Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats

Altered calcium (Ca²⁺) handling has been identified as a key contributor to diabetic cardiac dysfunction (DCD) [1, 2]. Ca²⁺ is a critical second messenger in cardiac muscle, and thus changes in Ca²⁺ handling have acute and chronic consequences on cardiac function [3, 4]. Increased resting intracellular Ca²⁺ levels, attenuated sarcoplasmic reticulum Ca²⁺ release and re-uptake, and delayed recovery of the intracellular Ca²⁺ transient have been observed in rodent models of both type 1 and type 2 diabetes, ultimately resulting in reduced contractility and relaxation of cardiac muscles [5‐8]. However, the key mechanisms underpinning perturbations of contractility in the diabetic heart remain to be determined.

One emerging mediator of cardiac contractility is the myocardial isoform of Ca²⁺/calmodulin-dependent protein kinase II (CaMKIIδ) [9‐11]. This multifunctional serine/threonine protein kinase regulates proteins associated with cardiac Ca²⁺ flux, including ryanodine receptors [12], phospholamban [13], and L-type Ca²⁺ channels [14], as well as proteins integral to sarcomere structure [15] and cross bridge cycling [16]. Therefore, CaMKIIδ plays a pivotal role in cardiac contraction and relaxation. Persistent activation of CaMKIIδ is linked to a number of cardiac pathologies, including maladaptive hypertrophy [17, 18] apoptosis [19, 20] and arrhythmogenic events [21, 22].

Inhibition of CaMKIIδ has emerged as a potential therapeutic strategy for the prevention of cardiac dysfunction [23]. Previous studies using non-diabetic animal models with global deletion of CaMKIIδ have reported protection against cardiac hypertrophy [24], apoptosis [25], and Ca²⁺ leak induced arrhythmia [26]. Pharmacological inhibition of CaMKIIδ has also shown promising results both in vitro and in vivo. For example, acute CaMKIIδ inhibition in isolated cardiac muscle taken from non-diabetic patients with ischemic heart failure resulted in increased contractility and reduced Ca²⁺ spark frequency [27]. Recent studies have demonstrated that CaMKIIδ activity is increased in myocytes subjected to hyperglycemia or myocytes isolated from animal models of type 1 diabetes [28, 29]. However, the effects of CaMKIIδ inhibition on cardiac function in type 2 diabetes, which comprises about 95% of diabetes diagnoses in human patients, have not been determined.

Thus, the present study examined the link between CaMKIIδ activation and cardiac contractile function in a rat model of type 2 diabetes with preserved ejection fraction (EF). We found that the phosphorylated form of CaMKIIδ was upregulated in the hearts of patients with type 2 diabetes, even prior to the development of altered heart function. We then used isolated ventricular trabeculae from non-diabetic (nDM) and type 2 diabetic (DM) Zucker Diabetic Fatty (ZDF) rats to measure contraction and relaxation properties of the cardiac muscle with and without CaMKIIδ inhibitors. Our data demonstrate that CaMKIIδ inhibition attenuated the reduced force development and impaired rates of contraction and relaxation associated with type 2 diabetes, and moreover that these effects are independent of Ca²⁺ flux properties, including transient amplitude and Ca²⁺ load. These findings extend our understanding of the role of CaMKIIδ in regulating the function of the heart in the context of diabetes and suggest a novel therapeutic potential for CaMKIIδ inhibition to reverse impaired contractility and prevent heart failure progression in diabetic patients.

Activation of the multifunctional kinase CaMKIIδ has emerged as a key nodal point in the translation of cellular stresses into downstream alterations to cardiac physiology [5, 11, 38]. Recent publications have extended the role of CaMKIIδ into hyperglycemia and type 1 diabetes [28, 29]. Here, we show that CaMKIIδ plays a critical role in reduced cardiac contractility associated with type 2 diabetes. Critically, we show that CaMKIIδ activation is enhanced in type 2 diabetes and alters contraction and relaxation prior to the development of a heart failure phenotype. Thus, these findings suggest that altered metabolism in the diabetic heart activates CaMKIIδ and alters function at the myocyte level, eventually leading to the development of systolic and diastolic dysfunction.

