Short communication: ischemia/reperfusion tolerance is time-of-day-dependent: mediation by the cardiomyocyte circadian clock.

RATIONALE: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown. OBJECTIVE: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice. METHODS AND RESULTS: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following 1 day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (zeitgeber time [ZT]12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following 1 month of reperfusion, prior ischemic event at ZT12 versus ZT0 resulted in significantly greater infarct volume, fibrosis, and adverse remodeling, as well as greater depression of contractile function. Genetic ablation of the cardiomyocyte circadian clock (termed cardiomyocyte-specific circadian clock mutant [CCM] mice) attenuated/abolished time-of-day variations in I/R outcomes observed in wild-type hearts. Investigation of Akt and glycogen synthase kinase-3beta in wild-type and CCM hearts identified these kinases as potential mechanistic ties between the cardiomyocyte circadian clock and I/R tolerance. CONCLUSIONS: We expose a profound time-of-day dependence for I/R tolerance, which is mediated by the cardiomyocyte circadian clock. Further understanding of I/R tolerance rhythms will potentially provide novel insight regarding the etiology and treatment of ischemia-induced cardiac dysfunction.

Doppler velocity measurements from large and small arteries of mice.

With the growth of genetic engineering, mice have become increasingly common as models of human diseases, and this has stimulated the development of techniques to assess the murine cardiovascular system. Our group has developed nonimaging and dedicated Doppler techniques for measuring blood velocity in the large and small peripheral arteries of anesthetized mice. We translated technology originally designed for human vessels for use in smaller mouse vessels at higher heart rates by using higher ultrasonic frequencies, smaller transducers, and higher-speed signal processing. With these methods one can measure cardiac filling and ejection velocities, velocity pulse arrival times for determining pulse wave velocity, peripheral blood velocity and vessel wall motion waveforms, jet velocities for the calculation of the pressure drop across stenoses, and left main coronary velocity for the estimation of coronary flow reserve. These noninvasive methods are convenient and easy to apply, but care must be taken in interpreting measurements due to Doppler sample volume size and angle of incidence. Doppler methods have been used to characterize and evaluate numerous cardiovascular phenotypes in mice and have been particularly useful in evaluating the cardiac and vascular remodeling that occur following transverse aortic constriction. Although duplex ultrasonic echo-Doppler instruments are being applied to mice, dedicated Doppler systems are more suitable for some applications. The magnitudes and waveforms of blood velocities from both cardiac and peripheral sites are similar in mice and humans, such that much of what is learned using Doppler technology in mice may be translated back to humans.

Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy.

Hypertrophic growth is a risk factor for mortality in heart diseases. Mechanisms are lacking for this global increase in RNA and protein per cell, which underlies hypertrophy. Hypertrophic signals cause phosphorylation of the RNA polymerase II C-terminal domain, required for transcript elongation. RNA polymerase II kinases include cyclin-dependent kinases-7 (Cdk7) and Cdk9, components of two basal transcription factors. We report activation of Cdk7 and -9 in hypertrophy triggered by signaling proteins (Galphaq, calcineurin) or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by endothelin. A preferential role for Cdk9 was shown in RNA polymerase II phosphorylation and growth induced by endothelin, using pharmacological and dominant-negative inhibitors. All four hypertrophic signals dissociated 7SK small nuclear RNA, an endogenous inhibitor, from cyclin T-Cdk9. Cdk9 was limiting for cardiac growth, shown by suppressing its inhibitor (7SK) in culture and preventing downregulation of its activator (cyclin T1) in mouse myocardium.Note: In the AOP version of this article, the numbering of the author affiliations was incorrect. This has now been fixed, and the affiliations appear correctly online and in print.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy.

