Effect of Chronic Heart Rate Reduction by I(f) Current Inhibitor Ivabradine on Left Ventricular Remodeling and Systolic Performance in Middle-Aged Rats With Postmyocardial Infarction Heart Failure.

BACKGROUND: A large myocardial infarction (MI) initiates progressive cardiac remodeling that leads to systolic heart failure (HF). Long-term heart rate reduction (HRR) induced by the I f current inhibitor ivabradine (IVA) ameliorates left ventricular (LV) remodeling and improves systolic performance in young post-MI rats. However, the beneficial effects of chronic IVA treatment in middle-aged rats remain to be determined. METHODS: A large MI was induced in 12-month-old rats by left coronary artery ligation. Rats were treated with IVA via osmotic pumps intraperitoneal in a dose of 10.5 mg/kg/d (MI + IVA) and compared with MI and sham-operated animals 12 weeks after MI. RESULTS: Heart rate in MI + IVA rats was on average 29% lower than that of rats in the MI group. Left ventricular remodeling was comparable between post-MI groups, although MI + IVA rats did not show the compensatory thickening of the noninfarcted myocardium. Chronic HRR had no effect on transverse cardiac myocyte size and capillary growth, but it reduced the collagen content in noninfarcted myocardium. Left ventricular systolic performance remained similarly impaired in MI and MI + IVA rats. Moreover, abrupt IVA withdrawal led to worsening HF and reduction of coronary reserve. CONCLUSION: Our data reveal that chronic IVA-induced HRR does not provide sustainable benefits for LV systolic performance in middle-aged rats with post-MI HF.

Coronary vessels and cardiac myocytes of middle-aged rats demonstrate regional sex-specific adaptation in response to postmyocardial infarction remodeling.

BACKGROUND: An increasing body of evidence indicates that left ventricular (LV) remodeling, especially the degree of reactive myocardial hypertrophy after myocardial infarction (MI), differs in males and females. Surprisingly, to date, the sex-specific post-MI alterations of the coronary vasculature remain undetermined. Therefore, we tested the hypothesis that adaptive coronary arteriolar and capillary modifications occurring in response to reactive myocyte hypertrophy differ between middle-aged male and female post-MI rats. METHODS: A large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague-Dawley rats by ligation of the left coronary artery. Four weeks after surgery, rats with transmural infarctions, greater than 50% of the LV free wall (FW), were evaluated. Sham-operated male (M-Sham) and female (F-Sham) rats served as an age-matched controls. RESULTS: F-MI and M-MI rats had similar sized infarcts (61.3% ± 3.9% vs. 61.5% ± 1.2%) and scale of LV remodeling, as indicated analogous remodeling indices (1.41 ± 0.11 vs. 1.39 ± 0.09). The degree of reactive post-MI myocardial hypertrophy was adequate to normalize LV weight-to-body weight ratio in both sexes; however, the F-MI rats, in contrast to males, showed no myocyte enlargement in the LVFW epimyocardium. At the same time, a greater than 50% expansion of myocyte area in the male epimyocardium and in the female endomyocardium was accompanied by a 23% (P < 0.05) increase in capillary-to-myocyte ratio, indicative of adaptive angiogenesis. Based on arteriolar length density in post-MI hearts, the resistance vessels grew in the male LVFW as well as the septum by 24% and 29%, respectively. In contrast, in females, a significant (30%) expansion of arteriolar bed was limited only to the LVFW. Moreover, in F-MI rats, the enlargement of the arteriolar bed occurred predominantly in the vessels with diameters <30 µm, whereas in M-MI rats, a substantial (two- to threefold) increase in the density of larger arterioles (30 to 50 µm in diameter) was also documented. CONCLUSION: Our data reveal that while both sexes have a relatively similar pattern of global LV remodeling and adaptive angiogenesis in response to a large MI, male and female middle-aged rats differ markedly in the regional scale of reactive cardiac myocyte hypertrophy and adaptive arteriogenesis.

Embryonic coronary vasculogenesis and angiogenesis are regulated by interactions between multiple FGFs and VEGF and are influenced by mesenchymal stem cells.

