Pradeep P.A. Mammen

Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family

We recently identified a brief time period during postnatal development when the mammalian heart retains significant regenerative potential after amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-d-old mice and found that this results in extensive myocardial necrosis and systolic dysfunction. Remarkably, the neonatal heart mounted a robust regenerative response, through proliferation of preexisting cardiomyocytes, resulting in full functional recovery within 21 d. Moreover, we show that the miR-15 family of microRNAs modulates neonatal heart regeneration through inhibition of postnatal cardiomyocyte proliferation. Finally, we demonstrate that inhibition of the miR-15 family from an early postnatal age until adulthood increases myocyte proliferation in the adult heart and improves left ventricular systolic function after adult MI. We conclude that the neonatal mammalian heart can regenerate after myocardial infarction through proliferation of preexisting cardiomyocytes and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity.

Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, α-myosin heavy chain promoter (α-MHC). In unstressed mice, forced expression of hMCIP1 resulted in a 5–10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy that otherwise result from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibited hypertrophic responses to β-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure.

Assessment of Myocardial Perfusion by Harmonic Power Doppler Imaging at Rest and During Adenosine Stress Comparison With 99mTc-Sestamibi SPECT Imaging

BACKGROUND Harmonic power Doppler imaging (HPDI) is a novel technique for assessing myocardial perfusion by contrast echocardiography in humans. The purpose of this study was to compare myocardial perfusion by HPDI with that obtained by (99m)Tc-sestamibi single photon emission computed tomography (SPECT) during rest and pharmacological stress. METHODS AND RESULTS HPDI was performed on 123 patients who were referred for SPECT imaging for known or suspected coronary artery disease. Images were obtained at baseline and during adenosine infusion (0.14 mg. kg(-)(1). min(-)(1)x6 minutes) in 3 apical views. Myocardial perfusion by HPDI was graded for each coronary territory as absent, patchy, or full. The persistence of absent or patchy myocardial perfusion by HPDI between rest and adenosine was interpreted as a fixed defect, whereas any decrease in perfusion grade was interpreted as a reversible defect. Overall concordance between HPDI and SPECT was 83 (81%) of 103 for normal versus abnormal perfusion. Agreement between the 2 methods for each of the 3 coronary territories was 81% (kappa=0.57) for the left anterior descending artery, 76% (kappa=0.52) for the right coronary artery, and 72% (kappa=0.40) for the left circumflex artery. Discrepancies between the 2 techniques were most notable in the circumflex territory, where fixed defects were observed in 33% by HPDI but in only 14% by SPECT (chi(2)=15.8, P=0.0001). CONCLUSIONS This study demonstrates that HPDI can reliably detect myocardial perfusion during pharmacological stress, although there was a significantly higher number of falsely abnormal results in the circumflex territory.

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.

Association of cystatin C with left ventricular structure and function: The Dallas Heart Study

Background—Cystatin C, a novel marker of renal function, has been associated with heart failure and cardiovascular mortality in older individuals. We tested the hypothesis that cystatin C is associated with preclinical cardiac structural and functional abnormalities in a younger population-based sample. Methods and Results—The study included participants in the Dallas Heart Study (ages 30 to 65 years) who had measurements of cystatin C and cardiac MRI. The associations of cystatin C with left ventricular (LV) mass, LV end-systolic and -diastolic volumes, concentricity (LV mass/LV end-diastolic volume), LV wall thickness, and LV ejection fraction were evaluated. Cystatin C levels ranged from 0.46 to 6.55 mg/L. In univariable analyses, increasing levels of cystatin C correlated with higher LV mass, concentricity, and wall thickness (P<0.001), but not with LV end-systolic volume, LV end-diastolic volume, or LV ejection fraction. After adjustment with traditional covariates and estimated glomerular filtration rate by the modification of diet in renal disease formula, log-transformed cystatin C remained independently associated with LV mass (P<0.001), concentricity (P=0.027), and wall thickness (P<0.001). These associations persisted when creatinine or estimated glomerular filtration rate by the Cockcroft-Gault formula were included in the models. Conclusions—Higher levels of cystatin C were associated with increased LV mass and a concentric LV hypertrophy phenotype. These findings were independent of potential confounding variables including standard measurements of renal function, supporting the hypothesis that cystatin C may be useful to identify individuals with preclinical structural heart abnormalities.

