2.2.2. Calcium Cycling Defects in DCM

DCM-causing mutations affect multiple aspects of calcium handling within the myocyte, from altering calcium sensitivity of the myofilament seen with sarcomeric mutations, to mislocalization and altered expression or function of calcium handling proteins seen with cytoskeletal mutations. In DCM, there is a decrease in peak height of the calcium transient in systole and a decreased rate of calcium reuptake in diastole [57]. Increased calcium leak from RyR2 can be a contributing factor, particularly in DMD cardiomyopathy [58]. The resulting decrease in SR calcium load decreases contractile function in systole [59]. Decreased calcium cycling and decreased SR calcium load can also occur via a decrease in the expression and/or activity of Serca2a [59–61]. PLN expression is not decreased to the same extent in DCM, thus increasing the ratio of PLN to Serca2a [60,61], which results in increased PLN inhibition of Serca2a. Additionally, β-adrenergic desensitization results in reduced PLN phosphorylation, which further increases its inhibition of Serca2a [54,60] (Figure 3).

**Figure 3.** Mechanisms of calcium overload in dystrophin-deficient cardiac myocytes. The absence of dystrophin destabilizes the sarcolemma and leads to stress-induced membrane damage/micro-tears and calcium influx (**a**). Excessive reactive oxygen species (ROS) production in cardiac myocytes leads to further membrane damage and increased calcium influx via stretch-activated channels (SACs) and ryanodine receptor 2 (RyR2) (**b**). Increased L-type calcium channel (LTCC/DHPR) current also contributes to increased intracellular calcium (**c**). Calcium leak from RyR2 (**d**), decreased Serca2a expression (**e**) and increased phospholamban (PLN) inhibition of Serca2a decrease sarcoplasmic reticulum (SR) calcium load, subsequently decreasing calcium transient peak height and decay rate and inhibiting contractile function in later stages of Duchenne muscular dystrophy (DMD) cardiomyopathy (dark lines in transients). Increased cytosolic calcium leads to mitochondrial cell death pathways (**f**). \* Indicates points of abnormal calcium entry into the myocyte. NCX: Sodium calcium exchanger; NOX-2: NADPH oxidase 2; NOS: Nitric oxide synthase; MCU: Mitochondrial calcium uniporter; NCLX: Mitochondrial sodium calcium exchanger.

#### **3. Molecular Mechanisms of DMD Cardiomyopathy**

The lack of dystrophin in cardiac myocytes leads to instability of the sarcolemma, resulting in calcium overload and oxidative stress. Over time, excess calcium and reactive oxygen species (ROS) activate cell death pathways, fibrosis, and dilation [12]. How dystrophin functions to stabilize the cell membrane is not fully understood, but two prevailing ideas are supported by the literature. First, dystrophin, as part of the DGC, connects the extracellular matrix and the intracellular cytoskeleton and acts as a "shock absorber" for the sarcolemma during the repeated stress of contraction and relaxation [62–64]. Sarcolemmal stress in the absence of dystrophin leads to muscle membrane damage or micro-tears, causing extracellular calcium influx and calcium overload [20,62,63] (Figure 3). Secondly, dystrophin acts as a scaffolding protein to localize and normalize function of proteins involved in intracellular calcium and redox homeostasis. Lack of dystrophin can cause mislocalization and abnormal expression/activity of these proteins, leading to calcium mishandling and oxidative stress [65]. Oxidative stress damages the sarcolemma and increases intracellular calcium entry through stretch-activated channels (SACs) in the sarcolemma [20] and RyR2 in the SR [65,66]. (Figure 3).

#### *3.1. Dystrophin as a Membrane Stabilizer*

When dystrophin is absent, as in DMD, the sarcolemma of cardiac myocytes is less compliant, which is revealed during passive length distension [41]. This leads to increased sarcolemma damage evidenced by lactate dehydrogenase (LDH) release under normal preload and afterload conditions in isolated working hearts [67]. This damage is even more pronounced under stress with isoproterenol or partial aortic constriction [67]. Damage is thought to occur via small membrane disruptions (micro-tears) that can lead to transient extracellular calcium influx [62] (Figure 3). Extracellular calcium influx raises intracellular calcium concentration, subsequently activating calcium release from the SR and increasing calcium concentration even further. Calcium overload ultimately results in myocyte hypercontracture and cell death [41,68].

Evidence for membrane destabilization as a primary cause of calcium overload and cell death in DMD comes from studies employing membrane stabilizers in the context of dystrophin deficiency. Membrane stabilizers, most notably the tri-block copolymer P188, have shown efficacy in reducing stress-induced calcium overload, hypercontracture, and cell death in animal models of DMD [41,68]. Membrane stabilizer studies provide evidence that dystrophin confers protection against stress-induced mechanical damage to the sarcolemma, and stabilizing damaged portions of membranes can prevent calcium overload and myocyte death.

