The mechanism of PLB action on SERCA and its relevance in cardiac muscle physiology has been studied extensively over the past several years. Studies using genetically altered mouse models have given important insight into the role of PLB in cardiac physiology. Using a PLB knockout mouse model [41
], Dr. Kranias and colleagues provided the crucial evidence that PLB is an important regulator of the SERCA2a. They showed that absence of PLB enhanced SR calcium uptake and increased rates of contraction and relaxation. This was associated with an increase in SERCA2a affinity for calcium [41
]. On the other hand, over-expression of PLB in the heart resulted in a decrease in SR Ca2+
uptake and depressed cardiac contractile performance in vivo
] These studies revealed that a shift in PLB: SERCA ratio leads to a corresponding shift in SERCA affinity for Ca2+
, so that an increase in the PLB: SERCA ratio leads to decreased Ca2+
affinity. Thus, an alteration in the PLB: SERCA ratio can affect SR Ca2+
transport. More recently, studies in human suggest that mutations in PLB [43
] or the absence of PLB [45
] can cause far more serious functional consequences, culminating in human heart failure. It is tempting to speculate that, in larger mammals PLB is essential for maintaining heart function, unlike in mouse. These studies underscore the importance of understanding species differences with regard to the role of PLB and SR Ca2+
transport, in general, between small and larger animals.
The physiological relevance of SLN in the heart was only recently identified with the help of two transgenic mouse models developed independently by the MacLennan lab and our lab. In the first case, Asahi et al [46
] used rabbit cDNA to overexpress SLN in the mouse, by targeting a single copy of the α-MHC driven SLN construct into the Hprt
locus of the X-chromosome. This resulted in heterogeneous expression of SLN in female mice due to X-chromosome inactivation. Therefore, only males were used in this study. Overexpression of SLN reduced the apparent Ca2+
affinity of the SERCA2a. In vivo
measurements of cardiac function showed a significant decrease in +dP/dt and −dP/dt and led to ventricular hypertrophy. The inhibitory effect of SLN was reversed by treatment with the β-adrenergic agonist, isoproterenol, which restored contractile function. They also reported that basal phosphorylation of PLB was decreased in the SLN transgenic hearts and in the presence of isoproterenol, PLB phosphorylation was restored to the level seen in wild-type controls. This was interpreted as an enhanced PLB phosphorylation, resulting in the dissociation of SLN from PLB and leading to the restoration of contractile function in the SLN transgenic hearts during β-adrenergic stimulation. By co-immunoprecipitation analysis using microsomes prepared from transgenic hearts, it was observed that SLN was bound to both SERCA2a and PLB, forming a ternary complex. These data suggested that SLN mediates its inhibitory effect on SERCA2a through stabilization of the SERCA2a-PLB complex and through the inhibition of PLB phosphorylation.
Our lab used the cardiac specific α-MHC promoter to overexpress mouse SLN in the atria and ventricles [47
]. To study the role of SLN, the SLN: SERCA2a ratio was increased in the ventricle, where the level of SLN is naturally low. Overexpression of mouse SLN in the mouse ventricle did not lead to hypertrophy. The development of hypertrophy observed by Asahi et al. [46
] is probably due to the overexpression of rabbit SLN in the mouse heart, which differs from mouse SLN at the N-terminus. SLN overexpression in the ventricle leads to decreased SERCA2a affinity for calcium, Ca2+
transient amplitude and shortening, and slowed relaxation. Consistent with Asahi et al [46
] the +dP/dt and −dP/dt were significantly decreased, due to SLN overexpression. Similar results were found in myocytes and muscle preparations from mice overexpressing SLN, in comparison to the wild-type littermates. The inhibitory effect of SLN on SERCA2a was reversed upon β-adrenergic stimulation, suggesting that SLN is a reversible inhibitor of SERCA2a, similar to the role of PLB. In this study, we observed that an increase in SLN level does not affect PLB levels, PLB monomer to pentamer ratio and its phosphorylation status, and we concluded that the effect of SLN on SERCA2a is direct and is not mediated by a change in PLB monomer levels or its phosphorylation status. This was further confirmed by Gramolini et al. [48
] by expressing SLN in the PLB null (−/−) background. This was achieved by mating the SLN transgenic mice, with cardiac specific overexpression of SLN, with the PLB KO mice. Overexpression of SLN in the absence of PLB led to a decrease in the affinity of SERCA2a for Ca2+
, impaired contractility, reduced calcium transient amplitude and slower decay kinetics, compared to PLB (−/−) animals. Further, in the SLN/PLB (−/−), mice isoproterenol restored the calcium dynamics to the levels seen in PLB (−/−) mice, suggesting that SLN could mediate the β-adrenergic response. The ventricular myocytes from PLB−/− mice did not show an increase in calcium handling in response to isoproterenol (ISO) which is consistent with the lack of PLB and its phosphorylation effects. Where as ventricular myocytes from SLN/PLB (−/−) showed an increased calcium transient amplitude as well as increased calcium decay kinetics, which suggests that SLN could be a mediator of β-adrenergic response and this response is independent of PLB. The lack of ISO -response in the PLB−/− ventricular myocytes, as well as other data showing very low levels of SLN suggests that SLN has little physiological role in the normal ventricle. But this does not rule out the possibility of its role in certain diseased conditions where the levels of SLN are altered. However, such conditions are yet to be reported to date. Further research in this area will determine the role of SLN in ventricular patho-physiology. These data suggest that SLN can mediate its inhibitory effect on SERCA2a independent of PLB, and could also be an important mediator of β-adrenergic response in the heart.
We recently developed a SLN knock out mouse model. Preliminary studies carried out using SLN null mice showed that loss of SLN leads to an increase in SR calcium uptake function and enhanced cardiac contractility. These studies further indicate that SLN is an important regulator of SERCA2a function and cardiac contractility [Babu et al unpublished data].
Based on the studies using genetically engineered mouse models it could be interpreted that an increase in the apparent ratio of either PLB or SLN, with respect to SERCA2a, may lead to depressed Ca2+ transport kinetics and contractile parameters in the mammalian heart.