Our findings show that in human right atrial myocardium from type 2 diabetic patients, with CAD and preserved EF, the contractile state is preserved, whereas relaxation is impaired, despite an increase in SERCA2a:PLB expression ratio. This is associated with an increased level of atrial myocardial fibrosis. The increase in SERCA2a:PLB ratio may suggest a compensatory mechanism in an attempt to maintain diastolic function at rest despite impaired relaxation in the fibrotic diabetic atrial myocardium. We also show for the first time that human right atrial cardiac muscles from patients with type 2 diabetes and preserved EF are unresponsive to β-adrenergic stimulation without a change in expression level of the β1-adrenoreceptors, suggesting impaired downstream β-adrenergic modulation. Our study provides novel information on the underlying pathology of diastolic dysfunction in the diabetic myocardium, strengthening the knowledge base for the development of therapeutics, which are currently very limited for diastolic dysfunction[5, 22].
Impaired relaxation in human right atrial diabetic myocardium at rest
Our in vivo echocardiography data and our ex vivo cardiac muscle data confirmed both preserved contractile function with no development of structural cardiac hypertrophy in diabetic patients with preserved EF. Echocardiography of the left ventricle also revealed a slower early filling time (prolonged deceleration time) and higher filling pressures during early filling (increased E/e’) with a tendency to slower early and late filling velocities (E and A, respectively), which in the isolated right atrial muscles were accompanied by prolongations of early and late relaxation (decreased -dF/dtmin and prolonged Tau). Together these data indicate reduced diastolic function due to impaired relaxation and increased myocardial stiffness of the heart in the diabetic population with preserved EF. This is consistent with previous echocardiography findings in type 1 and type 2 diabetic patients[2, 23] and with functional myocardial studies in type 1 and type 2 diabetic animal models, however the myocardial diastolic dysfunction observed in animal models was always accompanied by systolic dysfunction. On the contrary, Reuter et al. have reported no difference in the rate of relaxation or the contractile function of right atrial cardiac muscles from type 2 diabetic patients undergoing CABG. However, the contractile and relaxation measurements reported in that study were much lower in magnitude when compared to values reported in other studies using human right atrial cardiac muscles[27, 28] and our study (Table 2). This may be related to tissue degradation in their samples due to prolonged loading with a fluorescence indicator, while our experimental measurements were obtained within 90 minutes after dissection.
The heart has the ability to increase its cardiac efficiency via intrinsic means, such as volume. Several studies reported that the Frank-Starling mechanism is maintained in patients with reduced EF[28, 30]. More recently, patients with preserved EF showed a preserved static (intrinsic to the heart) Frank-Starling mechanism, whereas the beat-to-beat dynamic (including the heart-arterial interaction) Frank-Starling mechanism was impaired in these patients. By increasing the length of our isolated cardiac muscles we mimicked the myocardial stretch and recorded a similar increase in force development in both our non-DM and DM groups. This indicates a preserved Frank-Starling mechanism in diabetic patients with preserved EF, and supports the concept that arterial stiffening might be more important for the observed impaired dynamic Frank-Starling mechanism during preserved EF than myocardial stiffening.
