Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Elevated expression of activated Na+/H+ exchanger protein induces hypertrophy in isolated rat neonatal ventricular cardiomyocytes

The plasma membrane protein the Na(+)/H(+) exchanger isoform1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. Levels of NHE1 protein and activity are elevated in cardiac disease; however, the mechanism by which these factors contribute to the accompanying hypertrophy in the myocardium is still not clear. To investigate the mechanism of NHE1-induced hypertrophy in the myocardium we constructed two adenoviral vectors expressing either wild type NHE1 protein or a constitutively active NHE1 protein. Infection of neonatal rat ventricular cardiomyocytes (NRVM) resulted in elevated expression of both wild type NHE1 or constitutively active NHE1. Only expression of activated NHE1 protein resulted in an increase in cell size and in an increase in protein synthesis in isolated cardiomyocyte cells. The results demonstrate that expression of activated NHE1 promotes cardiac hypertrophy in isolated cardiac cells and that simple elevation of levels of wild type NHE1 protein does not have a significant hypertrophic effect in NRVM. The results suggest that regulation of NHE1 activity is a critical direct effector of the hypertrophic effect induced in the myocardium by the NHE1 protein.     

Na+/H+ Exchanger Isoform 1-Induced Osteopontin Expression Facilitates Cardiomyocyte Hypertrophy

Enhanced expression and activity of the Na⁺/H⁺ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.     

Gender-specific effects of exercise on cardiac pathology in Na+/H+ exchanger overexpressing mice

The Na(+)/H(+) exchanger isoform 1 (NHE1) has been implicated in various cardiac pathologies including ischemia/reperfusion damage to the myocardium and cardiac hypertrophy. It is known that NHE1 levels increase in cardiac disease and we have recently demonstrated that expression of an activated NHE1 protein promotes cardiac hypertrophy in the mouse myocardium. We examined the gender-specific effects of exercise in combination with elevated cardiac expression of NHE1 on the myocardium in mice. Control mice and transgenic mice expressing elevated levels of wild type NHE1 and activated NHE1 were examined. There were gender-specific differences in the effects of NHE1 with exercise. Exercised wild type male mice showed a tendency toward increased heart weight. This was not apparent in female mice expressing elevated NHE1 levels. In some transgenic female mice, there was a significant decrease in the size of the exercised hearts, which was different from what occurred with male mice. Body weight was maintained in exercised control and transgenic male mice; however, it decreased in female mice with exercise more so in transgenic female mice expressing elevated levels of NHE1. Female mice expressing activated NHE1 had elevated HW/BW ratios compared to males, and this was exaggerated by exercise. These results suggest that gender-specific activation of NHE1 may be critical and that NHE1 plays a more critical role in promoting some types of hypertrophy in females in comparison with males.