Aortic banding produces pressure overload cardiac hypertrophy in mice, leading to decompensated heart failure in four to eight weeks, but the effects on coronary blood flow velocity and reserve are unknown. To determine whether coronary flow reserve (CFR) was reduced, we used noninvasive 20-MHz Doppler ultrasound to measure left main coronary flow velocity at baseline (B) and at hyperemia (H) induced by low (1%) and high (2.5%) concentrations of isoflurane gas anesthesia. Ten mice were studied before (Pre) and at 1 d, 7 d, 14 d and 21 d after constricting the aortic arch to 0.4 mm diameter distal to the innominate artery. We also measured cardiac inflow and outflow velocities at the mitral and aortic valves and velocity at the jet distal to the aortic constriction. The pressure drop as estimated by 4V2 at the jet was 51 +/- 5.1 (mean +/- SE) mm Hg at 1 d, increasing progressively to 74 +/- 5.2 mm Hg at 21 d. Aortic and mitral blood velocities were not significantly different after banding (p = NS), but CFR, as estimated by H/B, dropped progressively from 3.2 +/- 0.3 before banding to 2.2 +/- 0.4, 1.7 +/- 0.3, 1.4 +/- 0.2 and 1.1 +/- 0.1 at 1 d, 7 d, 14 d and 21 d, respectively (all p < 0.01 vs. Pre). There was also a significant and progressive increase the systolic/diastolic velocity ratio (0.17 Pre to 0.92 at 21 d, all p < 0.01 vs. Pre) suggesting a redistribution of perfusion from subendocardium to subepicardium. We show for the first time that CFR, as estimated by the hyperemic response to isoflurane and measured by Doppler ultrasound, can be measured serially in mice and conclude that CFR is virtually eliminated in banded mice after 21 d of remodeling and hypertrophy. These results demonstrate that CFR is reduced in mice as in humans with cardiac disease but before the onset of decompensated heart failure.

Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis.

Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors,and not on gene deletion. Here we used ROCK1knockout (ROCK1-/-) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1-/- mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1-/- mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFbeta2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of Gq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli.

Cardiac function in young and old Little mice.

We studied cardiac function in young and old, wild-type (WT), and longer-living Little mice using cardiac flow velocities, echocardiographic measurements, and left ventricular (LV) pressure (P) to determine if enhanced reserves were in part responsible for longevity in these mice. Resting/baseline cardiac function, as measured by velocities, LV dimensions, +dP/dt(max), and -dP/dt(max), was significantly lower in young Little mice versus young WT mice. Fractional shortening (FS) increased significantly, and neither +dP/dt(max) nor -dP/dt(max) declined with age in Little mice. In contrast, old WT mice had no change in FS but had significantly lower +dP/dt(max) and -dP/dt(max) versus young WT mice. Significant decreases were observed in the velocity indices of old Little mice versus old WT mice, but other parameters were unchanged. The magnitude of dobutamine stress response remained unchanged with age in Little mice, while that in WT mice decreased. These data suggest that while resting cardiac function in Little mice versus WT mice is lower at young age, it is relatively unaltered with aging. Additionally, cardiac function in response to stress was maintained with age in Little mice but not in their WT counterparts. Thus, some mouse models of increased longevity may not be associated with enhanced reserves.

CCL2/Monocyte Chemoattractant Protein-1 regulates inflammatory responses critical to healing myocardial infarcts.

