Characterization of Near Full-Length Transmitted/Founder HIV-1 Subtype D and A/D Recombinant Genomes in a Heterosexual Ugandan Population (2006-2011).

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5' and 3' half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag/pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses (n = 3) and inter-subtype mosaic recombinants (n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Transgenic mouse model with deficient mitochondrial polymerase exhibits reduced state IV respiration and enhanced cardiac fibrosis.

Mitochondria produce the energy required for proper cardiac contractile function, and cardiomyocytes that exhibit reduced mitochondrial electron transport will have reduced energy production and decreased contractility. Mitochondrial DNA (mtDNA) encodes the core subunits for the protein complexes of the electron transport chain (ETC). Reduced mtDNA abundance has been linked to reduced ETC and the development of heart failure in genetically engineered mice and in human diseases. Nucleoside reverse-transcriptase inhibitors for HIV/AIDS are used in antiretroviral regimens, which cause decreased mtDNA abundance by inhibiting the mitochondrial polymerase, pol-γ, as a limiting side effect. We explored consequences of AZT (1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione) exposure on mtDNA abundance in an established transgenic mouse model (TG) in which a cardiac-targeted mutant form of pol-γ displays a dilated cardiomyopathy (DCM) phenotype with increased left ventricle (LV)-mass and increased LV-end diastolic dimension. TG and wild-type littermate mice received 0.22 mg per day AZT or vehicle for 35 days, and were subsequently analyzed for physiological, histological, and molecular changes. After 35 days, Y955C TGs exhibited cardiac fibrosis independent of AZT. Reduced mtDNA abundance was observed in the Y955C mouse; AZT treatment had no effect on the depletion, suggesting that Y955C was sufficient to reduce mtDNA abundance maximally. Isolated mitochondria from AZT-treated Y955C hearts displayed reduced mitochondrial energetic function by oximetric measurement. AZT treatment of the Y955C mutation further reduced basal mitochondrial respiration and state IV(0) respiration. Together, these results demonstrate that defective pol-γ function promotes cardiomyopathy, cardiac fibrosis, mtDNA depletion, and reduced mitochondrial energy production.

Cardiomyocyte-Restricted Deletion of PPARß/δ in PPARα-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation.

It is well documented that PPARα and PPARß/δ share overlapping functions in regulating myocardial lipid metabolism. However, previous studies demonstrated that cardiomyocyte-restricted PPARß/δ deficiency in mice leads to severe cardiac pathological development, whereas global PPARα knockout shows a benign cardiac phenotype. It is unknown whether a PPARα-null background would alter the pathological development in mice with cardiomyocyte-restricted PPARß/δ deficiency. In the present study, a mouse model with long-term PPARß/δ deficiency in PPARα-null background showed a comparably reduced cardiac expression of lipid metabolism to those of single PPAR-deficient mouse models. The PPARα-null background did not rescue or aggravate the cardiac pathological development linked to cardiomyocyte-restricted PPARß/δ deficiency. Moreover, PPARα-null did not alter the phenotypic development in adult mice with the short-term deletion of PPARß/δ in their hearts, which showed mitochondrial abnormalities, depressed cardiac performance, and cardiac hypertrophy with attenuated expression of key factors in mitochondrial biogenesis and defense. The present study demonstrates that cardiomyocyte-restricted deletion of PPARß/δ in PPARα-null mice causes impaired mitochondrial biogenesis and defense, but no further depression of fatty acid oxidation. Therefore, PPARß/δ is essential for maintaining mitochondrial biogenesis and defense in cardiomyocytes independent of PPARα.

Inhibition of NF-kappaB signaling reduces virus load and gammaherpesvirus-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disorder of unknown etiology. Several studies have demonstrated an association between pulmonary infection with a herpesvirus and IPF. Based on those observations, we have developed a mouse model in which interferon (IFN)gammaR(-/-) mice infected intranasally with murine gammaherpesvirus 68 (MHV68) develop lung fibrosis. We hypothesize that viral load was a critical factor for the development of fibrosis. Because nuclear factor (NF)-kappaB signaling is required to efficiently establish gammaherpesvirus, latency we infected IFNgammaR(-/-) mice with a MHV68 virus that expresses a mutant dominant inhibitor of the NF-kappaB signaling pathway, called IkappaBalphaM. Striking differences were observed at the onset of the chronic infection, which correlated with a decreased virus load in mice infected with MHV68-IkappaBalphaM compared with mice infected with control MHV68 (MHV68-MR). IFNgammaR(-/-) mice infected with MHV68-IkappaBalphaM lacked vasculitis and fibrosis 15 to 120 days post infection. Inhibition of NF-kappaB in MHV68-infected cells of the lungs diminished the expression of the fibrocyte recruiting chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL12, ameliorated macrophage expression of markers of alternative activation, and failed to increase expression of the integrin alphavbeta6, which is implicated in the activation of the profibrotic factor TGF-beta. Thus, the inhibition of NF-kappaB signaling in the infected lung cells of IFNgammaR(-/-) mice reduces virus persistence and ameliorates profibrotic events. Host determinants of latency might therefore represent new therapeutic targets for gammaherpesvirus-associated pulmonary fibrosis.

