Prasanna Krishnamurthy

Invariant natural killer T (iNKT) cells are a unique subset of innate immune cells activated by T cell receptor (TCR) signaling through CD1d molecules presenting lipid antigens. By localizing to specific tissues, iNKT cells play critical roles in maintaining homeostasis and regulating pathophysiology, making them attractive therapeutic targets for inflammatory diseases. However, their frequency in the heart and potential roles in mitigating inflammation-induced cardiac damage remain poorly understood. In this study, we examined the distribution of cardiac iNKT subsets and their response to TCR-directed alpha Galactosyl ceramide/cluster of differentiation 1d (αGalCer/CD1d) complexes in a mouse model of Doxorubicin (Dox)-induced cardiotoxicity. In healthy mice, the heart harbored distinct iNKT subsets, dominated by pro-inflammatory iNKT1 cells, followed by iNKT2 subsets. Treatment with αGalCer/CD1d complexes preferentially expanded the iNKT1 subset. In Dox-induced cardiotoxicity, this treatment unexpectedly increased both IFN-γ, a hallmark cytokine of iNKT1 cells, and IL-10, a key anti-inflammatory mediator typically associated with regulatory iNKT subsets. Despite the expansion of iNKT1 cells and elevated IFN-γ levels, αGalCer/CD1d treatment did not exacerbate cardiac damage and showed modest clinical improvements compared to controls. These findings highlight the complexity of iNKT-mediated immune regulation in the heart and emphasize the need for modified TCR-directed immunotherapeutics to skew immune responses toward a regulatory phenotype. Such approaches could hold promise for treating inflammatory cardiac diseases, including Dox-induced myocarditis, myocardial infarction, and autoimmune myocarditis.

Exosomes are small lipid bilayer-encapsulated nanosized extracellular vesicles of endosomal origin. Exosomes are secreted by almost all cell types and are a crucial player in intercellular communication. Exosomes transmit cellular information from donor to recipient cells in the form of proteins, lipids, and nucleic acids and influence several physiological and pathological responses. Due to their capacity to carry a variety of cellular cargo, low immunogenicity and cytotoxicity, biocompatibility, and ability to cross the blood-brain barrier, these nanosized vesicles are considered excellent diagnostic tools and drug-delivery vehicles. Despite their tremendous potential, the progress in therapeutic applications of exosomes is hindered by inadequate isolation techniques, poor characterization, and scarcity of specific biomarkers. The current research in the field is focused on overcoming these limitations. In this chapter, we have reviewed conventional exosome isolation and characterization methods and recent advancements, their advantages and limitations, persistent challenges in exosome research, and future directions.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps to chromosome 2 (locus 2q31.1), near Titin (TTN), which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in failing human heart tissue (HF) compared to non-failing hearts (NF). Using the Sanger sequencing method, we characterized the human OLA1 gene and screened for mutations in the OLA1 gene in patients with failing and non-failing hearts. Among failing and non-failing heart patients, we found 15 different mutations in the OLA1 gene, including two transversions, one substitution, one deletion, and eleven transitions. All mutations were intronic except for a non-synonymous 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results demonstrate that this PCR test can effectively screen for OLA1 mutation-associated cardiomyopathy in human patients using easily accessible cells or tissues, such as blood cells. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Cardiomyopathy, disease of the heart muscle, is a significant contributor to heart failure. The pathogenesis of cardiomyopathy is multifactorial and involves genetic, environmental, and lifestyle factors. Identifying and characterizing novel genes that contribute to cardiac pathophysiology are crucial for understanding cardiomyopathy and effective therapies. In this study, we investigated the role of a novel gene, Obg-like ATPase 1 ( Ola1 ), in cardiac pathophysiology using a cardiac-specific knockout mouse model as well as a Drosophila model. Our previous work demonstrated that OLA1 modulates the hypertrophic response of cardiomyocytes through the GSK-beta/beta-catenin signaling pathway. Furthermore, recent studies have suggested that OLA1 plays a critical role in organismal growth and development. For example, Ola1 null mice exhibit increased heart size and growth retardation. It is not known, however, if loss of function for Ola1 leads to dilated cardiomyopathy. We generated cardiac-specific Ola1 knockout mice (OLA1-cKO) to evaluate the role of OLA1 in cardiac pathophysiology. We found that Ola1 -cKO in mice leads to dilated cardiomyopathy (DCM) and left ventricular (LV) dysfunction. These mice developed severe LV dilatation, thinning of the LV wall, reduced LV function, and, in some cases, ventricular wall rupture and death. In Drosophila, RNAi-mediated knock-down specifically in developing heart cells led to the change in the structure of pericardial cells from round to elongated, and abnormal heart function. This also caused significant growth reduction and pupal lethality. Thus, our findings suggest that OLA1 is critical for cardiac homeostasis and that its deficiency leads to dilated cardiomyopathy and dysfunction. Furthermore, our study highlights the potential of the Ola1 gene as a therapeutic target for dilated cardiomyopathy and heart failure.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy. Abstract Figure:

