CRISPR-Cas9-induced IGF1 gene activation as a tool for enhancing muscle differentiation via multiple isoform expression.

Muscle wasting, or muscle atrophy, can occur with age, injury, and disease; it affects the quality of life and complicates treatment. Insulin-like growth factor 1 (IGF1) is a key positive regulator of muscle mass. The IGF1/Igf1 gene encodes multiple protein isoforms that differ in tissue expression, potency, and function, particularly in cellular proliferation and differentiation, as well as in systemic versus localized signaling. Genome engineering is a novel strategy for increasing gene expression and has the potential to recapitulate the diverse biology seen in IGF1 signaling through the overexpression of multiple IGF1 isoforms. Using a CRISPR-Cas9 gene activation approach, we showed that the expression of multiple IGF1 or Igf1 mRNA variants can be increased in human and mouse skeletal muscle myoblast cell lines using a single-guide RNA (sgRNA). We found increased IGF1 protein levels in the cell culture media and increased cellular phosphorylation of AKT1, the main effector of IGF1 signaling. We also showed that the expression of Class 1 or Class 2 mRNA variants can be selectively increased by changing the sgRNA target location. The expression of multiple IGF1 or Igf1 mRNA transcript variants in human and mouse skeletal muscle myoblasts promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro. Our findings suggest that this novel approach for enhancing IGF1 signaling has potential therapeutic applications for treating skeletal muscle atrophy.

Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells.

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

Selective inhibition of HDAC2 by magnesium valproate attenuates cardiac hypertrophy.

The regulatory paradigm in cardiac hypertrophy involves alterations in gene expression that is mediated by chromatin remodeling. Various data suggest that class I and class II histone deacetylases (HDACs) play opposing roles in the regulation of hypertrophic pathways. To address this, we tested the effect of magnesium valproate (MgV), an HDAC inhibitor with 5 times more potency on class I HDACs. Cardiac hypertrophy was induced by partial abdominal aortic constriction in Wistar rats, and at the end of 6 weeks, we evaluated hypertrophic, hemodynamic, and oxidative stress parameters, and mitochondrial DNA concentration. Treatment with MgV prevented cardiac hypertrophy, improved hemodynamic functions, prevented oxidative stress, and increased mitochondrial DNA concentration. MgV treatment also increased the survival rate of the animals as depicted by the Kaplan-Meier curve. Improvement in hypertrophy due to HDAC inhibition was further confirmed by HDAC mRNA expression studies, which revealed that MgV decreases expression of pro-hypertrophic HDAC (i.e., HDAC2) without altering the expression of anti-hypertrophic HDAC5. Selective class I HDAC inhibition is required for controlling cardiac hypertrophy. Newer HDAC inhibitors that are class I inhibitors and class II promoters can be designed to obtain "pan" or "dual" natural HDAC "regulators".

C.B-17 SCID mice develop epicardial calcinosis with unaltered cardiac function.

Inbred mouse strains are the most widely used mammalian model organism in biomedical research owing to ease of genetic manipulation and short lifespan; however, each inbred strain possesses a unique repertoire of deleterious homozygous alleles that can make a specific strain more susceptible to a particular disease. In the current study, we report dystrophic cardiac calcinosis (DCC) in C.B-17 SCID male mice at 10 weeks of age with no significant change in cardiac function. Acquisition of DCC was characterized by myocardial injury, fibrosis, calcification, and necrosis of the tissue. At 10 weeks of age, 38% of the C.B-17 SCID mice from two different commercial colonies exhibited significant calcinosis on the ventricular epicardium, predominantly on the right ventricle. The frequency of calcinosis was more than 50% for mice obtained from Taconic's Cambridge City colony and 25% for mice obtained from Taconic's German Town colony. Interestingly, the DCC phenotype did not affect cardiac function at 10 weeks of age. No differences in echocardiography or electrocardiography were observed between the calcinotic and non-calcinotic mice from either colony. Our findings suggest that C.B-17 SCID mice exhibit DCC as early as 10 weeks of age with no significant impact on cardiac function. This strain of mice should be cautiously considered for the study of cardiac physiology.

Cardioprotective effects of magnesium valproate in type 2 diabetes mellitus.

We have evaluated the effect of magnesium valproate (210 mg/kg/day, p.o.) in type 2 diabetes induced cardiovascular complications induced by streptozotocin (STZ, 90 mg/kg, i.p.) in neonatal wistar rats. Various biochemical, cardiovascular and hemodynamic parameters were measured at the end of 8 weeks of treatment. STZ produced significant hyperglycaemia, hypoinsulinemia and dyslipidemia, which was prevented by magnesium valproate treatment. STZ produced increase in Creatinine Kinase, C-reactive protein and lactate dehydrogenase levels and treatment with magnesium valproate produced reduction in these levels. STZ produced increase in cardiac and LV hypertrophy index, LV/RV ratio, LV collagen deposition and LV cardiomyocyte diameter which were decreased by magnesium valproate treatment. Magnesium valproate also prevented STZ induced hemodynamic alterations and oxidative stress. These results were further supported by histopathological studies in which magnesium valproate showed marked reduction in fibrosis and cardiac fiber disarray. In conclusion, our data suggests that magnesium valproate is beneficial as an anti-diabetic agent in type-2 diabetes mellitus and also prevents its cardiac complications.

Evaluation of buspirone on streptozotocin induced type 1 diabetes and its associated complications.

We have evaluated the effect of buspirone (1.5 mg/kg/day, p.o.) type 1 diabetes induced cardiovascular complications induced by streptozotocin (STZ, 45 mg/kg, i.v.) in Wistar rats. Various biochemical, cardiovascular, and hemodynamic parameters were measured at the end of 8 weeks of treatment. STZ produced significant hyperglycaemia, hypoinsulinemia, and dyslipidemia, which was prevented by buspirone treatment. STZ produced increase in serum creatinine, urea, lactate dehydrogenase, creatinine kinase, and C-reactive protein levels and treatment with buspirone produced reduction in these levels. STZ produced increase in cardiac and LV hypertrophy index, LV/RV ratio, and LV collagen, which were decreased by buspirone treatment. Buspirone also prevented STZ induced hemodynamic alterations and oxidative stress. These results were further supported by histopathological studies in which buspirone showed marked reduction in fibrosis and cardiac fiber disarray. In conclusion, our data suggests that buspirone is beneficial as an antidiabetic agent in type 1 diabetes mellitus and also prevents its cardiac complications.

Therapeutic implications of small interfering RNA in cardiovascular diseases.

Cardiovascular diseases (CVDs) place a heavy burden on the economies of low- and middle-income countries. Comprehensive action requires combining approaches that seek to reduce the risks throughout the entire population with strategies that target individuals at high risk or with established disease. Small interfering RNA (siRNA) as a functional mediator for regulation of gene expression has been evaluated for potential therapeutic targets for the treatment of various cardiovascular diseases such as hypertension, atherosclerosis, heart failure etc. The present review attempts have been made to provide a brief outline of the current understanding of the mechanism of RNAi and the delivery system and describe the therapeutic application of siRNAs and their potential for treating CVDs which are taking a heavy toll on human life.