High-fat feeding in cardiomyocyte-restricted PPARdelta knockout mice leads to cardiac overexpression of lipid metabolic genes but fails to rescue cardiac phenotypes.

Peroxisome proliferator-activated receptor delta (PPARdelta) is an essential determinant of basal myocardial fatty acid oxidation (FAO) and bioenergetics. We wished to determine whether increased lipid loading affects the PPARdelta deficient heart in transcriptional regulation of FAO and in the development of cardiac pathology. Cardiomyocyte-restricted PPARdelta knockout (CR-PPARdelta(-/-)) and control (alpha-MyHC-Cre) mice were subjected to 48 h of fasting and to a long-term maintenance on a (28 weeks) high-fat diet (HFD). The expression of key FAO proteins in heart was examined. Serum lipid profiles, cardiac pathology, and changes of various transduction signaling pathways were also examined. Mice subjected to fasting exhibited upregulated transcript expression of FAO genes in the CR-PPARdelta(-/-) hearts. Moreover, long-term HFD in CR-PPARdelta(-/-) mice induced a strikingly greater transcriptional response. After HFD, genes encoding key FAO enzymes were expressed remarkably more in CR-PPARdelta(-/-) hearts than in those of control mice. Despite the marked rise of FAO gene expression, corresponding protein expression remained low in the CR-PPARdelta(-/-) heart, accompanied by abnormalities in sarcomere structures and mitochondria that were similar to those of CR-PPARdelta(-/-) hearts with regular chow feeding. The CR-PPARdelta(-/-) mice displayed increased expression of PPARgamma co-activator-1alpha (PGC-1alpha) and PPARalpha in the heart with deactivated Akt and p42/44 MAPK signaling in response to HFD. We conclude that PPARdelta is an essential determinant of myocardial FAO. Increased lipid intake activates cardiac expression of FAO genes via PPARalpha/PGC-1alpha pathway, albeit it is not sufficient to improve cardiac pathology due to PPARdelta deficiency.

Cardiac peroxisome proliferator-activated receptor gamma is essential in protecting cardiomyocytes from oxidative damage.

OBJECTIVES: Peroxisome proliferator-activated receptors (PPAR) alpha and beta/delta are essential transcriptional regulators of fatty acid oxidation in the heart. However, little is known about the roles of PPARgamma in the heart. The present study is to investigate in vivo role(s) of PPARgamma in the heart. METHODS: A Cre-loxP mediated cardiomyocyte-restricted PPARgamma knockout line was investigated. In these mice, exon 1 and 2 of PPARgamma were targeted to eliminate PPARgamma from cardiomyocytes. RESULTS: PPARgamma null mice exhibited pathological changes around 3 months of age, featuring progressive cardiac hypertrophy with mitochondrial oxidative damage. Most mice died from dilated cardiomyopathy. Cardiac expression of Sod2 (encoding manganese superoxide dismutase; MnSOD), a mitochondrial antioxidant enzyme was downregulated both in transcript and protein levels in cardiac samples in PPARgamma knockout mice independent of pathological changes. Promoter analyses revealed that Sod2 is a target gene of PPARgamma. Consequently, myocardial superoxide content in PPARgamma knockout mice was increased, leading to extensive oxidative damage. Treatment with a SOD mimetic compound, MnTBAP, prevented superoxide-induced cardiac pathological changes in PPARgamma knockout mice. CONCLUSIONS: The present study demonstrates that PPARgamma is critical to myocardial redox homeostasis. These findings should provide new insights into understanding the roles of PPARgamma in the heart.

Adiponectin and its receptors are expressed in adult ventricular cardiomyocytes and upregulated by activation of peroxisome proliferator-activated receptor gamma.