Pathological fibrosis contributes to progression of various diseases, for which the therapeutic options are limited. Idiopathic pulmonary fibrosis (IPF) is one such progressive and fatal interstitial fibrotic disease that is often characterized by excessive accumulation of extracellular matrix (ECM) proteins leading to stiff lung tissue and impaired gas exchange. However, the molecular mechanisms underlying IPF progression remain largely unknown. In this study, we determined the role of Runt‐related transcription factor 1 (RUNX1), an evolutionarily conserved transcription factor, in the differentiation of human lung fibroblasts (HLFs) in vitro and in an animal model of bleomycin (BLM)‐induced lung fibrosis. We observed that the expression of RUNX1 was significantly increased in the lungs of BLM‐injected mice as compared to saline‐treated mice. Furthermore, HLFs stimulated with transforming growth factor β (TGF‐β) showed significantly higher RUNX1 expression at both mRNA and protein levels, and compartmentalization in the nucleus. Inhibition of RUNX1 in HLFs (using siRNA) showed a significant reduction in the differentiation of fibroblasts into myofibroblasts as evidenced by reduced expression of alpha‐smooth muscle actin (α‐SMA), TGF‐β and ECM proteins such as fibronectin 1 (FN1), and collagen 1A1 (COL1A1). Mechanistic studies revealed that the increased expression of RUNX1 in TGF‐β‐stimulated lung fibroblasts is due to enhanced mRNA stability of RUNX1 through selective interaction with the RNA‐binding profibrotic protein, human antigen R (HuR). Collectively, our data demonstrate that increased expression of RUNX1 augments processes involved in lung fibrosis including the differentiation of fibroblasts into collagen‐synthesizing myofibroblasts. Our study suggests that targeting RUNX1 could limit the progression of organ fibrosis in diseases characterized by abnormal collagen deposition.

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Objective and design Phagocytosis and clearance of apoptotic cells are essential for inflammation resolution, efficient wound healing, and tissue homeostasis. MicroRNAs are critical modulators of macrophage polarization and function. The current study aimed to investigate the role of miR-181c-5p in macrophage phagocytosis. Materials and methods miR-181c-5p was identified as a potential candidate in microRNA screening of RAW264.7 macrophages fed with apoptotic cells. To investigate the role of miR-181c-5p in phagocytosis, the expression of miR-181c-5p was assessed in phagocyting bone marrow-derived macrophages. Phagocytosis efficiency was measured by fluorescence microscopy. Gain- and loss-of-function studies were performed using miR-181c-5p-specific mimic and inhibitor. The expression of the phagocytosis-associated genes and proteins of interest was evaluated by RT² profiler PCR array and western blotting, respectively. Results miR-181c-5p expression was significantly upregulated in the phagocyting macrophages. Furthermore, mimic-induced overexpression of miR-181c-5p resulted in the increased phagocytic ability of macrophages. Moreover, overexpression of miR-181c-5p resulted in upregulation of WAVE-2 in phagocyting macrophages, suggesting that miR-181c-5p may regulate cytoskeletal arrangement during macrophage phagocytosis. Conclusion Altogether, our data provide a novel function of miR-181c-5p in macrophage biology and suggest that targeting macrophage miR-181c-5p in injured tissues might improve clearance of dead cells and lead to efficient inflammation resolution.

Rationale: After myocardial ischemic injury, improper phagocytic clearance of dying cardiac cells and the ensuing lack of inflammation resolution results in adverse cardiac remodeling and dysfunction that might lead to heart failure. Therefore, therapeutic strategies to ameliorate immune cell phagocytic function are critical for augmenting cardiac repair after injury. Objective: To determine whether mesenchymal stem cell–derived exosomes (MSC-Exos) act as opsonin for apoptotic cells or trigger eat-me phagocytic signaling in resident/recruited phagocytes after myocardial ischemic injury. Methods and Results: We evaluated MSC-Exo–mediated opsonization of apoptotic cardiomyocytes and in vitro and in vivo effects of MFGE8 (milk fat globule epidermal growth factor-factor VIII)-deficient mouse MSC-Exo on macrophage engulfment of apoptotic cardiomyocytes and its implications on cardiac remodeling, repair, and function. Microscopy and FACS analyses show that opsonization of apoptotic cardiomyocytes with MSC-Exo enhances their engulfment by macrophages. Furthermore, preincubation of macrophages with MSC-Exo reprogrammed the signaling pathways involved in phagocytosis and expression of proreparative cytokines. Protein analysis of MSC-Exo reveals expression of MFGE8—a glycoprotein that bridges externalized phosphatidylserine on the apoptotic cell surface to αVβ3 or αVβ5 integrins on the phagocyte. Most intriguingly, siRNA inhibition of MFGE8 significantly reduced the MSC-Exo–mediated augmentation of dead cell engulfment, associated signaling, and proreparative phenotype. After myocardial ischemic injury, intramyocardial administration of MSC-Exo increases macrophage uptake of apoptotic bodies in the border zone of infarct and is associated with reduced proinflammatory response, increase in neovascularization, lower infarct size, and an improvement in cardiac function and MFGE8-deficient MSC-Exo administration failed to protect mice against myocardial infarction. Conclusions: Our data demonstrate that exosome-associated MFGE8 on one hand enhances opsonization of dead cells and on the other activates phagocytic signaling, thus augmenting removal of apoptotic cells, resolution of inflammation, and, therefore, efficient cardiac recovery after injury.