GENETIC ANALYSIS IN CORONARY HEART DISEASE PATIENTS AND DYNAMICS OF NUCLEAR CALCIUM IN ISOLATED CARDIOMYOCYTES

Abstract

Coronary Heart Disease (CHD) is one of the leading killers of human beings in Pakistan. Outcome of complex diseases like CHD is determined by interaction between multiple factors including the genetic factors, most importantly. To scrutinize the association of various genetic polymorphisms [rs7025486(A/G), rs1333049(G/C), rs6922269(G/A), rs2943634(C/A), rs599839(A/G), rs17465637(C/A), rs501120(C/T) and rs17228212(C/T)] with the risk of CHD in Pakistani population, the present investigation was designed. Although these genetic variants have been already explored for their association with CHD in European populations, the unique architecture and genetic composition of Pakistani population warrant an independent trial in this population. Samples for patients were collected from different hospitals in Lahore and Gujranwala. Samples for controls were randomly collected from various Government and private institutes and hospitals. A detailed questionnaire was administered for acquisition of desired information and for taking informed consent. A total of 645 samples (435 cases and 210 controls) were collected out of which 6% were excluded based on positive screening for Hepatitis B, Hepatitis C or HIV. The remaining 606 samples (403 cases and 203 controls) were utilized for biochemical (lipid profile) and genetic analysis. Results for the lipid profiling imply that the levels of various lipoproteins and triglycerides were impaired in a higher percentage of cases as compared to controls pointing towards an association between dyslipidaemia and the atherosclerosis which underlies CHD. Findings of the present exploration pertaining to the genetic polymorphisms indicate that for rs7025486(A/G), increased risk of CHD was on margin level in GA carriers (Odds ratio: 1.72; 95% Confidence interval: 1.223- 2.428) and frequency of risk allele [A] was higher in cases as compared to controls (Cases: 0.45; Controls: 0.41). For rs1333049(G/C), those carrying CC genotype were at margin of increased risk for CHD (Odds ratio: 1.65; 95% Confidence interval: 1.096-2.476). Carriers of GC were experienced to be at risk margin of CHD only in men (Odds ratio: 1.18; 95% Confidence interval: 0.805-1.730) and in the combined data analysis (Odds ratio: 1.12; 95% Confidence interval: 0.800-1.571). The frequency of risk allele [C] was observed as being elevated in case subjects than xiv controls (Cases: 0.55; Controls: 0.45). For rs6922269(G/A), elevated risk of CHD was at margin level in bearers of AA genotype (Odds ratio: 1.36; 95% Confidence interval: 0.523-3.523). In addition, those possessing GA genotype were also found to be at risk margin of CHD (Odds ratio: 1.76; 95% Confidence interval: 1.183-2.630). Besides, frequency of risk allele [A] was comparatively raised in case subjects (Cases: 0.20; Controls; 0.13). Results pertinent to rs2943634(C/A) indicate that carriers of CC were at increased risk margin of CHD (Odds ratio: 1.53; 95% Confidence interval: 1.058-2.221) and that, the risk allele frequency [C] was lower in controls than in cases (Cases: 0.87; Controls; 0.80). With regards to rs599839(A/G), increased risk margin of CHD was experienced in AA carriers (Odds ratio: 1.72; 95% Confidence interval: 1.156-2.566) and the risk allele frequency [A] observed a decrease in controls as compared to cases (Cases: 0.90; Controls; 0.84). As far as rs17465637(C/A) is concerned, vulnerability to CHD was revealed to be at margin level in CC carriers (Odds ratio: 1.40; 95% Confidence interval: 0.993-1.961). This observation stayed the same when the data was analyzed for MI patients only (Odds ratio: 1.68; 95% Confidence interval: 1.163-2.442) In addition, only the female AA carriers were divulged to be at an increased risk margin of CHD (Odds ratio: 1.13; 95% Confidence interval: 0.367-3.471). Further, frequency of the risk allele [C] was higher in case subjects than in control subjects (Cases: 0.65; Controls; 0.60). In relation to rs501120(C/T), TT genotype carriers were found to be at elevated risk margin of CHD (Odds ratio: 1.29; 95% Confidence interval: 0.917-1.816). Besides, CT carriers were also at margin of increased risk of CHD (Odds ratio: 1.05; 95% Confidence interval: 0.750-1.484). Furthermore, cases experienced a comparatively increased frequency of the risk allele [T] (Cases: 0.68; Controls; 0.61). For rs17228212(C/T), frequency of the CT carriers was extremely low (< 5 %) in our sample population and CC carriers were completely absent implying that the results were of little potential significance keeping in view the low frequency of C allele. Besides the genetic analysis of CHD patients, some of the experiments included here pertinent to dynamics of nuclear calcium in isolated cardiomyocytes were conducted as part of a project entitled "Nuclear Calcium and Gene Regulation in the Remodeled Heart" which aimed at investigating the role of nuclear calcium in cardiac remodeling and pursuing it as a therapeutic approach. The results highlight that xv nuclear calcium transient manifests a slower release time but a similar uptake time when compared with cytoplasmic calcium. Independent release of nuclear calcium was also observed. Addition of 2-APB, a blocker of IP3 receptor mediated calcium release, lead to a decrease in the amplitude of calcium transient in the cytoplasm but not in the nucleus at both 1 and 2 Hz. No effect of the drug could be ascertained on calcium release or reuptake time. Furthermore, 2-APB caused a shortening of the calcium transient in both nucleus and cytoplasm of isolated cardiomyocytes. Isolation of adult mouse ventricular cardiomyocytes done as a part of the work on mice expressing VSFP2.3 targeted at the investigation of this protein as an optogenetic tool for studying cardiac activity is also a part of the present research. The findings relevant to this research indicate that genetic polymorphisms play a significant role in modifying the risk of CHD and that, modulation of nuclear calcium can be pursued as an effective target for attenuation of cardiac remodeling which is associated with CHD.