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Examines the effects of postnatal hypo- or hyperthyroidism on sarcolemmal Na[sup +]/Ca[sup +] exchanger (NCX) function and sarcoendoplasmic reticulum Ca[sup 2+]-ATPase (SERCA2) function. Elevation of NCX mRNA and protein in hypothyroidism; Lack of change in the levels of NCX and SERCA2 mRNA and protein in hyperthyroidism; Decline in noninhibitory phosphorylated phospholamban.

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

Background: Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca(2+)-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca(2+) uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart., Objective: To explore whether enhanced Ca(2+) cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca(2+) (I(Ca-L)) currents., Methods: The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals., Results: The amplitudes of I(Ca-L) currents at depolarizing pulses ranging from -45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I(Ca-L) showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I(Ca-L) currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca(2+) with BAPTA., Conclusions: These findings indicate that enhanced Ca(2+) cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I(Ca-L) currents. Modified I(Ca-L) kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.

The bradykinin-forming enzyme kallikrein-1 is expressed in the heart. To examine whether contractile performance and sarcoplasmic reticulum Ca2+ transport of the diabetic heart can be rescued by targeting the kallikrein-kinin system, we studied left ventricular function and sarcoplasmic reticular Ca2+ uptake after induction of streptozotocin-induced diabetes mellitus in transgenic rats expressing the human tissue kallikrein-1 gene. Six weeks after a single injection of either streptozotocin (70 mg/kg ip) or vehicle, left ventricular performance was determined using a Millar-Tip catheter system. The Ca2+-transporting activity of reticulum-derived membrane vesicles was determined in left ventricular homogenates as oxalate-supported 45Ca2+ uptake. Western blot analysis was used to quantify the reticular Ca2+-ATPase SERCA2a, phospholamban, and the phosphorylation status of the latter. Contractile performance and Ca2+ uptake activity were similar in nondiabetic wild-type and transgenic rats. Severely diabetic wild-type animals exhibited impaired left ventricular performance and decreased reticular Ca2+ uptake (-39% vs. wild-type rats, P<0.05, respectively). These changes were attenuated in diabetic transgenic rats that, in addition, exhibited a markedly increased phospholamban phosphorylation at the Ca2+/calmodulin kinase-specific site threonine17 (2.2-fold vs. diabetic wild-type rats, P<0.05). These transgene-related effects were abolished after treatment with the bradykinin B2 receptor antagonist icatibant (Hoe 140). The SERCA2-to-phospholamban ratio, phosphoserine16-phospholamban levels, and the apparent affinity for Ca2+ of the uptake reaction did not differ between the groups. Increasing the activity of the kallikrein-kinin system by expressing a human kallikrein-1 transgene protects rat heart against diabetes-induced contractile and reticular Ca2+ transport dysfunctions. An increased phosphorylation of the SERCA2 regulatory protein phospholamban at threonine17 via a B2 receptor-mediated mechanism is thereby involved.

Slowed relaxation in diabetic cardiomyopathy (CM) is partially related to diminished expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on SR Ca2+ handling in diabetic CM, we 1) generated transgenic rats harboring a human cytomegalovirus enhancer/chicken beta-actin promotor-controlled rat SERCA2 transgene (SERCA2-TGR), 2) characterized their SR phenotype, and 3) examined whether transgene expression may rescue SR Ca2+ transport in streptozotocin-induced diabetes. The transgene was expressed in all heart chambers. Compared to wild-type (WT) rats, a heterozygous line exhibited increased SERCA2 mRNA (1.5-fold), SERCA2 protein (+26%) and SR Ca2+ uptake (+37%). Phospholamban expression was not altered. In SERCA2-TGR, contraction amplitude (+48%) and rates of contraction (+34%) and relaxation (+35%) of isolated papillary muscles (PM) were increased (P2+ uptake and SERCA2 protein of SERCA2-TGR were 1.3-fold higher (P2+ uptake, accelerates relaxation and compensates, in part, for depressed Ca2+ uptake in diabetic CM. Therefore, SERCA2 expression might constitute an important therapeutic target to rescue cardiac SR Ca2+ handling in diabetes.