Impaired relaxation during physiological challenge
Although contractile function at rest was not altered by diabetes, the relaxation of the right atrial diabetic muscles was impaired over the entire range of myocardial stretch, and both the inotropic and lusitropic responses to β-adrenergic stimulation were completely absent in cardiac muscles from DM patients. A reduced but still responsive myocardium to β-adrenergic stimulation has been observed in many diabetic animal models[33, 34] and has been related to reduced expression levels of β1-adrenoreceptors. Surprisingly, cardiac muscles from our cohort of diabetic patients failed to respond to the β-adrenergic stimulation, even at very high (10-5 M) concentrations, which would indicate a lack of contractile reserve even in these patients with normal resting contractile function. In addition, our new data emphasizes that caution should be taken with translating animal data directly to the human situation. It would be easy to attribute this to the use of β-adrenoceptor blocking agents, however almost all patients, both the diabetic and the non-diabetic, were on β-blocker treatment (Table 1). Secondly, although the hemodynamic responses to dobutamine are reduced in diabetic patients they normally still do respond. Consequently the non-responsiveness to β-adrenergic stimulation in the isolated right atrial cardiac muscles in our study should reside within the myocardium. Thirdly, although a reduced expression of β1-adrenoreceptors has been reported in myocardial tissue from diabetic patients undergoing CABG in the absence of β-blockers, to the best of our knowledge expression of β1-adrenoreceptors has never been reported in human myocardial tissue from diabetic patients with preserved EF. Interestingly, we did not observe a statistically significant reduction in β1-adrenoreceptors expression levels (Figure 3F) in the cardiac tissue from our diabetic cohort. Therefore, we suggest that the reduced β-adrenergic inotropic and lusitropic responses in the diabetic myocardium from patients with preserved EF are related to alterations in downstream β-adrenergic signaling pathways. Phosphorylation of PLB is an important downstream target of β-adrenergic signaling and we observed a markedly reduced PLB protein expression in the diabetic myocardium. Indirectly this implies there is less PLB to be phosphorylated and therefore this could be the reason for the reduced β-adrenergic inotropic and lusitropic responses in the diabetic myocardium from patients with preserved EF. Alternatively, Daniels et al. suggested that reduced β-adrenergic responsiveness in diabetes is related to metabolic changes leading to impaired energy conversion, which becomes apparent during physiological challenge. Additionally, it was recently shown that although exercise limitations were similar between patients with reduced and preserved EF, significant differences occurred in their exercise-induced changes in systolic and diastolic properties, again reflecting their different underlying pathologies.
Underlying mechanism of impaired relaxation
The underlying mechanism of the impaired relaxation may be related to impaired Ca2+ cycling within the cardiomyocytes. In human diabetic myocardium from patients with reduced EF a decreased level of RyR2 mRNA was observed. In contrast, the human atrial myocardium from diabetic patients with preserved EF in our study showed no change in expression of the SR Ca2+ release channel RyR2, which is consistent with the preserved contractile function observed. Interestingly, in human non-diabetic and type 2 diabetic patients with preserved EF, it was reported that both SERCA2a and PLB expression were similar, and recently mRNA of SERCA was also shown to not be different between diabetic and non-diabetic spontaneously hypertensive rats, which is in contrast to our study showing an increased SERCA2a protein expression and a decrease in PLB protein expression. However, an altered mRNA level does not always equate to altered protein expression. Moreover, our observed changes in SERCA2a and PLB expression lead to an increase in the SERCA2a:PLB ratio in myocardial tissue of DM patients. From our data, which suggest accelerated SR Ca2+ uptake, it is tempting to speculate that the changes in protein density reflect a compensatory mechanism to attempt to normalize the impaired relaxation at rest. This is supported by the findings of Selby et al. who suggested that in myocardial muscles obtained from non-diabetic patients with preserved EF, the impaired relaxation is accompanied by a disproportionate increase in SR Ca2+ content due to an increase in SR Ca2+ uptake. Notably, they found that increased SR Ca2+ content did not translate into a stronger contraction. In addition, our results are supported by studies using rodent diabetic models that have reported an up-regulation of SERCA2a, a down-regulation of PLB and an improved contractile function following insulin treatment[19, 41]. These authors showed that expression of SERCA2a mRNA in cardiomyocytes is under control of insulin, associated with changes in SR-Ca2+ uptake and phosphorylation of Akt, and inhibited by phosphoinositide 3 (PI3)-kinase inhibitor wortmanin. This indicates that the underlying cellular mechanism for upregulation of SERCA2a could be mediated by the PI3-kinase-Akt-SERCA2a signaling cascade, suggesting that subtle changes in Ca2+ regulation, which promote diastolic dysfunction prior to overt systolic dysfunction, may be common to early stages of type 2 diabetes involving insulin resistance[19, 42]. Moreover, it was shown recently that elevated oxidative stress may induce oxidative modifications of SERCA2a that could contribute to abnormal function in high-sucrose fed rats mimicking the metabolic syndrome heart.
