Sangita Choudhury

Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window

The growth factor neuregulin stimulates heart muscle repair in newborn mice and heart muscle from human infants if given during a specific therapeutic time period. Young at heart: Restoring cardiac function in children When children are given adult roles in TV commercials, the results range from adorable to brilliant to simply hilarious. But when children with heart disease were given adult medicines in clinical trials, the results were disappointing—and the need for pediatric-specific treatment regimens became clear. In adult mice, the recombinant growth factor neuregulin-1 (rNRG1) stimulates heart regeneration by driving the proliferation of heart muscle cells (cardiomyocytes). Because young mice bear more proliferation-competent cardiomyocytes than do adult animals, Polizzotti et al. asked whether rNRG1 might put cardiomyocyte proliferation into overdrive if given to mice during the neonatal period. To test their hypothesis, the authors treated newborn mice with rNRG1 at various times after heart injury and found that early treatment starting at 1 day of age boosted cardiomyocyte cell division and heart function in a persistent manner relative to treatment regimens that began at 4 days after birth. rNRG1 also drove cardiomyocyte proliferation in heart muscle isolated from human infants with heart disease who were less than 6 months of age, but not in tissue from older pediatric patients. These findings suggest that rNRG1 administration during the neonatal period might be a new therapeutic strategy for pediatric heart disease. Now that would be brilliant. Therapies developed for adult patients with heart failure have been shown to be ineffective in pediatric clinical trials, leading to the recognition that new pediatric-specific therapies for heart failure must be developed. Administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates regeneration of heart muscle cells (cardiomyocytes) in adult mice. Because proliferation-competent cardiomyocytes are more abundant in growing mammals, we hypothesized that administration of rNRG1 during the neonatal period might be more effective than in adulthood. If so, neonatal rNRG1 delivery could be a new therapeutic strategy for treating heart failure in pediatric patients. To evaluate the effectiveness of rNRG1 administration in cardiac regeneration, newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity. Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars. In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). We also assessed the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease who were less than 6 months of age. Our results identify an effective time period within which to execute rNRG1 clinical trials in pediatric patients for the stimulation of cardiomyocyte regeneration.

Preventing progression of cardiac hypertrophy and development of heart failure by paricalcitol therapy in rats

AIMS Vitamin D deficiency is associated with cardiac hypertrophy and heart failure, and vitamin D therapy prevents the progression of cardiac hypertrophy in animal models. Here, we examine whether vitamin D therapy prevents progression of pre-existing cardiac hypertrophy and development of heart failure. METHODS AND RESULTS When male Dahl salt-sensitive rats were fed a high salt (HS) diet, all rats developed cardiac hypertrophy after 5 weeks. Thereafter, rats were treated with vehicle (V), paricalcitol (PC, an active vitamin D analogue, at 200 ng, IP 3x/week), enalapril (EP, 90 μg/day), and PC + EP. All groups were continued on the HS diet and evaluated after 4 weeks of therapy. The PC and PC + EP groups, but not the V and EP only groups, showed significant prevention of progression of pre-existing cardiac hypertrophy. The signs of decompensated heart failure were evident in the vehicle-treated group; these heart failure parameters significantly improved with PC, EP or PC + EP therapy. The expression of PKCα, which is regulated by Ca(2+)and known to stimulate cardiac hypertrophy, was significantly increased in the vehicle group, and PC, EP or PC + EP effectively decreased PKCα activation. We also observed normalization of genetic alterations during progression to heart failure with PC treatment. CONCLUSION PC treatment resulted in both the prevention of progression of pre-existing cardiac hypertrophy and the development of heart failure, compared with improvement in progression to heart failure by EP alone. These beneficial findings in heart were associated with inhibition of PKCα activation and reversal of gene alterations.

Role of AIF in cardiac apoptosis in hypertrophic cardiomyocytes from Dahl salt-sensitive rats

AIMS The caspases are thought to be central mediators of the apoptotic program, but recent data indicate that apoptosis may also be mediated by caspase-independent mechanisms such as apoptosis-inducing factor (AIF). The role of AIF-induced apoptosis in heart, however, is currently not well understood. The aim of this study was to investigate the presence of and conditions for AIF-induced cardiac apoptosis in vitro. METHODS AND RESULTS Hypertrophic cardiomyocyte (H-CM) cultures were prepared from the hearts of Dahl salt-sensitive rats fed a high salt diet. Apoptotic stimulation induced by hypoxia/reoxygenation or staurosporine (1 microM) enhanced AIF release in H-CMs compared with non-hypertrophic cardiomyocytes (N-CMs). Caspase inhibition using zVAD.fmk (25 microM) or overexpression of CrmA using recombinant adenovirus only partially protected N-CMs from apoptosis (63 +/- 0.93%) and provided no significant protection against apoptosis in hypertrophic cells (23 +/- 1.03%). On the other hand, poly-ADP-ribose polymerase inhibition using 4-AN (20 microM) during apoptotic stimulation blocked the release of AIF from mitochondria and significantly improved cell viability in hypertrophied cardiomyocytes (74 +/- 1.18%). CONCLUSION A caspase-dependent, apoptotic pathway is important for N-CM death, whereas a caspase-independent, AIF-mediated pathway plays a critical role in H-CMs.