In embryonic hearts explanted on collagen gels, epicardial cells delaminate and form vascular tubes, thus providing a model for coronary tubulogenesis. Using this model, we show that fibroblast growth factors (FGFs) 1, 2, 4, 8, 9, and 18 contribute to tubulogenesis and that the availability of multiple FGFs provides the optimal tubulogenic response. Moreover, the FGF effects are vascular endothelial growth factor (VEGF) -dependent, while VEGF-induced tubulogenesis requires FGF signaling. The number of endothelial cells (ECs) is increased by all of the FGFs, while EC migration is significantly enhanced only by FGF-2 and FGF-18. Finally, addition of embryonic mesenchymal stem cells (EMSC) to the explants markedly enhances EC numbers and a 23-fold increase in stromal derived factor-1α (SDF-1α), which is FGF dependent. Both explants and EMSCs produce SDF-1α. In conclusion, coronary tubulogenesis of embryonic epicardium: (1) is responsive to many FGF family members, (2) requires both FGF and VEGFA signaling, and (3) is responsive to EMSCs.

Chronic heart rate reduction facilitates cardiomyocyte survival after myocardial infarction.

Chronic heart rate reduction (HRR) therapy following myocardial infarction, using either the pure HRR agent ivabradine or the beta-blocker atenolol, has been shown to preserve maximal coronary perfusion, via reduction of perivascular collagen and a decrease in renin-angiotensin system activation. In addition ivabradine, but not atenolol, treatment attenuated the decline in ejection fraction and decreased left ventricular wall stress. In this study, we tested the hypothesis that cell survival within the infarct region was enhanced by these two pharmacological agents. Four weeks after ligating the left anterior descending coronary artery, the percentage of the LV that contained the infarct was similar in the untreated (MI) rats and those chronically treated with ivabradine (MI + IVA) or atenolol (MI + ATEN). However, the mean thickness (mm) of the ventricular wall containing the scar was significantly greater in the MI + IVA, 1.54 (P < or = 0.01) and the MI + ATEN 1.32, compared to 1.1 in the MI group, due to a 2-fold greater area of surviving cardiomyocytes (P < or = 0.01) in the treated rats compared to the untreated group. Regions of cell survival were usually in the subepicardium, with cardiomyocytes surrounding veins or venules. However, some hearts displayed surviving cells along the endocardium. These data suggest that HRR by either ivabradine or atenolol facilitates a more favorable O2 microenvironment via improved venous flow and decreased O2 demand. We conclude that chronic HRR by these agents may serve to limit infarct expansion and wall thinning and may serve to reduce the potential for ventricular rupture.

Postmyocardial infarction remodeling and coronary reserve: effects of ivabradine and beta blockade therapy.

We compared the effects of heart rate reduction (HRR) by the hyperpolarization-activated pacemaker current (I(f)) channel inhibitor ivabradine (MI+Iva) and the beta(1)-blocker atenolol (MI+Aten) on ventricular remodeling and perfusion after myocardial infarction (MI) in middle-aged (12 mo) Sprague-Dawley rats. Mean HRR was virtually identical in the two treated groups (19%). Four weeks after coronary artery ligation, maximal myocardial perfusion fell in the MI group but was preserved in infarcted rats treated with either Iva or Aten. However, coronary reserve in the remodeled hearts was preserved only with Iva, since Aten treatment elevated baseline perfusion in response to a higher wall stress. The higher maximal perfusion noted in the two treated groups was not due to arteriogenesis or angiogenesis. Plasma levels of angiotensin (ANG) II and myocardial ANG type 1 (AT(1)) receptor and transforming growth factor (TGF)-beta1 were reduced during the first week of treatment by both Iva and Aten. Moreover, treatment also reduced arteriolar perivascular collagen density. Despite these similar effects of Iva and Aten on vascularity and ANG II, Iva, but not Aten, attenuated the decline in ejection fraction and lowered left ventricular (LV) end-diastolic volume (LVEDV)-to-LV mass ratio, determined by echocardiography. In conclusion, 1) Iva has advantages over Aten in postinfarction therapy that are not due to differential effects of the drugs on heart rate, and 2) age limits growth factor upregulation, angiogenesis, and arteriogenesis in the postinfarcted heart.

Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses.

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.

Temporally expressed PDGF and FGF-2 regulate embryonic coronary artery formation and growth.

OBJECTIVE: PDGF and FGF-2 are important regulators of vascular wall assembly. We tested the hypothesis that their embryonic temporal expression facilitates 2 specific events: (1) the endothelial invasion of the aortic root to form the coronary artery stems and (2) the subsequent growth and development of the arterial tree. METHODS AND RESULTS: Addition of FGF-2 and PDGF-BB proteins to embryonic quail heart explants stimulated a 3- and 7-fold increase, respectively, in tubulogenesis, whereas neutralizing antibodies to these growth factors attenuated tubulogenesis by 40%. Anti-FGF-2 and anti-PDGF neutralizing antibodies were then introduced in ovo via the vitelline vein at various embryonic (E) days. When injections occurred before coronary ostial formation, the embryos usually developed only 1 coronary artery or lacked coronary arteries. When 1 or both major coronary arteries formed: (1) their branches had a thinner tunica media, and (2) smooth muscle investment did not progress as far distally as in shams. Other anomalies included smaller diameter coronary artery stems in some hearts. Inhibition of VEGF via injections of aflibercept (VEGF-Trap, a VEGFR-1 and -2 chimera), previously shown to be essential for coronary stem formation, limited development of the coronary arteries even though introduced after formation of coronary ostia (at E9 or EI0). CONCLUSIONS: Our data (1) document a role for FGF-2 and PDGF in the temporal regulation of coronary artery stem formation and growth of the coronary arterial tree and (2) reveal that VEGF expression is required for normal artery/arterial formation, even after coronary artery stem formation.

Preservation of coronary reserve by ivabradine-induced reduction in heart rate in infarcted rats is associated with decrease in perivascular collagen.

We tested the hypothesis that chronically reducing the heart rate in infarcted middle-aged rats using ivabradine (IVA) would induce arteriolar growth and attenuate perivascular collagen and, thereby, improve maximal perfusion and coronary reserve in the surviving myocardium. Myocardial infarction (MI) was induced in 12-mo-old male Sprague-Dawley rats, which were then treated with either IVA (10.5 mg.kg(-1).day(-1); MI + IVA) or placebo (MI) via intraperitoneal osmotic pumps for 4 wk. Four weeks of IVA treatment limited the increase in left ventricular end-diastolic pressure and the decrease in ejection fraction but did not affect the size of the infarct, the magnitude of myocyte hypertrophy, or the degree of arteriolar and capillary growth. However, treatment reduced interstitial and periarteriolar collagen in the surviving myocardium of MI + IVA rats. The reduced periarteriolar collagen content was associated with improvement in maximal myocardial perfusion and coronary reserve. Although the rates of proliferation of periarteriolar fibroblasts were similar in the MI and MI + IVA groups, the expression levels of the AT(1) receptor and transforming growth factor (TGF)-beta(1) in the myocardium, as well as the plasma level of the ANG II peptide, were lower in treated rats 14 days after MI. Therefore, our data reveal that improved maximal myocardial perfusion and coronary reserve in MI + IVA rats are most likely the result of reduced periarteriolar collagen rather than enhanced arteriolar growth.

Bradycardia stimulates vascular growth during gradual coronary occlusion.

OBJECTIVE: In cultured endothelium, stretch induces release of growth factors that contribute to angiogenesis. We tested the hypothesis that bradycardia, which prolongs ventricular diastolic filling time and volume, promotes collateral vessel growth. METHODS AND RESULTS: An ameroid occluder was placed on coronary arteries of dogs with normal heart rates (AM) or bradycardia (55 bpm; AM+BC). A third group had normal heart rates and no ameroid (control [CON]). Four weeks after occluder placement, myocardial blood flow at rest and maximal vasodilation (adenosine) at equivalent heart rates and vascular morphometry of hearts were measured. In AM dogs, conductance (myocardial flow/diastolic pressure) of collateral-dependent myocardium was similar to collateral-independent myocardium during rest but increased to only one third of CON during maximal vasodilation. In contrast, in AM+BC dogs, conductance was similar in collateral-dependent and -independent regions during maximal vasodilation. Arteriolar length density in collateral-dependent myocardium was 80% greater in AM+BC than AM dogs. Capillary length density in collateral-dependent region of AM dogs was lower than CON but normal in AM+BC dogs. The angiopoietin receptor Tie-2 increased in collateral-dependent regions of AM and AM+BC groups, whereas vascular endothelial growth factor increased in collateral-dependent and -independent regions only in AM+BC dogs. CONCLUSIONS: Chronic bradycardia during gradual coronary artery occlusion facilitates angiogenesis/arteriogenesis in collateral-dependent myocardium and preserves maximal perfusion.