In the present study, DM patients had an increase in hyperglycemic markers (glucose and HbA1c), despite all being on hyperglycemic control medication (metformin and insulin), and therefore changes in the expression of calcium-handling proteins might still be due to altered glycemic control. These changes in Ca2+ regulation may over time increase the energy demands of the diabetic myocardium and have important ramifications for future diabetic myocardium remodeling, potentially further impairing relaxation and eventually leading to myocardial dysfunction. Therefore, the increased SERCA2A:PLB expression ratio may be compensatory and beneficial for relaxation at resting conditions, but once the myocardium is challenged, there is little reserve for an increase in Ca2+ cycling leading to the lack of response to β-adrenergic stimulation observed in our study. Alternatively, in rodent models, experimental diabetes changed the phosphorylation of the sarcomeric protein troponin I[44, 45] and also markedly shifted the myosin heavy chain (MHC) from the fast (V1) to the slow (V3) isoform[46, 47], although the later might be less important in humans as most MHC already is predominantly in the slow isoform. However, these studies indicate that myocardial relaxation can also be affected by alterations in regulatory myofilament proteins of the cardiac actomyosin system of which upon the effects of diabetes in humans are unknown.
A well-known underlying cause of diastolic dysfunction is increased myocardial fibrosis. The extracellular matrix remodeling plays an important role in cardiac fibrosis and the amount of extracellular collagen is caused by an imbalance between synthesis and degradation of collagen[49, 50] and formation of advanced glycation end-products. Cardiac fibrosis leads to increased myocardial stiffness, eventually resulting in both systolic and diastolic dysfunction. Increased fibrosis is commonly observed in diabetic animal models (see review), whereas reports in human cardiac tissue are conflicting[8, 49]. In our study, the elevated collagen deposition in the right atrium of diabetic patients is indicative of increased fibrosis. Alternatively, increased diastolic myocardial stiffness could result from an increased cardiomyocyte stiffness, which relates to the elasticity of the giant cytoskeletal protein titin and is determined by its isoform expression and/or posttranslational modification[53, 54]. Changes in isoform expression of titin have been observed in type 1 diabetic animal models[47, 55, 56]. Recently in a metabolic animal model hyperphosphorylation of titin contributed importantly to underlying diastolic dysfunction. However the specific role of titin regulating diastolic (dys) function, especially in type 2 diabetes in humans, is still not clear.
Due to the use and access to human tissue, there are a number of limitations to our study. CAD is known to be a co-morbidity and a specific causal factor for the transition from diastolic to systolic failure. As all our patients had CAD for inclusion into the study, our data were not compared to healthy human myocardium, so it is unclear if our findings would translate to patients with DM but without CAD. Non-transplanted donor heart tissue is the closest available “non-diseased’ human myocardial tissue, and our force values obtained in right atrial cardiac muscles from non-DM patients with CAD and preserved EF were similar to data from LV myocardial tissue of non-transplanted donor hearts, although caution should still be taken that the function of the “non-diseased” hearts might still be affected by the cause of death (such as accident trauma), the use of cardioplegic solutions and the variable elapsed time between removal of the heart and the dissection of cardiac muscles. Nevertheless, diabetic patients with CAD are an extensive cohort and are known to have double the risk of progressing to HF compared to non-diabetic CAD patients, and therefore our findings provide important novel and relevant knowledge to a clinically large and important patient group.
Another limitation is that differences in the ultrastructure and Ca2+ dynamics between the atria and ventricular myocardium exists , which need to be carefully considered when translating right atrial findings to the left ventricular myocardium or the whole heart. For instance it has been shown that atrial tissue expresses relatively more SERCA2a and less PLB compared to ventricular tissue. On the other hand, cardiac muscles obtained from right atrial appendages have been extensively used to study the function of human myocardium[9, 26–28].