Abstract Heart responds to pathological hemodynamic stress by increasing cardiac myocyte size, reprogramming gene expression and enhancing contractile protein synthesis. Neurohumoral factors mediate hypertrophic adaptation either directly via specific receptors or indirectly by increasing blood pressure and cardiac load. The aim of this study was to evaluate the role of angiotensin II (Ang II) in the atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression during cardiac overload. Furthermore, the mechanisms of action of Ang II in regulating cardiac gene expression were studied. Hemodynamic stress was produced by Ang II or nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) administration in conscious rats. Despite hypertension and increased left ventricular ANP and BNP mRNA levels, L-NAME administration for 8 weeks did not induce left ventricular hypertrophy. Ang II type 1 receptor (AT1) antagonism decreased significantly L-NAME-induced hypertension and ventricular ANP gene expression. Ang II-induced cardiac overload produced significant increase in ventricular ANP and BNP mRNA levels at 12 and 72 h, respectively. To study whether the factors synthesized by adrenals modulate the response of Ang II, the effects of adrenalectomy were studied. In Ang II-treated rats, adrenalectomy either abolished or blunted the early activation of ANP and BNP gene expression, respectively. Ang II infusion for 2 weeks increased cardiac mass and blood pressure measured by telemetry, and produced changes in diastolic function detected by echocardiography. By using direct plasmid DNA injections into the rat myocardium, BNP promoter activity was observed to increase at 2 h and remain up-regulated up to 2 weeks of Ang II infusion, except at 12 h. BNP mRNA levels increased at 2 h but decreased to basal levels after 72 h. Mutation of GATA elements of the BNP promoter and DNA binding assays revealed that GATA4 mediates the Ang II-responsiveness of the BNP gene. These results indicate that Ang II plays an important role in regulating natriuretic peptide gene expression during cardiac overload. ANP and BNP gene expression in the rat heart is modulated by the adrenal factors during Ang II-stimulated hemodynamic stress and the AT1 receptor antagonism in NO-deficient hypertension. Moreover, ventricular BNP gene expression in Ang II-induced hypertension in vivo is controlled by posttranscriptional mechanisms and GATA elements.

Abstract Cardiac hypertrophy is an adaptive response of the heart to a variety of mechanical, hemodynamic, neurohumoral, and pathologic stimuli. Prolonged pathophysiological load leads to development of left ventricular hypertrophy and ultimately to heart failure. The natriuretic peptides including the B-type natriuretic peptide (BNP) provide the physiological feedback mechanism to suppress the load signal. The aim of the present study was to evaluate the cis elements within the BNP promoter that mediate the cardiac load responses in vivo, and to study the involvement of paracrine factors, such as endothelin-1 (ET-1) and angiotensin II (Ang II) in activating these transcription factors. In this study, cardiac overload was produced by bilateral nephrectomy, and infusions of arginine8-vasopressin (AVP) or Ang II. In isolated perfused rat heart, the direct wall stretch was achieved by inflating the left ventricular balloon. To identify the cis elements within the BNP promoter that mediate hemodynamic overload response, the approach of DNA injection into the myocardium was used. Mutation or deletion of proximal BNP GATA sites abrogated the response to nephrectomy. AVP-induced acute pressure overload increased left ventricular BNP mRNA and peptide levels. In gel mobility shift assays, pressure overload produced rapid activation of transcription factor GATA4 DNA binding, which was completely inhibited by the ET-1 receptor antagonist bosentan. Both ET-1 and Ang II receptor antagonism inhibited the wall stretch-induced increases in left ventricular GATA4 and AP-1 binding activities in isolated perfused heart preparation. BNP promoter activity and BNP mRNA and peptide levels were regulated distinctly in chronic hemodynamic overload produced by Ang II. In conclusion, GATA4 appears to be necessary and sufficient to confer transcriptional activation of BNP gene during hemodynamic stress in vivo. ET-1 is a signaling molecule mediating the cardiac response to acute pressure overload in vivo. In isolated rat heart, Ang II and ET-1 are required for the stimulation of GATA4 and AP-1 binding activity in response to direct left ventricular wall stretch. Finally, posttranscriptional mechanisms play an important role in the regulation of BNP gene expression in pressure overload produced by Ang II in vivo.

