compared with the same region of the sham-operated rat, although the ACE activity in the IF region increased 10-fold. The denmsity of Ang II receptor was also increased in NI compared with sham-operated rats. The administration of E-4177 significantly reduced the ratio of IVS to BW, collagen concentration, and ACE activity in NI myocardium, and also reduced the collagen concentration in the IF region of the LV. Na+-Ca2+ exchange activity (Na+-dependent Ca2+ uptake) in sarco-lemmal membrane was significantly decreased in NI myocardium at 8 and 12 weeks after MI compared with that sham-operated rats. By E-4177 administration, Na+-Ca2+exchange activity was significantly increased in MI rats compared with untreated MI rats. When we determined the relationship between Na+-Ca2+ exchange and cardiac ACE activities in sham-operated, treated, and untreated hearts in NI myocardium, a significant negative correlation was obtained. These results suggest that the AT-1 antagonist, E-4177, inhibited the development of reactive hypertrophy and improved its Ca2+ transport activity. Therefore, E-4177 may be clinically useful for the treatment of the development of left ventricular remodeling and hypertrophy after MI.

Heart disease remains the leading cause of death in the industrialised world, accounting for 50% of all deaths in North America. Overwhelmingly the most important form of heart disease is ischaemic heart disease (IHD), which usually (90% of cases) takes the form of a myocardial infarction (MI) as a result of artherosclerosis (AS). Fissuring of an AS plaque results in thrombosis, which can cause blockage of the coronary arteries leading to ischaemia. Prolonged ischaemia (20 minutes) leads to irreversible damage to the heart. If the initial ischaemic event is survived, significant modifications of the extracellular matrix (ECM) of the heart become apparent after about 7 days, leading to pronounced scar formation after 6–8 weeks. As the ECM plays an important role in maintaining the mechanical and electrical properties of the heart, modifications of the ECM have pronounced pathological effects, resulting in reduced compliance of the left ventricle and potentially fatal arrhythmias (due to modification of conduction properties of the tissues)., 6th European Conference on the Spectroscopy of Biological Molecules, September 3–8, 1995, Villeneuve d’Ascq, France

The major protein components of cardiac extracellular matrix include collagen, fibronectin, and elastin. Collagen is by far the predominant component of the cardiac extracellular matrix, and it has been shown that heart muscle contains five isoforms of this protein, including types I, III, IV, V, and VI. Of these, collagen types I and III are most abundant and represent more than 90% of all collagen proteins in the myocardium. Recent investigation of regulation of these proteins indicates that the synthesis and degradation of cardiac collagens are mediated by several key peptide and cytokine factors. Cardiac collagen concentration (fibrosis) is dependent upon the balance struck between the synthetic and degradative pathways, which are assessed by transcriptional and translational studies of specific collagen genes and cardiac collagenase activity, respectively. Recent studies indicate that the extracellular matrix plays an important role in the remodeling of the myocardium in various types of experimental heart failure. Clinical studies also demonstrate that extracellular matrix proteins are altered in patients suffering from end-stage heart failure of different etiologies. It is suggested that increased deposition of extracellular matrix may impair normal heart function. In this regard, inappropriate deposition of collagen may reduce right and left ventricular chamber compliance, and may thereby affect both positive and negative cardiac inotropism. As these changes may accelerate the development of heart failure, pharmacologic management of synthetic/degradative pathways of extracellular matrix components should be a priority for effective treatment of heart failure.

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca(2+)-channels, Na(+)-Ca2+ exchange and Ca(2+)-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca(2+)-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development +(dP/dt)max and pressure fall -(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and -(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca(2+)-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na(+)-K+ ATPase were decreased whereas the Mg(2+)-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na(+)-K+ ATPase activity, whereas Ca(2+)-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg(2+)-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na(+)-Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca(2+)-transport processes is regulated by thyroid hormones and the modification of Ca(2+)-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure.

The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, alpha and beta, that exist in tandem array on chromosome 14q12. We have previously sequenced the entire human cardiac beta-MyHC gene and now report the complete nucleotide sequence of the human cardiac alpha-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translation initiation codon. With the exception of the 13th intron of the human cardiac beta-MyHC, which is not present within the alpha-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the alpha-MyHC gene include four Alu repeats, a single (GT)n element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human alpha-MyHC cDNA sequence reveals several potential polymorphisms.