The CC chemokine Monocyte Chemoattractant Protein (MCP)-1/CCL2 has potent mononuclear cell chemo-attractant properties, modulates fibroblast and endothelial cell phenotype and may play an important role in wound healing. In order to examine whether MCP-1 critically regulates myocardial infarct healing, we studied the effects of MCP-1 gene disruption and antibody neutralization in a closed-chest model of reperfused murine myocardial infarction. MCP-1-/- mice had decreased and delayed macrophage infiltration in the healing infarct and demonstrated delayed replacement of injured cardiomyocytes with granulation tissue. In contrast, the time course and density of neutrophil infiltration was similar in MCP-1 null and wild-type animals. MCP-1-/- infarcts had decreased mRNA expression of the cytokines TNF-alpha, IL-1beta, TGF-beta2, -beta3, and IL-10 and demonstrated defective macrophage differentiation evidenced by decreased Osteopontin-1 expression. MCP-1 deficiency diminished myofibroblast accumulation but did not significantly affect infarct angiogenesis. Despite showing delayed phagocytotic removal of dead cardiomyocytes, MCP-1-/- mice had attenuated left ventricular remodeling, but similar infarct size when compared with wild-type animals. MCP-1 antibody inhibition resulted in defects comparable with the pathological findings noted in infarcted MCP-1-/- animals without an effect on macrophage recruitment. MCP-1 has important effects on macrophage recruitment and activation, cytokine synthesis and myofibroblast accumulation in healing infarcts. Absence of MCP-1 results in attenuated post-infarction left ventricular remodeling, at the expense of a prolonged inflammatory phase and delayed replacement of injured cardiomyocytes with granulation tissue.

Effects of isoflurane on coronary blood flow velocity in young, old and ApoE(-/-) mice measured by Doppler ultrasound.

The commonly used anesthetic agent isoflurane (ISO) is a potent coronary vasodilator that could potentially be used in the assessment of coronary reserve, but its effects on coronary blood flow in mice are unknown. Coronary reserve is reduced by age, coronary artery disease and other cardiac pathologies in man, and some of these conditions can now be modeled in mice. Accordingly, we used Doppler ultrasound to measure coronary flow velocity in mice anesthetized with low (1%) and high (2.5%) levels of ISO to generate baseline (B) and elevated hyperemic (H) coronary flows, respectively. A 20-MHz Doppler probe was mounted in a micromanipulator and pointed trans-thoracically toward the origin of the left main coronary arteries of 10 6-wk (Young [Y]), 10 2-y (Old [O]) and 20 2-y apolipoprotein-E null (ApoE(-/-)) atherosclerotic (A) mice. In each mouse, we measured (B) and (H) peak diastolic velocities. B was 35.4 +/- 1.4 cm/s (Y), 24.8 +/- 1.6 (O) and 51.7 +/- 6.4 (A); H was 83.5 +/- 1.3 (Y), 86.5 +/- 1.9 (O) and 120 +/- 16.9 (A) and H/B was 2.4 +/- 0.1 (Y), 3.6 +/- 0.2 (O) and 2.5 +/- 0.2 (A). The differences in baseline velocities and H/B between O and Y and between A and O were significant (p < 0.01), whereas the differences in hyperemic velocities were not (p > 0.05). H/B was higher in old mice as a result of decreased baseline flow rather than increased hyperemic flow velocity. In contrast, ApoE(-/-) mice have increased baseline and hyperemic velocities, perhaps because of coronary lesions. The differences in baseline velocities between young and old mice could be the result of age-related changes in basal metabolism or to differential sensitivity to isoflurane. We conclude that Doppler ultrasound combined with coronary vasodilation via isoflurane could provide a convenient and noninvasive method to estimate coronary reserve in mice, but also that care must be taken when assessing coronary flow in mice under isoflurane anesthesia because of its potent coronary vasodilator properties.

Critical role of endogenous thrombospondin-1 in preventing expansion of healing myocardial infarcts.