Heart failure is responsible for one in twenty deaths in the UK, and as the average age of the general population increases that number is predicted to rise over the coming years. Hypertrophic growth is believed to be an adaptive response to a chronic increase in workload under circumstances such as hypertension, yet it is also known to contribute to the pathological progression into heart failure. Abnormal calcium handling is known to play a critical role in determining disease progression, not only through its function as the driving force behind myocardial contraction and relaxation but also through directing the signals which regulate hypertrophic growth. Both isoforms 1 and 4 of the diastolic calcium extrusion pump plasma membrane calcium ATPase (PMCA) are present in the heart, yet unlike in other cell types their contribution to overall calcium clearance is only small; however their role in the disease process is yet to be defined. A novel mouse line was generated in which both PMCA1 and 4 were deleted from the myocardium (PMCA1:4dcko mice). Through comparison with PMCA1 knockout mice (PMCA1cko) this thesis set out to identify the specific function of each pump under normal conditions and during the development of pathological hypertrophy induced by pressure overload through transverse aortic constriction (TAC).Under basal conditions each isoform functioned independently, PMCA1 to extrude calcium during diastole and PMCA4 to regulate calcium levels during systole; however the loss of neither isoform impacted significantly on cardiac function. In response to TAC, PMCA1cko mice progressed rapidly into decompensation and displayed signs of systolic failure after just 2 weeks, whilst cardiac function was preserved in TAC controls. Calcium handling analysis revealed that prior to the onset of failure PMCA1cko mice displayed a distinct lack of adaptive changes to calcium cycling which were present in controls. In stark contrast, the additional loss of PMCA4 led to an attenuated hypertrophic response to TAC in PMCA1:4dcko mice which remarkably preserved cardiac function despite the absence of PMCA1. This adds to accumulating evidence which suggests that the inhibition of PMCA4 may be protective during the development of pathological hypertrophy, whilst highlighting the possibility for a novel role for PMCA1 in coordinating essential adaptations required to enhance calcium cycling in response to the increased demands imposed on the left ventricle during pressure overload.

Background: Heart attack or myocardial infarction (MI) is a major cause of death worldwide. MI is generally attributed to the detrimental effects of myocardial ischemia/reperfusion (I/R) injury. I/R injury induces cell death and reduces heart function. To compensate, the heart remodels with an associated increase in cell death, fibrosis, and hypertrophy, which can further compromise heart function. Ubiquitin (UB) is an evolutionarily conserved protein. Our lab has shown that pre-I/R injury treatment with exogenous UB preserves heart function and reduces fibrosis 3-days post-I/R in mice. A major objective of this study is to analyze the long-term cardioprotective potential of UB post-I/R injury. Here the UB treatment was continued until 28 days post-I/R to include the entire remodeling period. To enhance the clinical applicability, UB treatment was started at the time of reperfusion. Methods: C57BL/6 mice (aged ~3 months) underwent myocardial I/R surgery. Mice were anesthetized and the left anterior descending coronary artery (LAD) was ligated for 45 minutes. The ligature was then removed for reperfusion. Mice were treated with UB (1µg/g body weight; intraperitoneal (IP) injection) or saline at the time of reperfusion; followed by 3-days of saline or UB IP treatment twice per day. The mice were then implanted with micro-osmotic pumps containing UB (1 μg·g−1·h−1) or saline to continue treatment 28-days post I/R. Mice were sacrificed at 28-days post I/R injury. Sham animals underwent the same surgery without LAD ligation. Heart functional parameters (percent ejection fraction and fractional shortening) were analyzed by echocardiography in a time-dependent manner (3, 7, 14 and 28 days post-I/R). Extracted hearts were embedded in paraffin. Heart sections (5µm) were stained with Mason’s Trichrome to measure fibrosis, TUNEL to measure apoptosis, and fluorescein-conjugated wheat germ agglutinin to measure hypertrophy. Index of fibrosis was quantified as a percentage of total left ventricular area, apoptosis was quantified as a percentage of the total number of nuclei, and hypertrophy was quantified by measuring the myocyte cross-sectional area. Major findings: 1) I/R+saline exhibited a significant decrease in the functional parameters of the heart at 3, 7, 14 and 28 days post-I/R vs sham (n=4-12). No significant decrease in heart function observed between I/R+UB vs sham, and heart function was significantly lower in I/R+saline compared to UB+I/R (n=7-12); 2) I/R surgery significantly increased fibrosis in the myocardium of I/R+saline vs sham. No significant difference was observed between UB+I/R and sham, and fibrosis was significantly lower in UB+I/R vs I/R+saline (n=4-6); 3) Apoptosis was significantly higher in I/R+saline vs sham (p4) Myocyte hypertrophy was significantly higher in I/R+saline vs sham (pConclusion: Long-term UB treatment has the potential to preserve heart function with effects on myocardial fibrosis, myocyte apoptosis, and hypertrophy following myocardial I/R injury.

Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca2+-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H2O2 generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca2+ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca2+ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca2+ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.

The purpose of this study was to investigate the role of osteopontin (OPN) in diabetic hearts. Diabetes was induced in wild-type (WT) and OPN knockout (KO) mice by using streptozotocin (150 mg/kg) injection. Left ventricular (LV) structural and functional remodeling was studied 30 and 60 days after induction of diabetes. Induction of diabetes increased OPN expression in cardiac myocytes. Heart weight-to-body weight ratio was increased in both diabetic (D) groups. Lung wet weight-to-dry weight ratio was increased only in the WT-D group. Peak left ventricular (LV) developed pressures measured using Langendorff perfusion analyses were reduced to a greater extent in WT-D versus KO-D group. LV end-diastolic pressure-volume curve exhibited a significant leftward shift in WT-D but not in KO-D group. LV end-diastolic diameter, percent fractional shortening, and the ratio of peak velocity of early and late filling (E/A wave) were significantly reduced in WT-D mice as analyzed by echocardiography. The increase in cardiac myocyte apoptosis and fibrosis was significantly higher in the WT-D group. Expression of atrial natriuretic peptide and transforming growth factor-β1 was significantly increased in the WT-D group. Induction of diabetes increased protein kinase C (PKC) phosphorylation in both groups. However, phosphorylation of PKC-βII was significantly higher in the WT-D group, whereas phosphorylation of PKC-ζ was significantly higher in the KO-D group. Levels of peroxisome proliferator-activated receptor-γ were significantly decreased in the WT-D group but not in the KO-D group. Thus increased expression of OPN may play a deleterious role during streptozotocin-induced diabetic cardiomyopathy with effects on cardiac fibrosis, hypertrophy, and myocyte apoptosis.

Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed protein kinase with key roles in controlling proliferation, differentiation and survival of a wide variety of mammalian cells. In most cells, GSK3 is active in the absence of growth factor signaling and acts to inhibit cell proliferation and induce apoptosis. In cardiomyocytes, GSK3 plays a novel role as a negative regulator of cardiac hypertrophy, and it appears that GSK3 plays a central role as an inhibitor of cardiac hypertrophy induced by a variety of stimuli. In the present study, we sought to further elucidate the role of GSK3 in cardiomyocyte hypertrophy by studying the effects of inhibition of GSK3 in the absence of other hypertrophic stimuli. By combining global expression profiling with computational predictions and experimental analysis of transcription factor binding sites, we have identified hypertrophy-related genes that are controlled directly by GSK3 and have found that CREB is a major transcriptional target of GSK3 in cardiomyocytes. In addition, we find that inhibition of GSK3 is sufficient to induce the re-expression of fetal development genes characteristic of hypertrophy, but not sufficient to induce the full hypertrophic phenotype of cardiomyocyte growth.