It is now well established that Na+-K+ ATPase is primarily localized in the sarcolemmal membrane in the myocardium (1,2). This enzyme is intimately involved in the transport of Na+ and K+ and is thus considered to maintain the electrochemical gradient across the cell membrane which allows depolarization to occur. An inhibition of Na+-K+ ATPase activity has been shown to increase the intracellular concentration of Na+, which then increases the cytoplasmic level of Ca 2+ through Na +-Ca 2+ exchange system in the plasma membrane and thus has been proposed to serve as a mechanism of the positive inotropic action of cardiac glycosides (digitalis). Although Na+-K+ ATPase is known to possess high and low affinity sites for cardiac glycosides, the exact significance of these sites is not clear at present (3). Since the intracellular concentration of Na + has been shown to regulate myocardial contractility (4), any change in the operation of sarcolemmal Na+-K+ ATPase activity can be seen to alter Ca2+ ion movements in the cell and thus modify the heart function. Accordingly, a great deal of attention has been focussed on elucidation of the function of this enzyme in both health and disease.

Moderate and severe stages of congestive heart failure due to the loss of myocardium upon coronary occlusion in rats was associated with an increase in alpha-adrenergic receptors and a decrease in beta-adrenergic receptors in the viable left ventricle. However, at early stages of heart failure the number of beta-adrenergic receptors was decreased without any changes in the number of alpha-adrenergic receptors. The affinities of these receptors to alpha receptor antagonist (3H-prazosin) and beta receptor antagonist (3H-dihydroalprenolol) were not altered in the failing hearts. On the other hand, the pattern of changes in both alpha- and beta-adrenergic receptors in heart membranes treated with oxygen free radical generating system was different from that seen in the failing hearts. In particular, the affinities for these receptors were decreased whereas the number of beta-receptors was increased and the number of alpha-receptors was decreased or unchanged. These results indicate that alterations in the adrenergic receptors in heart failure are not due to the formation of oxygen free radicals.

Because Na(+)-Ca2+ exchange and Ca2+ pump are thought to play a role in sarcolemmal Ca2+ movements, we examined the Na(+)-dependent Ca(2+)-uptake and ATP-dependent Ca(2+)-uptake activities in failing heart after myocardial infarction in rats. The left coronary artery was ligated, and the viable left ventricle was used 4, 8, and 16 wk later; sham-operated animals served as controls. Increased left ventricular diastolic pressure and decreased positive and negative change in pressure over time were observed in experimental animals at 4, 8, and 16 wk; these changes were associated with accumulation of fluid in the abdominal cavity. The sarcolemmal Na(+)-dependent Ca2+ uptake was depressed in 4-, 8-, and 16-wk experimental hearts. The decrease in sarcolemmal Na(+)-dependent Ca2+ uptake in failing hearts was seen when the activity was assayed either as a function of time or Ca2+ concentration; a depression of maximal velocity without any change in activity constant for Ca2+ was observed. No alteration in the Ca2+ pump (ATP-dependent Ca2+ accumulation and Ca(2+)-stimulated adenosinetriphosphatase) activities was evident in the 4-, 8-, and 16-wk experimental groups. These data suggest that changes in the Na(+)-dependent Ca2+ handling by the sarcolemmal membrane may be associated with contractile abnormalities in this model of congestive heart failure.

Rats, subsequent to loss of a large amount of left ventricular free wall due to surgically-induced myocardial infarction, form a good model of congestive heart failure. Since depressed cardiac pump function is the hallmark of heart failure, it is suspected that decreased influx of Ca2+ into the cardiac cell is responsible for depressed contractile function. Because Ca2+ movements in the sarcolemmal membrane are known to involve Ca2+-channels, Na+-Ca2+ exchange, Ca2+-pump, Na+-K+ ATPase, β-adrenoceptors and α-adrenoceptors directly or indirectly, the status of these mechanisms was examined by employing rats at different degrees of congestive heart failure. The left coronary artery was ligated and hearts were examined 4, 8, and 16 weeks later; sham-operated animals served as controls. The number of Ca2+ channels in the myocardium was depressed in moderate and severe stages of heart failure. Furthermore, depressions in sarcolemmal Na+-Ca2+ exchange activity and β-adrenoceptor number were associated with the development of early stages of heart failure, whereas sarcolemmal Na+-K+ ATPase activity was decreased and the number of aadrenoceptors was increased at moderate and severe stages. The Ca2+-pump activities were not altered in failing hearts. Thus it appears that changes in Na+-Ca2+ exchange as well as β-adrenoceptors and Ca2+ channels may contribute towards decreasing Ca2+ influx at early and moderate stages of congestive heart failure, respectively. On the other hand, changes in α-adrenoceptors and Na+-K+ ATPase may act as compensatory mechanisms for maintaining Ca2+ influx at moderate and late stages of congestive heart failure.