BACKGROUND: Matricellular proteins are extracellular matrix proteins that do not contribute directly to tissue integrity but are capable of modulating cell function. We hypothesized that the matricellular protein thrombospondin (TSP)-1, a potent inhibitor of angiogenesis and activator of transforming growth factor (TGF-beta), is induced in healing myocardial infarcts and plays a role in suppressing the postinfarction inflammatory response, inhibiting local angiogenesis, and limiting expansion of granulation tissue into the noninfarcted area. METHODS AND RESULTS: We used a canine and a murine model of reperfused infarction. TSP-1 mRNA was induced in canine infarcts after 1 hour of ischemia and 3 to 7 days of reperfusion. TSP-1 protein showed a strikingly selective localization in the extracellular matrix, microvascular endothelium, and a subset of mononuclear cells of the infarct border zone after 5 to 28 days of reperfusion. Isolated canine venous endothelial cells showed low-level constitutive expression of TSP-1 mRNA, which was markedly induced by TGF-beta, and basic fibroblast growth factor. Murine infarcts also had marked TSP-1 deposition in the border zone. Infarcted TSP-1-/- mice exhibited sustained upregulation of the chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and interferon-gamma-inducible protein-10/CXCL10 and the cytokines interleukin-1beta, interleukin-6, and TGF-beta, suggesting an enhanced and prolonged postinfarction inflammatory response. In addition, TSP-1-/- mice had markedly increased macrophage and myofibroblast density in infarcts and in remodeling noninfarcted myocardial areas neighboring the myocardial scar, suggesting expansion of granulation tissue formation into the noninfarcted territory. TSP-1-/- animals had more extensive postinfarction remodeling than wild-type mice, although infarct size was similar in both groups. CONCLUSIONS: The infarct border zone may be capable of modulating the healing process through its unique extracellular matrix content. The selective endogenous expression of TSP-1 in the infarct border zone may serve as a "barrier," limiting expansion of granulation tissue and protecting the noninfarcted myocardium from fibrotic remodeling.

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.

Nuclear factor-kappaB protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction.

BACKGROUND: Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor kappaB (NF-kappaB) and given that NF-kappaB mediates cytoprotective responses, we asked whether NF-kappaB activation conferred cytoprotective responses in acute myocardial ischemia/infarction. METHODS AND RESULTS: We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated IkappaBalpha protein (IkappaBalphaDeltaN) that prevents nuclear translocation of NF-kappaB in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was approximately 50% greater (P<0.02) in the IkappaBalphaDeltaN mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P<0.008) in the IkappaBalphaDeltaN mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the IkappaBalphaDeltaN mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity. CONCLUSIONS: Transgenic mice with a defect in activation of NF-kappaB have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-kappaB are mediated, at least in part, by Bcl-2 or c-IAP1.

Disruption of Rho signaling results in progressive atrioventricular conduction defects while ventricular function remains preserved.

Recent studies suggest that RhoA and Rac1 mediate hypertrophic signals in cardiac myocyte hypertrophy. However, effects on cardiac function caused by inhibition of their activity in the heart have yet to be evaluated. Cardiac-specific inhibition of Rho family protein activities was achieved by expressing Rho GDIalpha, an endogenous specific GDP dissociation inhibitor for Rho family proteins, using the alpha-myosin heavy-chain promoter. Increased expression of Rho GDIalpha led to atrial arrhythmias and mild ventricular hypertrophy in adult mice (4-7 months). However, left ventricular systolic and diastolic function was largely preserved before and after the development of cardiac hypertrophy, indicating that Rho GTPases are not required to maintain ventricular contractile function under basal physiological condition. Electrocardiography and intracardiac electrophysiological studies revealed first-degree atrioventricular (AV) block in the transgenic heart at 1 week of age, which further progressed into second-degree AV block at 4 weeks of age before the development of cardiac hypertrophy. Expression of connexin 40 dramatically decreased from 1 week to 4 weeks of age in the transgenic heart, which may contribute in part to the conduction defects in the transgenic mice. This study provides novel evidence for an important role of Rho GTPases in regulating AV conduction.

Pulsed Doppler signal processing for use in mice: applications.