Reduction of heart rate by chronic beta1-adrenoceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted heart.

Adequate growth of coronary vasculature in the remaining left ventricular (LV) myocardium after myocardial infarction (post-MI) is a crucial factor for myocyte survival and performance. We previously demonstrated that post-MI coronary angiogenesis can be stimulated by bradycardia induced with the ATP-sensitive K(+) channel antagonist alinidine. In this study, we tested the hypothesis that heart rate reduction with beta-blockade may also induce coronary growth in the post-MI heart. Transmural MI was induced in 12-mo-old male Sprague-Dawley rats by occlusion of the left anterior descending coronary artery. Bradycardia was induced by administration of the beta-adrenoceptor blocker atenolol (AT) via drinking water (30 mg/day). Three groups of rats were compared: 1) control/sham (C/SH), 2) MI, and 3) MI + AT. In the MI + AT rats, heart rate was consistently reduced by 25-28% compared with C/SH rats. At 4 wk after left anterior descending coronary ligation, infarct size was similar in MI and MI + AT rats (67.1 and 61.5%, respectively), whereas a greater ventricular hypertrophy occurred in bradycardic rats, as indicated by a higher ventricular weight-to-body weight ratio (3.4 +/- 0.1 vs. 2.8 +/- 0.1 mg/g in MI rats). Analysis of LV function revealed a smaller drop in ejection fraction in the MI + AT than in the MI group ( approximately 24 vs. approximately 35%). Furthermore, in MI + AT rats, maximal coronary conductance and coronary perfusion reserve were significantly improved compared with the MI group. The better myocardial perfusion indexes in MI + AT rats were associated with a greater increase in arteriolar length density than in the MI group. Thus chronic reduction of heart rate induced with beta-selective blockade promotes growth of coronary arterioles and, thereby, facilitates regional myocardial perfusion in post-MI hearts.

Stretch induces upregulation of key tyrosine kinase receptors in microvascular endothelial cells.

We previously demonstrated that cyclic stretch of cardiac myocytes activates paracrine signaling via vascular endothelial growth factor (VEGF) leading to angiogenesis. The present study tested the hypothesis that cyclic stretch upregulates tyrosine kinase receptors in rat coronary microvascular endothelial cells (RCMEC) and human umbilical vein endothelial cells (HUVEC). VEGF receptor-2 (Flk-1) protein levels increased in HUVEC and RCMEC in a time-dependent manner, but the increase occurred much earlier in RCMEC than in HUVEC. The enhancement of Flk-1 protein level was not inhibited by addition of VEGF neutralizing antibodies, indicating that VEGF is not involved in stretch-induced Flk-1 expression. VEGF receptor-1 (Flt-1) protein and mRNA were not changed by stretch. However, Tie-2 and Tie-1 protein levels increased in RCMEC. Angiopoietin-1 and -2, the ligands for Tie-2, increased in cardiac myocytes subjected to cyclic stretch but were not affected by stretch in endothelial cells (EC). Stretch or incubation of RCMEC with VEGF increased cell proliferation moderately, whereas stretch + VEGF had an additive effect on proliferation. Mechanical stretch induces upregulation of the key tyrosine kinase receptors Flk-1, Tie-2, and Tie-1 in vascular EC, which underlies the increase in sensitivity of EC to growth factors and, therefore, facilitates angiogenesis. These in vitro findings support the concept that stretch of cardiac myocytes and EC plays a key role in coronary angiogenesis.