Adiponectin is a protein hormone involved in maintaining energy homeostasis in metabolically active tissues. It enhances glucose and lipid metabolism via activation of AMP-dependent kinase (AMPK) in skeletal muscle and liver. Energy homeostasis is vital for the heart to work as a pump. In this study, we investigated whether adiponectin and its receptors are expressed in adult ventricular cardiomyocytes. We observed adiponectin transcript and protein in cultured ventricular cardiomyocytes isolated from adult rat, by quantitative real-time PCR, ELISA assays, Western blots, and immunofluorescent staining. In addition, we detected adiponectin receptor (AdipoR1 and AdipoR2) expression in the heart. AdipoR1 was expressed in rat myocardium at a level of approximately 50% of that in skeletal muscle; whereas adipoR2 was expressed at a similar level to that in liver. Rosiglitazone, a Peroxisome proliferator activated receptor gamma (PPARgamma) activator, substantially elevated expression of adiponectin in cultured cardiomyocytes and its secretion into cultured media. Rosiglitazone also increased adipoR1 and adipoR2 expression in cardiomyocytes. Treatment of recombinant globular adiponectin in cultured cardiomyocytes increased fatty acid oxidation and glucose uptake via activation of AMPK, suggesting a role for adiponectin in cardiac energy metabolism. Together, these data establish the existence of a local cardiac-specific adiponectin system that is regulated by PPARgamma. Moreover, these findings indicate a role for adiponectin on normal myocardial energy homeostasis, in part, through the activation of AMPK.

PPARdelta modulates lipopolysaccharide-induced TNFalpha inflammation signaling in cultured cardiomyocytes.

Peroxisome proliferator activated receptors (PPARs: PPARalpha, gamma and delta) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Tumor necrosis factor alpha (TNFalpha) is one of the important pathological factors in inflammatory responses during the pathological progression of myocardial ischemic/reperfusion and hypertrophy. Accumulating evidence shows that synthetic ligands of PPARalpha and PPARgamma suppress myocardial inflammatory responses, such as the production of TNFalpha, thus exerting beneficial effects in animals who had undergone ischemia/reperfusion injury or cardiac hypertrophy. However, it remains obscured if PPARdelta and its ligands exert any effect on the production of TNFalpha, thus influencing cardiac inflammatory responses. In this study, we investigated the effects of PPARdelta and its synthetic ligand GW0742 on TNFalpha production in cultured cardiomyocytes. Our studies indicate that a PPARdelta-selective ligand inhibited lipopolysaccharide (LPS)-induced TNFalpha production from cardiomyocytes. Adenoviral-mediated overexpression of PPARdelta substantially inhibited TNFalpha expression in cultured cardiomyocytes compared to controls, whereas overexpression of a PPARdelta mutant with ablated ligand binding domain did not show similar effect. Conversely, absence of PPARdelta in cardiomyocytes further exaggerated LPS-induced TNFalpha production. Moreover, activation of PPARdelta abrogated LPS-induced degradation of IkappaBs, thus suppressing LPS-induced nuclear factor kappaB (NF-kappaB) activities. Therefore, PPARdelta is an important determinant of TNFalpha expression via the NF-kappaB signaling pathway, thus serving as therapeutic targets to attenuate inflammation that are involved in cardiac pathological progression.

Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy.

Fatty acid oxidation (FAO) is a primary energy source for meeting the heart's energy requirements. Peroxisome proliferator-activated receptor-delta (PPAR-delta) may have important roles in FAO. But it remains unclear whether PPAR-delta is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-delta in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-delta deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-delta is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-delta is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.

Peroxisome proliferator-activated receptor delta activates fatty acid oxidation in cultured neonatal and adult cardiomyocytes.

Peroxisome proliferator-activated receptors (PPARalpha, -gamma and -delta) are nuclear receptors involved in transcriptional regulations of lipid metabolism. The effect of PPARalpha in regulation of cardiac fatty acid oxidation has been well characterized. Whether PPARdelta also independently regulates fatty acid oxidation in the heart remains unclear. In this study, we tested the hypothesis that PPARdelta activates fatty acids oxidation in cardiomyocytes through transcriptional activation that are independent of PPARalpha. Our results first indicate that PPARdelta abundantly expresses in nucleus of cardiomyocytes. Palmitate oxidation rates were significantly increased in both neonatal and adult cardiomyocytes after treatment of a PPARdelta-selective ligand (GW0742). Further increases of fatty acid oxidation were evident when the treatment was applied to cardiomyocytes overexpressing a wild type PPARdelta, but not a mutant PPARdelta that lacks the intact carboxyl ligand-binding domain. Furthermore, genes of fatty acid oxidation enzymes were significantly upregulated in cultured rat neonatal cardiomyocytes when exposed to GW0742. GW0742 can restore partly the expression of certain key genes of fatty acid oxidation in mouse adult cardiomyocytes isolated from PPARalpha knockout mice. Therefore, while active crosstalk between PPARdelta and -alpha may exist, PPARdelta regulates cardiac fatty acid oxidation in the heart at least partly independent of PPARalpha. We conclude that PPARdelta may play a key role in cardiac energy balance and may serve as a "sensor" of fatty acid of other endogenous ligands in controlling fatty acids oxidation levels in the hearts under normal and pathological conditions.