We have developed a high-frequency, high-resolution Doppler spectrum analyzer (DSPW) and compared its performance against an adapted clinical Medasonics spectrum analyzer (MSA) and a zero-crossing interval histogram (ZCIH) used previously by us to evaluate cardiovascular physiology in mice. The aortic velocity (means +/- SE: 92.7 +/- 2.5 versus 82.2 +/- 1.8 cm/s) and aortic acceleration (8194 +/- 319 versus 5178 +/- 191 cm/s2) determined by the DSPW were significantly higher compared to those by the MSA. Aortic ejection time was shorter (48.3 +/- 0.9 versus 64.6 +/- 1.8 ms) and the isovolumic relaxation was longer (17.6 +/- 0.6 versus 13.5 +/- 0.6 ms) when determined by the DSPW because it generates shorter temporal widths in the velocity spectra when compared to the MSA. These data indicate that the performance of the DSPW in evaluating cardiovascular physiology was better than that of the MSA. There were no significant differences between the aortic pulse wave velocity determined by using the ZCIH (391 +/- 16 cm/s) and the DSPW (394 +/- 20 cm/s). Besides monitoring cardiac function, we have used the DSPW for studying peripheral vascular physiology in normal, transgenic, and surgical models of mice. Several applications such as the detection of high stenotic jet velocities (> 4 m/s), vortex shedding frequencies (250 Hz), and subtle changes in wave shapes in peripheral vessels which could not obtained with clinical Doppler systems are now made possible with the DSPW.

Morphological characteristics of the microvasculature in healing myocardial infarcts.

Myocardial infarction (MI) is associated with an angiogenic response, critical for healing and cardiac repair. Using a canine model of myocardial ischemia and reperfusion, we examined the structural characteristics of the evolving microvasculature in healing MI. After 7 days of reperfusion, the infarcted territory was rich in capillaries and contained enlarged, pericyte-poor "mother vessels" and endothelial bridges. During scar maturation arteriolar density in the infarct increased, and a higher percentage of microvessels acquired a pericyte coat (60.4 +/- 6.94% after 28 days of reperfusion vs 30.17 +/- 3.65% after 7 days of reperfusion; p<0.05). The microvascular endothelium in the early stages of healing showed intense CD31/PECAM-1 and CD146/Mel-CAM immunoreactivity but weak staining with the Griffonia simplicifolia lectin I (GS-I). In contrast, after 28 days of reperfusion, most infarct microvessels demonstrated significant lectin binding. Our findings suggest that the infarct microvasculature undergoes a transition from an early phase of intense angiogenic activity to a maturation stage associated with pericyte recruitment and formation of a muscular coat. In addition, in the endothelium of infarct microvessels CD31 and CD146 expression appears to precede that of the specific sugar groups that bind the GS-I lectin. Understanding of the mechanisms underlying the formation and remodeling of the microvasculature after MI may be important in designing therapeutic interventions to optimize cardiac repair.

Vascular mural cells in healing canine myocardial infarcts.

Angiogenesis is a critical process in healing of myocardial infarcts, leading to the formation of highly vascular granulation tissue. However, effective cardiac repair depends on mechanisms that inhibit the angiogenic process after a mature scar is formed, preventing inappropriate expansion of the fibrotic process. Using a canine model of reperfused myocardial infarction, we demonstrated that maturation of the infarct leads to the formation of neovessels, with a thick muscular coat, that demonstrate distinct morphological characteristics. Many of these "neoarterioles" lack a defined internal elastic lamina and demonstrate irregular deposits of extracellular matrix in the media. Vascular mural cells in healing infarcts undergo phenotypic changes, showing minimal expression of desmin during the proliferative phase (1 hr occlusion/7 days reperfusion) but in the mature scar (8 weeks reperfusion) acquire a phenotype similar to that of vascular smooth muscle cells in control areas. Non-muscle myosin heavy chains A and B are induced in infarct endothelial cells and myofibroblasts, respectively, but are not expressed in neovascular mural cells. Recruitment of a muscular coat and formation of neoarterioles in mature scars may inhibit endothelial cell proliferation and vascular sprouting, stabilizing the infarct vasculature.

Cardiac muscle plasticity in adult and embryo by heart-derived progenitor cells.