Delayed activation of caspase-independent apoptosis during heart failure in transgenic mice overexpressing caspase inhibitor CrmA

Although caspase activation is generally thought to be necessary to induce apoptosis, recent evidence suggests that apoptosis can be activated in the setting of caspase inhibition. In this study, we tested the hypothesis that caspase-independent apoptotic pathways contribute to the development of heart failure in the absence of caspase activation. Acute cardiomyopathy was induced using a single dose of doxorubicin (Dox, 20 mg/kg) injected into male wild-type (WT) and transgenic (Tg) mice with a cardiac-specific expression of cytokine response modifier A (CrmA), a known caspase inhibitor. Early (6 day) survival was significantly better in CrmA Tg (81%) than WT (38%) mice. Twelve days after Dox injection, however, the mortality benefit had dissipated, and increased cardiac apoptosis was observed in both groups. There was, however, a significantly greater release of apoptosis-inducing factor (AIF) from mitochondria to cytosol in CrmA Tg compared with WT mice, which suggests that an enhancement of activation in caspase-independent apoptotic pathways had occurred. The administration of a poly(ADP-ribose) polymerase-1 inhibitor, 4-amino-1,8-naphthalimide (4-AN), to Dox-treated mice resulted in significantly improved cardiac function, a significant blockade of AIF released from mitochondria, and decreased cardiac apoptosis. There were also significantly improved survival in WT (18% without 4-AN vs. 89% with 4-AN) and CrmA Tg (13% without 4-AN vs. 93% with 4-AN) mice 12 days after Dox injection. In conclusion, these findings suggest that apoptosis can be induced in the heart lacking caspase activation via caspase-independent pathways and that enabling the inhibition of AIF activation may provide a significant cardiac benefit.

Abnormal Calcium Handling and Exaggerated Cardiac Dysfunction in Mice with Defective Vitamin D Signaling

Aim Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction. Methods and Results We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase−/−) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase−/− mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase−/− mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca2+) transient demonstrated profound Ca2+ handling abnormalities in 1α-OHase−/− mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D3 analog, significantly attenuated defective Ca2+ handling in 1α-OHase−/− CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase−/− mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca2+ handling abnormalities and cardiac function compared to the vehicle treated animals. Conclusions Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca2+ handling and defective vitamin D signaling in 1α-OHase−/− mice.

Thoracic aortic aneurysm in patients with loss of function Filamin A mutations: Clinical characterization, genetics, and recommendations

The frequency and gender distribution of thoracic aortic aneurysm as a cardiovascular manifestation of loss‐of‐function (LOF) X‐linked FilaminA (FLNA) mutations are not known. Furthermore, there is very limited cardiovascular morbidity or mortality data in children and adults. We analyzed cardiac data on the largest series of 114 patients with LOF FLNA mutations, both children and adults, with periventricular nodular heterotopia (PVNH), including 48 study patients and 66 literature patients, median age of 22.0 years (88 F, 26 M, range: 0–71 years), with 75 FLNA mutations observed in 80 families. Most (64.9%) subjects had a cardiac anomaly or vascular abnormality (80.8% of males and 60.2% of females). Thoracic aortic aneurysms or dilatation (TAA) were found in 18.4% (n = 21), and were associated with other structural cardiac malformations in 57.1% of patients, most commonly patent ductus arteriosus (PDA) and valvular abnormalities. TAA most frequently involved the aortic root and ascending aorta, and sinus of Valsalva aneurysms were present in one third of TAA patients. Six TAA patients (28.5%) required surgery (median age 37 yrs, range 13–41 yrs). TAA with its associated complications was also the only recorded cause of premature, non‐accidental mortality in adults (2 M, 2 F). Two adult patients (1 F, 1 M, median 38.5 yrs), died of spontaneous aortic rupture at aortic dimensions smaller than current recommendations for surgery for other aortopathies. Data from this largest series of LOF FLNA mutation patients underscore the importance of serial follow‐up to identify and manage these potentially devastating cardiovascular complications.

Antibodies against potassium channel interacting protein 2 induce necrosis in isolated rat cardiomyocytes.

Auto‐antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C‐terminal part of KChIP2 (anti‐KChIP2 [C‐12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti‐KChIP2‐mediated cell death. Rat cardiomyocytes were treated with anti‐KChIP2 (C‐12). KChIP2 RNA and protein expressions, nuclear NF‐κB, mitochondrial membrane potential Δψm, caspase‐3 and ‐9 activities, necrotic and apoptotic cells, total Ca2+ and K+ concentrations, and the effects on L‐type Ca2+ channels were quantified. Anti‐KChIP2 (C‐12) induced nuclear translocation of NF‐κB. Anti‐KChIP2 (C‐12)‐treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti‐KChIP2 (C‐12) induced nuclear translocation of NF‐κB after 1 h. After 6 h, Δψm and caspase‐3 and ‐9 activities were not significantly changed. After 24 h, anti‐KChIP2 (C‐12)‐treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty‐six ± 1% of experimental buffer‐treated cells were viable. Anti‐KChIP2 (C‐12) induced significant increases in total Ca2+ (plus 11 ± 2%) and K+ (plus 18 ± 2%) concentrations after 5 min. Anti‐KChIP2 (C‐12) resulted in an increased Ca2+ influx through L‐type Ca2+ channels. In conclusion, our results suggest that anti‐KChIP2 (C‐12) enhances cell death of rat cardiomyocytes probably due to necrosis. J. Cell. Biochem. 115: 678–689, 2014.