Bradycardia induces angiogenesis, increases coronary reserve, and preserves function of the postinfarcted heart.

BACKGROUND: We tested the hypothesis that induction of chronic bradycardia would trigger an upregulation of key growth factors and receptors, which would then lead to angiogenesis and improve coronary reserve in the left ventricle after myocardial infarction. METHODS AND RESULTS: Bradycardia was induced in rats by administering alinidine via osmotic pumps beginning 1 day after coronary artery ligation. Echocardiographic analysis was conducted before and after treatment. Morphometric analysis was used in perfusion-fixed hearts to document arteriolar and capillary growth. Western blots were used to evaluate growth factor and receptor changes. During the first week of treatment, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), and basic fibroblast growth factor proteins were higher in the treated group, whereas VEGF receptor 2 (Flk-1), angiopoietin-1, and angiopoietin-2 were not affected by treatment. After 3 weeks, VEGF protein remained elevated, and bradycardia was associated with a higher capillary length density in the border (40%) and remote (14%) regions and a higher arteriolar length density in the septum (62%), despite a greater increase in left ventricular mass. Although arteriolar length density increased in all size classes, the greatest increase occurred in the smallest (terminal) arterioles. This vascular growth was associated with a 23% greater coronary reserve. Echocardiography revealed a smaller increase in ventricular volume and a greater preservation of ejection fraction in rats treated with bradycardia. CONCLUSIONS: Pharmacologic induction of bradycardia enhances vascularity and coronary reserve, preserves function of surviving myocardium, and therefore, is a noninvasive, therapeutic avenue that provides an alternative to gene therapy.

Estrogen therapy induces collateral and microvascular remodeling.

Estrogen increases proliferation and migration of cultured endothelial cells and perfusion of ischemic hindlimbs of rabbits. We tested the hypothesis that estrogen is angiogenic and arteriogenic in the heart during progressive coronary occlusion. Ovariectomized (OVX) and 17beta-estradiol (1 mg.kg(-1).wk(-1) im)-treated OVX (OVX-ES) female New Zealand White rabbits were instrumented with an ameroid occluder on a proximal coronary artery. Four weeks after implantation of an ameroid occluder, we measured myocardial perfusion with microspheres at rest and during adenosine-induced maximal vasodilation. The heart was fixed by perfusion at physiological pressure, and capillary angiogenesis and remodeling were assessed by image analysis of tissue sections in collateral-dependent myocardium. Coronary conductance was higher at rest and during maximal vasodilation in collateral-dependent myocardium of OVX-ES than OVX rabbits. Estrogen treatment increased the wall-to-lumen ratio of collateral vessels while it decreased the wall-to-lumen ratio of noncollateral arteries in normal regions. In normal and collateral-dependent myocardium, mean capillary diameter and capillary volume density were greater in OVX-ES rabbits. However, estrogen had no effect on capillary length density in either region of the myocardium. These data suggest that estrogen induces remodeling of the collateral vasculature and may stimulate growth of the resistance vessels, thereby providing protection during development of a gradual coronary occlusion.

DITPA stimulates bFGF, VEGF, angiopoietin, and Tie-2 and facilitates coronary arteriolar growth.

Previous studies from our laboratory and those of others have shown thyroxine to be a stimulator of coronary microvascular growth. The present study tested the hypothesis that 3,5-diiodothyropropionic acid (DITPA), a thyroid hormone analog with inotropic but not chronotopic characteristics, is angiogenic in the nonischemic heart. Daily injections (3.75 mg/kg sc) of DITPA to Sprague-Dawley rats affected protein increases in vascular endothelial growth factor (VEGF)(164), VEGF(188,) basic fibroblast growth factor (bFGF) (FGF-2), angiopoietin-1, and Tie-2 during the first few days of treatment. After 3 wk of treatment, arteriolar length density and the relative number of terminal arterioles (<10 microm diameter) increased in the left ventricle as determined by image analysis of perfuse-fixed hearts. These findings occurred in hearts that did not undergo changes in mass nor in increases in capillary length density. We conclude that DITPA, which is known to improve ventricular function after infarction, is angiogenic in normal nonischemic hearts.