The evidence of cardiomyocyte proliferation in damaged heart implied cardiac regeneration might occur by resident or extra cardiac stem cells. However, the specification and origin of these cells remain unknown. Here, we report using fluorescence-activated cell sorting that cardiac progenitor cells resided in adult heart and colocalized with small capillary vessels, within the stem cell antigen (Sca-1) population expressing high telomerase activity. Notably, hematopoietic stem cells capable of efflux Hoechst 33342, termed side population cells, also were identified within the heart-derived cells. The cardiac progenitor cells (CD45(-)/CD34(-)) express neither cardiac muscle nor endothelial cell markers at an undifferentiated stage. The exposure of 5-azacytidine induced cardiac differentiation, which depends, in part, on Bmpr1a, a type IA receptor for bone morphogenetic protein (BMP). The capability of adult Sca1(+) cells to adopt a cardiac muscle in embryogenesis was substantiated by blastocyst injection, using progenitors from the adult hearts of transgenic mice that harbor a bacterial artificial chromosome expressing GFP via the Nkx-2.5 locus. Intravenously injected progenitors, shortly after ischemic/reperfusion, homed and functionally differentiated 3.5% of total left ventricle in the host myocardium. Differentiation included both fusion-independent and fusion-associated components, proved by the Cre/loxP donor/recipient system. Our studies suggest that endogenous cardiac progenitors reside in the adult heart, regenerate cardiomyocytes functionally, and integrate into the existing heart circuitry.

Effect of age on peripheral vascular response to transverse aortic banding in mice.

The placement of a ligature to constrict the transverse aorta has become a standard procedure to induce cardiac hypertrophy in mice. Apart from cardiac response, there are adaptive changes in the proximal and distal arterial system that function to maintain adequate peripheral perfusion. The purpose of this study was to characterize the peripheral vascular response by measuring the carotid blood flow using noninvasive Doppler methods, and to investigate the effect of aging on the adequacy and timing of the response after aortic banding in mice. Five 16-month-old and 9 4-month-old male B6D2F1 mice underwent transverse aortic banding. Blood flow velocity was measured with Doppler in the right and left carotid arteries (RCA and LCA) before, 1 day after, and 7 days after, banding. Pulsatility index defined as (peak - minimum)/mean velocity was used to estimate local compliance and distal arterial resistance. The RCA/LCA mean velocity ratio was lower and pulsatility index ratio was higher at 1 day after banding in older mice. However, at 7 days, the RCA/LCA mean velocity ratio and pulsatility index ratio were similar between the 2 age groups. Our data indicate that there is an age-related delay in the development of vascular adaptations in carotid arteries after aortic banding. Older mice take a longer time for adaptation to establish adequate and equal mean flow velocity in the carotid arteries.

Increasing access to medical education for students from medically underserved communities: one program's success.

The Premedical Honors College (PHC) is an eight-year, BS-MD program created in 1994 by Baylor College of Medicine (BCM) and The University of Texas-Pan American (UT-PA) to increase the number of physicians addressing the health care needs of underserved populations in Texas. The PHC targets South Texas, a 13-county, medically underserved area with a population that is 82% Hispanic. To date, the PHC has had 159 matriculants and 71 graduates, of whom 60 (84.5%) have matriculated into medical school. These results are significant considering that in 1996, only four students from all five South Texas colleges (combined enrollment of 30000 students) were accepted to medical school. An outcomes study comparing PHC matriculants with students of similar academic ability, ethnicity, and interest in medicine revealed that the odds of medical school matriculation were seven times higher for PHC students than for non-PHC students. The PHC's initial success has been acknowledged by the Texas legislature, which recently passed a bill to promote the PHC's replication. In addition, the number of PHC students-of whom 95% are Mexican American-who matriculate into medical school annually is significant nationally. In 2001, only 386 U.S. medical school matriculants (2.3% of all matriculants) were Mexican American; 17 of these students (4.4%) were PHC graduates. If current trends continue, the PHC could significantly expand the number of physicians serving minority and medically underserved populations in Texas and the nation. Also, the PHC provides an opportunity for research on programs designed to create pathways from high school to medical school.