Although cardiac dysfunction due to ischemia-reperfusion injury is considered to involve oxygen free radicals, the exact manner by which this oxidative stress affects the myocardium is not clear. As the occurrence of intracellular Ca2+ overload has been shown to play a critical role in the genesis of cellular damage due to ischemia-reperfusion, this study was undertaken to examine whether oxygen free radicals are involved in altering the sarcolemmal Ca2(+)-transport activities due to reperfusion injury. When isolated rat hearts were made globally ischemic for 30 min and then reperfused for 5 min, the Ca2(+)-pump and Na(+)-Ca2+ exchange activities were depressed in the purified sarcolemmal fraction; these alterations were prevented when a free radical scavenger enzymes (superoxide dismutase plus catalase) were added to the reperfusion medium. Both the Ca2(+)-pump and Na(+)-Ca2+ exchange activities in control heart sarcolemmal preparations were depressed by activated oxygen-generating systems containing xanthine plus xanthine oxidase and H2O2; these changes were prevented by the inclusion of superoxide dismutase and catalase in the incubation medium. These results support the view that oxidative stress during ischemia-reperfusion may contribute towards the occurrence of intracellular Ca2+ overload and subsequent cell damage by depressing the sarcolemmal mechanisms governing the efflux of Ca2+ from the cardiac cell.

Although the occurrence of intracellular Ca2+ overload is known to be an important factor in hypoxia-reoxygenation injury, the exact mechanisms for this abnormality are not presently clear. Since Na+-Ca2+ exchange in the sarcolemmal membrane is considered to be involved in Ca2+ efflux, this study was undertaken to examine the effect of hypoxia reoxygenation on this system. Isolated rat hearts were made hypoxic by perfusing with a substrate-free medium gassed with 95% N2-5% CO2 and then reperfused with oxygenated normal medium. Hypoxia was found to markedly increase the resting tension and depress the ability of the heart to generate contractile force; reoxygenation resulted in partial recovery of these parameters. Sarcolemmal vesicles were isolated from control, hypoxic, and hypoxia-reoxygenated hearts, and the Na+-dependent Ca2+ uptake activity was measured at different times of incubation as well as at different concentrations of calcium. Sarcolemmal ATP-dependent Ca2+ accumulation was also measured for the purpose of comparison. A significant decrease in Na+-dependent Ca2+ uptake was observed in preparations from hearts made hypoxic for 10 min. Reoxygenation of 10-min hypoxic hearts resulted in a further depression of Na+-Ca2+ exchange activity. ATP-dependent Ca2+ accumulation was also depressed in hypoxic as well as reoxygenated hearts. These results suggest a defect in the Na+-Ca2+ exchange system and the ATP-dependent Ca2+ pump in the heart sarcolemmal membrane, and this may contribute to the occurrence of intracellular Ca2+ overload and functional abnormalities due to hypoxia-reoxygenation injury.

Global ischemia in guinea-pig hearts for 60 to 90 min depressed microsomal and mitochondrial Ca 2+ uptake activities. Reperfusion of the 60 min ischemic hearts resulted in incomplete recovery of contractile function and calcium uptake activities of both mitochondrial and microsomal fractions. On the other hand, reperfusion of the 90 min ischemic hearts further depressed the microsomal Ca 2+ uptake activity. Coronary occlusion for 90 min in dog hearts was found to decrease microsomal Ca 2+ -pump and sarcolemmal Na + K + ATPase activities. Reperfusion of these regional ischemic hearts further depressed the microsomal Ca 2+ uptake and Ca 2+ -stimulated ATPase as well as sarcolemmal Na + K + ATPase activities whereas mitochondrial Ca 2+ uptake was increased. Perfusion of rat hearts for 60 min with hypoxic medium resulted in depression of the sarcolemmal Na + -dependent Ca 2+ uptake and ATP-dependent Ca 2+ uptake activities. Reperfusion of these hypoxic hearts failed to recover the sarcolemmal Na + Ca 2+ exchange and Ca 2+ -pump activities. These results demonstrate that membrane defects with respect to Ca 2+ transport processes in ischemic/hypoxic hearts may be associated with irreversible injury.

Comparison of mid- and near-infrared spectra of control and infarcted rat ventricular tissue reveals the presence of absorptions in infarcted tissue which are highly characteristic of collagen, indicating large scale deposition of type I collagen in the myocardium following infarction. These results demonstrate that IR spectroscopy may be used to rapidly monitor the modifications of the extracellular matrix associated with myocardial infarction.