Impaired contractility underpins reduced cardiac performance and contributes to enhanced susceptibility to heart failure in the diabetic myocardium [39]. Thus, one of the goals in the treatment of diabetes is targeting cellular pathways that modulate cardiac contractility. Here, we provide the first evidence that inhibition of CaMKIIδ restores both the force and rate of contraction in ventricular trabeculae from rats with type 2 diabetes. These findings are consistent with a previous study demonstrating that CaMKIIδ inhibition improves contractility in cardiac muscle during heart failure [27]. The previous study showed improvement in force generation and contraction, but not in relaxation parameters, following CaMKIIδ inhibition in trabeculae from patients with heart failure. Interestingly, our study showed a positive effect for CaMKIIδ inhibition on both contraction and relaxation parameters in the diabetic myocardium. This difference may be attributable to alterations in expression and/or activity of proteins associated with cardiac relaxation or alterations to myofilament function during diabetes and/or heart failure. For example, sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) expression was reduced in heart failure patients [27], whereas Lamberts et al. [40] showed in diabetic patients with preserved EF that myocardial SERCA2a expression was increased and phospholamban expression was decreased.

An important finding of this study was that CaMKIIδ inhibition in healthy nDM rats resulted in reduced F_dev (Fig. 3), thus confirming the role of CaMKIIδ in the positive modulation of inotropy in the healthy heart. These findings mirror previously published observations that inhibition of CaMKIIδ in the healthy myocardium disrupts the beneficial effects of exercise on cardiac function [41]. In addition a study from Kemi et al. [42] investigating aerobic interval training in healthy mice also suggested that the improved inotropy and lusitropy they observed in cardiomyocytes after exercise training was due to an increase CaMKIIδ activation. Taken together, these studies point to an important and positive role for CaMKIIδ in the healthy heart, a role that becomes pathological during chronic cardiac stress as occurs in, for example, type 2 diabetes.

Another important finding from this study was the observation that AIP, which inhibits both calmodulin-dependent and autonomous CaMKIIδ activation, is more effective for rescuing cardiac function in diabetic tissue than KN93, which inhibits only calmodulin-dependent activation of CaMKIIδ. This study showed that AIP was able to restore F_dev and dF/dt_max in the diabetic trabeculae to similar levels as observed in the nDM trabeculae and even improve dF/dt_min to levels above that in the nDM. Therefore AIP was able to fully restore contraction and relaxation in the DM trabeculae. AIP inhibits CaMKIIδ by blocking substrate binding at the catalytic domain and thus inhibits the autonomously active form of CaMKIIδ that is frequently associated with cardiac pathology [43]. Prolonged Ca²⁺ elevations at high frequency are a hallmark of cardiac stress and result in autonomous activation of CaMKIIδ via auto-phosphorylation [9]. Moreover, CaMKIIδ activity is enhanced in diabetic models due to post-translational modification of the kinase [28, 29], while other post-translational modifications of CaMKIIδ are emerging [44]. Our data using the two inhibitors is consistent with these prior studies, as only the AIP would be expected to inhibit CaMKIIδ after post-translational modification. More work is necessary to delineate the roles of various modifications in CaMKIIδ-induced alterations to contraction and relaxation in the type 2 diabetic heart.

One of the most widely acknowledged roles of CaMKIIδ in the heart is modulation of Ca²⁺ flux [45‐48]. Interestingly, our data indicate that neither the differences in contractile performance between nDM and DM trabeculae, nor the restoration of contractility by CaMKIIδ inhibition, are derived from alterations to Ca²⁺ transients or SR load. However, a number of alternative explanations remain unexplored. First, while baseline Ca²⁺ handling was not altered, these results do not preclude the possibility that spontaneous Ca²⁺ release events may be altering contractility in the diabetic myocardium. Indeed, CaMKIIδ inhibition has previously been shown to reduce Ca²⁺ sparks and ameliorate arrhythmogenic events in the diabetic heart [28]. Another potential target for alteration of cardiac contractility during diabetes is the myofilament itself. Previous studies have shown that diabetes triggers impaired function at the myofilament [49, 50], and CaMKIIδ is known to phosphorylate proteins associated with the myofilament [51]. Future work will need to focus on the mechanism by which diabetes reduces contractility.