#### *3.2. Dystrophin as a Sca*ff*old Protein*

In addition to its function as a membrane stabilizer, accumulating evidence suggests a role for dystrophin in regulating ROS production and calcium handling within the myocyte. Dystrophin serves as a scaffold, helping to localize multiple proteins involved in calcium and oxidative homeostasis within the cell [65]. This allows for spatiotemporal control of ROS production and downstream signaling [66]. ROS products are important signaling molecules within the myocyte that regulate calcium cycling during physiological changes in cardiac load [69] (Figure 2). Abnormal or excessive ROS signaling in the absence of dystrophin [70] may contribute to cardiac pathology through aberrant calcium handling [70] (Figure 3).

Increased ROS production in DMD contributes to calcium overload from intracellular sources, namely the SR [66]. Indeed, ROS was found to underlie the hypersensitivity of RyR2 to increasing intracellular calcium concentrations in *mdx* cardiac myocytes, a mouse model of DMD [71]. Physiological stretch of isolated cardiac myocytes was found to induce a localized, rapid and transient increase in RyR2 calcium spark production, and this effect was amplified in *mdx* cardiac myocytes, leading to calcium waves [72]. Antioxidant treatment, microtubule depolymerization, and inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) all abrogated this effect. It was concluded from this study that myocyte stretch caused microtubule activation of NOX-2, which increased NOX-2 ROS production, sensitized RyR2 and subsequently increased calcium spark frequency [72]. This is a physiologically important mechanism to increase calcium release during increased cardiac load. However, in the context of the dystrophin-deficient myocyte, this effect is amplified [72] and may be a contributing factor to increased diastolic calcium concentration [18] (Figure 3). *Mdx* mice have increased expression and density of microtubules [73], increased expression and activity of NOX-2 [72], and a compromised endogenous reducing system [66], all of which may lead to enhanced production of ROS and increased RyR2 calcium leak. Taken together, these studies reveal an important role for dystrophin in proper function and activity of proteins involved in both ROS and calcium handling within the myocyte.

#### *3.3. Calcium Overload Leading to Myocyte Death, Fibrosis, and Dilation*

Increased intracellular calcium in the context of dystrophin deficiency is a key mediator in myocyte death and fibrotic development [12]. As discussed above, excess calcium originates from both intracellular stores through hypersensitivity of RyR2 and from extracellular influx via sarcolemmal micro-tears and increased SAC activity [20]. Additionally, there is evidence for increased L-type calcium channel (LTCC) activity, which increases calcium cycling to compensate for myocyte loss and decreased β-adrenergic activity in young *mdx* mice [22]. This is an additional avenue for intracellular calcium overload and a potential cause of arrhythmias in DMD [74]. Increased calcium concentration in the cytosol leads to activation of calcium-dependent proteases and protein degradation [12,75] (Figure 3).

Increased intracellular calcium also causes an increase in mitochondrial uptake of calcium. Increased mitochondrial calcium concentration occurs via the mitochondrial uniporter (MCU) as a consequence of RyR2 calcium leak [76] or increased LTCC calcium current [77]. Additionally, high cytosolic calcium can inhibit mitochondrial calcium extrusion via the mitochondrial sodium-calcium exchanger (NCLX) [78]. Increased mitochondrial calcium leads to enhanced mitochondrial ROS production, depolarization of the mitochondrial membrane, opening of the mitochondrial permeability transition pore (MPTP) [79,80] and decreased ATP production. Additionally, it was found that impaired communication between the LTCC and mitochondria in the absence of dystrophin decreases mitochondrial membrane potential and energy production [81]. The end result of these processes is cell death via necrosis and apoptosis [12,75,79,80] (Figure 3). Mitochondrial-mediated cell death was found to be an important contributor to disease progression and fibrosis development in multiple animal models of muscular dystrophy [82]. Deletion or chemical inhibition of cyclophilin-D, an enzyme that regulates mitochondrial-mediated necrosis resulting from excess calcium [83], improved the dystrophic phenotype and decreased the replacement of healthy myocytes with fibrotic tissue [82].

Myocyte death, as a result of calcium overload, leads to the release of intracellular components and enzymes that initiate an inflammatory response. Clinically, DMD myocardium displays alternating areas of myocyte hypertrophy, atrophy/necrosis and fibrosis with replacement of heart muscle by connective tissue and fat [84–86]. DCM progression in DMD is characterized by a distinctive pattern of fibrosis, initially affecting the posterobasal myocardium of the left ventricular free wall, progressing to the ventricular septum, and extending transmurally to affect the outer half of the ventricular wall [87]. There is likely a long subclinical phase of progressive fibrosis that starts early in the course of the disease [88–90]. The development of progressive fibrosis will eventually lead to overt cardiac disease, dilation, and decreased pump function.