Indiana University-Purdue University Indianapolis (IUPUI) / The ability to preserve, protect, or grow skeletal muscle would greatly benefit patients in health and disease. Understanding the molecular pathways that regulate muscle size is necessary to develop interventions. The extracellular signal-related kinase (ERK) and Hedgehog signaling pathways each play necessary roles in skeletal muscle development. The ERK pathway has been shown to both stimulate and inhibit muscle development at different stages, while Hedgehog signaling is vital for embryonic muscle development. Thus, these pathways represent prime targets for manipulation in diseases associated with muscle loss. In prior studies, cancer patients treated with the ERK inhibitor, Selumetinib, experienced significant gains in lean body mass. To study the mechanisms responsible, we tested the potential of Selumetinib to protect against muscle wasting in muscle cell cultures and in mice with experimental lung cancer. Selumetinib was able to induce hypertrophy of cultured muscle cells. In mice, we observed a reduction in tumor mass and in circulating mediators of muscle wasting including inflammatory cytokines. However, Selumetinib treatment did not prevent cancer-induced muscle loss. Together, these data suggest a diversity in the underlying molecular mechanisms and the need for careful consideration when extrapolating results across different disease states, clinical trials, and model systems. In separate studies, we found that the Hedgehog pathway was increased in mice and patients with cancer-associated muscle wasting and inflammation. In a series of studies in muscle cell cultures, in genetically modified mice, and in mice bearing tumors, we found that inflammatory cytokines activated Hedgehog expression in muscle. Hedgehog signaling promoted the replication of muscle stem cells but reduced the expression of genes that specify mature muscle. Inhibiting Hedgehog signaling promoted muscle growth, while activating it caused muscle wasting. Furthermore, we identified unique properties of two proteins activated by Hedgehog, Gli1 and Gli2, where Gli1 appears to promote muscle stem cell proliferation and Gli2 mature muscle gene expression. These data implicate the Hedgehog pathway, GLI1 and GLI2 as targets for treatment of muscle wasting diseases. / 2 years

Increases in cardiac afterload caused by disease conditions results in remodeling of heart structure by hypertrophy and alterations in the molecular regulation of contractile performance. These adaptations can be regulated by various Ca2+-dependent signaling processes. STIM1 is an important regulator of Ca2+ signaling in different cell types by sensing endoplasmic reticular Ca2+ levels and coupling to plasma membrane Orai channels. The role of STIM1 in heart is not well understood, given the robust Ca2+ regulatory machinery present within cardiac myocytes. Previous reports indicate that STIM1 may play a role in regulation of cardiac hypertrophy. The goal of this work is to understand how STIM1 can affect contractile Ca2+ regulation in normal and diseased myocytes. We induced cardiac hypertrophy by slow progressive pressure overload in adult cats. Isolated adult feline ventricular myocytes (AFMs) exhibited increased STIM1 expression and activity, which correlated with altered Ca2+ handling. Use of BTP2 to block Orai channels resulted in a reduction of action potential (AP) duration and diastolic spark rate of hypertrophied myocytes, without affecting myocytes from sham-operated animals. Overexpressed STIM1 in cultured AFMs caused persistent Ca2+ influx that resulted in increased diastolic spark rates and prolonged APs, similar to myocytes from banded animals. STIM1 mediated Ca2+ influx could load the sarcoplasmic reticulum and activated CaMKII, which increased spark rates and lead to spontaneous APs. Importantly, STIM1 operated by associating with Orai channels because these effects could be blocked with either BTP2 or with a dominant negative Orai construct. Prolonged Ca2+ entry through this pathway eventually causes cell death. In conclusion, the work presented in this thesis establishes a role for STIM1-Orai in contractile Ca2+ regulation. / Biomedical Sciences

Activation of TLRs mediated the NF-κB signaling pathway plays an important pathophysiological role in cardiac hypertrophy. Triad3A, a ubiquitin E3 ligase, has been reported to negatively regulate NF-κB activation pathway via promoting ubiquitination and degradation of TLR4 and TLR9 in innate immune cells. The role of Triad3A in cardiac hypertrophic development remains unknown. The present study investigated whether there is a link between Triad3A and TLR4 and TLR9 in pressure overload induced cardiac hypertrophy. We observed that Triad3A levels were markedly reduced following transverse aortic constriction (TAC) induced cardiac hypertrophy. Similarly, stimulation of neonatal rat cardiac myocytes (NRCMs) with angiotensin-II (Ang II) significantly decreased Triad3A expression. To determine the role of Triad3A in TAC-induced cardiac hypertrophy, we transduced the myocardium with adenovirus expressing Triad3A followed by induction of TAC. We observed that increased expression of Triad3A significantly attenuated cardiac hypertrophy and improved cardiac function. To investigate the mechanisms by which Triad3A attenuated cardiac hypertrophy, we examined the Triad3A E3 ubiquitination on TLR4 and TLR9. We found that Triad3A promoted TLR4 and TLR9 degradation through ubiquitination. Triad3A mediated TLR4 and TLR9 degradation resulted in suppression of NF-κB activation. Our data suggest that Triad3A plays a protective role in the development of cardiac hypertrophy, at least through catalyzing ubiquitination-mediated degradation of TLR4 and TLR9, thus negatively regulating NF-κB activation.