Type 2 diabetes is a worldwide epidemic such that, by the year 2035, 592 million people are projected to suffer from the disease [52]. The landmark Framingham study established a now well-recognized link between diabetes and cardiovascular disease [53], which remains the leading cause of mortality in diabetic patients. Inhibition of CaMKIIδ has been proposed as an exciting new avenue in the treatment of cardiovascular disease. The data in this study provide novel evidence of CaMKIIδ activation as mediator of cardiac contractility in the type 2 diabetic heart and provide novel evidence and point to the potential for targeting CaMKIIδ as a therapeutic approach in diabetic patients to prevent structural remodeling and subsequent heart failure that arise from diabetes-induced impairment of contractile performance. Indeed, a new generation of CaMKIIδ inhibitors is currently in development that may have significant impact on the treatment of DCD and other forms of cardiovascular disease [43, 54]. In addition to pharmacological CaMKIIδ inhibitors, exercise is emerging as a therapeutic approach that may ablate the negative effects of CaMKIIδ in the diabetic heart [55]. The benefits of exercise for diabetic patients are well known, such as improving glycemic control, blood lipid profiles and cardiovascular function [56]. Interestingly, aerobic interval training in type 2 diabetic mice has been shown to reduce the activation of CaMKIIδ and improve cardiac function [57]. Therefore the combination of pharmacological CaMKIIδ inhibitors and exercise-based approaches in diabetic patients warrants further investigation.

In this study, we showed that CaMKIIδ phosphorylation and O-GlcNAc modification were increased in RAA samples from DM patients compared to those from nDM patients. While the use of human tissue is critical for establishing that the underlying mechanisms that drive CaMKIIδactivation are similar between DM ZDF rats and diabetic human patients, we must acknowledge a couple limitations of this study. First, the number of patients in each cohort was small (7 per group). While this group size was sufficiently large to see significant differences in CaMKIIδ modification, it is critical to consider that small human cohorts will have a great deal of heterogeneity. Second, all tissue was donated by patients undergoing coronary artery bypass graft surgery, and therefore all patients (DM and nDM) were not fully healthy. Importantly, we were still able to detect significant CaMKIIδ activation by phosphorylation and by O-GlcNAc modification in the diabetic patients that rose above the non-diabetic cohort, even with the presence of confounding health issues. Finally, we were not able to match the tissues used between the human and rat experiments, as we did not have access to RV human tissue. This limitation raises the possibility that there are heart chamber differences in our echo and Western blot data between the two species. The current literature regarding biochemical features of different heart chambers is split, with examples of differences in expression of proteins between chambers [58] and examples of no differences between chambers [59]. Critically, the alterations in phosphorylation and O-GlcNAc modification brought about by diabetes were similar in direction and magnitude for the RAA and LV, consistent with the hypothesis that CaMKIIδmodification is increased throughout the diabetic heart.

Here, we show that cardiac tissue both from type 2 diabetic patients and from DM ZDF diabetic rats has elevated CaMKIIδ activation, independent of heart failure, compared to nDM controls. Moreover, we show that trabeculae from DM ZDF diabetic rats have reduced contraction and relaxation performance compared to trabeculae from nDM rats, indicating that the altered metabolic state of diabetes directly impacts contractile performance of cardiac muscle independent of nerve input, catecholaminergic effects, or other factors. Finally, we show that two different inhibitors of cardiac CaMKIIδ are both able to restore contractile performance of trabeculae from DM ZDF rats to levels similar to nDM controls. Our data demonstrate that activation of CaMKIIδ underlies reduced force development and impaired contractility in the diabetic heart. Further, it suggests that inhibition of CaMKIIδ is a potential clinical approach to prevent heart failure associated with type 2 diabetes.