The Capsaicin-Sensitive Afferent Neuron in Skeletal Muscle Is Abnormal in Heart Failure

Background—In heart failure, the cardiovascular response to activation of the skeletal muscle exercise pressor reflex (EPR) is exaggerated. Group IV afferent neurons, primarily stimulated by the metabolic by-products of skeletal muscle work, contribute significantly to the EPR. Therefore, it was postulated that alterations in the activity of group IV neurons contribute to the EPR dysfunction manifest in heart failure. Methods and Results—Group IV afferent fibers were ablated in neonatal Sprague-Dawley rats by subcutaneous administration of capsaicin. In neonatal capsaicin-treated adult animals, selective activation of the EPR, by electrically induced static muscle contraction, recapitulated the exaggerated increases in heart rate and blood pressure observed in rats with dilated cardiomyopathy (DCM). Furthermore, compared with control animals, both neonatal capsaicin-treated and DCM rats displayed a decreased pressor response to the intra-arterial administration of capsaicin within the hindlimb, a maneuver that selectively excites group IV afferent neurons. Moreover, expression of mRNA for the capsaicin receptor TRPv1, a marker of group IV fibers, was downregulated in DCM animals compared with controls. Conclusions—These findings suggest that EPR dysfunction in heart failure results in part from functional and molecular alterations in group IV fibers. Furthermore, the responsiveness of these metabolically sensitive neurons appears to be blunted in DCM, indicating that their contribution to the EPR may be reduced. This occurs despite an overall exaggeration of the EPR in heart failure. These insights into the basic mechanisms of EPR dysfunction are essential to the development of effective therapeutic strategies aimed at improving exercise capacity in heart failure.

Role of the Exercise Pressor Reflex in Rats With Dilated Cardiomyopathy

Background—In heart failure, there is a sympathetically mediated hyperkinetic cardiovascular response to exercise that limits tolerance to physical activity. Alterations in skeletal muscle morphology and metabolism have led to the hypothesis that the exercise pressor reflex (EPR) becomes hyperactive after the development of cardiomyopathy and contributes to the exaggerated circulatory response elicited. Methods and Results—To test this hypothesis, Sprague-Dawley rats were divided into the following groups: control, sham, and dilated cardiomyopathy (DCM, induced by ischemic injury). Using transthoracic echocardiography, left ventricular fractional shortening was 47±2%, 44±1%, and 24±2% in control, sham, and DCM rats, respectively. Activation of the EPR by electrically induced static muscle contraction resulted in significantly larger increases in mean arterial pressure and heart rate in DCM animals (32±2 mm Hg, 13±1 bpm) compared with control (20±1 mm Hg, 8±1 bpm) and sham (20±2 mm Hg, 8±1 bpm) rats. Comparable results were obtained with selective stimulation of the mechanically sensitive component of the EPR by passive muscle stretch. The augmentations in EPR and mechanoreflex activity in DCM occurred progressively over a 10-week period, becoming greater as the severity of left ventricular dysfunction increased. Conclusions—In DCM, the potentiated cardiovascular response to static muscle contraction is mediated, in part, by an exaggerated EPR. The muscle mechanoreflex contributes significantly to the EPR dysfunction that develops.

Hypoxia reprograms calcium signaling and regulates myoglobin expression

Myoglobin is an oxygen storage molecule that is selectively expressed in cardiac and slow-twitch skeletal muscles that have a high oxygen demand. Numerous studies have implicated hypoxia in the regulation of myoglobin expression as an adaptive response to hypoxic stress. However, the details of this relationship remain undefined. In the present study, adult mice exposed to 10% oxygen for periods up to 3 wk exhibited increased myoglobin expression only in the working heart, whereas myoglobin was either diminished or unchanged in skeletal muscle groups. In vitro and in vivo studies revealed that hypoxia in the presence or absence of exercise-induced stimuli reprograms calcium signaling and modulates myoglobin gene expression. Hypoxia alone significantly altered calcium influx in response to cell depolarization or depletion of endoplasmic reticulum calcium stores, which inhibited the expression of myoglobin. In contrast, our whole animal and transcriptional studies indicate that hypoxia in combination with exercise enhanced the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors triggered by caffeine, which increased the translocation of nuclear factor of activated T-cells into the nucleus to transcriptionally activate myoglobin expression. The present study unveils a previously unrecognized mechanism where the hypoxia-mediated regulation of calcium transients from different intracellular pools modulates myoglobin gene expression. In addition, we observed that changes in myoglobin expression, in response to hypoxia, are not dependent on hypoxia-inducible factor-1 or changes in skeletal muscle fiber type. These studies enhance our understanding of hypoxia-mediated gene regulation and will have broad applications for the treatment of myopathic diseases.

Emerging roles for myoglobin in the heart

Myoglobin (Mb) is an intensely studied hemoprotein that is restricted mainly to the heart and oxidative myofibers in skeletal muscle. Previous physiologic and pharmacologic studies have supported a role for Mb in facilitated oxygen transport or as an oxygen reservoir in striated muscle. Transgenic and gene disruption technologies have been utilized to produce mice that lack Mb. Studies utilizing these transgenic mouse models support the notion that Mb may have multiple, diverse functions in the heart. Future studies using these emerging technologies will further enhance the understanding of the role of Mb and other hemoproteins in cardiovascular biology.